Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1375

Immunoglobulin Gene Analysis inDifferent B cell Lymphomas

With Focus on Cellular Origin and Antigen Selection

BY

MIA THORSÉLIUS

ACTA UNIVERSITATIS UPSALIENSISUPPSALA 2004

”Science is built up with facts, as a house is with stones. But a collection of facts is no more a science than a heap of stones is a house.”

Jules Henri Poincaré

List of Papers

I Thorsélius M, Walsh S, Eriksson I, Thunberg U, Johnson A, Backlin C, Enblad G, Sundström C, Roos G, Rosenquist R. So-matic hypermutation and VH gene usage in mantle cell lym-phoma. Eur J Haematol. 2002; 68: 217-24.

II Walsh SH, Thorsélius M, Johnson A, Söderberg O, Jerkeman M, Björck E, Eriksson I, Thunberg U, Landgren O, Ehinger M, Löfvenberg E, Wallman K, Enblad G, Sander B, Porwit-MacDonald A, Dictor M, Olofsson T, Sundström C, Roos G, Rosenquist R. Mutated VH genes and preferential VH3-21 use de-fine new subsets of mantle cell lymphoma. Blood 2003; 101: 4047-54.

III Thorsélius M, Kröber A, Thunberg U, Tobin G, Bühler A, Kienle D, Vilpo J, Döhner H, Stilgenbauer S, Rosenquist R. Strikingly homologous VH3-21/V 2-14 gene rearrangements in chronic lymphocytic leukemia despite different geographical origin. Manuscript.

IV Thorsélius M, Walsh SH, Thunberg U, Hagberg H, Sundström C, Rosenquist R. Heterogeneous somatic hypermutation status con-founds the cell of origin in hairy cell leukaemia. Leukemia Re-search, in press 2004.

V Thunberg U, Bånghagen M, Bengtsson M, Christensen LD, Geisler CH, Gimsing P, Lenhoff S, Mortensen BT, Olofsson T, Simonsson B, Andersen NS, Sundström C, Swedin A, Sällström JF, Thuresson B, Westin J, Carlson K. Linear reduction of clonal cells in stem cell enriched grafts in transplanted multiple mye-loma. Br J Haematol. 1999; 104: 546-52.

Reprints were made with permission from the publishers.

Contents

Introduction...................................................................................................11B cell differentation..................................................................................11

Antigen independent B cell maturation in the bone marrow ...............12Antigen dependent B cell development in the periphery.....................13

The immunoglobulin genes ......................................................................15Immunoglobulin organization .............................................................16Rearrangement of the IgH locus ..........................................................16Rearrangement of the IgL loci .............................................................17Somatic hypermutation........................................................................18Class switch recombination .................................................................19Diversity of the immunoglobulins .......................................................20

Ig rearrangement as a clonal marker ........................................................20B cell lymphomas.....................................................................................21

Classification .......................................................................................21Mantle cell lymphoma .........................................................................21Chronic lymphocytic leukemia............................................................23Hairy cell leukemia..............................................................................25Multiple myeloma................................................................................26

Stem cell transplantation ..........................................................................27Autologous transplantation..................................................................27Allogeneic transplantation ...................................................................27

PCR quantification of tumor content........................................................28Origin of B cells lymphomas....................................................................29VH gene usage and antigen selection........................................................31

Aims..............................................................................................................34

Material & Methods......................................................................................35Patients and tumor specimens ..................................................................35PCR amplification and sequencing of Ig gene rearrangements................36Analyzes of the Ig sequences ...................................................................37Quantitative PCR......................................................................................37

Results & Discussion ....................................................................................38VH gene usage and somatic hypermutation in MCL ................................38Further characterization of the VH3-21+ CLL subset ................................40

Somatic hypermutation status and indication of antigen selection in HCL..................................................................................................................41Quantification of tumor content in MM autotransplants..........................43

Conclusions...................................................................................................45

Acknowledgements.......................................................................................46

References.....................................................................................................48

Abbreviations

AID Activation-induced cytidine deaminase ALL Acute lymphoblastic leukemia ASO Allele specific oligonucleotide ATM Ataxia telangiectasia mutated BCR B cell receptor BCL B cell lymphoma C Constant CDR Complementarity determining region CLL Chronic lymphocytic leukemia CSR Class switch recombination D Diversity DLBCL Diffuse large B cell lymphoma FDC Follicular dendritic cell FL Follicular lymphoma GC Germinal center HCL Hairy cell leukemia Ig Immunoglobulin IgH Ig heavy chain IgL Ig light chain J Joining KDE Kappa deleting element MCL Mantle cell lymphoma MCL-BV MCL blastoid variant MM Multiple myeloma NHEJ Nonhomologous DNA end-joining PCR Polymerase chain reaction Q-PCR Quantitative polymerase chain reaction RAG Recombination activating genes RSS Recombination signal sequence SHM Somatic hypermutation TdT Terminal deoxyribonucleotidyl transferase V Variable VH Variable heavy chain VL Variable light chain

Kappa Lambda

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Introduction

Leukemias and lymphomas are tumors derived from cells in the immune system. Since both lymphatic leukemias and lymphomas can originate from a number of different lymphoid cells in the immune system, these diseases consist of many entities and are very heterogeneous tumors both regarding morphological, molecular and clinical features1. The focus of this thesis will be on lymphomas originating from B cells, i.e. B cell lymphomas (BCLs).

During development the B cell goes through several dangerous genetic events where the normal differentiation can fail and transform the cell into a tumor cell. This has been described as the dark side of B cell differentiation2.In particular, the process of producing a functional and specific antibody, through gene rearrangement of the immunoglobulin (Ig) genes and somatic hypermutation (SHM), puts the cell at a high risk of undergoing malignant transformation. However, the enormous diversity of antibodies created in B cells via unique Ig gene rearrangements and SHM offers a tool to monitor tumors derived from these cells. Furthermore, since the Ig gene rearrange-ments can undergo genetic changes during B cell development, for instance introduction of SHM, studies of the Ig genes can provide information at which developmental stage the normal cell transformed into a tumor cell3.

To understand the origin of BCLs it is essential to know the basics of the normal B cell development and Ig gene rearrangement process, which will be outlined in the following sections.

B cell differentation B cell development is the programmed process in which a stem cell matures by passing a number of cell stages to eventually become a memory B cell that expresses Ig molecules on its surface, or a plasma cell, which secretes antibodies4,5 (Figure 1). In both these cell types the produced Ig molecules have achieved a high degree of antigen affinity during differentiation. The specific maturation stages in B cell development have been defined by cell size, growth properties and Ig gene rearrangement status.

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Figure 1. B cell development - from a self renewing stem cell to a terminally differ-entiated Ig secreting plasma cell. GC=germinal center.

Antigen independent B cell maturation in the bone marrow The first stage of B cell maturation is antigen independent and starts in the bone marrow where the self renewing hematopoietic stem cells are located. The hematopoietic stem cell differentiates into a lymphoid stem cell express-ing the surface marker CD34 and on some co-expression of CD10. The ear-liest identifiable B cell progenitor is the pro-B cell, which displays CD34, CD19 and CD10 on the cell surface6. The rearrangement process of the Ig heavy chain (IgH) locus is initiated at this stage, and expression of enzymes involved in Ig rearrangement, such as the nuclear terminal deoxyribonucleo-tidyl transferase (TdT) together with the recombinase enzymes recombina-tion activating genes (RAG) 1 and RAG-2 are also characteristic of the pro-B cell stage7. The rearrangement process of the Ig genes will be outlined in further detail in a subsequent section.

The differentiation of a pro-B cell into a precursor B cell (pre-B) requires the presence of bone marrow stromal cells, which directly interact with the early B cells and secrete various cytokines thereby promoting their matura-tion8. The pre-B cells are defined by loss of CD34 and the cessation of TdT expression. They also express the pre-B cell receptor (pre-BCR) complex consisting of the newly produced heavy chain Ig, a surrogate light chain encoded by the Vpre-B and 5 genes and the / heterodimers6. At this stage, the rearrangement of the Ig light chain (IgL) loci occurs. For contin-ued development of a pre-B cell into an immature B cell, one of the two IgL loci, kappa ( or lambda ( has to be successfully rearranged and the sur-rogate light chain should be replaced by the functional light chain6. The im-mature B cells can then express membrane IgM on their cell surface and have no expression of TdT or RAG1/2 enzymes9. The expression of a func-tional BCR on the cell surface marks the end of the antigen independent

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stage, and the immature B cell can exit the bone marrow and migrate to the periphery.

Antigen dependent B cell development in the periphery A change in RNA processing of the heavy chain primary transcript makes it possible for the cell to express both IgM and IgD on the cell surface. The cell has now become a mature B cell, also known as a naïve B cell, which is capable of interacting with antigen10. The mature B cells that have left the bone marrow recirculate between blood and the lymphatic system. For the cells to be activated, antigen is required. If antigen is encountered affinity maturation of the Ig molecule occurs, after which the cell can differentiate into either a memory B cell or a plasma cell. However, in the absence of antigen encounter, naïve B cells in the periphery have a short life span of only a few weeks before dying by apoptosis10,11.

Antigen encounter and germinal centers Antigens are molecules or substances which can bind a BCR or a T cell re-ceptor and start an immune response either independently, or with help of a carrier12. Foreign antigens that are introduced into the body become concen-trated in various peripheral lymphoid organs, such as lymph nodes, spleen

Figure 2. Schematic picture of a lymph node.

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and mucosa-associated tissue. In the lymph node (Figure 2), more than 90% of the antigens become trapped as the lymph passes through. When a B cell circulates through the node and encounters the antigen it is specific for, the lymph node becomes a site for intense B cell activation12.

Figure 3. Schematic figure of a GC. The GC is composed of three areas: 1) the dark zone where the activated centroblasts divide rapidly and may undergo SHM; 2) the light zone, where the centrocytes are exposed to antigens presented by FDCs. If antigen binding fails the B cell undergoes apoptosis, whereas if the antigen receptor shows a high affinity for the antigen the centrocyte survives and can mature into a memory B cell or a plasma cell after interaction with T cells; and 3) the mantle zonewhich surrounds the light zone and is mainly populated with naïve B cells.

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Antigens are presented to B cells by two major populations of antigen presenting cells; interdigitating dendritic cells located in the extra follicular areas and follicular dendritic cells (FDCs) in the follicles of the lymph node12. The activation of antigen-specific B cells occurs in association with T cells and interdigitating dendritic cells in the extra follicular areas. The activated B cell, now known as a centroblast, migrates into the follicle and gives rise to a germinal center (GC)13 (Figure 3).

The GC consists of three zones of varying composition, the dark zone, the light zone and the mantle zone12. In the dark zone rapid proliferation of the centroblasts occurs, thereby creating the dense structure. The surface Ig ex-pression is down-regulated on these centroblasts, making it possible for the SHM process to be initiated and the introduction of point mutations in the Ig genes to occur (see further below)13. The intense proliferation of centroblasts displaces the other recirculating follicular B cells, which instead form the follicular mantle zone. As the cell enters the light zone surface Ig expression is up-regulated and the cell becomes smaller. At this stage, the descendant cells originating from the centroblasts are known as non-dividing centro-cytes. These cells make up the structure of the less dense light zone together with a network of FDCs and T cells13. In the light zone, the centrocytes are exposed to different antigens presented by FDCs, and at this point the fate of the cell is decided. If the centrocyte fails to bind the immune complexes held on the surface of the FDC it will die by apoptosis, whereas if the surface Ig instead has a high affinity for the immune complex, the cell will be rescued from apoptosis by up-regulating survival signals (e.g. Bcl-2)12. Interaction between centrocytes and T helper cells could also lead to an Ig isotype switch, described in a following section. The centrocyte will thereafter dif-ferentiate to either a memory B cell, with expression of surface Ig, or a plasmablast, which exits the GC to become an Ig secreting plasma cell12.

The immunoglobulin genes All Ig molecules share structural features and are built up by four polypep-tide chains, two identical heavy chains and two identical light chains that are joined by disulphide bridges. The molecule can be divided into two main functional regions, the variable (V) region and the constant (C) region found in both the heavy and light chain Ig. The V region varies greatly between different Ig molecules and is responsible for antigen binding, while the C region is more conserved and is involved in the biological functions of the molecule14.

The membrane-bound Ig molecule does not constitute the entire antigen binding receptor on B cells, rather it is accompanied by two accessory pro-teins, Ig and Ig , to form a complete BCR. Upon activation by foreign

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antigen, the heterodimer Ig /Ig is required for interaction with intracellular signaling molecules10.

Immunoglobulin organization The IgH locus is located on chromosome 14q32 and the two IgL loci ( and

) on chromosomes 2 and 22, respectively. The IgH locus consists of clus-ters of VH, diversity (D), joining (JH) and C germline gene segments. There are approximately 123 VH gene segments15, of which 51 represent functional genes and the remaining 72 are non-functional pseudo-genes. The IgH locus also contains approximately 30 D gene segments, 6 JH genes and a series of C segments16. Based on sequence homology the functional VH genes are divided into seven families (VH1-VH7), with at least 80% homology within each VH gene family. The VH3 family is the largest, consisting of 22 gene segments, followed by the VH1 and VH4 families consisting of 11 members each16.

The two IgL loci are organized in a similar way to the IgH locus, although they do not contain any D gene segments and thus only consist of variable (VL), joining (JL) and constant (CL) gene segments. Between 36 and 40 func-tional V and between 29 and 33 functional V gene segments have been identified, together with 5 functional J and 7 functional J gene seg-ments17,18. The light chain is expressed in approximately 60% of mature B cells, while is found in the remaining 40%.

Rearrangement of the IgH locus A functional heavy chain molecule is created by recombination of the differ-ent VH, D and JH segments, a process which can be divided into three differ-ent stages: recognition, cleavage and rejoining19,20.

The germline VH, D and JH gene segments are flanked by unique recom-bination signal sequences (RSSs), which are recognized by the recombina-tion system and are required for cleavage. The RSSs are located 3´ to each VH gene segment, 5´ to each JH and on both sides of each D segment. Each RSS is composed of a conserved heptamer and an AT-rich nonamer se-quence separated by a spacer sequence of 12 or 23 base pairs, corresponding to one or two turns of the -helix14. A signal sequence having a one helix turn spacer, such as the D segment RSS, can only be combined with a se-quence that has a two turn spacer, such as the VH and JH RSSs14. This en-sures that the VH, D and JH segments rearrange in the proper order and that recombination of segments of the same type does not occur.

The rearrangement of the IgH locus begins with the joining of the D and JH gene segments, followed by VH joining to the DJH segment (Figure 4A). The RSSs flanking the D and JH segments are recognized by the RAG1/2 enzymes which introduce a nick to one of the DNA strands, thereby initiat-

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ing the rearrangement process21,22. This leads to the formation of a hairpin structure, followed by a double stand break with removal of the intervening DNA. The same event occurs during VH to DJH joining. The cutting of the hairpin generates a site for the insertion of palindromic (P) nucleotides23.Addition of random nucleotides can also occur at the cut ends of V, D and J coding sequences, the so-called N-region, via the enzyme TdT23. The DNA is then joined together by the nonhomologous end-joining (NHEJ) factors (Ku70, Ku80, XRCC4, DNA ligase 4, DNA-PKcs and Artemis), which are proteins known to repair double stand breaks24. Addition of nucleotides be-fore ligation of the strands can cause an out-of-frame sequence resulting in a non-functional protein, but it may also lead to an increased variability of the rearrangement. The most hypervariable region of the V(D)J sequence is the N-D-N junction, known as the complementarity determining region (CDR) 3. Together with the CDR1 and CDR2, which are located in the VH region, the CDR3 encodes the sequence of the Ig molecule most important in anti-gen recognition. The sequences in between the CDRs exhibit far less varia-tion and are known as framework regions (FRs) (Figure 4B)14.

When the V(D)J segment is combined with a C gene segment the IgH lo-cus rearrangement is completed. If a non-functional IgH rearrangement has been produced the second IgH allele starts to rearrange, if however the IgH is functional the rearrangement of one of the IgL loci begins25,26.

Rearrangement of the IgL loci The IgL rearrangement process is similar to the one that occurrs at the IgH locus, but since the light chain loci do not contain any D segments only one recombination step between the VL and JL genes takes place25,26 (Figure 4A). The IgL loci are rearranged in a hierarchic order, starting with rearrangement of one of the Ig alleles. If the first Ig allele fails to produce a functional light chain, the rearrangement can be deleted by rearrangement of the kappa deleting element (KDE) before the next V allele is rearranged27.

The KDE is located approximately 24 kb downstream of the C gene segment and mediates a deletion process of the Ig locus by a site-specific rearrangement28-31. Two different KDE recombinations can inactivate a non-functional V rearrangement. The RSS at the 5´ flank of the KDE can either rearrange to the RSS in the J -C junction, deleting the C region of the Iglocus, or to a RSS flanking the V at the 3´side, leading to deletion of the Jand C regions28-31. In both alternatives the V rearrangement becomes in-complete and non-functional. If the rearrangements of both V alleles are deleted by the KDE, rearrangement of the Ig locus will follow. However, it is not known for definite whether V gene rearrangement and KDE involve-ment is required for the Ig locus to begin rearrangement. B cells with rear-rangement of the Ig locus have been observed, although the Ig allelesremained in germline configuration26,32.

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A

B

Figure 4A) Rearrangement of the IgH and IgL loci and the resulting antibody com-posed of two identical heavy chains and two identical light chains. B) Structure of the rearranged IgH and IgL loci. The CDRs encodes the sites involved in antigen binding, where CDR3 represents the most variable region containing the joining regions with inserted N-nucleotides. CDR: complementarity determining region, FR: framework region

Somatic hypermutation When a B cell encounters antigen, the Ig molecule can undergo affinity maturation, which generally occurs in the GC. Affinity maturation alters the sequence of the Ig genes, by the SHM mechanism, to gain a higher affinity for the antigen. The target for SHM are the rearranged V regions within the IgH and IgL loci where these mutations occur predominantly in the CDRs of

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the VL and VH genes14. The rate of introduced changes into the V gene se-quences is close to 10-3 mutations per base pair per generation, which is 106

times higher in comparison with the frequency of spontaneous mutations occurring in other genes33. The most common change introduced by the SHM mechanism is nucleotide substitutions, but insertions, duplications and deletions also occur, although infrequently34. The activity of the SHM proc-ess is dependent on cell proliferation, yet the mechanism is not fully under-stood35-38. Activation-induced cytidine deaminase (AID) has been shown to be an essential component of the SHM mechanism, where it deaminates the cytosines involved in the initiation of the mutation process33. A mismatch repair system has been suggested to trigger the second part of the SHM mechanism, effected by an error-prone repair of mismatched DNA33. The mutations are introduced in a non-random manner in that the V region muta-tions primarily occur within certain DNA sequence motifs targeted by AID, known as ‘hot-spot’ motifs: RGYW and its complement, WRCY (R=A/G, Y=C/T and W=A/T) and the recently refined motif DGYW/WRCH (D=A/G/T and H=T/C/A)39. The introduction of a mutation can either result in a change of the amino acid sequence, known as a replacement mutation or a nucleotide change without alteration of the amino acids, a so-called silent mutation. A higher frequency of replacement mutations compared to silent mutations in the CDRs is considered to be characteristic of antibodies influ-enced by antigen40. It is currently not resolved whether the SHM process continues when the second main event of antibody maturation, class switch recombination (CSR), occurs.

Class switch recombinationThe CSR process renders a change in the isotype of the Ig molecule from IgM to IgD, IgG, IgA or IgE, giving rise to different effector mechanisms of the antibody41 and like SHM this process takes place in the GC. The CH gene segments, that encode the isotype of the antibody, are located downstream of the JH region, and each CH, except C , is flanked by a switch region located 5´of the gene segment. The CSR is initiated by a cytokine signal which de-termines which particular isotype the cell will express after switching42.

According to the conventional switching model, two switch regions are brought together creating a loop of intervening CH region genes which will be deleted42. AID has been shown to be required for the CSR mechanism, most likely in the beginning of this process33. The DNA breaks occurring in the switch regions have sticky ends which are believed to be processed by an error prone repair mechanism before they are ligated. Ligation of the switch ends is probably performed by the NHEJ proteins, which are also active during V(D)J recombination43-45.

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Diversity of the immunoglobulins The above described processes can result in production of an antibody with high specificity for its particular antigen. In summary the following mecha-nisms contribute to the diversity when an antibody is produced:

1. Different VHDJH joining combinations 2. Different VLJL joining combinations 3. Different combinations of rearranged IgH and IgL genes 4. Insertion of N and P nucleotides in the junctional regions 5. SMH6. CSR

The probability that two B cells have identical antibodies without being de-rived from the same ancestor or being exposed to antigen selection is ex-tremely low. The likelihood that two IgH loci recombine the same V, D, J gene segments at random is 1/8262 (1/51[VH] x 1/27[D] x 1/6[JH]) and for the and rearrangements the probability is 1/200 (1/40[V ] x 1/5[J ]) and 1/231 (1/33[V ] x 1/7[J ]), respectively. Furthermore, the probability that a particular VHDJH rearrangement would compose a BCR in combination with a certain V J in two unselected cells is only 1 in 1.6 million. Insertion of nucleotides in the joining region together with three different reading frames for the D segment increase the diversity even more and diminish the likeli-hood of producing two identical antibodies to less than 10-12 46,47. The high specificity, which might be further increased by somatic mutation and iso-type switch, makes the Ig genes very suitable clonal markers for analyzing B cell tumors.

Ig rearrangement as a clonal marker B cell derived malignancies arise from a clonal expansion of a single B cell. In BCLs, the cell which has given rise to the tumor has passed the early stages of development and carries both specific IgH and IgL rearrangements. Furthermore, the Ig genes may also have undergone SHM, resulting in a rearrangement unique for that specific cell and its clonal progeny3. The IgH and IgL rearrangements can therefore be used as specific markers for the tumor population.

Since the Ig gene rearrangements can underego SHM and CSR during B cell differentiation it is possible to detect where in its development the cell transformed into a tumor cell. By comparing the V region sequence to the germline sequence, the presence or absence of SHM will be revealed, indi-cating whether the cell of origin has encountered antigen in the GC or not3.A deviation of 2% or more from the closest germline sequence has typically

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been used as a cut off when determining a mutated VH gene. This is done to ensure that polymorphisms and Taq enzyme errors are not counted as muta-tions48-50. Analysis of the Ig gene rearrangement can also be used to follow clonal evolution of a tumor as well as be applied to detect ongoing SHM. Another application of Ig rearrangement analysis is as a marker of the tumor clone in detection of minimal residual disease by quantitative polymerase chain reaction (Q-PCR).

B cell lymphomas Classification As leukemias and lymphomas consist of a very heterogenous group of tu-mors, the classification of tumors derived from hematopoietic and lymphoid tissue has constantly been changing. Early classification was based on mor-phology, of which pathologists sometimes disagreed51. Later on immuno-phenotype analysis has become the standard complement to morphologicl evaluation, especially for leukemia classification. The classification used today is the WHO classification1 which is a modified and extended version of the “Revised European-American Classification of Lymphoid Neo-plasms” (REAL) classification from 199452. This classification uses all available information to define a disease entity – morphology, immunophe-notype, genetic features and clinical features. With the WHO classification, the former categorization of malignant lymphomas into either of two main groups; Hodgkin’s disease and non-Hodgkin’s lymphoma, became outdated. Instead, three main groups were defined, T cell lymphoma, BCL and Hodg-kin’s lymphoma, of which the majority of tumors derived from B cells (80%)1. There are currently approximately 20 categories of mature BCLs in the WHO classification. In Sweden about 2000 lymphomas are diagnosed every year, representing approximately 5% of all malignant tumors53. The following sections will focus on four types of BCL: chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), mantle cell lymphoma (MCL) and multiple myeloma (MM)

Mantle cell lymphomaIn Sweden 70 new cases of MCL are diagnosed every year, which corresponds to approximately 4% of all lymphomas53.The disease is much more common in men than women (3:1 ratio) and the median age of diagnosis is 65 years 54,55. MCL almost invariably presents with advanced disease with bone

marrow involvement (Ann Arbor stage IV), lymphadenopathy and splenomegaly. Other involved extranodal sites include the gastrointestinal

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tract and the Waldeyer’s ring54-56. The disease has a poor prognosis with a median survival of 3-4 years, although survival rates have improved as new treatment alternatives have emerged (see below)57.

MCL is comprised of two morphological subtypes, the typical MCL and MCL blastoid variant (MCL-BV)1. Tumor cells of the MCL-BV resemble lymphoblasts or larger and more pleomorphic cells58. The MCL-BV has a high proliferative activity contributing to a more aggressive clinical course compared to typical MCL, with a median survival of less than 2 years59. The typical variant consists of small to intermediate sized cells, with irregular nuclei and scarce cytoplasm (Figure 5A). There are also three different histo-logical growth patterns in MCL. In the mantle zone pattern, tumor cells infil-trate the mantle zone, whilst with a nodular growth pattern a more extensive infiltration, resembling a follicular structure, is observed. In the diffuse growth pattern the architecture of the GC can be totally obliterated due to the large number of invading tumor cells.

The immunophenotype in MCL corresponds to a mature B cell with ex-pression of CD19, CD20, CD22, CD5, CD79a and high expression of sur-face IgM, with a peculiar strong predominance of light chain expres-sion56,59. The immunophenotype of MCL and CLL are rather similar with the mature B cell markers and CD5 expression found in both diseases, but it is possible to distinguish between them due to the lack of CD23 and the over-expression of cyclin D1in MCL57.

The translocation t(11;14)(q13;q32) is present in almost all MCL cases and is characteristic of MCL. It involves the Bcl-1 gene and the IgH locus, resulting in an overexpression of cyclin D1 which can be detected in a ma-jority of cases60-63. Thus, detection of overexpression of cyclin D1 or alterna-tively, the presence of the translocation is required for the MCL diagnosis1.Cyclin D1 plays an important role in the pathogenesis of MCL, since it is involved in cell cycle regulation. A constant overexpression of cyclin D1 leads to an increase in cell replication, although additional alterations are probably required to induce lymphoma development63. Another genetic ab-normality found in 20-40% of MCL cases is the 11q22-23 deletion involving the ataxia telangiectasia mutated (ATM) gene59,64-67. ATM is involved in the signaling pathway that responds to double strand breaks. It phosphorylates p53 in the presence of DNA damage, which results in cell cycle arrest, be-fore repair of the damage has occurred. The loss of ATM increases the risk of other chromosomal aberrations, which have been shown in MCL68.

Recent microarray studies have revealed a group of MCL patients with shorter survival showing a high expression of genes involved in prolifera-tion69. Patients belonging to this group with poor survival can be identified by the proliferation rate measured either by the mitotic index or by expres-sion of Ki-6769,70.

MCL is the lymphoma entity considered to have the worst prognosis of all lymphomas and to be an incurable disease. During the last couple of

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years the prognosis associated with this disease has changed, with the intro-duction of high dose chemotherapy and addition of anti-CD20 monoclonal antibody (rituximab) in the induction treatment71. Outcome is currently in level with other high grade lymphomas but a cure has still not been achieved71,72. Also, for elderly patients the outcome is improved with the use of multi-drug combinations and rituximab73. The only curative treatment for MCL is allogeneic bone marrow transplantation but transplantation related complications and the limited availability of suitable donors make this pro-cedure an option only for a minority of patients57. Autologous stem cell transplantations with different myeloablative regimens is another treatment approach which shows promising results although this treatment strategy is mainly applicable in younger patients74-76.

Chronic lymphocytic leukemia CLL is the most common leukemia among adults, with 400-500 newly diagnosed cases in Sweden every year. The majority of affected patients are men, with a male:female ratio of 2:1. It is occurs mostly in the aging population, with a median age at diagnosis of 65 years1. Many CLL patients are

asymptomatic at the time of diagnosis and the disease is detected inciden-tally by routine full blood count77, but it may also present with lymphade-nopathy, infections, hemolysis or non-specific symptoms such as weight loss, fatigue due to anemia and night sweats1,77. The most common sites of involvement are the bone marrow, peripheral blood and lymph nodes78.

CLL is defined as a clonal expansion of small monomorphic B cells (Fig-ure 5B), with expression of CD19, CD5 and CD23 and low levels of surface IgM79. The clonal expansion of tumor cells in CLL is associated with in-creased cell survival where the cells avoid apoptosis rather than with a high proliferation rate63. The anti-apoptotic protein Bcl-2 has been shown to be overexpressed in CLL cells and expression of other genes involved in apop-tosis are also deregulated, whereas genes involved in cell proliferation (eg. Ki67, cyclin A, cyclin B1 and p16) have been shown not to be expressed in CLL80. The survival rate in CLL has a very wide range. In the most aggres-sive cases survival may be less than 2 years81, whereas in indolent form, patients can survive without treatment for more than 20 years82.

During the last two decades the Rai and Binet staging system has been the standard in prognosis evaluation83,84. However, owing to heterogeneity in the clinical behaviour of the disease it is of great importance to identify new prognostic factors that are better at predicting the outcome of CLL. It has recently been revealed that CLL is comprised of at least two clinical entities, with a clear difference in survival depending upon whether the VH genes of the Ig rearrangement contain SHM or not49,50. In the initial studies, cases with somatically mutated VH genes were shown to have a better prognosis

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with either a median survival of 25 years or greater as compared to the un-mutated CLLs that had a median survival of only 8 to 9 years49,50. The usage of the VH gene mutation status as a prognostic marker has thereafter been confirmed in several multivariate studies85,86. Another interesting marker is ZAP-70, which has been proposed to correlate with the VH gene mutation status, since CLLs with unmutated VH genes show expression of ZAP-70, while CLLs with mutated VH genes are mainly negative for ZAP-7087,88.ZAP-70 can be detected by flow cytometry87,88, which is efficient from a diagnostic point of view as compared to the more time-consuming VH gene analysis. CD38 has also been suggested as a surrogate marker for the muta-tion status in CLL, however the expression has been demonstrated to vary over time and a poor correlation to mutation status has been shown, although CD38 may serve as an independent prognostic marker50,86,89-92.

Figure 5. Immunostainings of (A) MCL (B) CLL (C) HCL and (D) MM

There is no characteristic translocation in CLL, but deletions are fre-quently occurring in this disease. The most often found is the deletion of 13q14 (~50%)93,94, which has been correlated to an improved survival93,95.

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Other detected aberrations are deletion of 11q22-23 (including the ATM gene), trisomy of chromosome 12 and 17p deletion (harbouring the p53 gene)64,66,93-96, where the 11q and 17p deletions correlate with a worse out-come64,93,96.

Since CLL is considered an incurable disease, asymptomatic patients are generally not treated77. For elderly patients with a non aggressive disease, chlorambucil is often used as the initial treatment77. The introduction of purine analogues has improved the treatment, in particularly in younger pa-tients and patients with a more aggressive disease46. Lately, monoclonal antibodies against CD20 (rituximab) and CD52 (Campath1H) have shown increased remission rates and prolonged survival especially in combination with chemotherapy (e.g. fludarabine and cyclophosphamide)97-99. Althoughhigh dose chemotherapy with autologous stem cell transplantation is another alternative in treatment of CLL it has not been used extensively due to diffi-culties in the clearing of tumor cells from blood and bone marrow46. Also, allogeneic stem cell transplantation, with reduced induction therapy, is an alternative with encouraging results, but is currently still under investiga-tion100.

Hairy cell leukemia HCL is a slowly growing leukemia, in which the increasing amount of tumor cells is due to a prolonged cell survival rather than a high proliferation rate101. It is an uncommon disease contributing to ~1.5% of the lymphomas. In Sweden approximately 25 new cases of HCL are diagnosed every

year53. HCL predominatly affects men, the male female ratio being 4:1, and the mean age at diagnosis is approximately 50 years102.

The disease often presents with non-specific symptoms such as weakness, weight loss and dyspnoea102. Due to the infiltration of tumor cells into the bone marrow and spleen, which are the major sites of involvement, pancyto-penia and splenomegaly are common early findings in patients with HCL. The tumor cells have a characteristic morphology with irregular cytoplasmic projections (Figure 5C), and they also contain large amounts of tartare-resistant acid phosphatase (TRAP), which is not found in any other circulat-ing blood cells102. The postulated cell of origin is a post-GC B cell, although the hairy cell belongs to the tumor cells of which the normal counterpart is not clearly defined. The immunophenotype correlates to a mature B cell, with expression of CD19, CD20 and FMC-7, but the cells also express the monocyte marker CD11c, IL-2 receptor CD25 and the -chain of integrin B-Ly7, CD103, which are used as markers to confirm a HCL diagnosis79. No cytogenetic abnormalities have been found to be characteristic of HCL103,yet the most common cytogenetic alteration in HCL is, as in many other lymphomas, a structural abnormality involving chromosome 14, engaging

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the IgH locus104,105. Trisomy of chromosome 5 and structural abnormalities involving band 5q13 have also been reported as rather common amongst the cytogenetic findings in HCL103 in contrast to other leukemias and lympho-mas.

The most often used treatments of HCL nowadays are purine analogues (nucleosides) such as chlorodeoxyadenosine and deoxycoformycin. -interferon, which previously was the treatment of choice, is still used, but not to the same extent. Another treatment alternative is the anti-CD20 mono-clonal antibody, rituximab, which has fewer side effects compared to other treatments106. It is especially useful in patients resistant to nucleosides and -interferon. Due to the newer effective treatments, the survival in HCL has increased from median 50 months102 to approximately 80% of the patients still living after 10 years107.

Multiple myeloma MM is a plasma cell malignancy located in the bone mar-row. It is one of the most frequently occurring lymphomas with 500 new diagnosed cases in Sweden every year. The median age at diagnosis is 70 years and it is more common in men than women53. MM often presents with bone pain, due to the bone destruction caused by the infiltrating tumor

cells. Also pathologic fractures, because of bone lesions, are common in patients with MM as well as renal failure, hypercalcemia, anemia and recur-rent infections108-110.

The normal counterpart of the myeloma cell is a terminally differentiated B cell which has passed the GC, and has consequently undergone SHM and isotype switching, and reached the plasma cell stage before transformation (Figure 5D). The tumor cells express CD38, CD79a and CD138 (syndecan-1), which is normally found on plasma cells, but lack expression of CD19 and CD201. Another deviation from the normal counterpart is expression of CD56/58111. Myeloma cells secrete Ig, normally IgG or IgA, which can be detected in serum and urine. The genetic instability in MM is high and they generally display more chromosomal aberrations than other lymphomas110.The most frequently occurring chromosomal aberration involves the IgH locus on chromosome 14q32, which recurrently translocates to 11q13 (cyk-lin D1), 4p16 (FGFR3 & MMSET), 6p21 (cyklin D3), 16q23 (c-maf) and 20q11 (maf b)112-114. Translocations of 8q24 (c-myc)114 and monosomy of chromosome 13115 are further aberrations present in MM, and both correlate with poor prognosis116-118.

MM is still an incurable, fatal lymphoma and although treatment has im-proved, the median survival is only 3 years119. The standard treatment in elderly patients is melphalan in combination with prednisolone110,120. Che-motherapy with different agents gives a higher response rate but the survival

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rate does not differ120. Some of the chemotherapy regimens i.e. vincristine, adriamycin and dexametasone, are often used as induction therapy because their effects on stem cells are less toxic110. This is of great importance since autologous peripheral blood stem cell transplantation followed by high dose melphalan is considered to be the standard treatment in younger patients with MM121. Autologous transplantation has been shown to prolong survival and for these patients the median survival is 4-5 years119. New treatment strategies with promising results involve; thalidomide, thalidomide ana-logues, proteasome inhibitors and mini-allogeneic transplantations119,121.

Stem cell transplantation There are two major stem cell transplantation alternatives in hematological malignancies, autologous and allogeneic. In this section the basic concepts of the two categories will be described, with focus on their use in MM.

Autologous transplantation As mentioned above autologous peripheral blood stem cell transplantation has become the standard treatment in younger patients with MM. In this type of transplantation the stem cells originate from the patient, with the advan-tage that there is no problem finding a donor and the risk of a rejection of the graft is eliminated.

Stem cells are normally located in the bone marrow but with the use of chemotherapy and hematopoietic growth factors stem cells can be mobilized into peripheral blood where they are easier to harvest. This is the most com-monly used procedure in the harvesting of stem cell for autologous trans-plantations to date. After collection of the peripheral blood stem cells the patient is treated with high dose chemotherapy to remove remaining tumor cells from the body. This therapy not only affects the tumor cells but also knocks out normal hematopoiesis. The harvested cells are transplanted back into the patient to reconstitute the hematopoesis. Autologous transplantation, in combination with high dose chemotherapy has improved response rates and prolonged survival in patients with myeloma119. The disadvantage of this approach is that tumor cells from the patient can remain in the graft and con-tribute to relapse. Furthermore, there is no graft versus myeloma effect in autologus transplantation, as seen in allogeneic transplantation.

Allogeneic transplantation The source of the stem cells in an allogeneic transplantation is a HLA-matched donor. The procedure is similar to the autologous transplantation where a high dose chemotherapy treatment is followed by transplantation of

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stem cells as a rescue in this approach also. The major drawback with al-logeneic transplantations is primarily the difficulty of finding a matching donor. Although even with a so-called perfect match the procedure is still considered a high risk treatment. In MM the mortality rate associated with allogeneic transplantations is high (~40%) primarily as a consequence of infection or graft versus host disease122. Despite the advantages of allogeneic transplantations such as the absence of contaminating tumor cells and a po-tential graft versus myeloma effect119,123, allogeneic transplantation plays a small role in myeloma due to the high complication risk. In the so-called mini-allotransplants, nonmyeloablative conditioning regimens are combined with donor lymphocyte infusions in an attempt to achieve a graft versus myeloma effect with a lower toxicity than in full allogeneic transplanta-tion119,124. Yet the frequency of graft versus host disease is lower in this pro-cedure it is still a major concern affecting 45-55% of transplanted patients119.

PCR quantification of tumor contentQ-PCR, with the IgH rearrangement as a marker of the malignant B cells, is a highly sensitive method to detect small amounts of tumor cells in periph-eral blood or bone marrow samples125,126. It is constructed as a two step PCR where in the first step a consensus primer recognizing the sequence of the VHgene is used together with a consensus primer for the JH gene segment. In the second PCR the JH gene primer is replaced by an allele specific oligo (ASO) primer constructed to be unique for the tumor clone VH gene rearrangement. Two different methods are used to carry out the quantification, limiting dilu-tion Q-PCR and real-time Q-PCR. The limiting dilution assay is based on the ability to detect a single IgH copy from a tumor cell within a sample contain-ing a background of normal hematopoietic cells127. This is accomplished by diluting a sample in a series of different concentrations, which are then am-plified and analyzed for all-or-none positive reactions. The number of tumor cells in the sample could then be calculated based on Poisson distribution statistics128. In the real-time Q-PCR method, quantification is performed by automated colorimetric detection, monitoring the PCR products by measur-ing fluorescence in each cycle of the PCR amplification. PCR products are quantified in the exponential phase of the PCR using this method, which is in contrast to limiting dilution assay where end-point analysis is applied127.

Q-PCR has been used in acute myelogenous leukemia to monitor the tu-mor clone and predict relapses. A tumor clone reduction of less than 2-log after induction therapy or a change in the PCR amplification result from negative to positive is highly indicative of relapse129,130. In childhood acute lymphoblastic leukemia (ALL) detection of minimal residual disease by Q-PCR has been shown to be an independent prognostic factor131-133. Patients without detectable tumor cells after initial therapy have a very good out-

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come. This is in contrast to those displaying high levels of tumor cells at this time point, which have a high risk of relapse131,133,134. Q-PCR has also been used in other hematological malignancies for example in MM where it has been applied for evaluation of tumor content in autografts and for detection of residual tumor cells in bone marrow after transplantation127,135.

Origin of B cells lymphomas BCLs are tumors derived from normal B cells which have transformed into malignant cells during the maturation process. Throughout normal B cell differentiation the B cell undergoes morphological and immunophenotypical changes. Based on these changes it has been possible to classify the lym-phomas and to postulate their cell of origin2. The B cell neoplasms can de-rive from most differentiation stages from early B cells e.g. pre-B ALL to MM which resembles a fully differentiated plasma cell, although most lym-phomas appear to be derived from GC or post-GC cells (Figure 6). The many entities of mature BCLs could be explained by the rapid cell prolifera-tion that takes place in the GC and also by the molecular events engaging the Ig genes. Both SHM and CSR create double strand breaks in the DNA se-quence, which sometimes lead to translocations of proto-oncogenes to the IgH locus2.

The IgH locus can also be used to determine at which developmental stage transformation occurred, since it can undergo constant changes as the B cell develops. Tumors with germline VH genes, such as pre-B ALL are, as previously mentioned, thought to derive from an early B cell which has not encountered antigen. Conversely, follicular lymphomas (FLs) are thought to originate from a more differentiated cell, since the VH genes contain somatic mutations and show signs of ongoing mutation 136. This type of lymphoma has, apart from mutated VH genes, a growth pattern that resembles the GC structure and the cells express the GC marker CD10, indicating a GC cell origin of the tumor136,137.

Another lymphoma that is thought to originate from GC or post GC cells is the diffuse large B cell lymphoma (DLBCL) which, in resemblance to FLs, has mutated VH genes and a subset that displays signs of ongoing muta-tions138. DLBCL has recently been divided into two main subgroups based on their gene expression profiles. The GC B cell-like DLBCL has somati-cally mutated VH genes with ongoing mutation and a gene expression corre-lating with a GC origin. In contrast, the second subset, activated B cell-like DLBCL, does not express genes associated with the GC, but contains mu-tated VH genes without intraclonal diversity, which makes a post-GC deriva-tion more likely139,140.

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Figure 6. Postulated cellular origin of different B cell neoplasms. ALL= acute lym-phoblastic leukemia; BL= Burkitt´s lymphoma; CLL= chronic lymphocytic leuke-mia; ABC-DLBCL = activated B cell like diffuse large B cell lymphoma; GCB-DLBCL = germinal center B cell like diffuse large B cell lymphoma; FL= follicular lymphoma; GC= germinal center; MCL=mantle cell lymphoma. Both CLL and DLBCL are divided into two subsets, which are thought to derive of B cells from different maturation stages.

CLL is also a disease with a heterogenous origin. It was previously thought to derive from a naïve B cell with unmutated VH genes, but some years ago studies of the IgH locus revealed that the disease consists of two subsets with different mutation status, and possibly different origin49,50.Since then, new theories regarding the cell of origin in CLL have arisen. One proposal is that the mutated subset has derived from a GC cell that has been exposed to the SHM mechanism, while the unmutated subset originates from a B cell activated outside the GC by a T cell independent antigen or alterna-tively, by a superantigen81. Another explanation is that both subsets derive from marginal zone B cells which can also be activated by a T cell inde-pendent pathway, resulting in one subset of unmutated antigen-experienced cells and one subset of mutated memory B cells78. Recent microarray studies have revealed a characteristic gene expression in CLL, regardless of muta-tion status, but which differs from other lymphomas and from normal GC B cells63,139,141. These results indicate that the two subsets most likely have a common cell of origin or alternatively, undergo a common event when trans-forming into a malignant cell80. In one of the studies both the mutated as well as the unmutated CLL cells were found to resemble the phenotype of memory B cells141.

A lymphoma that shares some of the morphological and genetical charac-teristics of CLL is MCL. This disease is presumed to originate from a naïve

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pre-GC B cell, due to the expression of CD5 and unmutated VH genes. How-ever, studies have indicated that some MCLs have somatically mutated VHgenes and may have been exposed to the GC environment142-145.

The normal counterpart of HCL is not yet fully clarified. The morphology does not resemble that of any type of normal B cell, but on account of the immunophenotype expressed by HCL the cell of origin is thought to be a mature B cell that is not terminally differentiated. Only smaller VH gene studies have been published regarding the mutation status in this malignancy and these reports have indicated the presence of mutated VH genes in the majority of cases146-148.

VH gene usage and antigen selection There are 51 functional VH genes which theoretically could be used to the same extent in the rearranged Ig genes of the normal B cell population. However, studies of normal B cells have shown that this is not the case and that the VH gene selection is a non-random process149-153.

As mentioned before, VH3 is the largest VH family representing 43% of all functional VH genes, followed by VH1 (21%) and VH4 (21%). The genes of the VH3 family were also found to be the most frequently rearranged VHgenes in CD5+/IgM+ and CD5-/IgM+B cells followed by VH4150,154. However, in this study the high frequency of VH3 rearrangements was not due to a balanced distribution of all 22 functional VH3 genes, but rather to a preferen-tial usage of a small number of VH genes including VH3-23, VH3-30 and VH3-07 in both cell types. This was also seen among the VH4 genes where VH4-59, VH4-39 and VH4-34 were displayed to a higher extent than ex-pected150. In a study focusing on the presence of individual VH genes in the VH3 and VH4 families of pro-B cells, the same VH genes were found to be most frequently rearranged, except VH3-07 which was not detected to such high extent152.

The selections of VH genes have also been reported to vary with age; in young adults VH3 family genes are more often rearranged, while members of the VH4 family are more frequently observed in the elderly153. Both in the young and the elderly overrepresentation of the VH3-23 and VH1-2 genes were demonstrated, but the individual VH genes VH4-34, VH4-59 and VH1-69 were found to a higher extent in the elderly153.

The non-random selection of VH genes could be caused by different mechanisms. It has been shown that the RSS, the site for the recombinase enzymes RAG1/RAG2, has a natural variation in the heptamer and nonamer sequence, which could influence the binding of the enzymes and thereby play a major role in the VH gene segment selection155. Remodeling of the chromatin structure can change gene segments from an inaccessible to an accessible state where gene rearrangement can occur. Modification of the

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histone proteins by acetylation makes the nucleosomal structure less dense and the DNA more accessible to transcription factors. This has been shown to occur as a global regulation mechanism for recombination of D-JH gene segments and for VH-DJH recombination at the pro-B cell stage156,157, but is also proposed to locally regulate the accessibility of certain VH genes157. The skewed VH utilization could also be a result of an antigen driven selection. B cells expressing a BCR with rearrangement of a certain VH gene may be positively selected by antigen resulting in prolonged survival, while unse-lected cells with lower affinity for the antigen undergo apoptosis.

In autoimmune disease, where the immune system responds to self anti-gens, certain VH genes have been found to be utilized to a higher extent than others. One example is in systemic lupus erythematosus where autoantibod-ies (cold agglutinins) encoded by the VH4-34 gene segment, react against antigens on red blood cells158. In Sjögrens syndrome and in rheumatoid ar-thritis the VH3-21 gene has been shown to be involved in production of autoantibodies159,160. In different B cell malignancies an overrepresentation of certain VH genes has also been reported, such as in CLL (VH1-69, VH3-07, VH3-21 and VH4-34)49,149,161-165, MCL (VH4-34)166, nodal marginal zone BCL (VH1-69 and VH 4-34)167, salivary gland mucosa associated lymphoid tissue lymphoma (VH1-69)168 and MM (VH1-69, VH3-9, VH3-23 and VH3-30)169.

This indicates that antigens can be involved in lymphoma development, possibly by stimulating proliferation of B cells that express Igs encoded by certain VH genes, leading to increased risk of transforming events. Another indication of antigen involvement in tumor development is the VH1-69 and VH3-21 genes in CLL, which apart from being among the most frequently rearranged VH gene segments in CLL also display unique molecular fea-tures163-165,170. The VH1-69 CLL rearrangements are unmutated, show a pref-erential usage of certain D and JH genes and also display a longer CDR3 (~19 codons) compared to normal cells with VH1-69 rearrangement and CLLs with rearrangement of other VH genes161,163,165,171. Lately, small subsets of VH1-69 using cases with very homologous CDR3s have been shown that also display a preferential VL gene usage48,170. VH3-21 gene rearrangement are more frequently detected in mutated CLLs, but can also be found in un-mutated CLLs. Many of these cases show highly homologous CDR3s, but with a shorter amino acid sequence (~10 codons) than average164,165. A re-stricted V usage of the V 2-14 gene is another of the features displayed by VH3-21 CLLs and VH3-21 usage has further been shown to correlate with poor prognosis164,165. These findings strongly indicate that antigen plays a role in VH3-21+ CLL development, but have so far mainly been reported from the Scandinavian countries. The likelihood that two identical VHDJHrearrangements would occur at random is, as mentioned, rather low 1/8262 and that identical VH and VL rearrangements, as in the VH1-69 and VH3-21 subsets, would join together in two BCRs, in two different patients without any selection is almost negligible (1 in 1.9 million). Since the CDR3 is the

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most variable antigen binding site of the BCR172, high homology in this re-gion in both VH1-69 and VH3-21 CLLs points towards the theory that tumor cells expressing similar BCRs have been positively selected by a common antigen. CLL is one of the tumors where VH gene usage has been extensively studied, whereas much remains to be resolved about this in other lympho-mas.

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Aims

The general aims of this study were, firstly to gain a greater insight into the biology of different mature BCLs, by analyzing the VH and VL rearrange-ments of the Ig gene and secondly to quantify the tumor content in stem cell mobilized peripheral blood from MM patients with the Ig gene as a clonal marker. In detail the aims were as follows:

To evaluate the SHM status of the VH genes in MCL and HCL with the aim of better defining the normal cellular counterpart of these lymphomas.

To study the extent of restricted VH gene usage in MCL and HCL in order to reveal if antigen selection could be involved in lym-phoma development.

To investigate if prognosis is influenced by the SHM status or VHgene usage in MCL.

To characterize VH/VL gene rearrangements in an extended VH3-21+ CLL material from three different countries and to determine whether these cases have similarly restricted BCR features in dif-ferent geographical regions.

To analyze the purging effect in stem cell enriched peripheral blood autotransplants from MM patients, by measuring the number of tumor cells before and after the enrichment with a highly sensitive Q-PCR.

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Material & Methods

Patients and tumor specimens In paper I-IV the tumor samples were diagnosed based on morphologic and immunophenotypic criteria according to the WHO classification1 Tumor material was mainly obtained from frozen tissue, but in paper I-II 10 samples from paraffin-embedded tissue were also included. The specimens were as-sembled from the archives of the following University Hospitals: Paper I included tumor samples from 51 MCL patients collected at the University Hospitals of Uppsala and Umeå, Sweden. In paper II 110 MCL cases were included from the University Hospitals of Umeå, Lund, Karolinska and Huddinge, Sweden. In paper III samples from 67 patients diagnosed with CLL utilizing the VH3-21 gene were retrieved from the University Hospitals of Umeå, Linköping, and Huddinge, Sweden, the University Hospital of Tampere, Finland and the University Hospital of Ulm, Germany. In paper IV tumor material from 32 HCL patients was collected from Uppsala University Hospital. The characteristics of the different patient material are detailed in each separate paper.

Inclusion criteria in paper I-IV were as follows; samples should (1) be di-agnosed according to the WHO classification and (2) have amplifiable DNA for the PCR analyzes and sequencing. Also in papers I-II over-expression of cyclin D1 confirmed by immunohistochemistry, or alternatively, a transloca-tion of the Bcl-1 gene, t(11;14), detected by fluorescence in situ hybridiza-tion (FISH) were required for inclusion. Furthermore, in paper III, all CLL cases should display rearrangement of the VH3-21 gene.

In paper V, tumor samples from 30 younger patients (41-60 years) newly diagnosed with MM and scheduled for peripheral blood stem cell transplan-tation were collected at the three Scandinavian bone marrow transplantation centres: Uppsala University Hospital, Lund University Hospital, Sweden and the Rigshospitalet in Copenhagen, Denmark. Criteria for inclusion in paper V were; patients that (1) were newly diagnosed with MM, aged 18-65 years and (2) had no disease progression after VAD therapy173. Furthermore, it was necessary that (3) both sequencing of the VH rearrangement and the design of an ASO primer for the subsequent MRD analyzes were possible and that (4) myeloma cells could be detectable by the Q-PCR method in the peripheral blood stem cell harvest, before selection of stem cells (CD34+)was preformed.

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PCR amplification and sequencing of Ig gene rearrangementsHigh-molecular weight DNA was prepared from fresh-frozen material or paraffin-embedded tissue using standard protocols including proteinase-K treatment. The PCR amplification of the VH and VL gene rearrangements was carried out using family-specific VH/JH, V /J and V /J consensus primers174,175. The PCR reaction for the VHDJH rearrangement analysis was carried out in a 50µl reaction volume containing 200ng of genomic DNA, 200µM each of dATP, dCTP, dGTP, dUTP, 1.5mM MgCl2, 50mM KCl., 10 mM Tris-HCl, 2.5U of Platinum® Taq (Invitrogen, Paisley, United King-dom), 0.125µM of each primer and 0.01% gelatin. The amplification was performed with an initial 5 min at 95°C, activating the enzyme, followed by 45 cycles of denaturation (90s at 94°C), annealing (30s at 61°C [65°C for VH3 and VH4]), elongation (80s at 72°C) and a final step of 5 min at 72°C. For some of the VH gene rearrangements in paper I-II and paper III, PCR amplification was performed as described by Thunberg et al.176 and accord-ing to Kröber et al.86, respectively.

The VL/JL gene rearrangement PCR analysis was either carried out as de-scribed by Li et al.177 or in a 50µl reaction volume containing 200ng DNA, 0.2mM of each dNTP, 2.0mM MgCl2, 2.5U Platinum® Taq (Invitrogen), 0.125µM of each primer and 1x PCR Rxn buffer (Invitrogen). The V /Jgene rearrangements were amplified with an initial 2 min denaturation at 95°C followed by 40 cycles of 30 sec at 95°C, 30 sec at 61°C and 30 sec at 72°C, ending with a 5 min elongation at 72°C. The V PCR was performed at 95°C for 2 min, 65°C for 1 min, 72°C for 1 min followed by the same cycling conditions as for V except for the elongation step which was 45 sec at 72°C. Single strand conformational polymorphism (SSCP) analysis was applied to confirm amplification of a monoclonal PCR product and to avoid misinterpretation of polyclonal products178.

In paper I-IV most clonal products from VH or VL PCR were direct se-quenced. Cloning was performed on samples for which direct sequencing was not possible and a minimum of 3 colonies were analyzed. Cloning was also performed for analyzes of intraclonal heterogeneity, using the proof reading enzyme pfu (Stratagene, La Jolla, CA) and the Zero Blunt TOPO PCR Cloning kit (Invitrogen, Paisley, UK). A minimum of 10 colonies were analyzed in paper I and II and at least 13 colonies in paper IV. The samples were sequenced with the BigDye terminator cycle sequencing kit (Applied Biosystems, Fostercity, CA) and analyzed in an automated sequencer (ABI 377 or 3700, Applied Biosystems).

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Analyzes of the Ig sequencesThe obtained VHDJH and VLJL sequences were submitted to three different databases GenBank, V-BASE and IMGT and aligned to the published germ-line gene segments. VH/VL sequences with <98% homology to the closest germline gene were considered mutated. To identify the D gene a minimum of 7 consecutive nucleotides aligned to the corresponding germline gene were required. Mutations found in hotspot regions were evaluated by count-ing the number of mutations occurring in the germline sequence motifs RGYW/WRCY and DGYW/WRCH39. In paper I and II intraclonal hetero-geneity was defined as the presence of the same base substitution in at least 2 of the minimum 10 clones analyzed. In paper IV a minimum of 13 colonies were sequenced for detection of intraclonal heterogeneity. Extra mutations within clones were considered as signs of intraclonal variation once the mu-tation rate exceeded the expected polymerase error rate. In paper III VH and VL sequences were converted into amino acid sequences aligned in the mul-tiple alignment software Clustal X (1.83) for Windows. The Bioedit software program (5.09) was used to prepare the alignments.

Quantitative PCR Q-PCR by limiting dilution of sample DNA was carried out with patient ASO primers in paper V, as described by Ouspenskaia et al.128, thereby de-termining the number of cells carrying the clonal VH rearrangement before and after stem cell enrichment. To construct the ASO primer, VH gene rear-rangements from the diagnostic sample was amplified and sequenced as described by Thunberg et al.179 with consensus primers for the VH families and JH gene segments, as described by Deane and Norton180,181. The ASO primers in each case were designed from the CDR3 of the tumor clone VHrearrangement to obtain as high specificity as possible. The tumor content was analysed, with the Q-PCR method, in autotransplant samples before and after a selection of CD34+ cells. Each sample was diluted stepwise to at least four different concentrations. For each concentration step PCR amplification was carried out in 10 separate reactions. In the first PCR, family specific VHprimers were used together with a JH consensus primer as described above. In the semi-nested amplification, an ASO primer specific for the clonal VHgene rearrangement replaced the JH primer. The fraction of all-or-none (i.e. positive or negative) reactions of the 4x10 PCR amplifications were re-corded and translated into figures using the von Krogh formula128.

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Results & Discussion

VH gene usage and somatic hypermutation in MCLThe postulated cell of origin in MCL has been questioned in earlier studies of MCL, particularly in the subset of MCL-BV where mutated cases have been reported144,182,183. Regarding VH gene usage in MCL, there have only been a few reports analyzing the VH genes previously and these were based on low patient numbers145,166. Therefore, we analyzed the VH genes in 51 MCL cases for presence of somatic mutations and VH gene usage using PCR amplification and nucleotide sequencing (paper I). In total, 53 VH gene rear-rangements were amplified, due to double rearrangements in two of the cases. The majority of cases showed unmutated VH genes (80%), while the remaining displayed mutated rearrangements (20%). The homology to the closest germline gene in the mutated VH gene rearrangements varied be-tween 95.3 and 97.8%. The typical MCL morphology was seen in 8 of 10 mutated cases and the MCL-BV morphology in the remaining 2 cases. The most often rearranged VH gene family were VH3 (43%), followed by VH4(32%). When compared to the distribution of VH gene families in normal peripheral blood CD5+ cells, this correlates to an overrepresentation of the VH4 and VH5 gene families and a minor underrepresentation of the VH3 fam-ily genes. VH4-34 (n=11), VH3-21 (n=8) and VH5-51 (n=6) were the three most frequently rearranged individual VH genes representing ~50% of the material. Significant differences in survival were neither found when the unmutated cases were compared with the mutated cases nor between the typical MCL and the MCL-BV. A shorter survival was seen in patients util-izing the VH5-51 gene segment, however these patients had a higher median age at diagnosis (76 years) compared to the median age (70 years) in all pa-tients.

To further characterize the VH genes of MCL and to assess the clinical impact of SHM and skewed VH gene usage, we extended the study to include 110 patients in paper II. Double rearrangements were detected in 7 of the cases resulting in amplification and sequencing of a total of 117 VH generearrangements. The two subsets described in the first study, defined accord-ing to mutation status, were confirmed in this extended material; unmutated VH genes were detected in 84% of the patients and 16% had mutated VHgenes, with a homology to the closest germline sequence varying between 93.3 and 97.7%. In paper II, we also analyzed unrearranged VH genes in T

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cells from five patients with rearranged VH genes having between 2.2 to 3.6% mutations. Analysis of unrearranged VH genes in T cells showed 100% homology to the published germline genes, thus confirming that mutations identified in the clonal rearrangements corresponded to true SHM and did not represent single nucleotide polymorphisms or Taq polymerase errors.

The results from our studies indicate that the subset with somatically mu-tated VH genes derives from a more mature B cell that has been exposed to the GC environment, rather than from the presumed naïve B cell. Another indication of GC exposure in the mutated subset is that 50% of the mutations were found in the hotspot regions (RGYW/WRCY motifs) known to be tar-geted by AID during the SHM process. There was no evidence of ongoing mutation in five analyzed mutated cases, which suggests that mutated MCL derives from a post-GC B cell. The unmutated subset, which represents the majority of the MCLs, is on the other hand most likely to be derived from pre-GC B cells residing in the follicular mantle.

Another alternative explanation for the two subsets, as mentioned, is that antigen stimulation of the B cells occurred before malignant transformation outside the GC. It has been suggested that cells in the marginal zone can be stimulated by T cell independent antigens184, which theoretically could result in two subgroups; antigen experienced cells with unmutated VH genes and memory B cells with mutated VH genes185. Furthermore, Weller et al. re-ported that patients unable to form GCs due to a defective CD40 ligand could display a subset of B cells (CD27+, IgM+, IgD+) whose VH genes car-ried a low degree of mutations (~1-2%)186. However, limited knowledge exists so far concerning this alternative pathway of SHM and also whether mutated/unmutated MCL could derive from marginal zone cells.

There was no difference in survival between MCLs with unmutated and mutated VH genes when applying the 2% mutation border. This cut off level is an empirical border mainly used to rule out the risk of Taq errors and polymorphisms being counted as SHM. In CLL the 2% cut off level is used as a prognostic factor, but to ensure that a different border would not be more appropriate in MCL we performed survival analysis using different cut offs (1-5%). The results were similar for the different levels and we conclude that the mutation status of the VH rearrangements does not constitute a prog-nostic factor in our MCL cohort. This is also supported by recent studies where the VH gene mutation status could not be correlated with overall sur-vival187-189.

In both paper I and II we found an overrepresentation of the VH3-21 and VH4-34 genes and, in paper II, it was shown that these genes were utilized in 19% and 17% of the patients, respectively. An increased usage of VH4-34 has been shown in other MCL studies where this gene segment was detected in 10-15% of the cases187-189. This gene was also found to a high extent in one earlier study where 28% of the cases displayed positive immunohisto-chemical stainings for VH4-34166. Our findings of VH3-21 overutilization in

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MCL have as well been recently supported in three studies187-189. The VH3-21rearrangements did not show homology between their CDR3s, as reported in VH3-21+ CLLs164,165, but were found to have a skewed V gene usage where the V 3-19 gene was detected in 16 of the 18 analyzed patients. In contrast to CLL, the survival analysis showed a prolonged survival in MCL patients rearranging the VH3-21 gene. This trend has also been indicted in other stud-ies187-189. One explanation for this improved survival could be the finding by Flordal Thelander et al. that VH3-21 MCLs demonstrate a lower frequency of chromosomal aberrations, which is generally correlated with better prog-nosis190.

The finding of a skewed VH usage indicates that antigens could be influ-encing the tumor development. This might occur by a positive selection of B cells expressing certain VH genes, which stimulates proliferation and thereby increases the risk of transformation into a malignant cell166,191-193. The possi-bility of antigenic involvement in MCL is further supported by the finding that of cases utilizing the VH3-21/V 3-19 gene segment combination. The likelihood that a certain combination of VH/V would be recombined at ran-dom in two B cell clones is extremely low and in this material identical heavy and light chain combinations were found in 16 cases.

Further characterization of the VH3-21+ CLL subsetA restricted usage of certain VH genes has been reported in CLL. As men-tioned, one of the most frequently rearranged genes is VH3-21, which has been found overutilised especially in the mutated subset. VH3-21 rearrange-ments have been reported to display specific genotypic and phenotypic char-acteristics among which homologous HCDR3s and preferential V gene usage, especially V 2-14, have been shown164,165. This subset also has poorer outcome despite the fact that many of the VH3-21 rearrangements contain somatic mutations, a feature which is known to correlate to improved sur-vival in CLL. However, the VH3-21 utilizing patients have mainly been re-ported in CLLs from the Scandinavian countries representing ~10% of the cases, while in other studies from Europe and USA the frequency has been lower (0-2.6%)49,161,164,165,194. This has raised the question as to whether this biased usage is influenced by an undetermined geographical factor 78. We therefore extended our Scandinavian VH3-21 utilizing CLL cohort to further include samples retrieved from Germany. Thus, in total, 67 cases with VH/VLrearrangments were analyzed regarding gene usage and CDR3 composition.

In parallel with our previous studies the VH3-21 subset contained mainly cases with mutated VH genes (61%), where the mutation rate was 3.2% (range 2.2-10%), but also cases without somatic mutation in their VH genes (39%). At the amino acid level, the HCDR3s of the VH3-21 CLLs were shorter (median 9 codons) than the average HCDR3 found in other CLLs (14

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codons). The short HCDR3s are due to rearrangement of VH3-21 to the JH6gene, without any identifiable D gene segment. This VH3-21/D-/JH6 combi-nation was detected in 55% of the analyzed cases, with representation of all three included countries. The HCDR3s also showed high homology between the tumors, thereby confirming our previous results. Greater than 75% HCDR3 homology was found in 30 of the cases, where a highly conserved amino acid motif ARDANGMDV (Ala-Arg-Asp-Ala-Asn-Gly-Met-Asp-Val) was detected in 9 of the cases (7 Swedish/2German) and with minor additional amino acid differences in the remaining 21 cases.

We could also verify a preferential usage of the V 2-14 gene which was found in 75% of the cases, most often joined with the J 3 gene (70%). In parallel with the HCDR3, the light chain CDR3s displayed a highly con-served motif consisting of 12 amino acids QVWDS[S/G]SDHPWV (Gln-Val-Trp-Asp-Ser-[Ser/Gly]-Ser-Asp-His-Pro-Trp-Val), which was detected in 36 cases.

The very high homology of the BCRs between different VH3-21 cases is a strong indication that antigen selection by a common antigen epitope has occurred in these CLLs, especially considering that the probability of two B cells using identical VHDJH rearrangement combined with a specific V gene rearrangement is very low. The finding of similar VH/VL gene rearrange-ments in different countries and in a substantial number of patients makes it even more unlikely that this could occur at random and strengthens the the-ory that these CLL precursor cells were selected by antigen(s). We therefore conclude that VH3-21 CLLs with homologous V gene rearrangements are not just a Swedish phenomenon, but could also be found in other parts of North-ern Europe. Some recent studies have also shown a higher usage (range 4-10%) of VH3-21 than previously reported195,196, further supporting the theory that this gene is commonly rearranged in CLL in different parts of the world.

Somatic hypermutation status and indication of antigen selection in HCL Thirty-two HCL patient samples were analyzed regarding mutation status and VH gene utilization. Three cases showed double rearrangements result-ing in a total of 35 amplified and sequenced rearrangements. Somatic muta-tions in the VH genes were found in the majority of cases (84%), displaying a homology to the closest germline sequence between 91 and 97.8% (mean 95%). A similar frequency of somatic mutation in the VH genes was found in the seven HCLs analyzed by Maluom et al.146, whereas Forconi et al. re-ported a somewhat lower mutation level in their five cases148.

Five of the VH gene rearrangements showed an unmutated VH region (98.4-100% homology) of which four had no detectable mutations i.e. the VH

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genes displayed germline configuration. This finding questions the postu-lated cell of origin of HCL, which had been thought to be a mature post GC cell and proposes a more complex picture than previously believed. This is furthermore supported by the recent correspondence by Forconi et al. report-ing that 5 of 13 analyzed HCL cases had unmutated VH genes, 2 of which showed germline configurations and 3 with >98% homology197. We consider two possible scenarios for the unmutated subset in HCL: the clonal cells (1) originate from a pre-GC B cell, which has not encountered antigen, or (2) pass through the GC without being targeted by the SHM mechanism.

In the mutated VH gene rearrangements, analysis showed that 37% of the mutations were located in the hypermutation hotspot motifs DGYW/WRCH and 29% in the less comprehensive RGYW/WRCY motifs, which indicates that mutated HCLs have been exposed to the SHM process. Eight of the mutated cases were further investigated regarding the presence of intraclonal heterogenity. All analyzed cases showed the presence of unique mutations ranging from 1 to 14 mutations, and five of the cases also displayed between 2 and 3 partially shared mutations. Since the error rate of the proof-reading enzyme Pfu is very low and the expected mutation frequency due to enzyme errors varied between 0.12 and 0.26, all mutations found in the different clones could be considered as true mutations. We could therefore conclude, as previously indicated in other studies146,148, that HCL displays intraclonal heterogeneity. This also contradicts the originally proposed cell of origin and indicates a closer association with the GC than previously assumed. In other GC-derived lymphomas, e.g. FLs and DLBCLs, the incidence of ongoing mutations is more extensive than in HCLs138,198. Our results indicate that the transformed B cell was frozen at a stage where the SHM mechanism was still active but not as active as in cells giving rise to FL and DLBCL. The theory of a GC derivation of HCL is further strengthened by a finding of multiple isotypes in tumor cells199, since B cells containing multiple isotypes are normally found in the GC and the switch process is completed upon exit from the GC.

The most frequently utilised VH gene family genes were VH3 (66%) and VH4 (23%), whilst the remaining VH families were detected at a frequency of 0-3% each. This correlates to an overrepresentation of the VH3 family and a lower usage than expected of the VH1 family gene segments. The most fre-quently used individual VH gene segment was VH3-30 found in 6 of the cases (19%), followed by VH3-33 in 4 cases. No preferential VHDJH combinations could be detected in this material.

VH3-30 is one of the most frequently rearranged gene segments in CD5- B cells, although to a lower extent (6-11%) in comparsion to our data in HCL150,154. Overutilization of this VH gene has also been indicated in a study of FL (16%)200, but has not been shown in other BCL entities138,165,187,201. A biased usage of the VH3-30 gene was not apparent in the previous studies of HCL, although in those reports the number of included HCL cases was

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lower. All of the VH3-30 rearrangements in our material displayed SHM, which indicates that a positive selection of these Ig genes might have oc-cured, possibly by antigens. If antigens influence the development of HCL it is likely that the most important antigen binding site in the VH3-30 cases is the CDR1, CDR2 or the FRs, since no specific D/JH combinations were rear-ranged together with the VH3-30 gene segments.

Quantification of tumor content in MM autotransplants Autologous stem cell transplantation is the standard treatment in younger patients with MM. A drawback with this transplantation type is that tumor contamination of the autograft is common and therefore strategies of purifi-cation have been developed to obtain tumor-free grafts. The surface mole-cule CD34 is a 105-120kD glycoprotein present on hematopoietic progenitor cells and has been used in large-scale clinical trials to achieve a positive selection of stem cells with a simultaneous reduction of contaminating tumor cells179,202-206. We investigated the tumor content in grafts from 30 patients scheduled for peripheral blood autologous stem cell transplantation, before and after stem cell enrichment based on CD34+ selection. The myeloma cells were quantified with a limited dilution Q-PCR assay, performed with ASO primers for the unique VH rearrangement of the tumor clone.

In all samples a reduction of tumor cells was found with a median reduc-tion of 2.15 log units. The total quantity of re-infused tumor cells was re-duced with a median of 99.3%, although in 29 of 30 samples, myeloma cells were still detectable after the CD34+ enrichment. The reduction rate of tumor cells correlates with findings in other studies206,207, but the number of con-taminated autografts after CD34+ enrichment was higher in our study com-pared to that of Schiller et al.206, which reported a contamination in only 21% of the samples. The difference between the studies might be explained by the high sensitivity of the Q-PCR used in the present study. The mean concentration of CD34+ cells in the autograft before enrichment was 3.4% (range 0.7%-14.2%) and after selection the concentration was 84.0% (range 65.4%-92.2%). The CellPro column used to select for the CD34+ cells en-riched these cells on average 38-fold and showed a mean depletion of CD34-

cells of 6.9 times (median 6.9 times), whereas the average depletion of tumor cells was somewhat lower (mean 5.7 times, median 2.0 times). This indicates that the tumor cells adhere to the column to a higher extent compared to CD34- cells, possibly due to their ability of non-specifically adhering to sur-faces. An alternative explanation could be that a very small fraction of mye-loma cells carry the CD34 surface marker as suggested by Pilarski & Jen-sen208 and Szczepek et al.209 and thus are selected together with the stem cells.

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We found a linear relationship between tumor cells in the autograft before and after CD34+ enrichment indicating that the purging effect was correlated with the amount of tumor cells in the initial fraction. Despite the reduction of clonal cells, this approach does not seem efficient enough to eliminate the tumor cells from the graft. The number of tumor cells was median 7.2 log in the initial fraction and the reduction was only 2.15 log (median), indicating that further purging would be needed to obtain a tumor-free graft.

Clinical follow-up studies have reported that transplantations with CD34 enriched autografts do not influence the event free survival or overall sur-vival compared to transplantations with unselected grafts210,211. Instead, the two most important prognostic factors in autologous transplantations are low levels of 2-microglobulin and a patient age of less than 60 years, which correlates to a longer survival210,212,213. Positive selection of CD34+ cells did not influence the neutrophil recovery210,211,214 although a delay in platelet recovery has been shown in the group with selected grafts213-216. No differ-ence in complete remission rates or molecular remission between groups has been detected210,211,214. Several studies therefore conclude that the clinical importance of positive CD34 selection is highly questionable210,211.

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Conclusions

Paper I and II MCL is a more heterogenous disease than previously thought. MCLs with mutated VH genes do not correlate to the postulated naïve B cell origin, but instead this subset most likely originates from a more mature B cell that has been exposed to the GC environment or, alternatively, undergone hypermu-tation outside the GC by a second diversification pathway.

An overrepresentation of the VH3-21 gene in MCL together with a re-stricted V 3-19 gene usage in many of the VH3-21+ cases strongly implies that antigens may be involved in the development of this disease subset. Furthermore, the VH3-21+ MCL cases showed superior survival compared to the remaining cases.

Paper III Restricted V /J gene usage, with similar LCDR3s, combined with homolo-gous HCDR3s was shown in the VH3-21+ CLL cases from all three countries included. These findings indicate the presence of highly homologous BCRs despite geographical origin, thus further supporting a common binding site and antigen involvement in CLL pathogenesis.

Paper IV The finding of intraclonal variation in mutated HCL and a subset with unmu-tated VH genes questions the postulated post-GC origin in HCL. The unmu-tated subgroup has either originated from a pre-GC B cell or has passed the GC without acquiring SHM. The cell of origin in the mutated subset is more closely related to the GC reaction than previously believed, since ongoing mutation was identified. A restricted usage of the VH3-30 gene indicates that antigen selection may have occurred in HCL development.

Paper V The stem cell (CD34) enrichment in autografts of MM patients led to a 2.15 log reduction of clonal cells as detected by the Q-PCR analysis and using the VH rearrangements as clonal marker. The reduction was shown as a linear relationship between the number of clonal cells before and after CD34 en-richment. However, stem cell enrichment could not completely eliminate the tumor cells from the graft, thus increasing the risk of relapse.

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Acknowledgements

This work was performed at the Department of Genetics and Pathology, Rudbeck laboratory, Uppsala University. I would like to express my sincere gratitude to colleagues, family and friends without whom I would never have been able to complete this thesis. Especially I would like to thank:

My supervisors Richard Rosenquist Brandell and Christer Sundström for giving me the opportunity to study at the department and for your huge knowledge in the lymphoma field. Richard also for your never-ending enthu-siasm and energy, as well as for pep talks and support.

Jan Sällström my former supervisor who introduced me to the world of molecular pathology.

All the people in ‘lymphoma group’ and everyone else in the oncology cor-ridor for making work such a funny and sometimes hysterical place to be in and generally creating a warm and pleasant atmosphere. Ulf for good col-laboration, discussions and for all the laughs you prolong my life with. Sarah, my ‘par-häst’, for fruitful collaboration, your great organizational ability, linguistic competence and for being such an amiable travelling com-panion. Gerard for always finding time for my questions. Marie for all sup-port during the writing process. Majlis for all those encouraging talks about children, life and work. Mattias, Ola, Ingrid T, Åsa, Daniel, Ingrid G, Fiona, Martin and Fredrik for being such lovely and kind people. Martinand Fredrik also for all help with the figures in the thesis. How would I have managed without you?

Fiona Murray for skilful linguistic revision

The many collaborators without whom this work would not have been possi-ble to accomplish. Especially Anna Laurell and Kristina Carlson at Upp-sala University Hospital for providing me with clinical data and samples and for making me look at things from at different angle.

The MCL people at Karolinska Hospital Emma Flordal-Thelander, Svet-lana Lagercrantz, Erik Björck for great collaboration and enjoyable travel company.

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Rose-Marie Amini and Alkwin Wanders at the pathology department for support and encouragement. Alkwin also for your photographic contribution to my posters and to this thesis.

Anna for all your support and encouragement and most of all for being such a dear friend. Helena, Cilla and Gao Ling for talks and laughter during lunch and coffee breaks.

Viktor, Peter and the computer units former ‘boss’ William for being such nice guys, without you I would have gone mad over the computer a long time ago.

All the people at the Rudbeck laboratory.

The hematopathology section, particularly Margareta Hallin and AnneliKraft for all your help in finding the right samples and your expertise on how to cut sections and do stainings.

The staff at the sequencing core facility for adept technical assistance and for always helping out.

Eta and Stefan, my parents-in-law, for all your considerate help and encour-agement.

My mother, Görel for your never-ending love, your care and support. With-out you I would not be here. Kenth for your care and for your belief in me.

David, my beloved husband, for your love, support and for being there for me.

Calle and Albin, my wonderful children, for making the sun rise everyday (some days too early), for bringing so much joy into my life and making everything worthwhile.

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References

1. Jaffe, E., Harris, N., Stein, H., Vardiman, J. & (eds.). Pathology and Genet-ics of Tumours of Haematopoietic and Lymphoid Tissues. World Health Organization Classification of Tumours., (IARC Press, Lyon, France, 2001).

2. Shaffer, A.L., Rosenwald, A. & Staudt, L.M. Lymphoid malignancies: the dark side of B-cell differentiation. Nat Rev Immunol 2, 920-32 (2002).

3. Kuppers, R., Klein, U., Hansmann, M.L. & Rajewsky, K. Cellular origin of human B-cell lymphomas. N Engl J Med 341, 1520-9 (1999).

4. Tarlinton, D. B-cell differentiation in the bone marrow and the periphery. Immunol Rev 137, 203-29 (1994).

5. Melchers, F., Rolink, A., Grawunder, U., Winkler, T.H., Karasuyama, H., Ghia, P. & Andersson, J. Positive and negative selection events during B lymphopoiesis. Curr Opin Immunol 7, 214-27 (1995).

6. Ghia, P., ten Boekel, E., Rolink, A.G. & Melchers, F. B-cell development: a comparison between mouse and man. Immunol Today 19, 480-5 (1998).

7. Li, Y.S., Hayakawa, K. & Hardy, R.R. The regulated expression of B line-age associated genes during B cell differentiation in bone marrow and fetal liver. J Exp Med 178, 951-60 (1993).

8. LeBien, T.W. B-cell lymphopoiesis in mouse and man. Curr Opin Immunol10, 188-95 (1998).

9. Burrows, P.D., Stephan, R.P., Wang, Y.H., Lassoued, K., Zhang, Z. & Cooper, M.D. The transient expression of pre-B cell receptors governs B cell development. Semin Immunol 14, 343-9 (2002).

10. Wang, L.D. & Clark, M.R. B-cell antigen-receptor signalling in lympho-cyte development. Immunology 110, 411-20 (2003).

11. Hertz, M. & Nemazee, D. Receptor editing and commitment in B lympho-cytes. Curr Opin Immunol 10, 208-13 (1998).

12. MacLennan, I.C. Somatic mutation. From the dark zone to the light. Curr Biol 4, 70-2 (1994).

13. Camacho, S.A., Kosco-Vilbois, M.H. & Berek, C. The dynamic structure of the germinal center. Immunol Today 19, 511-4 (1998).

14. Tonegawa, S. Somatic generation of antibody diversity. Nature 302, 575-81 (1983).

15. Matsuda, F., Ishii, K., Bourvagnet, P., Kuma, K., Hayashida, H., Miyata, T. & Honjo, T. The complete nucleotide sequence of the human immu-noglobulin heavy chain variable region locus. J Exp Med 188, 2151-62 (1998).

16. Cook, G.P. & Tomlinson, I.M. The human immunoglobulin VH repertoire. Immunol Today 16, 237-42 (1995).

49

17. Lefranc, M.P. Locus maps and genomic repertoire of the human Ig genes. The immunologist, 80 (2000).

18. Lefranc, M.P.L.G. The immunoglobulin factsbook, (Academic Press, Hart-court Publishers Limited, London, 2001).

19. Alt, F.W., Oltz, E.M., Young, F., Gorman, J., Taccioli, G. & Chen, J. VDJ recombination. Immunol Today 13, 306-14 (1992).

20. Zhu, C. & Roth, D.B. Mechanism of V(D)J recombination. Cancer Surv28, 295-309 (1996).

21. Oettinger, M.A., Schatz, D.G., Gorka, C. & Baltimore, D. RAG-1 and RAG-2, adjacent genes that synergistically activate V(D)J recombination. Science 248, 1517-23 (1990).

22. McBlane, J.F., van Gent, D.C., Ramsden, D.A., Romeo, C., Cuomo, C.A., Gellert, M. & Oettinger, M.A. Cleavage at a V(D)J recombination signal requires only RAG1 and RAG2 proteins and occurs in two steps. Cell 83,387-95 (1995).

23. Lewis, S.M. The mechanism of V(D)J joining: lessons from molecular, immunological, and comparative analyses. Adv Immunol 56, 27-150 (1994).

24. Jung, D. & Alt, F.W. Unraveling V(D)J recombination; insights into gene regulation. Cell 116, 299-311 (2004).

25. Korsmeyer, S.J., Hieter, P.A., Ravetch, J.V., Poplack, D.G., Waldmann, T.A. & Leder, P. Developmental hierarchy of immunoglobulin gene rear-rangements in human leukemic pre-B-cells. Proc Natl Acad Sci U S A 78,7096-100 (1981).

26. Coleclough, C., Perry, R.P., Karjalainen, K. & Weigert, M. Aberrant rear-rangements contribute significantly to the allelic exclusion of immu-noglobulin gene expression. Nature 290, 372-8 (1981).

27. Hieter, P.A., Korsmeyer, S.J., Waldmann, T.A. & Leder, P. Human immu-noglobulin kappa light-chain genes are deleted or rearranged in lambda-producing B cells. Nature 290, 368-72 (1981).

28. Siminovitch, K.A., Bakhshi, A., Goldman, P. & Korsmeyer, S.J. A uniform deleting element mediates the loss of kappa genes in human B cells. Nature316, 260-2 (1985).

29. Klobeck, H.G. & Zachau, H.G. The human CK gene segment and the kappa deleting element are closely linked. Nucleic Acids Res 14, 4591-603 (1986).

30. Siminovitch, K.A., Moore, M.W., Durdik, J. & Selsing, E. The human kappa deleting element and the mouse recombining segment share DNA sequence homology. Nucleic Acids Res 15, 2699-705 (1987).

31. Graninger, W.B., Goldman, P.L., Morton, C.C., O'Brien, S.J. & Kors-meyer, S.J. The kappa-deleting element. Germline and rearranged, dupli-cated and dispersed forms. J Exp Med 167, 488-501 (1988).

32. Claverie, J.-M. & Langman, R. Models for the rearrangements of immu-noglobulin genes: a computer view. Trends in Biochemical Sciences 9, 293-296 (1984).

33. Martin, A. & Scharff, M.D. AID and mismatch repair in antibody diversifi-cation. Nat Rev Immunol 2, 605-14 (2002).

50

34. Wilson, P.C., de Bouteiller, O., Liu, Y.J., Potter, K., Banchereau, J., Capra, J.D. & Pascual, V. Somatic hypermutation introduces insertions and dele-tions into immunoglobulin V genes. J Exp Med 187, 59-70 (1998).

35. Bergthorsdottir, S., Gallagher, A., Jainandunsing, S., Cockayne, D., Sutton, J., Leanderson, T. & Gray, D. Signals that initiate somatic hypermutation of B cells in vitro. J Immunol 166, 2228-34 (2001).

36. Faili, A., Aoufouchi, S., Gueranger, Q., Zober, C., Leon, A., Bertocci, B., Weill, J.C. & Reynaud, C.A. AID-dependent somatic hypermutation occurs as a DNA single-strand event in the BL2 cell line. Nat Immunol 3, 815-21 (2002).

37. Inui, S., Maeda, K., Hua, D.R., Yamashita, T., Yamamoto, H., Miyamoto, E., Aizawa, S. & Sakaguchi, N. BCR signal through alpha 4 is involved in S6 kinase activation and required for B cell maturation including isotype switching and V region somatic hypermutation. Int Immunol 14, 177-87 (2002).

38. Weller, S., Faili, A., Aoufouchi, S., Gueranger, Q., Braun, M., Reynaud, C.A. & Weill, J.C. Hypermutation in human B cells in vivo and in vitro. Ann N Y Acad Sci 987, 158-65 (2003).

39. Rogozin, I.B. & Diaz, M. Cutting edge: DGYW/WRCH is a better predic-tor of mutability at G:C bases in Ig hypermutation than the widely accepted RGYW/WRCY motif and probably reflects a two-step activation-induced cytidine deaminase-triggered process. J Immunol 172, 3382-4 (2004).

40. Chang, B. & Casali, P. The CDR1 sequences of a major proportion of hu-man germline Ig VH genes are inherently susceptible to amino acid re-placement. Immunol Today 15, 367-73 (1994).

41. Chaudhuri, J. & Alt, F.W. Class-switch recombination: interplay of tran-scription, DNA deamination and DNA repair. Nat Rev Immunol 4, 541-52 (2004).

42. Snapper, C.M., Marcu, K.B. & Zelazowski, P. The immunoglobulin class switch: beyond "accessibility". Immunity 6, 217-23 (1997).

43. Casellas, R., Nussenzweig, A., Wuerffel, R., Pelanda, R., Reichlin, A., Suh, H., Qin, X.F., Besmer, E., Kenter, A., Rajewsky, K. & Nussenzweig, M.C. Ku80 is required for immunoglobulin isotype switching. Embo J 17, 2404-11 (1998).

44. Manis, J.P., Gu, Y., Lansford, R., Sonoda, E., Ferrini, R., Davidson, L., Rajewsky, K. & Alt, F.W. Ku70 is required for late B cell development and immunoglobulin heavy chain class switching. J Exp Med 187, 2081-9 (1998).

45. Manis, J.P., Dudley, D., Kaylor, L. & Alt, F.W. IgH class switch recombi-nation to IgG1 in DNA-PKcs-deficient B cells. Immunity 16, 607-17 (2002).

46. Keating, M.J., Chiorazzi, N., Messmer, B., Damle, R.N., Allen, S.L., Rai, K.R., Ferrarini, M. & Kipps, T.J. Biology and treatment of chronic lym-phocytic leukemia. Hematology (Am Soc Hematol Educ Program), 153-75 (2003).

47. Ghiotto, F., Fais, F., Valetto, A., Albesiano, E., Hashimoto, S., Dono, M., Ikematsu, H., Allen, S.L., Kolitz, J., Rai, K.R., Nardini, M., Tramontano,

51

A., Ferrarini, M. & Chiorazzi, N. Remarkably similar antigen receptors among a subset of patients with chronic lymphocytic leukemia. J Clin In-vest 113, 1008-16 (2004).

48. Widhopf II, Rassenti, L.Z., Toy, T.L., Gribben, J.G., Wierda, W.G. & Kipp, T.J. Chronic Lymphocytic Leukemia B Cells of Over One Percent of Patients Express Virtually Identical Immunoglobulins. Blood (2004).

49. Hamblin, T.J., Davis, Z., Gardiner, A., Oscier, D.G. & Stevenson, F.K. Unmutated Ig V(H) genes are associated with a more aggressive form of chronic lymphocytic leukemia. Blood 94, 1848-54 (1999).

50. Damle, R.N., Wasil, T., Fais, F., Ghiotto, F., Valetto, A., Allen, S.L., Buchbinder, A., Budman, D., Dittmar, K., Kolitz, J., Lichtman, S.M., Schulman, P., Vinciguerra, V.P., Rai, K.R., Ferrarini, M. & Chiorazzi, N. Ig V gene mutation status and CD38 expression as novel prognostic indica-tors in chronic lymphocytic leukemia. Blood 94, 1840-7 (1999).

51. Stevenson, F.K., Sahota, S.S., Ottensmeier, C.H., Zhu, D., Forconi, F. & Hamblin, T.J. The occurrence and significance of V gene mutations in B cell-derived human malignancy. Adv Cancer Res 83, 81-116 (2001).

52. Harris, N.L., Jaffe, E.S., Stein, H., Banks, P.M., Chan, J.K., Cleary, M.L., Delsol, G., De Wolf-Peeters, C., Falini, B. & Gatter, K.C. A revised Euro-pean-American classification of lymphoid neoplasms: a proposal from the International Lymphoma Study Group. Blood 84, 1361-92 (1994).

53. Socialstyrelsen. Cancer incidence in Sweden 2002. (2003). 54. Bosch, F., Lopez-Guillermo, A., Campo, E., Ribera, J.M., Conde, E., Piris,

M.A., Vallespi, T., Woessner, S. & Montserrat, E. Mantle cell lymphoma: presenting features, response to therapy, and prognostic factors. Cancer 82,567-75 (1998).

55. Argatoff, L.H., Connors, J.M., Klasa, R.J., Horsman, D.E. & Gascoyne, R.D. Mantle cell lymphoma: a clinicopathologic study of 80 cases. Blood89, 2067-78 (1997).

56. Decaudin, D., Bosq, J., Munck, J.N., Bayle, C., Koscielny, S., Boudjemaa, S., Bennaceur, A., Venuat, A.M., Naccache, P., Bendahmane, B., Ribrag, V., Carde, P., Pico, J.L. & Hayat, M. Mantle cell lymphomas: characteris-tics, natural history and prognostic factors of 45 cases. Leuk Lymphoma 26,539-50 (1997).

57. Lenz, G., Dreyling, M. & Hiddemann, W. Mantle cell lymphoma: estab-lished therapeutic options and future directions. Ann Hematol 83, 71-7 (2004).

58. Campo, E. & Jaffe, E.S. Mantle cell lymphoma. Accurate diagnosis yields new clinical insights. Arch Pathol Lab Med 120, 12-4 (1996).

59. Bertoni, F., Zucca, E. & Cotter, F.E. Molecular basis of mantle cell lym-phoma. Br J Haematol 124, 130-40 (2004).

60. Rosenberg, C.L., Wong, E., Petty, E.M., Bale, A.E., Tsujimoto, Y., Harris, N.L. & Arnold, A. PRAD1, a candidate BCL1 oncogene: mapping and ex-pression in centrocytic lymphoma. Proc Natl Acad Sci U S A 88, 9638-42 (1991).

52

61. Williams, M.E., Swerdlow, S.H. & Meeker, T.C. Chromosome t(11;14)(q13;q32) breakpoints in centrocytic lymphoma are highly local-ized at the bcl-1 major translocation cluster. Leukemia 7, 1437-40 (1993).

62. de Boer, C.J., Schuuring, E., Dreef, E., Peters, G., Bartek, J., Kluin, P.M. & van Krieken, J.H. Cyclin D1 protein analysis in the diagnosis of mantle cell lymphoma. Blood 86, 2715-23 (1995).

63. Korz, C., Pscherer, A., Benner, A., Mertens, D., Schaffner, C., Leupolt, E., Dohner, H., Stilgenbauer, S. & Lichter, P. Evidence for distinct pathome-chanisms in B-cell chronic lymphocytic leukemia and mantle cell lym-phoma by quantitative expression analysis of cell cycle and apoptosis-associated genes. Blood 99, 4554-61 (2002).

64. Dohner, H., Stilgenbauer, S., James, M.R., Benner, A., Weilguni, T., Bentz, M., Fischer, K., Hunstein, W. & Lichter, P. 11q deletions identify a new subset of B-cell chronic lymphocytic leukemia characterized by extensive nodal involvement and inferior prognosis. Blood 89, 2516-22 (1997).

65. Monni, O., Zhu, Y., Franssila, K., Oinonen, R., Hoglund, P., Elonen, E., Joensuu, H. & Knuutila, S. Molecular characterization of deletion at 11q22.1-23.3 in mantle cell lymphoma. Br J Haematol 104, 665-71 (1999).

66. Stilgenbauer, S., Winkler, D., Ott, G., Schaffner, C., Leupolt, E., Bentz, M., Moller, P., Muller-Hermelink, H.K., James, M.R., Lichter, P. & Dohner, H. Molecular characterization of 11q deletions points to a pathogenic role of the ATM gene in mantle cell lymphoma. Blood 94, 3262-4 (1999).

67. Schaffner, C., Idler, I., Stilgenbauer, S., Dohner, H. & Lichter, P. Mantle cell lymphoma is characterized by inactivation of the ATM gene. Proc Natl Acad Sci U S A 97, 2773-8 (2000).

68. Camacho, E., Hernandez, L., Hernandez, S., Tort, F., Bellosillo, B., Bea, S., Bosch, F., Montserrat, E., Cardesa, A., Fernandez, P.L. & Campo, E. ATM gene inactivation in mantle cell lymphoma mainly occurs by truncating mu-tations and missense mutations involving the phosphatidylinositol-3 kinase domain and is associated with increasing numbers of chromosomal imbal-ances. Blood 99, 238-44 (2002).

69. Rosenwald, A., Wright, G., Wiestner, A., Chan, W.C., Connors, J.M., Campo, E., Gascoyne, R.D., Grogan, T.M., Muller-Hermelink, H.K., Sme-land, E.B., Chiorazzi, M., Giltnane, J.M., Hurt, E.M., Zhao, H., Averett, L., Henrickson, S., Yang, L., Powell, J., Wilson, W.H., Jaffe, E.S., Simon, R., Klausner, R.D., Montserrat, E., Bosch, F., Greiner, T.C., Weisenburger, D.D., Sanger, W.G., Dave, B.J., Lynch, J.C., Vose, J., Armitage, J.O., Fisher, R.I., Miller, T.P., LeBlanc, M., Ott, G., Kvaloy, S., Holte, H., Dela-bie, J. & Staudt, L.M. The proliferation gene expression signature is a quantitative integrator of oncogenic events that predicts survival in mantle cell lymphoma. Cancer Cell 3, 185-97 (2003).

70. Wiestner, A. & Staudt, L.M. Towards molecular diagnosis and targeted therapy of lymphoid malignancies. Semin Hematol 40, 296-307 (2003).

71. Howard, O.M., Gribben, J.G., Neuberg, D.S., Grossbard, M., Poor, C., Janicek, M.J. & Shipp, M.A. Rituximab and CHOP induction therapy for newly diagnosed mantle-cell lymphoma: molecular complete responses are

53

not predictive of progression-free survival. J Clin Oncol 20, 1288-94 (2002).

72. Forstpointner, R., Dreyling, M., Repp, R., Hermann, S., Haenel, A., Metzner, B., Pott, C., Hartmann, F., Rothmann, F., Rohrberg, R., Boeck, H.P., Wandt, H., Unterhalt, M. & Hiddemann, W. The addition of rituxi-mab to a combination of fludarabine, cyclophosphamide, mitoxantrone (FCM) significantly increases the response rate and prolongs survival as compared to FCM alone in patients with relapsed and refractory follicular and mantle cell lymphomas - results of a prospective randomized study of the German low grade lymphoma study group (GLSG). Blood (2004).

73. Kaufmann, H., Raderer, M., Woehrer, S., Puespoek, A., Bankier, A., Zielinski, C., Chott, A. & Drach, J. Anti-tumor activity of rituximab plus thalidomide in patients with relapsed/refractory mantle cell lymphoma. Blood (2004).

74. Khouri, I.F., Saliba, R.M., Okoroji, G.J., Acholonu, S.A. & Champlin, R.E. Long-term follow-up of autologous stem cell transplantation in patients with diffuse mantle cell lymphoma in first disease remission: the prognostic value of beta2-microglobulin and the tumor score. Cancer 98, 2630-5 (2003).

75. Gianni, A.M., Magni, M., Martelli, M., Di Nicola, M., Carlo-Stella, C., Pilotti, S., Rambaldi, A., Cortelazzo, S., Patti, C., Parvis, G., Benedetti, F., Capria, S., Corradini, P., Tarella, C. & Barbui, T. Long-term remission in mantle cell lymphoma following high-dose sequential chemotherapy and in vivo rituximab-purged stem cell autografting (R-HDS regimen). Blood 102,749-55 (2003).

76. Mangel, J., Leitch, H.A., Connors, J.M., Buckstein, R., Imrie, K., Spaner, D., Crump, M., Pennell, N., Boudreau, A. & Berinstein, N.L. Intensive chemotherapy and autologous stem-cell transplantation plus rituximab is superior to conventional chemotherapy for newly diagnosed advanced stage mantle-cell lymphoma: a matched pair analysis. Ann Oncol 15, 283-90 (2004).

77. Oscier, D., Fegan, C., Hillmen, P., Illidge, T., Johnson, S., Maguire, P., Matutes, E. & Milligan, D. Guidelines on the diagnosis and management of chronic lymphocytic leukaemia. Br J Haematol 125, 294-317 (2004).

78. Ferrarini, M. & Chiorazzi, N. Recent advances in the molecular biology and immunobiology of chronic lymphocytic leukemia. Semin Hematol 41,207-23 (2004).

79. Matutes, E., Morilla, R., Owusu-Ankomah, K., Houliham, A., Meeus, P. & Catovsky, D. The immunophenotype of hairy cell leukemia (HCL). Pro-posal for a scoring system to distinguish HCL from B-cell disorders with hairy or villous lymphocytes. Leuk Lymphoma 14 Suppl 1, 57-61 (1994).

80. Guipaud, O., Deriano, L., Salin, H., Vallat, L., Sabatier, L., Merle-Beral, H. & Delic, J. B-cell chronic lymphocytic leukaemia: a polymorphic family unified by genomic features. Lancet Oncol 4, 505-14 (2003).

81. Hamblin, T. Chronic lymphocytic leukaemia: one disease or two? Ann Hematol 81, 299-303 (2002).

54

82. Dighiero, G. & Binet, J.L. When and how to treat chronic lymphocytic leukemia. N Engl J Med 343, 1799-801 (2000).

83. Rai, K.R., Sawitsky, A., Cronkite, E.P., Chanana, A.D., Levy, R.N. & Pasternack, B.S. Clinical staging of chronic lymphocytic leukemia. Blood46, 219-34 (1975).

84. Binet, J.L., Lepoprier, M., Dighiero, G., Charron, D., D'Athis, P., Vaugier, G., Beral, H.M., Natali, J.C., Raphael, M., Nizet, B. & Follezou, J.Y. A clinical staging system for chronic lymphocytic leukemia: prognostic sig-nificance. Cancer 40, 855-64 (1977).

85. Oscier, D.G., Gardiner, A.C., Mould, S.J., Glide, S., Davis, Z.A., Ibbotson, R.E., Corcoran, M.M., Chapman, R.M., Thomas, P.W., Copplestone, J.A., Orchard, J.A. & Hamblin, T.J. Multivariate analysis of prognostic factors in CLL: clinical stage, IGVH gene mutational status, and loss or mutation of the p53 gene are independent prognostic factors. Blood 100, 1177-84 (2002).

86. Krober, A., Seiler, T., Benner, A., Bullinger, L., Bruckle, E., Lichter, P., Dohner, H. & Stilgenbauer, S. V(H) mutation status, CD38 expression level, genomic aberrations, and survival in chronic lymphocytic leukemia. Blood 100, 1410-6 (2002).

87. Orchard, J.A., Ibbotson, R.E., Davis, Z., Wiestner, A., Rosenwald, A., Thomas, P.W., Hamblin, T.J., Staudt, L.M. & Oscier, D.G. ZAP-70 expres-sion and prognosis in chronic lymphocytic leukaemia. Lancet 363, 105-11 (2004).

88. Crespo, M., Bosch, F., Villamor, N., Bellosillo, B., Colomer, D., Rozman, M., Marce, S., Lopez-Guillermo, A., Campo, E. & Montserrat, E. ZAP-70 expression as a surrogate for immunoglobulin-variable-region mutations in chronic lymphocytic leukemia. N Engl J Med 348, 1764-75 (2003).

89. Hamblin, T.J., Orchard, J.A., Gardiner, A., Oscier, D.G., Davis, Z. & Ste-venson, F.K. Immunoglobulin V genes and CD38 expression in CLL. Blood 95, 2455-7 (2000).

90. Jelinek, D.F., Tschumper, R.C., Geyer, S.M., Bone, N.D., Dewald, G.W., Hanson, C.A., Stenson, M.J., Witzig, T.E., Tefferi, A. & Kay, N.E. Analy-sis of clonal B-cell CD38 and immunoglobulin variable region sequence status in relation to clinical outcome for B-chronic lymphocytic leukaemia. Br J Haematol 115, 854-61 (2001).

91. Thunberg, U., Johnson, A., Roos, G., Thorn, I., Tobin, G., Sallstrom, J., Sundstrom, C. & Rosenquist, R. CD38 expression is a poor predictor for VH gene mutational status and prognosis in chronic lymphocytic leukemia. Blood 97, 1892-4 (2001).

92. Hamblin, T.J., Orchard, J.A., Ibbotson, R.E., Davis, Z., Thomas, P.W., Stevenson, F.K. & Oscier, D.G. CD38 expression and immunoglobulin variable region mutations are independent prognostic variables in chronic lymphocytic leukemia, but CD38 expression may vary during the course of the disease. Blood 99, 1023-9 (2002).

93. Dohner, H., Stilgenbauer, S., Benner, A., Leupolt, E., Krober, A., Bullin-ger, L., Dohner, K., Bentz, M. & Lichter, P. Genomic aberrations and sur-vival in chronic lymphocytic leukemia. N Engl J Med 343, 1910-6 (2000).

55

94. Stilgenbauer, S., Bullinger, L., Lichter, P. & Dohner, H. Genetics of chronic lymphocytic leukemia: genomic aberrations and V(H) gene muta-tion status in pathogenesis and clinical course. Leukemia 16, 993-1007 (2002).

95. Juliusson, G., Oscier, D.G., Fitchett, M., Ross, F.M., Stockdill, G., Mackie, M.J., Parker, A.C., Castoldi, G.L., Guneo, A., Knuutila, S. & et al. Prog-nostic subgroups in B-cell chronic lymphocytic leukemia defined by spe-cific chromosomal abnormalities. N Engl J Med 323, 720-4 (1990).

96. Geisler, C.H., Philip, P., Christensen, B.E., Hou-Jensen, K., Pedersen, N.T., Jensen, O.M., Thorling, K., Andersen, E., Birgens, H.S., Drivsholm, A., Ellegaard, J., Larsen, J.K., Plesner, T., Brown, P., Andersen, P.K. & Han-sen, M.M. In B-cell chronic lymphocytic leukaemia chromosome 17 ab-normalities and not trisomy 12 are the single most important cytogenetic abnormalities for the prognosis: a cytogenetic and immunophenotypic study of 480 unselected newly diagnosed patients. Leuk Res 21, 1011-23 (1997).

97. Hainsworth, J.D., Litchy, S., Burris, H.A., 3rd, Scullin, D.C., Jr., Corso, S.W., Yardley, D.A., Morrissey, L. & Greco, F.A. Rituximab as first-line and maintenance therapy for patients with indolent non-hodgkin's lym-phoma. J Clin Oncol 20, 4261-7 (2002).

98. Byrd, J.C., Peterson, B.L., Morrison, V.A., Park, K., Jacobson, R., Hoke, E., Vardiman, J.W., Rai, K., Schiffer, C.A. & Larson, R.A. Randomized phase 2 study of fludarabine with concurrent versus sequential treatment with rituximab in symptomatic, untreated patients with B-cell chronic lym-phocytic leukemia: results from Cancer and Leukemia Group B 9712 (CALGB 9712). Blood 101, 6-14 (2003).

99. Lundin, J., Porwit-MacDonald, A., Rossmann, E.D., Karlsson, C., Edman, P., Rezvany, M.R., Kimby, E., Osterborg, A. & Mellstedt, H. Cellular im-mune reconstitution after subcutaneous alemtuzumab (anti-CD52 mono-clonal antibody, CAMPATH-1H) treatment as first-line therapy for B-cell chronic lymphocytic leukaemia. Leukemia 18, 484-90 (2004).

100. Keating, M.J. Management of chronic lymphocytic leukemia: a changing field. Rev Clin Exp Hematol 6, 350-65; discussion 449-50 (2002).

101. Pettitt, A.R., Zuzel, M. & Cawley, J.C. Hairy-cell leukaemia: biology and management. Br J Haematol 106, 2-8 (1999).

102. Allsup, D.J. & Cawley, J.C. The diagnosis and treatment of hairy-cell leu-kaemia. Blood Rev 16, 255-62 (2002).

103. Haglund, U., Juliusson, G., Stellan, B. & Gahrton, G. Hairy cell leukemia is characterized by clonal chromosome abnormalities clustered to specific re-gions. Blood 83, 2637-45 (1994).

104. Brito-Babapulle, V., Pittman, S., Melo, J.V., Parreira, L. & Catovsky, D. The 14q+ marker in hairy cell leukaemia. A cytogenetic study of 15 cases. Leuk Res 10, 131-8 (1986).

105. Sambani, C., Trafalis, D.T., Mitsoulis-Mentzikoff, C., Poulakidas, E., Makropoulos, V., Pantelias, G.E. & Mecucci, C. Clonal chromosome rear-rangements in hairy cell leukemia: personal experience and review of lit-erature. Cancer Genet Cytogenet 129, 138-44 (2001).

56

106. Hagberg, H. & Lundholm, L. Rituximab, a chimaeric anti-CD20 mono-clonal antibody, in the treatment of hairy cell leukaemia. Br J Haematol115, 609-11 (2001).

107. Flinn, I.W., Kopecky, K.J., Foucar, M.K., Head, D., Bennett, J.M., Hutchi-son, R., Corbett, W., Cassileth, P., Habermann, T., Golomb, H., Rai, K., Eisenhauer, E., Appelbaum, F., Cheson, B. & Grever, M.R. Long-term fol-low-up of remission duration, mortality, and second malignancies in hairy cell leukemia patients treated with pentostatin. Blood 96, 2981-6 (2000).

108. Samson, D. & Singer, C. Multiple myeloma. Clin Med 1, 365-70 (2001). 109. Shaughnessy, J.D., Jr. & Barlogie, B. Interpreting the molecular biology

and clinical behavior of multiple myeloma in the context of global gene ex-pression profiling. Immunol Rev 194, 140-63 (2003).

110. Sirohi, B. & Powles, R. Multiple myeloma. Lancet 363, 875-87 (2004). 111. Van Camp, B., Durie, B.G., Spier, C., De Waele, M., Van Riet, I., Vela, E.,

Frutiger, Y., Richter, L. & Grogan, T.M. Plasma cells in multiple myeloma express a natural killer cell-associated antigen: CD56 (NKH-1; Leu-19). Blood 76, 377-82 (1990).

112. Shaughnessy, J., Jr., Gabrea, A., Qi, Y., Brents, L., Zhan, F., Tian, E., Saw-yer, J., Barlogie, B., Bergsagel, P.L. & Kuehl, M. Cyclin D3 at 6p21 is dys-regulated by recurrent chromosomal translocations to immunoglobulin loci in multiple myeloma. Blood 98, 217-23 (2001).

113. Zhan, F., Hardin, J., Kordsmeier, B., Bumm, K., Zheng, M., Tian, E., San-derson, R., Yang, Y., Wilson, C., Zangari, M., Anaissie, E., Morris, C., Muwalla, F., van Rhee, F., Fassas, A., Crowley, J., Tricot, G., Barlogie, B. & Shaughnessy, J., Jr. Global gene expression profiling of multiple mye-loma, monoclonal gammopathy of undetermined significance, and normal bone marrow plasma cells. Blood 99, 1745-57 (2002).

114. Barille-Nion, S., Barlogie, B., Bataille, R., Bergsagel, P.L., Epstein, J., Fenton, R.G., Jacobson, J., Kuehl, W.M., Shaughnessy, J. & Tricot, G. Ad-vances in biology and therapy of multiple myeloma. Hematology (Am Soc Hematol Educ Program), 248-78 (2003).

115. Shaughnessy, J., Tian, E., Sawyer, J., Bumm, K., Landes, R., Badros, A., Morris, C., Tricot, G., Epstein, J. & Barlogie, B. High incidence of chro-mosome 13 deletion in multiple myeloma detected by multiprobe inter-phase FISH. Blood 96, 1505-11 (2000).

116. Tricot, G., Barlogie, B., Jagannath, S., Bracy, D., Mattox, S., Vesole, D.H., Naucke, S. & Sawyer, J.R. Poor prognosis in multiple myeloma is associ-ated only with partial or complete deletions of chromosome 13 or abnor-malities involving 11q and not with other karyotype abnormalities. Blood86, 4250-6 (1995).

117. Avet-Loiseau, H. & Bataille, R. Detection of nonrandom chromosomal changes in multiple myeloma by comparative genomic hybridization. Blood92, 2997-8 (1998).

118. Ho, P.J., Campbell, L.J., Gibson, J., Brown, R. & Joshua, D. The biology and cytogenetics of multiple myeloma. Rev Clin Exp Hematol 6, 276-300 (2002).

57

119. Durie, B.G., Kyle, R.A., Belch, A., Bensinger, W., Blade, J., Boccadoro, M., Child, J.A., Comenzo, R., Djulbegovic, B., Fantl, D., Gahrton, G., Harousseau, J.L., Hungria, V., Joshua, D., Ludwig, H., Mehta, J., Morales, A.R., Morgan, G., Nouel, A., Oken, M., Powles, R., Roodman, D., San Mi-guel, J., Shimizu, K., Singhal, S., Sirohi, B., Sonneveld, P., Tricot, G. & Van Ness, B. Myeloma management guidelines: a consensus report from the Scientific Advisors of the International Myeloma Foundation. Hematol J 4, 379-98 (2003).

120. Combination chemotherapy versus melphalan plus prednisone as treatment for multiple myeloma: an overview of 6,633 patients from 27 randomized trials. Myeloma Trialists' Collaborative Group. J Clin Oncol 16, 3832-42 (1998).

121. Barlogie, B., Shaughnessy, J., Tricot, G., Jacobson, J., Zangari, M., Anais-sie, E., Walker, R. & Crowley, J. Treatment of multiple myeloma. Blood103, 20-32 (2004).

122. Bjorkstrand, B.B., Ljungman, P., Svensson, H., Hermans, J., Alegre, A., Apperley, J., Blade, J., Carlson, K., Cavo, M., Ferrant, A., Goldstone, A.H., de Laurenzi, A., Majolino, I., Marcus, R., Prentice, H.G., Remes, K., Sam-son, D., Sureda, A., Verdonck, L.F., Volin, L. & Gahrton, G. Allogeneic bone marrow transplantation versus autologous stem cell transplantation in multiple myeloma: a retrospective case-matched study from the European Group for Blood and Marrow Transplantation. Blood 88, 4711-8 (1996).

123. Kroger, N., Kruger, W., Renges, H., Zabelina, T., Stute, N., Jung, R., Wittkowsky, G., Kuse, R. & Zander, A. Donor lymphocyte infusion en-hances remission status in patients with persistent disease after allografting for multiple myeloma. Br J Haematol 112, 421-3 (2001).

124. Harousseau, J.L. & Attal, M. The role of stem cell transplantation in multi-ple myeloma. Blood Rev 16, 245-53 (2002).

125. Vescio, R.A., Han, E.J., Schiller, G.J., Lee, J.C., Wu, C.H., Cao, J., Shin, J., Kim, A., Lichtenstein, A.K. & Berenson, J.R. Quantitative comparison of multiple myeloma tumor contamination in bone marrow harvest and leu-kapheresis autografts. Bone Marrow Transplant 18, 103-10 (1996).

126. Voena, C., Ladetto, M., Astolfi, M., Provan, D., Gribben, J.G., Boccadoro, M., Pileri, A. & Corradini, P. A novel nested-PCR strategy for the detection of rearranged immunoglobulin heavy-chain genes in B cell tumors. Leuke-mia 11, 1793-8 (1997).

127. Fenk, R., Haas, R. & Kronenwett, R. Molecular monitoring of minimal residual disease in patients with multiple myeloma. Hematology 9, 17-33 (2004).

128. Ouspenskaia, M.V., Johnston, D.A., Roberts, W.M., Estrov, Z. & Zipf, T.F. Accurate quantitation of residual B-precursor acute lymphoblastic leukemia by limiting dilution and a PCR-based detection system: a description of the method and the principles involved. Leukemia 9, 321-8 (1995).

129. Jaeger, U. & Kainz, B. Monitoring minimal residual disease in AML: the right time for real time. Ann Hematol 82, 139-47 (2003).

130. Raanani, P. & Ben-Bassat, I. Detection of minimal residual disease in acute myelogenous leukemia. Acta Haematol 112, 40-54 (2004).

58

131. van Dongen, J.J., Seriu, T., Panzer-Grumayer, E.R., Biondi, A., Pongers-Willemse, M.J., Corral, L., Stolz, F., Schrappe, M., Masera, G., Kamps, W.A., Gadner, H., van Wering, E.R., Ludwig, W.D., Basso, G., de Bruijn, M.A., Cazzaniga, G., Hettinger, K., van der Does-van den Berg, A., Hop, W.C., Riehm, H. & Bartram, C.R. Prognostic value of minimal residual disease in acute lymphoblastic leukaemia in childhood. Lancet 352, 1731-8 (1998).

132. Biondi, A., Valsecchi, M.G., Seriu, T., D'Aniello, E., Willemse, M.J., Fasching, K., Pannunzio, A., Gadner, H., Schrappe, M., Kamps, W.A., Bar-tram, C.R., van Dongen, J.J. & Panzer-Grumayer, E.R. Molecular detection of minimal residual disease is a strong predictive factor of relapse in child-hood B-lineage acute lymphoblastic leukemia with medium risk features. A case control study of the International BFM study group. Leukemia 14,1939-43 (2000).

133. Nyvold, C., Madsen, H.O., Ryder, L.P., Seyfarth, J., Svejgaard, A., Clausen, N., Wesenberg, F., Jonsson, O.G., Forestier, E. & Schmiegelow, K. Precise quantification of minimal residual disease at day 29 allows iden-tification of children with acute lymphoblastic leukemia and an excellent outcome. Blood 99, 1253-8 (2002).

134. Cave, H., van der Werff ten Bosch, J., Suciu, S., Guidal, C., Waterkeyn, C., Otten, J., Bakkus, M., Thielemans, K., Grandchamp, B. & Vilmer, E. Clini-cal significance of minimal residual disease in childhood acute lymphoblas-tic leukemia. European Organization for Research and Treatment of Can-cer--Childhood Leukemia Cooperative Group. N Engl J Med 339, 591-8 (1998).

135. Bakkus, M.H., Bouko, Y., Samson, D., Apperley, J.F., Thielemans, K., Camp, B.V., Benner, A., Goldschmidt, H., Moos, M. & Cremer, F.W. Post-transplantation tumour load in bone marrow, as assessed by quantitative ASO-PCR, is a prognostic parameter in multiple myeloma. Br J Haematol126, 665-74 (2004).

136. Bahler, D.W. & Levy, R. Clonal evolution of a follicular lymphoma: evi-dence for antigen selection. Proc Natl Acad Sci U S A 89, 6770-4 (1992).

137. Bahler, D.W., Campbell, M.J., Hart, S., Miller, R.A., Levy, S. & Levy, R. Ig VH gene expression among human follicular lymphomas. Blood 78,1561-8 (1991).

138. Lossos, I.S., Alizadeh, A.A., Eisen, M.B., Chan, W.C., Brown, P.O., Bot-stein, D., Staudt, L.M. & Levy, R. Ongoing immunoglobulin somatic muta-tion in germinal center B cell-like but not in activated B cell-like diffuse large cell lymphomas. Proc Natl Acad Sci U S A 97, 10209-13 (2000).

139. Alizadeh, A.A., Eisen, M.B., Davis, R.E., Ma, C., Lossos, I.S., Rosenwald, A., Boldrick, J.C., Sabet, H., Tran, T., Yu, X., Powell, J.I., Yang, L., Marti, G.E., Moore, T., Hudson, J., Jr., Lu, L., Lewis, D.B., Tibshirani, R., Sher-lock, G., Chan, W.C., Greiner, T.C., Weisenburger, D.D., Armitage, J.O., Warnke, R., Levy, R., Wilson, W., Grever, M.R., Byrd, J.C., Botstein, D., Brown, P.O. & Staudt, L.M. Distinct types of diffuse large B-cell lym-phoma identified by gene expression profiling. Nature 403, 503-11 (2000).

59

140. Wright, G., Tan, B., Rosenwald, A., Hurt, E.H., Wiestner, A. & Staudt, L.M. A gene expression-based method to diagnose clinically distinct sub-groups of diffuse large B cell lymphoma. Proc Natl Acad Sci U S A 100,9991-6 (2003).

141. Klein, U., Tu, Y., Stolovitzky, G.A., Mattioli, M., Cattoretti, G., Husson, H., Freedman, A., Inghirami, G., Cro, L., Baldini, L., Neri, A., Califano, A. & Dalla-Favera, R. Gene expression profiling of B cell chronic lympho-cytic leukemia reveals a homogeneous phenotype related to memory B cells. J Exp Med 194, 1625-38 (2001).

142. Hummel, M., Tamaru, J., Kalvelage, B. & Stein, H. Mantle cell (previously centrocytic) lymphomas express VH genes with no or very little somatic mutations like the physiologic cells of the follicle mantle. Blood 84, 403-7 (1994).

143. Andersen, N.S., Donovan, J.W., Borus, J.S., Poor, C.M., Neuberg, D., Aster, J.C., Nadler, L.M., Freedman, A.S. & Gribben, J.G. Failure of im-munologic purging in mantle cell lymphoma assessed by polymerase chain reaction detection of minimal residual disease. Blood 90, 4212-21 (1997).

144. Du, M.Q., Diss, T.C., Xu, C.F., Wotherspoon, A.C., Isaacson, P.G. & Pan, L.X. Ongoing immunoglobulin gene mutations in mantle cell lymphomas. Br J Haematol 96, 124-31 (1997).

145. Welzel, N., Le, T., Marculescu, R., Mitterbauer, G., Chott, A., Pott, C., Kneba, M., Du, M.Q., Kusec, R., Drach, J., Raderer, M., Mannhalter, C., Lechner, K., Nadel, B. & Jaeger, U. Templated nucleotide addition and immunoglobulin JH-gene utilization in t(11;14) junctions: implications for the mechanism of translocation and the origin of mantle cell lymphoma. Cancer Res 61, 1629-36 (2001).

146. Maloum, K., Magnac, C., Azgui, Z., Cau, C., Charlotte, F., Binet, J.L., Merle-Beral, H. & Dighiero, G. VH gene expression in hairy cell leukae-mia. Br J Haematol 101, 171-8 (1998).

147. Miranda, R.N., Cousar, J.B., Hammer, R.D., Collins, R.D. & Vnencak-Jones, C.L. Somatic mutation analysis of IgH variable regions reveals that tumor cells of most parafollicular (monocytoid) B-cell lymphoma, splenic marginal zone B-cell lymphoma, and some hairy cell leukemia are com-posed of memory B lymphocytes. Hum Pathol 30, 306-12 (1999).

148. Forconi, F., Sahota, S.S., Raspadori, D., Mockridge, C.I., Lauria, F. & Stevenson, F.K. Tumor cells of hairy cell leukemia express multiple clonally related immunoglobulin isotypes via RNA splicing. Blood 98,1174-81 (2001).

149. Schroeder, H.W., Jr. & Dighiero, G. The pathogenesis of chronic lympho-cytic leukemia: analysis of the antibody repertoire. Immunol Today 15,288-94 (1994).

150. Brezinschek, H.P., Foster, S.J., Brezinschek, R.I., Dorner, T., Domiati-Saad, R. & Lipsky, P.E. Analysis of the human VH gene repertoire. Differ-ential effects of selection and somatic hypermutation on human peripheral CD5(+)/IgM+ and CD5(-)/IgM+ B cells. J Clin Invest 99, 2488-501 (1997).

151. Kraj, P., Rao, S.P., Glas, A.M., Hardy, R.R., Milner, E.C. & Silberstein, L.E. The human heavy chain Ig V region gene repertoire is biased at all

60

stages of B cell ontogeny, including early pre-B cells. J Immunol 158,5824-32 (1997).

152. Rao, S.P., Riggs, J.M., Friedman, D.F., Scully, M.S., LeBien, T.W. & Sil-berstein, L.E. Biased VH gene usage in early lineage human B cells: evi-dence for preferential Ig gene rearrangement in the absence of selection. JImmunol 163, 2732-40 (1999).

153. Wang, X. & Stollar, B.D. Immunoglobulin VH gene expression in human aging. Clin Immunol 93, 132-42 (1999).

154. Geiger, K.D., Klein, U., Brauninger, A., Berger, S., Leder, K., Rajewsky, K., Hansmann, M.L. & Kuppers, R. CD5-positive B cells in healthy elderly humans are a polyclonal B cell population. Eur J Immunol 30, 2918-23 (2000).

155. Feeney, A.J., Goebel, P. & Espinoza, C.R. Many levels of control of V gene rearrangement frequency. Immunol Rev 200, 44-56 (2004).

156. McMurry, M.T. & Krangel, M.S. A role for histone acetylation in the de-velopmental regulation of VDJ recombination. Science 287, 495-8 (2000).

157. Johnson, K., Angelin-Duclos, C., Park, S. & Calame, K.L. Changes in histone acetylation are associated with differences in accessibility of V(H) gene segments to V-DJ recombination during B-cell ontogeny and devel-opment. Mol Cell Biol 23, 2438-50 (2003).

158. Potter, K.N. Molecular characterization of cold agglutinins. Transfus Sci22, 113-9 (2000).

159. He, X., Goronzy, J.J., Zhong, W., Xie, C. & Weyand, C.M. VH3-21 B cells escape from a state of tolerance in rheumatoid arthritis and secrete rheuma-toid factor. Mol Med 1, 768-80 (1995).

160. Elagib, K.E., Tengner, P., Levi, M., Jonsson, R., Thompson, K.M., Natvig, J.B. & Wahren-Herlenius, M. Immunoglobulin variable genes and epitope recognition of human monoclonal anti-Ro 52-kd in primary Sjogren's syn-drome. Arthritis Rheum 42, 2471-81 (1999).

161. Fais, F., Ghiotto, F., Hashimoto, S., Sellars, B., Valetto, A., Allen, S.L., Schulman, P., Vinciguerra, V.P., Rai, K., Rassenti, L.Z., Kipps, T.J., Dighiero, G., Schroeder, H.W., Jr., Ferrarini, M. & Chiorazzi, N. Chronic lymphocytic leukemia B cells express restricted sets of mutated and unmu-tated antigen receptors. J Clin Invest 102, 1515-25 (1998).

162. Rosenquist, R., Lindstrom, A., Holmberg, D., Lindh, J. & Roos, G. V(H) gene family utilization in different B-cell lymphoma subgroups. Eur J Haematol 62, 123-8 (1999).

163. Johnson, T.A., Rassenti, L.Z. & Kipps, T.J. Ig VH1 genes expressed in B cell chronic lymphocytic leukemia exhibit distinctive molecular features. JImmunol 158, 235-46 (1997).

164. Tobin, G., Thunberg, U., Johnson, A., Thorn, I., Soderberg, O., Hultdin, M., Botling, J., Enblad, G., Sallstrom, J., Sundstrom, C., Roos, G. & Rosenquist, R. Somatically mutated Ig V(H)3-21 genes characterize a new subset of chronic lymphocytic leukemia. Blood 99, 2262-4 (2002).

165. Tobin, G., Thunberg, U., Johnson, A., Eriksson, I., Soderberg, O., Karls-son, K., Merup, M., Juliusson, G., Vilpo, J., Enblad, G., Sundstrom, C., Roos, G. & Rosenquist, R. Chronic lymphocytic leukemias utilizing the

61

VH3-21 gene display highly restricted Vlambda2-14 gene use and homolo-gous CDR3s: implicating recognition of a common antigen epitope. Blood101, 4952-7 (2003).

166. Funkhouser, W.K. & Warnke, R.A. Preferential IgH V4-34 gene segment usage in particular subtypes of B-cell lymphoma detected by antibody 9G4. Hum Pathol 29, 1317-21 (1998).

167. Marasca, R., Vaccari, P., Luppi, M., Zucchini, P., Castelli, I., Barozzi, P., Cuoghi, A. & Torelli, G. Immunoglobulin gene mutations and frequent use of VH1-69 and VH4-34 segments in hepatitis C virus-positive and hepatitis C virus-negative nodal marginal zone B-cell lymphoma. Am J Pathol 159,253-61 (2001).

168. Bahler, D.W., Miklos, J.A. & Swerdlow, S.H. Ongoing Ig gene hypermuta-tion in salivary gland mucosa-associated lymphoid tissue-type lymphomas. Blood 89, 3335-44 (1997).

169. Rettig, M.B., Vescio, R.A., Cao, J., Wu, C.H., Lee, J.C., Han, E., Der-Danielian, M., Newman, R., Hong, C., Lichtenstein, A.K. & Berenson, J.R. VH gene usage is multiple myeloma: complete absence of the VH4.21 (VH4-34) gene. Blood 87, 2846-52 (1996).

170. Tobin, G., Thunberg, U., Karlsson, K., Murray, F., Laurell, A., Willander, K., Enblad, G., Merup, M., Vilpo, J., Juliusson, G., Sundstrom, C., Soder-berg, O., Roos, G. & Rosenquist, R. Subsets with restricted immunoglobu-lin gene rearrangement features indicate a role for antigen selection in the development of chronic lymphocytic leukemia. Blood (2004).

171. Potter, K.N., Orchard, J., Critchley, E., Mockridge, C.I., Jose, A. & Steven-son, F.K. Features of the overexpressed V1-69 genes in the unmutated sub-set of chronic lymphocytic leukemia are distinct from those in the healthy elderly repertoire. Blood 101, 3082-4 (2003).

172. Xu, J.L. & Davis, M.M. Diversity in the CDR3 region of V(H) is sufficient for most antibody specificities. Immunity 13, 37-45 (2000).

173. Lenhoff, S., Hjorth, M., Holmberg, E., Turesson, I., Westin, J., Nielsen, J.L., Wisloff, F., Brinch, L., Carlson, K., Carlsson, M., Dahl, I.M., Gim-sing, P., Hippe, E., Johnsen, H., Lamvik, J., Lofvenberg, E., Nesthus, I. & Rodjer, S. Impact on survival of high-dose therapy with autologous stem cell support in patients younger than 60 years with newly diagnosed multi-ple myeloma: a population-based study. Nordic Myeloma Study Group. Blood 95, 7-11 (2000).

174. Kuppers, R., Zhao, M., Rajewsky, K. & Hansmann, M.L. Detection of clonal B cell populations in paraffin-embedded tissues by polymerase chain reaction. Am J Pathol 143, 230-9 (1993).

175. Kuppers, R., Willenbrock, K., Rajewsky, K. & Hansmann, M.L. Detection of clonal lambda light chain gene rearrangements in frozen and paraffin-embedded tissues by polymerase chain reaction. Am J Pathol 147, 806-14 (1995).

176. Thunberg, U., Rosenquist, R., Lindstrom, A., Lindh, J., Sundstrom, C., Roos, G. & Sallstrom, J. Comparative analysis of detection systems for evaluation of PCR amplified immunoglobulin heavy-chain gene rear-rangements. Diagn Mol Pathol 6, 140-6 (1997).

62

177. Li, A.H., Rosenquist, R., Forestier, E., Lindh, J. & Roos, G. Detailed clon-ality analysis of relapsing precursor B acute lymphoblastic leukemia: im-plications for minimal residual disease detection. Leuk Res 25, 1033-45 (2001).

178. Thunberg, U., Alemi, M., Sundstrom, C. & Sallstrom, J. Nonradioactive detection of monoclonal immunoglobulin heavy chain gene rearrangement with PCR-SSCP. Hematol Pathol 9, 141-53 (1995).

179. Thunberg, U., Banghagen, M., Bengtsson, M., Christensen, L.D., Geisler, C.H., Gimsing, P., Lenhoff, S., Mortensen, B.T., Olofsson, T., Simonsson, B., Andersen, N.S., Sundstrom, C., Swedin, A., Sallstrom, J.F., Thuresson, B., Westin, J. & Carlson, K. Linear reduction of clonal cells in stem cell en-riched grafts in transplanted multiple myeloma. Br J Haematol 104, 546-52 (1999).

180. Deane, M. & Norton, J.D. Immunoglobulin heavy chain variable region family usage is independent of tumor cell phenotype in human B lineage leukemias. Eur J Immunol 20, 2209-17 (1990).

181. Deane, M., McCarthy, K.P., Wiedemann, L.M. & Norton, J.D. An im-proved method for detection of B-lymphoid clonality by polymerase chain reaction. Leukemia 5, 726-30 (1991).

182. Pittaluga, S., Tierens, A., Pinyol, M., Campo, E., Delabie, J. & De Wolf-Peeters, C. Blastic variant of mantle cell lymphoma shows a heterogenous pattern of somatic mutations of the rearranged immunoglobulin heavy chain variable genes. Br J Haematol 102, 1301-6 (1998).

183. Laszlo, T., Nagy, M., Kelenyi, G. & Matolcsy, A. Immunoglobulin V(H) gene mutational analysis suggests that blastic variant of mantle cell lym-phoma derives from different stages of B-cell maturation. Leuk Res 24, 27-31 (2000).

184. MacLennan, I.C., Toellner, K.M., Cunningham, A.F., Serre, K., Sze, D.M., Zuniga, E., Cook, M.C. & Vinuesa, C.G. Extrafollicular antibody re-sponses. Immunol Rev 194, 8-18 (2003).

185. Chiorazzi, N. & Ferrarini, M. B cell chronic lymphocytic leukemia: lessons learned from studies of the B cell antigen receptor. Annu Rev Immunol 21,841-94 (2003).

186. Weller, S., Faili, A., Garcia, C., Braun, M.C., Le Deist, F.F., de Saint Basile, G.G., Hermine, O., Fischer, A., Reynaud, C.A. & Weill, J.C. CD40-CD40L independent Ig gene hypermutation suggests a second B cell diver-sification pathway in humans. Proc Natl Acad Sci U S A 98, 1166-70 (2001).

187. Kienle, D., Krober, A., Katzenberger, T., Ott, G., Leupolt, E., Barth, T.F., Moller, P., Benner, A., Habermann, A., Muller-Hermelink, H.K., Bentz, M., Lichter, P., Dohner, H. & Stilgenbauer, S. VH mutation status and VDJ rearrangement structure in mantle cell lymphoma: correlation with genomic aberrations, clinical characteristics, and outcome. Blood 102, 3003-9 (2003).

188. Camacho, F.I., Algara, P., Rodriguez, A., Ruiz-Ballesteros, E., Mollejo, M., Martinez, N., Martinez-Climent, J.A., Gonzalez, M., Mateo, M., Caleo, A., Sanchez-Beato, M., Menarguez, J., Garcia-Conde, J., Sole, F., Campo, E. &

63

Piris, M.A. Molecular heterogeneity in MCL defined by the use of specific VH genes and the frequency of somatic mutations. Blood 101, 4042-6 (2003).

189. Bertoni, F., Conconi, A., Cogliatti, S.B., Schmitz, S.F., Ghielmini, M., Cerny, T., Fey, M., Pichert, G., Bertolini, F., Ponzoni, M., Baldini, L., Jones, C., Auer, R., Zucca, E., Cavalli, F. & Cotter, F.E. Immunoglobulin heavy chain genes somatic hypermutations and chromosome 11q22-23 de-letion in classic mantle cell lymphoma: a study of the Swiss Group for Clinical Cancer Research. Br J Haematol 124, 289-98 (2004).

190. Flordal Thelander, E., Walsh, S.H., Thorselius, M., Laurell, A., Landgren, O., Larsson, C., Rosenquist, R. & Lagercrantz, S. Mantle cell lymphomas with clonal immunoglobulin V(H)3-21 gene rearrangements exhibit fewer genomic imbalances than mantle cell lymphomas utilizing other immu-noglobulin V(H) genes. Mod Pathol (2004).

191. Efremov, D.G., Ivanovski, M., Siljanovski, N., Pozzato, G., Cevreska, L., Fais, F., Chiorazzi, N., Batista, F.D. & Burrone, O.R. Restricted immu-noglobulin VH region repertoire in chronic lymphocytic leukemia patients with autoimmune hemolytic anemia. Blood 87, 3869-76 (1996).

192. Ivanovski, M., Silvestri, F., Pozzato, G., Anand, S., Mazzaro, C., Burrone, O.R. & Efremov, D.G. Somatic hypermutation, clonal diversity, and pref-erential expression of the VH 51p1/VL kv325 immunoglobulin gene com-bination in hepatitis C virus-associated immunocytomas. Blood 91, 2433-42 (1998).

193. Bahler, D.W. & Swerdlow, S.H. Clonal salivary gland infiltrates associated with myoepithelial sialadenitis (Sjogren's syndrome) begin as nonmalignant antigen-selected expansions. Blood 91, 1864-72 (1998).

194. Maloum, K., Davi, F., Merle-Beral, H., Pritsch, O., Magnac, C., Vuillier, F., Dighiero, G., Troussard, X., Mauro, F.F. & Benichou, J. Expression of unmutated VH genes is a detrimental prognostic factor in chronic lympho-cytic leukemia. Blood 96, 377-9 (2000).

195. Matthews, C., Catherwood, M., Morris, T.C. & Alexander, H.D. Routine analysis of IgVH mutational status in CLL patients using BIOMED-2 stan-dardized primers and protocols. Leuk Lymphoma 45, 1899-904 (2004).

196. Degan, M., Bomben, R., Bo, M.D., Zucchetto, A., Nanni, P., Rupolo, M., Steffan, A., Attadia, V., Ballerini, P.F., Damiani, D., Pucillo, C., Poeta, G.D., Colombatti, A. & Gattei, V. Analysis of IgV gene mutations in B cell chronic lymphocytic leukaemia according to antigen-driven selection iden-tifies subgroups with different prognosis and usage of the canonical so-matic hypermutation machinery. Br J Haematol 126, 29-42 (2004).

197. Forconi, F., Sahota, S.S., Lauria, F. & Stevenson, F.K. Revisiting the defi-nition of somatic mutational status in B-cell tumors: does 98% homology mean that a V(H)-gene is unmutated? Leukemia 18, 882-3 (2004).

198. Ottensmeier, C.H., Thompsett, A.R., Zhu, D., Wilkins, B.S., Sweetenham, J.W. & Stevenson, F.K. Analysis of VH genes in follicular and diffuse lymphoma shows ongoing somatic mutation and multiple isotype tran-scripts in early disease with changes during disease progression. Blood 91,4292-9 (1998).

64

199. Forconi, F., Sahota, S.S., Raspadori, D., Ippoliti, M., Babbage, G., Lauria, F. & Stevenson, F.K. Hairy cell leukemia: at the cross road of somatic mu-tation and isotype switch. Blood (2004).

200. Aarts, W.M., Bende, R.J., Steenbergen, E.J., Kluin, P.M., Ooms, E.C., Pals, S.T. & van Noesel, C.J. Variable heavy chain gene analysis of follicular lymphomas: correlation between heavy chain isotype expression and so-matic mutation load. Blood 95, 2922-9 (2000).

201. Walsh, S.H., Thorselius, M., Johnson, A., Soderberg, O., Jerkeman, M., Bjorck, E., Eriksson, I., Thunberg, U., Landgren, O., Ehinger, M., Lofven-berg, E., Wallman, K., Enblad, G., Sander, B., Porwit-MacDonald, A., Dic-tor, M., Olofsson, T., Sundstrom, C., Roos, G. & Rosenquist, R. Mutated VH genes and preferential VH3-21 use define new subsets of mantle cell lymphoma. Blood 101, 4047-54 (2003).

202. Bensinger, W.I., Buckner, C.D., Shannon-Dorcy, K., Rowley, S., Appel-baum, F.R., Benyunes, M., Clift, R., Martin, P., Demirer, T., Storb, R., Lee, M. & Schiller, G. Transplantation of allogeneic CD34+ peripheral blood stem cells in patients with advanced hematologic malignancy. Blood 88,4132-8 (1996).

203. Gazitt, Y., Reading, C.C., Hoffman, R., Wickrema, A., Vesole, D.H., Ja-gannath, S., Condino, J., Lee, B., Barlogie, B. & Tricot, G. Purified CD34+ Lin- Thy+ stem cells do not contain clonal myeloma cells. Blood 86, 381-9 (1995).

204. Gorin, N.C., Lopez, M., Laporte, J.P., Quittet, P., Lesage, S., Lemoine, F., Berenson, R.J., Isnard, F., Grande, M., Stachowiak, J. & et al. Preparation and successful engraftment of purified CD34+ bone marrow progenitor cells in patients with non-Hodgkin's lymphoma. Blood 85, 1647-54 (1995).

205. Lemoli, R.M., Fortuna, A., Motta, M.R., Rizzi, S., Giudice, V., Nannetti, A., Martinelli, G., Cavo, M., Amabile, M., Mangianti, S., Fogli, M., Conte, R. & Tura, S. Concomitant mobilization of plasma cells and hematopoietic progenitors into peripheral blood of multiple myeloma patients: positive se-lection and transplantation of enriched CD34+ cells to remove circulating tumor cells. Blood 87, 1625-34 (1996).

206. Schiller, G., Vescio, R., Freytes, C., Spitzer, G., Sahebi, F., Lee, M., Wu, C.H., Cao, J., Lee, J.C., Hong, C.H. & et al. Transplantation of CD34+ pe-ripheral blood progenitor cells after high-dose chemotherapy for patients with advanced multiple myeloma. Blood 86, 390-7 (1995).

207. Pico, J.L., Bourhis, J.H., Bennaceur, A.L., Beaujean, F., Bayle, C., Ibrahim, A., Girinski, T., Hayat, M., Dighiero, G. & Tchernia, G. Engraftment of CD34+ peripheral blood progenitor cells into multiple myeloma patients following total body irradiation. Nouv Rev Fr Hematol 37, 381-3 (1995).

208. Pilarski, L.M. & Jensen, G.S. Monoclonal circulating B cells in multiple myeloma. A continuously differentiating, possibly invasive, population as defined by expression of CD45 isoforms and adhesion molecules. Hematol Oncol Clin North Am 6, 297-322 (1992).

209. Szczepek, A.J., Bergsagel, P.L., Axelsson, L., Brown, C.B., Belch, A.R. & Pilarski, L.M. CD34+ cells in the blood of patients with multiple myeloma

65

express CD19 and IgH mRNA and have patient-specific IgH VDJ gene re-arrangements. Blood 89, 1824-33 (1997).

210. Lemoli, R.M., Martinelli, G., Zamagni, E., Motta, M.R., Rizzi, S., Ter-ragna, C., Rondelli, R., Ronconi, S., Curti, A., Bonifazi, F., Tura, S. & Cavo, M. Engraftment, clinical, and molecular follow-up of patients with multiple myeloma who were reinfused with highly purified CD34+ cells to support single or tandem high-dose chemotherapy. Blood 95, 2234-9 (2000).

211. Patriarca, F., Damiani, D., Fanin, R., Grimaz, S., Geromin, A., Cerno, M., Sperotto, A., Silvestri, F., Zaja, F. & Baccarani, M. High-dose therapy in multiple myeloma: effect of positive selection of CD34+ peripheral blood stem cells on hematologic engraftment and clinical outcome. Haema-tologica 85, 269-74 (2000).

212. Attal, M., Harousseau, J.L., Stoppa, A.M., Sotto, J.J., Fuzibet, J.G., Rossi, J.F., Casassus, P., Maisonneuve, H., Facon, T., Ifrah, N., Payen, C. & Bataille, R. A prospective, randomized trial of autologous bone marrow transplantation and chemotherapy in multiple myeloma. Intergroupe Fran-cais du Myelome. N Engl J Med 335, 91-7 (1996).

213. Horvath, N., Hahn, U., Joshua, D., Dyson, P., Gibson, J., Stevens, J., Rawl-ing, T., Barrow, L., Brown, R., Stephens, S., Gower, G., Norman, J., Mills, B. & To, L.B. Long-term follow up of sequential mobilisation and autolo-gous transplantation with CD34-selected cells in multiple myeloma: a mul-timodality approach. Intern Med J 34, 167-75 (2004).

214. Morineau, N., Tang, X.W., Moreau, P., Milpied, N., Mahe, B., Bataille, R. & Harousseau, J.L. Lack of benefit of CD34+ cell selected over non-selected peripheral blood stem cell transplantation in multiple myeloma: re-sults of a single center study. Leukemia 14, 1815-20 (2000).

215. Vescio, R., Schiller, G., Stewart, A.K., Ballester, O., Noga, S., Rugo, H., Freytes, C., Stadtmauer, E., Tarantolo, S., Sahebi, F., Stiff, P., Meharchard, J., Schlossman, R., Brown, R., Tully, H., Benyunes, M., Jacobs, C., Ber-enson, R., DiPersio, J., Anderson, K. & Berenson, J. Multicenter phase III trial to evaluate CD34(+) selected versus unselected autologous peripheral blood progenitor cell transplantation in multiple myeloma. Blood 93, 1858-68 (1999).

216. Gupta, D., Bybee, A., Cooke, F., Giles, C., Davis, J.G., McDonald, C., Armitage, S.E., McGuigan, D., Lyttelton, M.P., Kanfer, E.J., Apperley, J.F. & Samson, D. CD34+-selected peripheral blood progenitor cell transplanta-tion in patients with multiple myeloma: tumour cell contamination and out-come. Br J Haematol 104, 166-77 (1999).

Acta Universitatis UpsaliensisComprehensive Summaries of Uppsala Dissertations

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A doctoral dissertation from the Faculty of Medicine, Uppsala University,is usually a summary of a number of papers. A few copies of the completedissertation are kept at major Swedish research libraries, while the sum-mary alone is distributed internationally through the series Comprehen-sive Summaries of Uppsala Dissertations from the Faculty of Medicine.(Prior to October, 1985, the series was published under the title “Abstracts ofUppsala Dissertations from the Faculty of Medicine”.)