canaadian t jouhrnal ofolog y volume 6, issue 2 summer 2014canadianjournalofpathology.ca ›...

Canadian Journal of

Pathology

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Official Publication of the Canadian Association of Pathologists

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Volume 6, Issue 2 • Summer 2014

OOfficiafficiall PuPubbllicaicattionion ofof tthhee CanadianCanadian AssAssoociaciattionion ooff PPaattholholoogisgisttss

CAP-ACP Junior Scientist Award Lecture 2013: Molecular Classification of Endometrial Sarcoma

Kulcsar Lecture 2013: Pulmonary Cytology – Blowing New Life into Diagnostic Challenges

DISPLAY CLASSIFIED

Academic HematopathologistVancouver Acute (Vancouver General

Hospital, UBC, G.F. Strong)Dept. of Pathology and Laboratory

Medicine, Division of Hematopathology

Vancouver Acute is the largest tertiary care and teaching institution in western Canada and the Vancouver General

and transfusion medicine hub for Vancouver Coastal Health Laboratories. There is a wide spectrum of routine and

medicine cases, including support of the provincial leukemia-bone marrow transplant program and a large group of general haematologists. The successful applicant will provide clinical service and academic teaching of residents and fellows in blood and marrow interpretation,

successful applicant will work collaboratively with the Division Head/Regional Hematopathology Medical Leader, the Transfusion Medicine Regional Medical Leader and four other full-time hematopathologists.

Pathology and additional formal training, experience, or

Hematopathology, clinical epidemiology or medical

With a clinical appointment at the University of British Columbia (UBC) in the Department of Pathology and Laboratory Medicine the successful applicant will engage in Departmental research and teaching programs including undergraduates, graduate students and residents. Further, the successful applicant will collaborate on health authority initiatives pertinent to laboratory hematology, and participate in quality committees and research.

Salary and hospital rank will be commensurate with

Applications will be accepted and evaluated on an ongoing basis until a suitable candidate is found.

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however, in accordance with Canadian immigration

permanent residents of Canada.

Please submit a letter of application, current CV, and the contact information of three references to:

Hematopathology, Vancouver Acute, JPPN-1, 855 West 12th Avenue, Vancouver, BC V5Z 1M9 Fax: 604-875-4798

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Canadian Journal of P athology 35Summer 2014

Original Articles CAP-ACP Junior Scientist Award Lecture 2013: MolecularClassification of Endometrial SarcomaCheng-Han Lee, MD, PhD, FRCPC

Kulcsar Lecture 2013: Pulmonary Cytology – Blowing New Life intoDiagnostic ChallengesManon Auger, MD, FRCPC

Cyclin D1–Positive Plasma Cell Leukemia with LymphoplasmacyticMorphology and t(11;14)(q13;q32): Case Report and Brief LiteratureReviewIrfan K. Khan, MD, Fariborz Rashid-Kolvear, PhD, Adnan Mansoor, MD,Jay L. Patel, MD

Medulloblastoma – An Unusual Variant of PrimitiveNeuroectodermal Tumour Arising in a Mature Teratoma of Testis:Case ReportMichelle R. Downes, MB, MRCSI, MD, FRCPC, Theo van der Kwast, MD, PhD

Professional Development/Employment OpportunitiesVancouver Coastal HealthUHN - Toronto General HospitalHologicVancouver Coastal Health

Canadian Journal of Pathology • Volume 6, Issue 2, 2014

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Publications Agreement Number 40025049 • ISSN 1918-915X

EDITOR-IN-CHIEFJ. Godfrey Heathcote, MA, MB BChir, PhD, FRCPC

EDITORIAL BOARDManon Auger, MD, FRCPC, Cytopathology;

Calvino Cheng, BSc, MD, FRCPC, Pathology Informaticsand Quality Management;

Pierre Douville, MD, FRCPC, Medical Biochemistry;David K. Driman, MB ChB, FRCPC, Anatomical Pathology;

Lawrence Haley, MD, FRCPC, Hematopathology;Todd F. Hatchette, BSc, MD, FRCPC, Medical Microbiology;

Michael J. Shkrum, MD, FRCPC, Forensic Pathology;

FOUNDING EDITORJagdish Butany, MBBS, MS, FRCPC

MANAGING EDITORSusan Harrison

COPY EDITORMichael Peebles

PROOFREADERScott Bryant

ART DIRECTORAndrea Mulholland, [email protected]

TRANSLATORSMarie Dumont, Suzanne Legendre

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ACCOUNTINGSusan McClung

GROUP PUBLI SHERJohn D. Birkby, [email protected]

VOLUME 6, ISSUE 2, 2014

Contents

The cover image shows fine-needleaspirate of lung adenocarcinoma.Malignant cells contain vacuolatedcytoplasm and conspicuous nucleoli.

About the Cover

Summer 201436 Canadian Journal of P athology

CAP-ACP Junior Scientist Award Lecture 2013:Molecular Classification of Endometrial

Sarcoma

Cheng-Han Lee, MD, PhD, FRCPC, is a member of the Department of Laboratory Medicine and Pathology at the University ofAlberta and the Royal Alexandra Hospital, in Edmonton, Alberta. Correspondence may be directed to Cheng-Han Lee [email protected] article has been peer reviewed.Competing interests: None declared

Cheng-Han Lee, MD, PhD, FRCPC

ABSTRACTEndometrial stromal sarcomas (ESSs) are traditionally classified into low-grade and high-grade

groups. The diagnostic category of high-grade ESS was removed from the 2003 World Health

Organization tumour classification system and replaced by undifferentiated endometrial

sarcoma (UES) in recognition of the fact that many tumours defined as high-grade ESS were

pleomorphic sarcomas that bear no resemblance to endometrial stromal tissue. Consequently,

tumours that were previously considered as high-grade ESS have to be reclassified as low-grade

ESS or UES. Recent advances in our understanding of the genetics of ESS, however, provide

strong support for the existence of a group of histologically high-grade ESS, which is

characterized by YWHAE-NUTM2 genetic fusion resulting from the translocation

t(10;17)(q22;p13). High-grade ESS with YWHAE-NUTM2 fusion shows more frequent

recurrence and is associated with decreased survival compared to low-grade ESS (characterized

most commonly by JAZF1-SUZ12 fusion). It does, however, show significantly better disease-

specific survival compared to UES. The histological, immunophenotypical, and genetic features

of low-grade ESS, high-grade ESS, and UES, with emphasis on the diagnostic distinction

between these clinically different tumour types, are reviewed.

RÉSUMÉ Les sarcomes du stroma endométrial (SSE) sont habituellement classés en deux catégories : bas

grade et haut grade. La catégorie dia gnostique de SSE de haut grade a été retirée en 2003 du

système de classification des tumeurs de l’Organisation mondiale de la santé et remplacée par

le terme sarcome endométrial indifférencié (SEI) en reconnaissance du fait que de nombreuses

tumeurs définies comme des SSE de haut grade étaient des sarcomes pléomorphes qui ne

ressemblent en rien au tissu stromal endométrial. Par conséquent, les tumeurs qui étaient

antérieurement considérées comme des SSE de haut grade doivent être reclassées comme des

SSE de bas grade ou des SEI. Toutefois, les progrès récents dans notre compréhension de la

génétique des SSE apportent des arguments solides en faveur de l’existence d’un groupe de SSE

de haut grade confirmés histologiquement, qui est caractérisé par fusion génétique YWHAE-

NUTM2 résultant de la translocation t(10;17)(q22;p13). Les SSE de haut grade avec fusion

YWHAE-NUTM2 montrent une récidive plus fréquente et sont associés à un taux de survie

ORIGINAL ARTICLE


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Endometrial stromal sarcoma (ESS) is the second most

common type of uterine sarcoma. It is historically

separated into low-grade and high-grade groups. However, the

existence of high-grade ESS became in doubt after the

realization that the degree of mitotic activity in ESS, used by

some to determine tumour grade, does not predict more

aggressive behaviour independently of tumour stage.1,2

Furthermore, it was also apparent that many tumours

classified as high-grade ESS were pleomorphic uterine

sarcomas that showed no morphological resemblance to

endometrial stromal tissue. Therefore, the diagnostic category

of high-grade ESS was removed from the 2003 World Health

Organization (WHO) tumour classification system and

replaced by undifferentiated endometrial sarcoma, while all

instances of ESS were considered low grade.3 Thus, all tumours

that were previously categorized as high-grade ESSs are now

reclassified as either undifferentiated endometrial sarcomas or

low-grade ESSs. However, debate regarding the existence of

high-grade ESS persisted as there are tumours that cannot be

appropriately classified as either undifferentiated endometrial

sarcoma or low-grade ESS. More recently, an improved

understanding of the genetic basis of ESS has provided

objective evidence for the existence of histologically high-grade

ESS and has prompted consideration of the inclusion of the

diagnostic category of high-grade ESS in the upcoming version

of the WHO classification system. This review will discuss

these updates as well as the pathological and molecular features

of low-grade ESS, high-grade ESS, and undifferentiated

endometrial sarcoma.

Low-Grade Endometrial Stromal SarcomaIn the uterine corpus, low-grade ESS characteristically exhibits

prominent “fingerlike” myometrial infiltration with

lymphovascular permeation. In its “classic” form (as currently

defined by the WHO), ESS is composed of a proliferation of

small, round, monomorphic cells with scanty cytoplasm and

round-to-oval nuclei with smooth nuclear contours,

reminiscent of a non-neoplastic proliferative-phase

endometrial stroma (Figure 1A and 1B).1,2,4,5 The stroma

contains a rich arborizing small arteriolar network around

which the tumour cells are concentrically arranged. Mitotic

activity can vary but is typically less than five mitotic figures

(MFs) per ten high-power fields (HPFs). Atypical mitotic

figures are not seen, but a subset of classic low-grade ESS can

display a greater degree of mitotic activity (less than 10 MFs

per 10 HPFs). However, according to the earlier literature,1,2

this finding alone does not appear to be of independent

prognostic value in terms of patient survival if no additional

aggressive features (such as concurrent cytological atypia and

extrauterine spread) are present. Feng et al. recently reported

that increased mitotic activity in low-grade ESS treated with

ovary-preserving surgery is associated with an increased risk

of recurrence,6 but this association needs to be further

validated. Necrosis can also be present although it is typically

not extensive. Stromal hyalinization is frequently seen in the

form of hyaline bands and plaques although some cases can

exhibit extensive hyalinization (Figure 1C and 1D). Collections

of foamy stromal cells and conspicuous lymphocytic infiltrates

may also be present. Although most low-grade ESSs display

the above-described classic morphology, some may display one

of several well-recognized histological variants; these include

tumours with smooth muscle differentiation,7–12 glandular

differentiation,13,14 sex cord–like differentiation (Figure 1E),8

pseudopapillae formation,15 epithelioid morphology,16 and

extensive myxoid-to-fibromyxoid stromal changes (Figure

1F).1,10,11,17,18 Because of their low-grade histological appearance

and typically indolent clinical course (with >95% 5-year

disease-specific survival),6–19 these ESSs are regarded as low-

grade sarcoma.

Genetically, most low-grade ESSs harbour structural

chromosomal rearrangements that result in the fusion between

proteins/enzymes involved in chromatin remodelling. These

réduit comparativement aux SSE de bas grade (caractérisés le plus souvent par fusion JAZF1-

SUZ12). Cependant, ils sont associés à une survie spécifique à la maladie significativement

meilleure comparativement au SEI. Cet article passe en revue les caractéristiques histologiques,

immunophénotypiques et génétiques des SSE de bas grade, des SSE de haut grade et des SEI,

avec un accent sur la distinction diagnostique entre ces types de tumeurs cliniquement

différents.


MOLECULAR CLASSIFICATION OF ENDOMETRIAL SARCOMA

Figure 1. Histological features of low-grade endometrial stromal sarcomas (ESSs). A and B, Two low-grade ESSs (JAZF1-SUZ12) showingclassic low-grade morphology with bland monotonous tumour cells and arborizing stromal arteriolar/capillary network. C and D, Twolow-grade ESSs (JAZF1-SUZ12) showing prominent stromal hyalinization. E, A JAZF1-PHF1 low-grade ESS showing sex cord–likedifferentiation. F, A PHF1-rearranged low-grade ESS showing variant fibrous/fibroblastic morphology. (Hematoxylin and eosin)


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include JAZF1-SUZ12 (formerly known as JJAZ1) in an ESS

with translocation t(7;17),20 JAZF1-PHF1 and EPC1-PHF1 in

an ESS with a 6p rearrangement including t(6;7),21 and

MEAF6-PHF1 genetic fusion in an ESS with t(1;6)(p34;p21).22

The majority of low-grade ESSs with demonstrable genetic

fusions display the classic morphology although variant

histology with smooth muscle differentiation, sex cord

differentiation, or a fibromyxoid appearance has been

documented in JAZF1-SUZ12 and PHF1-rearranged ESS (see

Figure 1E and 1F).11,12,23–26 Clinically, there does not appear to

be any difference between these mechanistically equivalent

genotypes. Histologically, the different genotypes all exhibit

low-grade morphological features (bland monomorphic

nuclei and low mitotic activity) in the majority of cases.21,23

However, one study found more-frequent sex cord

differentiation with tumours characterized by PHF1 genetic

rearrangement.27

A small subset of low-grade ESSs with classic morphology

remains genetically undefined (i.e., lacks demonstrable known

genetic rearrangements). It is unclear whether these low-grade

ESSs are biologically equivalent to low-grade ESSs with genetic

rearrangements. However, there appears to be no difference

clinically, since both behave in the same indolent manner.

Exceptionally, JAZF1-SUZ12 low-grade ESSs can undergo

Figure 2. Histological features of undifferentiated endometrial sarcoma (UES). A, A UES showing a myopermeative growth pattern at theperiphery of the expansile tumour mass. B, A UES showing prominent nuclear pleomorphism. C and D, Two UESs (both negative forYWHAE-NUTM2 genetic rearrangements) showing uniform nuclear features. (Hematoxylin and eosin)



high-grade transformation (dedifferentiation) with the

development of histologically high-grade tumours arising in

a background of classic low-grade ESS. This is such a rare

phenomenon that among nearly 100 molecularly confirmed

ESSs with JAZF1-SUZ12 or equivalent genetic rearrangements

reported to date, only a single molecularly confirmed case has

been documented.28

Immunohistochemically, the majority of low-grade ESSs with

genetic rearrangements show a CD10-positive, estrogen

receptor (ER)–positive, and progesterone receptor (PR)–

positive immunophenotype irrespective of the genotype, and

the staining pattern is typically diffuse in adequately fixed

tumour samples.29–31 Even though this pattern of CD10, ER,

and PR immunostaining is not specific for low-grade ESS, it

does provide support for the diagnosis in the appropriate

histological context (classic or variant morphology). In

addition, low-grade ESS with smooth muscle differentiation

can show positive staining for smooth muscle actin,

caldesmon, and desmin. Cyclin D1 expression is typically weak

and focal in low-grade ESS,32 including both the low-grade and

the high-grade areas of the single reported case of

dedifferentiated JAZF1-SUZ12 ESS.28

Undifferentiated Endometrial SarcomaUndifferentiated endometrial sarcoma (UES) is defined as a

high-grade uterine sarcoma that lacks demonstrable lineage(s)

of cellular differentiation and bears no histological

resemblance to endometrial stroma. Similar to

undifferentiated pleomorphic sarcoma of soft tissue, UES

represents a diagnosis of exclusion that requires thorough

histological and immunohistochemical examination. Whereas

in most cases UES forms a large expansile uterine mass, it can

exhibit “tonguelike” permeation of the adjacent myometrium

at its periphery (Figure 2A). The shape of the tumour cells can

range from spindled to epithelioid, and the growth pattern can

range from nondescript sheets to fascicles. The stromal

vascular pattern is variable but typically lacks the arborizing

arteriolar or fine capillary networks seen in ESS (low-grade or

high-grade). Unlike low-grade ESS, the cytological features of

tumour cells in UES are unquestionably malignant, as they

display moderate-to-severe nuclear atypia typically

accompanied by significant mitotic activity (including atypical

mitotic figures) and conspicuous necrosis. There is prominent

nuclear pleomorphism in most tumours (Figure 2A and 2B),

with tumour giant cells, although a subset of UESs lacks

prominent nuclear pleomorphism (Figure 2C and 2D).

Kurihara et al. have termed the latter “UES with nuclear

uniformity” (UES-U) in contrast to “UES with nuclear

pleomorphism” (UES-P).28 The prognostic significance of such

a distinction remains unclear. The amount of cytoplasm

present varies, and the cytoplasm is typically faintly

eosinophilic in character, in contrast to the well-defined

densely eosinophilic cytoplasm of smooth muscle neoplasms.

The usual histological differential diagnosis includes

leiomyosarcoma, rhabdomyosarcoma, perivascular epithelioid

cell tumour (PEComa), undifferentiated carcinoma,

adenosarcoma, carcinosarcoma, and metastatic tumours.

Depending on the morphological features, a panel of

immunomarkers may be needed to confirm that a suspected

UES is truly undifferentiated. This usually involves a

combination of muscle (smooth muscle actin, caldesmon,

desmin, myogenin), epithelial (pankeratin, endomysial

antibody [EMA]) markers, and melanocytic (S100, HMB45)

markers. By definition, UES lacks expression of these markers

– except for focal, patchy staining for smooth muscle actin,

which is insufficient on its own to warrant the diagnosis of

leiomyosarcoma. CD10 can be expressed also by UES, and its

expression does not indicate endometrial stromal

differentiation.29,33 More important, thorough tumour

sampling is needed to exclude the presence of a pre-existing

component of adenosarcoma (adenosarcoma with

sarcomatous overgrowth) or a component of high-grade

carcinoma (sarcoma-predominant carcinosarcoma).

Little is currently known about UES genetically. Cytogenetic

analysis of UES/high-grade ESS tends to show a complex

karyotype with many structural and numerical chromosomal

aberrations.34While unproven, I suspect that UES as presently

defined is genetically and biologically heterogeneous and likely

encompasses a mixture of tumour types that include very

poorly differentiated or dedifferentiated homologous sarcomas

(i.e., leiomyosarcoma and ESS), sarcoma-predominant

carcinosarcoma, adenosarcoma overgrown by high-grade

sarcoma, undifferentiated carcinoma, and de novo

undifferentiated sarcoma.

High-Grade Endometrial Stromal SarcomaIt was recently shown that about half of tumours previously

designated as high-grade ESS or UES (with uniform nuclear


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Figure 3. Histological features of YWHAE-NUTM2 high-grade endometrial stromal sarcoma (ESS). A and B, YWHAE-NUTM2 ESSscharacteristically show a myopermeative growth pattern (A) and a fine stromal capillary network (B), which imparts a vaguely nestedappearance. C and D, Cells in the high-grade area of a YWHAE-NUTM2 ESS are round to epithelioid, and the amount of cytoplasm canvary. E, Some YWHAE-NUTM2 ESSs contain an admixed low-grade component in which the cells are ovoid to spindled. (A low-grade areais shown in the left lower corner, a high-grade area in right upper corner). F, The high-grade component of YWHAE-NUTM2 ESS showsdiffuse strong nuclear cyclin D1 immunostaining while the low-grade component shows variable and patchy immunostaining. (A to E,hematoxylin and eosin; F, immunoperoxidase)



features) harbour a recurrent chromosomal translocation,

t(10;17)(q22;p13), and this results in YWHAE-NUTM2

genetic fusion.35 NUTM2A/B was formerly known as

FAM22A/B, but the nomenclature was changed to reflect its

sequence homology to NUT protein (encoded by NUTM1)

involved in NUT midline carcinoma.36 The finding of frequent

YWHAE-NUTM2 genetic fusion in what is currently classified

as UES (with nuclear uniformity) was subsequently confirmed

by another group of researchers.37 Compared to JAZF1-SUZ12

ESS, YWHAE-NUTM2 ESS more often presents with high-

stage disease and is associated with more frequent – as well as

earlier – recurrence, either regionally or at distant sites such as

the lungs.38 Most important, YWHAE-NUTM2 ESS is

associated with a 5-year disease-specific survival rate of 70%,

compared to more than 95% for low-grade ESS (JAZF1-

SUZ12) and 30% for UES (UES-P and UES-U) lacking

YWHAE-NUTM2 genetic fusion.19 Therefore, it would not be

clinically appropriate to group YWHAE-NUTM2 ESS under

the category of UES.

All YWHAE-NUTM2ESSs reported to date originate from the

uterus although the extrauterine component may be larger

than the intrauterine component. Similar to classic low-grade

ESS, YWHAE-NUTM2ESS characteristically displays a highly

permeative growth pattern with extensive tonguelike

Figure 4. Histological features of the low-grade component of YWHAE-NUTM2 endometrial stromal sarcoma (ESS). A and B, The low-grade component of a YWHAE-NUTM2 ESS showing classic low-grade ESS-like morphology. C, The low-grade component of aYWHAE-NUTM2 ESS shows variant fibromyxoid low-grade ESS-like morphology. D, The low-grade component of a YWHAE-NUTM2 ESSshows spindle cell proliferation arranged in a fascicular growth pattern. (Hematoxylin and eosin)


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myoinvasion and vascular invasion (Figure 3A).31,35 A rich fine

arborizing stromal capillary network is consistently present;

small concentric arterioles are seen only occasionally (Figure

3B). In contrast to classic low-grade ESSs, all primary YWHAE-

NUTM2ESSs either partly or exclusively contain a high-grade

round cell area. These cells, while monomorphic in

appearance, demonstrate a combination of nucleomegaly

(four to six times the size of stromal lymphocyte nuclei and

with no tumour giant cells), irregular nuclear contour, and

increased mitotic activity (all cases showing greater than

10 MFs per 10 HPFs) (Figure 3C and 3D). The nucleoli are

typically not prominent, and atypical mitoses are not present.

The amount of cytoplasm seen in the high-grade round cells

can range from scanty to moderate. In areas with scanty

cytoplasm, the combination of high cellularity and high-grade

nuclear features imparts a malignant small round blue cell

appearance (see Figure 3D),39 whereas areas with a moderate

amount of faintly eosinophilic cytoplasm can assume an

epithelioid appearance with a vaguely nested pattern (see

Figure 3B and 3C). Furthermore, additional variant features

(including focal sex cord–like differentiation, focal

pseudoglandular and pseudopapillary appearances, and focal

rosettelike formations) have been described in YWHAE-

NUTM2 ESSs.31,39 To date, smooth muscle differentiation

found in a subset of JAZF1-SUZ12 ESS has not been observed

in YWHAE-NUTM2 ESSs.

In about half of YWHAE-NUTM2 ESSs, there is an admixed

histologically low-grade component in which the tumour cells

appear ovoid to spindled (Figure 3E and Figure 4). The low-

grade component usually contains fibrous-to-fibromyxoid

stroma. In contrast to the tumour cells in the high-grade

component, those in the low-grade component exhibit

smooth nuclear contours and lack significant mitotic activity

(up to 3 MFs per 10 HPFs). The low-grade component can

assume a number of different architectural patterns that

include a classic low-grade ESS-like pattern (Figure 4A and

4B), a fibrous/fibromyxoid low-grade ESS pattern (Figure 4C),

and a fascicular spindle cell growth pattern (Figure 4D). The

low-grade ESS-like area of YWHAE-NUTM2 ESS can be

morphologically and immunophenotypically indistinguishable

from the classic form and the fibrous/fibromyxoid variant of

low-grade ESS,17 except that a high-grade component is

invariably seen when the primary tumour is thoroughly

examined. The fascicular spindle cell low-grade area of

YWHAE-NUTM2 can mimic a smooth muscle tumour, but it

lacks well-defined eosinophilic cytoplasm and

immunohistochemical evidence of smooth muscle

differentiation. Because this low-grade component is present

in only some YWHAE-NUTM2 ESSs whereas the high-grade

component is invariably present, the terminology of

dedifferentiated high-grade ESS is not descriptively accurate

for this type of ESS.

The high-grade and low-grade components of YWHAE-

NUTM2 ESS display different immunophenotypes.31 The

low-grade area is consistently positive for ER, PR, and CD10,

in contrast to the high-grade area, which lacks significant ER,

PR, and CD10 immunoreactivity. In contrast, the high-grade

area consistently exhibits diffuse (>70%) and strong nuclear

cyclin D1 immunostaining whereas the low-grade area shows

weak and focal cyclin D1 expression (see Figure 3E and 3F).

Such diffuse strong cyclin D1 expression is very uncommon

among other types of uterine sarcomas, and diffuse cyclin D1

immunostaining coupled with negative CD10 and ER

immunostaining in a high-grade monomorphic endometrial

sarcoma strongly supports a diagnosis of YWHAE-NUTM2

ESS. Reports have found KIT and CD99 to be positive in

YWHAE-NUTM2ESSs,39 but the staining pattern has not been

systematically examined. YWHAE-NUTM2ESS is negative for

pankeratin, EMA, smooth muscle actin, desmin, caldesmon,

S100, HMB-45, and Melan-A.38,39

Proposed Classification of Endometrial SarcomaOur improved understanding of the genetic basis of ESS

supports the return of the diagnostic category of high-grade

ESS, which can now be objectively defined by YWHAE-

NUTM2 fusion. A proposed classification system that

integrates molecular findings is illustrated in Figure 5. The

distinction between YWHAE-NUTM2 high-grade ESS and

UES is clinically important. Our preliminary data show that a

comparable majority of patients with YWHAE-NUTM2 high-

grade ESSs and UESs received adjuvant chemotherapy or

radiation therapy or both. Even though a higher percentage of

YWHAE-NUTM2 high-grade ESSs showed evidence of

extrauterine tumour extension at presentation, patients with

YWHAE-NUTM2 high-grade ESSs had better disease-specific

survival overall than patients with UESs (70% versus 30% 5-

year disease-specific survival).19 This prognostic difference may

reflect not only a difference in the natural history but also a

potential difference in response to adjuvant therapy between

the two diseases (i.e., a greater response to adjuvant therapy

for YWHAE-NUTM2 high-grade ESS when compared to

UES). Diagnostically, the presence of significant nuclear

pleomorphism (readily appreciable at low magnification)

excludes the possibility of YWHAE-NUTM2 high-grade ESS.

In a tumour showing uniform nuclear features, thorough

sampling of a hysterectomy specimen – with attention to the

presence of an admixed low-grade component coupled with

the application of a panel of CD10, ER, and cyclin D1

immunostains in the high-grade area – should help to

differentiate between YWHAE-NUTM2 high-grade ESS and

UES. Genetic testing using reverse transcriptase polymerase

chain reaction (RT-PCR) or fluorescence in situ hybridization

(FISH) to evaluate the presence of YWHAE-NUTM2 genetic

fusion can be used for definitive confirmation in

morphologically and immuno-phenotypically equivocal

cases.40

The distinction between YWHAE-NUTM2 high-grade ESS

and JAZF1-SUZ12 (and other fusion variants) low-grade ESS

is also important not only for prognostication but also for the

choice of therapy. Given that the more proliferative high-grade

component of YWHAE-NUTM2 lacks ER expression, or PR

expression or both, YWHAE-NUTM2 high-grade ESS would

not be expected to respond to antiestrogenic hormonal

therapy in the same manner that ER- and PR-positive low-

grade ESS may. Again, thorough tumour sampling in a

hysterectomy specimen is necessary. In tumours that display

classic low-grade ESS morphology and appropriate low-grade

ESS immunophenotype without a coexisting high-grade

component, a diagnosis of low-grade ESS can be rendered

without the need for molecular confirmation as there is no

known clinical difference between low-grade ESS with and

without demonstrable genetic rearrangements. In tumours

that display variant low-grade ESS morphology (i.e., fibrous

to fibromyxoid) and appropriate low-grade ESS

immunophenotype without a coexisting high-grade

component, genetic testing may be considered, particularly if

extrauterine tumour spread is evident. The above diagnostic

recommendations pertain only to resection specimens (i.e.,

hysterectomies). If a diagnosis of ESS is favoured on a biopsy

sample (i.e., a core biopsy or endometrial biopsy sample), then

CD10, ER, and cyclin D1 immunohistochemistry as well as

genetic testing should be considered to evaluate the possibility

of YWHAE-NUTM2 high-grade ESS, as the degree of nuclear

atypia can be underestimated (particularly in a core biopsy

sample), due to crush artefact.

Future Directions: The Need for Greater Molecular IntegrationThe recognition of histologically high-grade ESS defined by

YWHAE-NUTM2 fusion as a distinct tumour type illustrates

the need for the integration of molecular findings into the

classification of uterine sarcoma. Similar to soft tissue

sarcomas, uterine sarcomas are genetically and biologically

diverse, encompassing both karyotypically simple sarcomas

with characteristic genetic rearrangements (low-grade and

high-grade ESS) and genetically complex sarcomas with no

demonstrable recurrent disease-defining genetic aberrations

(leiomyosarcoma and UES). This is further complicated by the

occurrence of mixed epithelial-mesenchymal tumours such as

adenosarcoma and carcinosarcoma, in which the sarcomatous

component can predominate and display heterologous

differentiation. Several questions remain regarding the biology

and classification of uterine sarcomatous neoplasms. For

instance, does a subset of UES represent sarcoma-dominant

carcinosarcoma, and if so, how can it be separated from UES

that arises in a de novo manner in order to determine the most

appropriate tumour staging and therapy? Do pleomorphic

rhabdomyosarcomas occur in a de novo manner in the uterine

corpus (as it does in soft tissue), or do they represent



Figure 5. Proposed classification for endometrial sarcoma. UES =undifferentiated endometrial sarcoma; HG ESS = high-gradeendometrial stromal sarcoma; LG ESS = low-grade endometrialstromal sarcoma

Postmenopausal Pre- and post-menopausal

Perimenopausal

(antiestrogenictherapy)

Poorprognosis(no effectivetreatment)

Intermediateprognosis

(adjuvant radiation/chemotherapy)

Goodprognosis

Complexkaryotype

YWHAE-NUTM2

JAZF1-SUZ12JAZF1-PHF1EPC1-PHF1MEAF6-PHF1

UES HG ESS L G ESS


LEE

carcinosarcomas with heterologous rhabdomyosarcomatous

differentiation (even if no carcinomatous component can be

identified histologically) and hence would not benefit from

soft tissue rhabdomyosarcoma-type chemotherapy? These are

clinically relevant questions that arise not infrequently during

interdisciplinary management rounds, and it is clear that

further research and more insight into molecular genetics will

be needed to address them.

References1. Chang KL, Crabtree GS, Lim-Tan SK, et al. Primary uterine endometrial stromal

neoplasms. A clinicopathologic study of 117 cases. Am J Surg Pathol 1990;14:415–38.

2. Evans HL. Endometrial stromal sarcoma and poorly differentiated endometrialsarcoma. Cancer 1982;50:2170–82.

3. Silverberg S, Kurman R, Nogales F, et al. Tumors of the breast and female genitalorgans: World Health Organization classification of tumours. Lyon, France: IARCPress, 2003.

4. Hendrickson MR, Tavassoli FA, Kempson RL. Mesenchymal tumours and relatedlesions. World Health Organization Classification of Tumours. Pathology andgenetics of tumours of the breast and female genital organ. Lyon, France: IARCPress, 2003.

5. Norris HJ, Taylor HB. Mesenchymal tumors of the uterus. I. A clinical andpathological study of 53 endometrial stromal tumors. Cancer 1966;19:755–66.

6. Feng W, Hua K, Gudlaugsson E, et al. Prognostic indicators in WHO 2003 low-grade endometrial stromal sarcoma. Histopathology 2013;62:675–87.

7. Franquemont DW, Frierson HF Jr, Mills SE. An immunohistochemical study ofnormal endometrial stroma and endometrial stromal neoplasms. Evidence forsmooth muscle differentiation. Am J Surg Pathol 1991;15:861–70.

8. Dionigi A, Oliva E, Clement PB, et al. Endometrial stromal nodules andendometrial stromal tumors with limited infiltration: a clinicopathologic study of50 cases. Am J Surg Pathol 2002;26:567–81.

9. Oliva E, Clement PB, Young RH, et al. Mixed endometrial stromal and smoothmuscle tumors of the uterus: a clinicopathologic study of 15 cases. Am J SurgPathol 1998;22:997–1005.

10. Yilmaz A, Rush DS, Soslow RA. Endometrial stromal sarcomas with unusualhistologic features: a report of 24 primary and metastatic tumors emphasizingfibroblastic and smooth muscle differentiation. Am J Surg Pathol 2002;26:1142–50.

11. Huang HY, Ladanyi M, Soslow RA. Molecular detection of JAZF1-JJAZ1 genefusion in endometrial stromal neoplasms with classic and variant histology:evidence for genetic heterogeneity. Am J Surg Pathol 2004;28:224–32.

12. Oliva E, de Leval L, Soslow RA, et al. High frequency of JAZF1-JJAZ1 gene fusionin endometrial stromal tumors with smooth muscle differentiation by interphaseFISH detection. Am J Surg Pathol 2007;31:1277–84.

13. Clement PB, Scully RE. Endometrial stromal sarcomas of the uterus with extensiveendometrioid glandular differentiation: a report of three cases that causedproblems in differential diagnosis. Int J Gynecol Pathol 1992;11:163–73.

14. McCluggage WG, Ganesan R, Herrington CS. Endometrial stromal sarcomas withextensive endometrioid glandular differentiation: report of a series with emphasison the potential for misdiagnosis and discussion of the differential diagnosis.Histopathology 2009;54:365–73.

15. McCluggage WG, Young RH. Endometrial stromal sarcomas with true papillaeand pseudopapillae. Int J Gynecol Pathol 2008;27:555–61.

16. Oliva E, Clement PB, Young RH. Epithelioid endometrial and endometrioidstromal tumors: a report of four cases emphasizing their distinction fromepithelioid smooth muscle tumors and other oxyphilic uterine and extrauterinetumors. Int J Gynecol Pathol 2002;21:48–55.

17. Oliva E, Young RH, Clement PB, et al. Myxoid and fibrous endometrial stromal

tumors of the uterus: a report of 10 cases. Int J Gynecol Pathol 1999;18:310–19.18. Regauer S, Emberger W, Reich O, et al. Cytogenetic analyses of two new cases of

endometrial stromal sarcoma – non-random reciprocal translocationt(10;17)(q22;p13) correlates with fibrous ESS. Histopathology 2008;52:780–3.

19. Ali R, Kurihara S, Endo M, et al. Genetic reclassification of undifferentiatedendometrial sarcoma: clinical relevance. Mod Pathol 2013;26(S2):263–301.

20. Koontz JI, Soreng AL, Nucci M, et al. Frequent fusion of the JAZF1 and JJAZ1genes in endometrial stromal tumors. Proc Natl Acad Sci U S A 2001;98:6348–53.

21. Micci F, Panagopoulos I, Bjerkehagen B, et al. Consistent rearrangement ofchromosomal band 6p21 with generation of fusion genes JAZF1/PHF1 andEPC1/PHF1 in endometrial stromal sarcoma. Cancer Res 2006;66:107–12.

22. Panagopoulos I, Micci F, Thorsen J, et al. Novel fusion of MYST/Esa1-associatedfactor 6 and PHF1 in endometrial stromal sarcoma. PLoS One 2012;7:e39354.

23. Chiang S, Ali R, Melnyk N, et al. Frequency of known gene rearrangements inendometrial stromal tumors. Am J Surg Pathol 2011;35:1364–72.

24. Hrzenjak A, Moinfar F, Tavassoli FA, et al. JAZF1/JJAZ1 gene fusion in endometrialstromal sarcomas: molecular analysis by reverse transcriptase-polymerase chainreaction optimized for paraffin-embedded tissue. J Mol Diagn 2005;7:388–95.

25. Kurihara S, Oda Y, Ohishi Y, et al. Endometrial stromal sarcomas and related high-grade sarcomas: immunohistochemical and molecular genetic study of 31 cases.Am J Surg Pathol 2008;32:1228-38.

26. Nucci MR, Harburger D, Koontz J, et al. Molecular analysis of the JAZF1-JJAZ1gene fusion by RT-PCR and fluorescence in situ hybridization in endometrialstromal neoplasms. Am J Surg Pathol 2007;31:65–70.

27. D’Angelo E, Ali RH, Espinosa I, et al. Endometrial stromal sarcomas with sex corddifferentiation are associated with PHF1 rearrangement. Am J Surg Pathol2013;37(4):514–21.

28. Kurihara S, Oda Y, Ohishi Y, et al. Coincident expression of beta-catenin and cyclinD1 in endometrial stromal tumors and related high-grade sarcomas. Mod Pathol2010;23:225–34.

29. McCluggage WG, Sumathi VP, Maxwell P. CD10 is a sensitive and diagnosticallyuseful immunohistochemical marker of normal endometrial stroma and ofendometrial stromal neoplasms. Histopathology 2001;39:273–8.

30. Chu PG, Arber DA, Weiss LM, et al. Utility of CD10 in distinguishing betweenendometrial stromal sarcoma and uterine smooth muscle tumors: animmunohistochemical comparison of 34 cases. Mod Pathol 2001;14:465–71.

31. Lee CH, Marino-Enriquez A, Ou W, et al. The clinicopathologic features ofYWHAE-FAM22 endometrial stromal sarcomas: a histologically high-grade andclinically aggressive tumor. Am J Surg Pathol 2012;36(5):641–53.

32. Lee CH, Ali RH, Rouzbahman M, et al. Cyclin D1 as a diagnostic immunomarkerfor endometrial stromal sarcoma with YWHAE-FAM22 rearrangement. Am J SurgPathol 2012;36:1562–70.

33. Toki T, Shimizu M, Takagi Y, et al. CD10 is a marker for normal and neoplasticendometrial stromal cells. Int J Gynecol Pathol 2002;21:41–7.

34. Gil-Benso R, Lopez-Gines C, Navarro S, et al. Endometrial stromal sarcomas:immunohistochemical, electron microscopical and cytogenetic findings in twocases. Virchows Arch 1999;434:307–14.

35. Lee CH, Ou WB, Marino-Enriquez A, et al. 14-3-3 fusion oncogenes in high-gradeendometrial stromal sarcoma. Proc Natl Acad Sci U S A 2012;109:929–34.

36. French CA, Miyoshi I, Kubonishi I, et al. BRD4-NUT fusion oncogene: a novelmechanism in aggressive carcinoma. Cancer Res 2003;63:304-7.

37. Croce S, Hostein I, Ribeiro A, et al. YWHAE rearrangement identified by FISHand RT-PCR in endometrial stromal sarcomas: genetic and pathologicalcorrelations. Mod Pathol 2013; 26(10):1390–400.

38. Lee CH, Marino-Enriquez A, Ou W, et al. The clinicopathologic features ofYWHAE-FAM22 endometrial stromal sarcomas: a histologically high-grade andclinically aggressive tumor. Am J Surg Pathol 2012;36:641–53.

39. Amant F, Tousseyn T, Coenegrachts L, et al. Case report of a poorly differentiateduterine tumour with t(10;17) translocation and neuroectodermal phenotype.Anticancer Res 2011;31:2367–71.

40. Isphording A, Ali RH, Irving J, et al. YWHAE-FAM22 endometrial stromalsarcoma: diagnosis by reverse transcription-polymerase chain reaction informalin-fixed, paraffin-embedded tumor. Hum Pathol 2013;44:837–43.

References: 1. Liverani CA, et al. Am J Transl Res 2012; 4(4): 452-457

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ADS-001016 ©2014 Hologic, Inc. All rights reserved. Hologic, Aptima, Panther, ThinPrep and associated logos are trademarks of Hologic, Inc. and/or its subsidiaries in the U.S. and/or other countries. For information on specific products available for sale in a particular country, please contact your local Hologic representative or email [email protected].

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This is a permanent part-time (0.5 FTE) service-oriented position, with a requirement for the successful applicant to provide leadership for the Gynecological or Gastrointestinal pathology service, or both, within our hospital. Subspecialty

Vancouver General Hospital serves as a provincial reference center for both complex gynecological cancer and hepatobiliary surgery. As well, the successful candidate will cover other areas of surgical pathology (including frozen sections and on-call) and autopsy pathology, on a rotational basis. This position will require excellent diagnostic surgical pathology skills and a commitment to resident education. An interest in Quality Management and/or molecular pathology would be an asset.

The successful candidate will be recommended for a faculty appointment at in the Department of Pathology and Laboratory Medicine in the Faculty of Medicine at the University of British Columbia (UBC). Faculty appointments at UBC will be at a rank determined by the Faculty Appointment, Reappointment, and Promotion Committee of the UBC Department of Pathology and Laboratory Medicine.

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Kulcsar Lecture 2013: Pulmonary Cytology – Blowing New Life into

Diagnostic Challenges

Manon Auger, MD, FRCPC, is a member of the Department of Pathology at the McGill University Health Center and at McGillUniversity, in Montreal, Quebec. Correspondence may be directed to Manon Auger at [email protected]. This article has been peer reviewed.Competing interests: Dr. Auger is editor of the cytopathology section of the Canadian Journal of Pathology.

Manon Auger, MD, FRCPC

ABSTRACTThis article is a synopsis of the Kulcsar Lecture presented at the Canadian Society of

Cytopathology symposium at the annual meeting of the Canadian Association of Pathologists

in June, 2013. Recent updates for pulmonary cytological specimens, particularly those related

to the diagnostic approach to non–small cell carcinoma in the era of targeted therapy, are

outlined. In addition, diagnostic problems and pitfalls are discussed.

RÉSUMÉ Cet article présente un résumé de la conférence de Kulcsar présentée dans le cadre du

symposium de la Canadian Society of Cytopathology (CSC) lors de la réunion annuelle de

l’Association canadienne des pathologistes en juin 2013. On y présente les mises à jour récentes

pour les spécimens cytologiques pulmonaires, en particulier celles qui se rapportent à l’approche

diagnostique aux carcinomes non à petites cellules dans l’ère de la thérapie ciblée. De plus, on

y aborde les problèmes et les embûches diagnostiques.

ORIGINAL ARTICLE

This review is a summary of the Kulcsar Lecture given on

June 10th, 2013, for the Canadian Society of

Cytopathology symposium at the annual meeting of the

Canadian Association of Pathologists in Quebec City, Quebec.

The Kulcsar Lecture is named after Dr. David Kulcsar, who in

1961 was one of the founding members of the Canadian

Cytology Council (CCC), the name of which was eventually

changed to the Canadian Society of Cytopathology (CSC). Of

interest, Dr. Kulcsar, who worked at the Royal Victoria Hospital

in Montreal, was not a pathologist but rather a gynecologist

with a keen interest in cytology. In 1961, he became the first

secretary-treasurer of the CCC and subsequently the CSC, a

position he held for five years. In 1978, Dr. Kulcsar initiated

and generously supported the CSC Trust Fund. In 1980, the

CSC was recognized as a charitable foundation with the aim of

promoting and providing support for educational endeavors in

cytology. In 1990, the David Kulcsar Lecture Series was created

to honour the man whose efforts played such an important role

in founding and developing the CSC.

Choice of General Diagnostic Categories for Pulmonary Ex-foliative Specimens versus Fine-Needle AspiratesThe choice of diagnostic categories differs slightly between

those for pulmonary exfoliative specimens and those for lung

fine-needle aspirates (FNAs) (Table 1). The general

diagnostic category is easy to choose whenever a lung FNA

consists of either malignant or atypical cells; for those, it

should be “abnormal.” Whenever benign features leading to

a specific benign diagnosis (e.g., hamartoma or

granulomatous inflammation) are present, the diagnostic

category “negative for malignancy/benign” should be

selected. The decision becomes more problematic when

benign but nonspecific features (e.g., unremarkable

respiratory cells or pulmonary macrophages or both) are


PULMONARY CYTOLOGY

seen in a lung FNA. Although such nonspecific benign

features can be diagnosed as “negative for

malignancy/benign” in exfoliative specimens, it is best to

diagnose them as “non-diagnostic” rather than “benign”

when they are found in lung FNAs.

Although some consider the terms “unsatisfactory” and

“non-diagnostic” to be synonyms, it is best not to use them

as such. “Unsatisfactory,” in the context of FNAs, should be

used whenever one cannot provide any useful information

(e.g., as in the case of an entirely or virtually acellular

specimen or a cellular specimen that cannot be interpreted

because of abundant obscuring blood or a marked air-drying

artifact). On the other hand, the term “non-diagnostic,”

which applies to FNAs only and not to exfoliative specimens,

should be used when at least some partial information can

be provided. The aforementioned example of a lung FNA

consisting of benign respiratory epithelium and pulmonary

macrophages is best categorized as “non-diagnostic” because

it could either represent a sampling problem (a missed

lesion) or an appropriate sample, depending on the

particular setting (e.g., round atelectasis). Another example

of a “non-diagnostic” lung FNA would be a lung FNA

consisting of only necrotic debris and for which the

underlying etiology cannot be determined from the

specimen. However, if a specific etiology for the necrosis can

be determined (e.g., by the presence of fungi or acid-fast

bacilli), a “non-diagnostic” specimen becomes diagnostic and

can be categorized as “benign.” Whenever the “non-

diagnostic” category is used, a description of the cytological

findings should be provided so that the clinician understands

what the sample consists of. In addition, the following

comment should always be added: “Recommend

clinical/radiological correlation to assess the adequacy of

sampling.” The need to re-sample ultimately depends on

careful clinical or radiological correlation. The appropriate

use of the “benign” category versus the “non-diagnostic”

category is crucial; using “benign” for nonspecific findings in

FNAs can reassure clinicians inappropriately.

Non–Small Cell Lung Carcinoma in the Era of Targeted TherapyApproximately 70% of lung cancers are diagnosed and staged

by small biopsies or by cytology rather than by surgical

resection.1–3 In particular, with the growing use of

endobronchial ultrasound-guided (EBUS) fine-needle

aspiration and endoscopic ultrasound-guided (EUS) fine-

needle aspiration, cytology plays an increasingly important

role in many institutions.

Although it was always suggested that one subtype non–small

cell lung carcinoma (NSCLC) in cytological specimens, it was

not always attempted by pathologists as there was no

therapeutic or management difference between squamous

carcinoma and adenocarcinoma. However, subtyping

NSCLC has become very important for any pulmonary

specimen, because the cell type (squamous carcinoma versus

adenocarcinoma) is the criterion for triaging lung cancer

specimens for predictive biomarker testing.1–4 The four major

therapeutic findings since 2004 that are driving the need for

subtyping NSCLC are these: (1) tyrosine kinase inhibitors

(TKIs) such as gefitinib and erlotinib are now first-line

therapy for patients with advanced lung adenocarcinoma

with epidermal growth factor receptor mutations; (2)

adenocarcinomas with anaplastic lymphoma kinase (ALK)

rearrangements respond to crizotinib; (3) patients with

adenocarcinoma or NSCLC not otherwise specified

(NSCLC-NOS) are more responsive to pemetrexed (a folate

anti-metabolite) than those with squamous cell carcinoma;

and (4) squamous cell carcinoma is associated with life-

threatening hemorrhage in patients treated with

bevacizumab (a monoclonal antibody against vascular

endothelial growth factor), which is therefore

contraindicated in patients with a diagnosis of pulmonary

squamous cell carcinoma.

Within the NSCLC group, a definitive diagnosis of squamous

carcinoma or adenocarcinoma can be reached by most

pathologists when the tumours are well to moderately

Table 1. Diagnostic Categories for Lung Fine-NeedleAspirates and Exfoliative Respiratory Cytological Specimens

Respiratory Diagnostic Category Lung FNAs Exfoliative CytologyUnsatisfactory Yes YesNondiagnostic Yes NoBenign/negative for malignancy Yes YesAtypical/suspicious Yes YesMalignant Yes YesFNAs = fine-needle aspirates.


AUGER

differentiated; however, specific diagnoses are more difficult

with poorly differentiated tumours. One must try to subtype

NSCLC and minimize the use of “NSCLC-NOS” as much as

possible. In small biopsy or cytology specimens, the very

limited amount of tissue obtained must be carefully rationed

for use in traditional microscopic examination,

immunocytochemical studies to confirm cell type and/or

primary site, and molecular studies of predictive biomarkers

that are needed to determine the optimal treatment protocol.

In cytological material, squamous carcinomas are divided

according to their degree of keratinization. In general, the

"keratinizing" type corresponds to the "well-differentiated"

type, whereas the “nonkeratinizing” type corresponds to the

moderately and poorly differentiated types. The cytological

features are similar in all types of specimens, with the

following exceptions: (1) cells from squamous carcinoma in

sputum are usually more keratinized than those found in

bronchial washing or brushing specimens of the same

tumour, and (2) large tissue fragments of malignant cells may

be seen in bronchial brushings. Keratinizing squamous

carcinoma is characterized by prominent single cells, but

some groups are also present. The cells are relatively large and

often polygonal, with sharply defined borders and rigid

abundant cytoplasm; they are often orangeophilic on

Papanicolaou stain and usually have pyknotic nuclei and

inconspicuous nucleoli (Figure 1). Epithelial pearls and

keratin debris can be seen. When the tumour is cavitating,

abundant acute inflammation and necrotic debris may be

present, particularly in FNA specimens. In contrast to the

keratinizing type, groups of malignant cells in

nonkeratinizing squamous carcinoma tend to be more

prominent than single cells, and the nucleoli are usually more

conspicuous (Figure 2).5,6

In well-differentiated adenocarcinoma, the tumour cells are

predominantly arranged in cohesive clusters and loose

groupings; single cells become increasingly frequent in the

less differentiated adenocarcinomas. In contrast to squamous

carcinomas, which tend to exhibit groups in flat sheets, those

in adenocarcinoma groupings are three-dimensional; the

groupings may be of acinar, cell ball, or papillary

configuration. The cells have a moderate amount of

cytoplasm (usually cyanophilic, foamy, or vacuolated), and

the nuclei are usually round (Figure 3). Prominent large

nucleoli are usually seen in many cells and may be multiple.

The tumour cells may be multinucleated in the less

differentiated adenocarcinomas.5,6

When the subtyping of NSCLC is in doubt between

squamous carcinoma and adenocarcinoma, ancillary studies

Figure. 1. Photomicrograph of a well-differentiated squamouscarcinoma in a fine-needle aspirate of the lung. Note the single-cell pattern of the abnormal keratinized cells exhibiting pyknoticnuclei and no nucleoli. (Papanicolaou stain)

Figure 2. Photomicrograph of a poorly-differentiated squamouscarcinoma in a fine-needle aspirate of the lung. Note the flat-sheetarrangement of the cohesive abnormal squamous cells with apolygonal shape, rigid cytoplasm, and occasional small butconspicuous nucleoli. (Papanicolaou stain)


PULMONARY CYTOLOGY

are often helpful; however, it is crucial to keep the workup to

the minimum to preserve as much tissue as possible for

molecular studies. For initial evaluation, the use of only one

adenocarcinoma marker and one squamous marker is

suggested.1 The best marker for adenocarcinoma seems to be

thyroid transcription factor-1 (TTF-1); it can also help

confirm a primary lung origin in 75–85% of lung

adenocarcinomas.1 An alternate adenocarcinoma marker is

napsin A.7–9 As a squamous carcinoma marker, p63 is

considered the best choice; however, p40 is emerging as a very

sensitive and specific marker for squamous differentiation.

Positivity for CK5/6 can help support squamous

differentiation but is considered second line only.3,7–9 As an

alternative, “cocktails” that combine adenocarcinoma and

squamous carcinoma markers with differential nuclear and

cytoplasmic staining can be used; the advantage of this is the

use of only one slide for two different immunomarkers.

Examples of such cocktails include TTF-1 (nuclear

staining)/CK5/6 (cytoplasmic staining) and p63 (nuclear

staining)/napsin A (cytoplasmic staining). Other newer

promising markers for squamous differentiation include

desmoglein-3 and desmocollin. In most cases, only one

immunomarker will be positive, making interpretation of the

immunostaining relatively easy. Occasionally, however, dual

staining may be encountered; in such cases, if an

adenocarcinoma marker is positive, the tumour should be

classified as “NSCLC, favour adenocarcinoma” despite

expression of squamous carcinoma markers.1 If

there is reactivity for adenocarcinoma and squamous carcinoma

markers in different populations of tumour cells, this may

suggest an adenosquamous carcinoma.1Table 2 summarizes the

distinguishing cytological features and ancillary studies of

squamous carcinoma and adenocarcinoma.

If a diagnosis of squamous carcinoma or adenocarcinoma

can be reached on the basis of cytological features alone, the

recommended diagnostic terms are “malignant: squamous

carcinoma” and “malignant: adenocarcinoma.” If the

diagnosis of squamous carcinoma or adenocarcinoma

cannot be reached on the basis of cytological features alone

and the NSCLC is positive for an adenocarcinoma or for a

squamous marker, the recommended diagnostic terms are

“non–small cell carcinoma, favour adenocarcinoma” and

“non–small cell carcinoma, favour squamous carcinoma”

respectively.1,10,11

Rationale for Change in Lung Adenocarcinoma in the 2011International Association for the Study of LungCancer/American Thoracic Society/European RespiratorySociety ClassificationWith a 50% rate of occurrence, adenocarcinoma is the most

common histological subtype of lung cancer in most

countries. However, a widely divergent clinical, radiological,

molecular, and pathological spectrum within lung

adenocarcinoma leads to some confusion. The rationale for

Figure 3. Photomicrograph of an adenocarcinoma in a fine-needleaspirate of the lung. Note the roundness of the malignant cells,which exhibit delicate vacuolated cytoplasm and conspicuousnucleoli. One mitotic figure is also present. (Papanicolaou stain)

Table 2. Features Distinguishing Squamous Carcinoma and AdenocarcinomaFeature Squamous Carcinoma AdenocarcinomaArchitecture Flat sheets Three-dimensional groupsCytoplasm Rigid and uniform Thin and vacuolatedIntercellular bridges Present AbsentChromatin Coarse Finely granularMucin stain – +TTF-1 — +Napsin A — +p63 + —TTF-1 = thyroid transcription factor-1.


AUGER

the change in classification comes from the need for

universally accepted criteria for the various adenocarcinoma

subtypes, especially for tumours formerly classified as

bronchioloalveolar carcinomas.1,10,11 In contrast to previous

classifications, the new one provides more specific guidance

for small biopsy and cytology specimens, as 70% of lung

cancers are diagnosed on the basis of such samples. Also,

unlike previous World Health Organization classifications,

wherein the primary diagnostic criteria for many tumour

types were based on hematoxylin-eosin (H & E)–stained

slides, the new classification emphasizes the use and

integration of immunohistochemical (e.g., TTF-1, p63),

histochemical (e.g., mucin), and molecular studies. Although

those techniques should be used whenever possible, it is

recognized that this may not always be possible. In the new

classification, the term “bronchioloalveolar carcinoma”

(BAC) is eliminated, and it should no longer be used.1,10,11

The abolition of the term stems from the fact that although

it was used for the “mucinous type” and the “non-mucinous

type,” there were many significant differences between the

two entities from the clinical, radiological, morphological,

and molecular standpoints.

With the new classification, BAC has been reclassified into

the following five different entities, which can be determined

on resection specimens rather than small biopsy specimens

or cytology: (1) adenocarcinoma in situ (tumours ≤ 3 cm

with pure lepidic growth), which is associated with a 100%

five-year survival rate if completely resected; (2) minimally

invasive adenocarcinoma (tumours ≤ 3 cm with

predominant lepidic growth and ≤ 0.5 cm foci of invasion),

which is associated with a nearly 100% five-year survival rate

if completely resected; (3) invasive adenocarcinoma with a

lepidic component; (4) invasive mucinous adenocarcinoma;

and (5) widespread advanced-stage adenocarcinoma with a

lepidic component, which is associated with a very poor

survival rate.1,10,11 The diagnostic terms suggested for the

former BAC category in cytological specimens is

“adenocarcinoma with lepidic pattern” (formerly “BAC,

nonmucinous type”), and “mucinous adenocarcinoma”

(formerly “BAC, mucinous type”).1 The cytological features

in FNA specimens suggesting a diagnosis of adenocarcinoma

with lepidic features include neoplastic cells, most frequently

arranged in flat monolayered sheets. Rarely, papillary

fragments may be present. The cells are polyhedral with

round nuclei larger than those of normal bronchiolar cells.

The chromatin is finely granular, and although prominent

nucleoli may be seen, they are infrequent. Nuclear grooves

and intranuclear inclusions are often present. Psammoma

bodies may be seen very infrequently.5,6 In summary, in lung

FNA specimens, the former BAC of the nonmucinous type

has an appearance very similar to that of papillary thyroid

carcinoma in thyroid FNA specimens (Figure 4).

The cytological features of FNA specimens suggesting a

diagnosis of mucinous adenocarcinoma include (as a major

clue) a prominent mucoid background. As well as being

present as individual cells, the tumour cells are arranged in

cohesive and loose groupings; sheets and papillary

arrangements are unusual in this type of tumour. The nuclei

are round and have fine chromatin, and prominent nucleoli

are common. The cytoplasm is abundant, often with a foamy

or clear appearance.5,6 In summary, in lung FNA specimens,

mucinous adenocarcinoma has an appearance very similar

to that of mucinous/colloid carcinoma in breast FNA

specimens. Of note, the immunoprofile of mucinous

adenocarcinoma of the lung is unusual; although mucinous

adenocarcinomas of the lung are usually positive for

Figure 4. Photomicrograph of an adenocarcinoma with lepidicpattern (formerly classified as bronchioloalveolar carcinoma,nonmucinous type) in a fine-needle aspirate of the lung. Note theflat-sheet arrangement of the abnormal cells, with only mildnuclear atypia, fine chromatin, and frequent nuclear grooves.(Papanicolaou stain)


PULMONARY CYTOLOGY

cytokeratin 7, they often co-express cytokeratin 20 and CDX-

2 and are usually negative for TTF-1.10

Guidelines for Epidermal Growth Factor Receptor and ALKTesting for Lung Cancer The reader is referred to the article of Linderman et al. for

full details on the guidelines for epidermal growth factor

receptor (EGFR) and ALK testing.4 These evidence-based

guidelines address when and how testing should be

performed, whether other genes should be routinely tested

in lung cancer, and how molecular testing of lung cancer

should be implemented. Only a summary is given here, with

particular attention to testing in cytological specimens.

It is not recommended that clinical characteristics (e.g.,

ethnicity, sex, age) be used to exclude patients for EGFR

mutation or ALK rearrangement testing, because there are

many exceptions despite associations, and this approach

would exclude significant numbers of patients who might

benefit from treatment. Tissue should be prioritized for

EGFR and ALK testing. It is recommended that testing for

EGFR mutations and ALK rearrangements be ordered at the

time of diagnosis for patients presenting with advanced-stage

(stage IV) disease who are suitable for therapy, or at the time

of recurrence or progression in patients who originally

presented with lower-stage disease but were not previously

tested. In most institutions in Canada, EGFR and ALK testing

is performed upon the request of the treating oncologist.

However, depending on the particular setting, pathologists

could order those tests routinely for all cases of lung

adenocarcinoma, as is already done in certain centres in the

United States. Such decisions are best made locally, taking

into consideration the relevant financial and human

resources that are available.

Pathologists should use formalin-fixed paraffin-embedded

specimens or fresh, frozen, or alcohol-fixed specimens for

polymerase chain reaction–based EGFR mutation tests.

Other tissue treatments (e.g., decalcifying solutions or acidic

or heavy metal fixatives) should be avoided in specimens

destined for EGFR testing. Cytological samples are also

suitable for EGFR and ALK testing; cell blocks are preferred

to smear preparations. For ALK testing, laboratories should

use an ALK fluorescence in situ hybridization (FISH) assay

using dual-labelled break-apart probes for selecting patients

for ALK TKI therapy. If carefully validated, ALK

immunohistochemistry may be considered as a screening

methodology to select specimens for ALK FISH testing.

Although there is potential for the future use of mutation-

specific antibodies that require less tissue and are cheaper

and faster than the molecular assay, the official guidelines

recommend molecular methods as the most appropriate way

to test for EGFR mutations.

Many studies have shown that molecular testing can be

performed successfully on cytological specimens (including

brushes, washes, lavages, FNAs, and EBUS FNAs) for

identifying mutations; however, the success rate, which

ranges from 67% to 98%, depends on the type of specimen.3

The minimum amount of material needed for molecular

studies is 700 cells. The tumour sample should not be diluted

with nonmalignant cells. Using an H & E–stained slide as a

guide, normal tissue can be trimmed out of a paraffin block

to enrich the sample being analyzed. If a cell block or a liquid-

based slide is not available, it is technically possible to scrape

tumour cells directly off the stained cytology slides; in the

latter instance, the slide should be scanned or photographed

to create a permanent record.

Neuroendocrine TumoursThe primary neuroendocrine tumours of the lung form a

Figure 5. Photomicrograph of a carcinoid in a fine-needle aspirateof the lung. Note the monotony and mild nuclear atypia of theneoplastic cells arranged in an organoid pattern. (Papanicolaoustain)


AUGER

spectrum ranging from low-grade (i.e., typical carcinoid) to

high-grade (e.g., small cell carcinoma and large cell

neuroendocrine carcinoma ([LCNEC]) with intermediate

tumours (i.e., atypical carcinoids) in between.

From a practical standpoint, it is usually quite easy to

recognize carcinoid and small cell carcinoma prospectively

in cytological specimens.5,6,12,13 However, reproducible

prospective cytological diagnosis of atypical carcinoid and

LCNEC is problematic; the diagnostic uncertainty of such

cases should be conveyed in the diagnostic report.14,15 It is

best to recommend surgical biopsy or excision of the tumor

for definitive diagnosis when the cytological diagnosis is

doubtful.

The cytology of carcinoid tumours is characterized by

growth patterns (organoid, trabecular, insular, palisading,

ribbon, and rosette-like) that suggest neuroendocrine

differentiation (Figure 5).5,6,12,13 Tumour cells have uniform

cytological features with moderate amounts of eosinophilic

and finely granular cytoplasm as well as nuclei with a finely

granular chromatin pattern. Cells are mostly isolated or in

loosely cohesive groups, or they form ribbons or trabeculae

with an anastomosing pattern. The cells are uniform, small,

and round to cuboidal with eccentric nuclei that are round,

oval, or spindle shaped with stippled chromatin and

inconspicuous nucleoli. The cytoplasm is well-defined, and

the background is clean.

The two key criteria for distinguishing carcinoid from

atypical carcinoid are the mitotic count per 10 high-power

fields (HPFs) and the presence of necrosis. A typical

carcinoid tumour has fewer than 2 mitoses per 2 mm2

(10 HPFs) and lacks necrosis, whereas an atypical carcinoid

has 2 to 10 mitoses per 2 mm2 (10 HPFs) and (or) foci of

necrosis. This distinction can be achieved only in histological

material because there is no standardized way to count

mitoses per 10 HPFs in cytological material. Therefore, if one

sees a few mitoses or foci of necrosis in a tumour (suggesting

a carcinoid in cytological material), one must convey this

diagnostic uncertainty in the report so that further

investigations are pursued.

Small cell carcinoma is defined as a malignant epithelial

tumour consisting of small cells (defined as less than the size

of three small resting lymphocytes) with scant cytoplasm,

finely granular nuclear chromatin, and absent or

inconspicuous nucleoli.5,6,12,16 Cytologically, small cell

carcinoma is characterized by isolated and loose clusters of

cells, very scant cytoplasm, granular-to-clumped chromatin

with indistinct nucleoli, frequent degenerating cells, necrotic

debris, and streaks of nuclear debris in the background and

nuclear moulding (Figure 6). Of note, occasional larger cells

scattered among the smaller cells with typical features of

small cell carcinoma do not prevent a diagnosis of small cell

carcinoma. However, if the proportion of large cells with

prominent nucleoli admixed with the small cells reaches

10%, a diagnosis of combined small cell and large cell

Figure 6. Photomicrograph of a small cell carcinoma in a fine-needle aspirate of the lung.Note the nuclear moulding and the pyknotic debris. (Papanicolaou stain)


PULMONARY CYTOLOGY

carcinoma should be considered.

Histological features such as organoid nesting, trabecular

growth, and rosettes, suggesting neuroendocrine

differentiation, occur in LCNEC.5,6,12 The tumour cells are

generally large, with moderate to abundant cytoplasm.

Nucleoli are frequent and prominent, and their presence

facilitates differentiation from small cell carcinoma.

Confirmation of neuroendocrine differentiation by at least

one immunohistochemical stain is required. Obtaining a

reproducible prospective cytological diagnosis of this entity

is difficult. Again, any diagnostic uncertainty should be

conveyed in the cytological report.

Causes of False-Positive and False-Negative DiagnosesThe most common causes of false-positive diagnoses include

granulomatous inflammation, radiation or chemotherapy,

reactive epithelial atypia (such as in a lung abscess or fungal

infections), viral changes, pulmonary infarct, organizing

pneumonia, and (rarely) overinterpretation of

normal/reactive hepatocytes if the liver is erroneously

sampled for right-sided “lung” aspirates.5,6,17–25 Occasionally,

hamartomas can also be a pitfall, leading to misdiagnoses of

carcinoid or well-differentiated adenocarcinoma.26

Creola bodies (three-dimensional pseudopapillary clusters

of reactive bronchial cells) may occasionally exhibit nuclear

atypia and prominent nucleoli. Cilia (usually present) and

the lack of single atypical cells aid in distinguishing creola

bodies from adenocarcinoma. Reactive endobronchial cells

that are not in creola body formations can also lead to false-

positive diagnoses; such cells can show atypical cellular

changes, such as nuclear pleomorphism. However, in contrast

to adenocarcinoma, they maintain good cohesion, having few

(if any) single atypical cells. In addition, there is a range of

atypia in benign conditions, whereas there is no such

transition in malignancy. Features favouring (benign) goblet

cell metaplasia over adenocarcinoma include well-oriented

cell clusters with little cellular overlap, ciliated cells admixed

with goblet cells, and mild nuclear atypia with inconspicuous

nucleoli.

Reserve cell hyperplasia occurs in response to a variety of

stimuli, and its importance lies in the danger of

misdiagnosing it as small cell carcinoma. It is seen in

exfoliative specimens only. It is characterized by uniform,

small, dark round nuclei; a thin rim of basophilic cytoplasm;

and a high nucleocytoplasmic ratio. Mild nuclear moulding

may be present (Figure 7). Although both reserve cell

hyperplasia and small cell carcinoma can show nuclear

moulding, it is much more marked in small cell carcinoma.

The cells of reserve cell hyperplasia are much smaller than

those of small cell carcinoma. The cells of reserve cell

hyperplasia are typically smaller than normal endobronchial

cells, whereas those of small cell carcinoma are larger than

normal endobronchial cells. The cells in reserve cell

hyperplasia are very cohesive (i.e., without single cells); those

in small cell carcinoma are very dyscohesive. Nuclear

pleomorphism is more marked in small cell carcinoma than

in reserve cell hyperplasia. Finally, pyknotic debris is typically

seen in small cell carcinoma but is absent from reserve cell

hyperplasia.

Loose clusters of macrophages, especially when reactive and

exhibiting prominent nucleoli, may lead to false-positive

diagnoses, particularly diagnoses of adenocarcinoma.

Features favouring macrophages over adenocarcinoma

include phagocytosed material within macrophages'

cytoplasm, few aggregates, reniform nuclei, and mild nuclear

atypia. When reactive (such as in the context of pulmonary

Figure 7. Photomicrograph of reserve cell hyperplasia in abronchial washing. Although some nuclear moulding is present,the cells are smaller than the surrounding normal endobronchialcells and are cohesive without pyknotic debris, features that pointto reserve cell hyperplasia rather than small cell carcinoma.(Papanicolaou stain)


AUGER

infarct, diffuse alveolar damage, or organizing pneumonia),

pneumocytes can mimic adenocarcinoma. The cytological

criteria that favour a benign process include mostly cohesive

groups with few single cells, a heterogenous cell population,

scalloped borders, a lower nucleocytoplasmic ratio,

background inflammation, and hemosiderin-laden

macrophages. Mesothelial cells are often seen in lung FNA

specimens, especially those from peripheral lesions. They

usually form flat sheets in a “mosaic” or “pavement-like”

arrangement. They exhibit abundant cytoplasm and round

large nuclei with nucleoli that are usually small but that can

become prominent when reactive. Mesothelial cells are a

cause of false-positive diagnoses, particularly diagnoses of

squamous carcinoma and of adenocarcinoma with a lepidic

pattern.

The cytological features that suggest chemotherapy or

radiation changes rather than malignancy include mostly

cohesive cell clusters with few single atypical cells, a

heterogenous cell population with atypical cells within the

same clusters with normal cells, cilia with or without

terminal bars, multinucleation, vacuolated cytoplasm

(especially if the vacuoles are large), and a normal or near-

normal nucleocytoplasmic ratio. Granulomatous

inflammation is also a well-known cause of false-positive

diagnoses.25 Cytologically, it is characterized by clumps of

epithelioid macrophages and lymphocytes with or without

multinucleated giant cells. False-positive diagnoses when

there is granulomatous inflammation may be due to the

associated reactive atypia of the surrounding alveolar,

bronchial, or squamous metaplastic epithelium or to the

misinterpretation of the granulomata themselves as

carcinoma because they appear as cohesive cellular groups.

A helpful feature for correctly identifying granulomata is the

polymorphous population within the cohesive clusters that

includes a mixture of lymphocytes and histiocytes; in

contrast, clusters in carcinoma are composed of only one

type of cell.

The most common causes of false-negative diagnoses in lung

FNAs are poor technique and sampling, small size or

inaccessibility of the lesion, inability to puncture the lesion,

necrosis or inflammation within a lesion, and (rarely)

incorrect interpretation (especially in regard to the tumour

formerly designated as BAC).5,6

Staging Non–Small Cell Lung CancerThe management of NSCLC depends on the stage; therefore,

evaluation of the mediastinal lymph nodes is essential for

decisions regarding treatment. Although mediastinoscopy

has been the gold standard, staging is increasingly done by

less invasive methods, particularly EBUS FNA and EUS FNA.

Both EUS and EBUS can sample the upper paratracheal

(station 2), lower paratracheal (station 4), and subcarinal

(station 7) lymph nodes. Only EBUS can sample the hilar

(station 10), interlobar (station 11), and lobar (station 12)

lymph nodes, whereas only EUS FNA, which is performed

via the esophagus, can sample the paraesophageal (station 8)

and pulmonary ligament (station 9) lymph nodes.27,28

When used for staging, the specimens should be considered

unsatisfactory if they contain only benign bronchial mucosal

cells or cartilage (for EBUS FNA) or benign squamous cells

(for EUS FNA), as such specimens represent sampling from

the needle tract (i.e., from the bronchial and esophageal

walls, respectively) instead of the targeted lymph node. If the

specimens are used for staging, the presence of lymphocytes

or anthracotic histiocytes should be noted in the report,

confirming the sampling of a lymph node. It is important to

exercise caution when interpreting EBUS FNA specimens,

because reactive endobronchial cells can be a cause of a false-

positive diagnoses of adenocarcinoma or carcinoid, while

squamous metaplasia can cause a false-positive diagnosis of

squamous carcinoma.

ConclusionThere is a growing opportunity for cytology to play a

prominent role in the arena of lung cancer, and

cytopathologists are in an ideal position to influence the

appropriate testing and treatment of non–small cell lung

carcinoma in the era of targeted therapy. Endobronchial and

endoscopic ultrasound-guided fine-needle aspirates are

increasingly important for mediastinal staging, allowing

extensive cytological sampling by much less invasive means.

Because molecular techniques cannot be applied in a

morphological vacuum, cytomorphology will continue to

play an essential role.

References1. Travis WD, Brambilla E, Noguchi M, et al. Diagnosis of lung cancer in small


PULMONARY CYTOLOGY

biopsies and cytology. Implications of the 2011 IASLC/ATS/ERS classification.

Arch Pathol Lab Med 2013;137:668–84.

2. Cagle PT, Olsen RJ. The proposed new classification of pulmonary

adenocarcinoma and the conservation of small tissue samples for testing. Arch

Pathol Lab Med 2013;137:453–4.

3. Zakowski MF. Lung cancer in the era of targeted therapy. A cytologist’s

perspective. Arch Pathol Lab Med 2013;137:1816–21.

4. Linderman NI, Cagle PT, Beasley MB, et al. Molecular testing guidelines for

selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors

– guideline for the College of American Pathologists, International Association

for the Study of Lung Cancer, and Association for Molecular Pathology. Arch

Pathol Lab Med 2013;137:828–60.

5. Cibas ES, Ducatman BS. Respiratory tract. In: Cibas ES, Ducatman BS.

Cytology: diagnostic principles and clinical correlates. 3rd ed. Philadelphia:

Saunders Elsevier, 2009: 65–103.

6. DeMay RM. Lung. In: DeMay RM. The art and science of cytopathology, 2nd

edition (Vol. 3). Chicago: ASCP Press, 2012:1155–245.

7. Mukhopadhyay S, Katzenstein ALA. Subclassification of non-small cell lung

carcinomas lacking morphologic differentiation on biopsy specimens: utility

of an immunohistochemical panel containing TTF-1, Napsin A, p63 and

CK5/6. Am J Surg Pathol 2011;35:15–25.

8. Whithaus K, Fukuoka J, Prihoda TJ, Jagirdar J. Evaluation of Napsin A,

cytokeratin 5/6, p63, and thyroid transcription factor 1 in adenocarcinoma

versus squamous cell carcinoma of the lung. Arch Pathol Lab Med

2012;136:155–62.

9. Aikawa E, Kawahara A, Hattori S, et al. Comparison of the expression levels of

Napsin A, thyroid transcription factor-1, and p63 in nonsmall cell lung cancer

using cytocentrifuged bronchial brushings. Cancer (Cancer Cytopathol)

2011;119:335–45.

10. Travis WD, Brambilla E, Noguchi M, et al. IASLC/ATS/ERS international

multidisciplinary classification of lung adenocarcinoma. J Thorac Oncol

2011;6:244–85.

11. Travis WD, Brambilla E, Noguchi M, et al. Diagnosis of lung adenocarcinoma

in resected specimens. Implications of the 2011 IASLC/ATS/ERS classification.

Arch Pathol Lab Med 2013;137:685–705.

12. Siddiqui M. Pulmonary neuroendocrine neoplasms: a review of

clinicopathologic and cytologic features. Diagn Cytopathol 2010;38:607–17.

13. Stoll LM, Johnston MW, Burroughs F, Li QK. Cytologic diagnosis and

differential diagnosis of lung carcinoid tumors: a retrospective study of 63 cases

with histologic correlation. Cancer (Cancer Cytopathol) 2010:118;457–67.

14. Wiatrowska BA, Krol J, Zakowski MF. Large cell neuroendocrine carcinoma of

the lung: proposed criteria for cytologic diagnosis. Diagn Cytopathol

2001;24:58–64.

15. Jimenez-Heffernan JA, Lopez-Ferrer P, Vicandi B, et al. Fine-needle aspiration

cytology of large cell neuroendocrine carcinoma of the lung. A cytohistologic

correlation study of 11 cases. Cancer (Cancer Cytopathol) 2008;114:180–6.

16. Renshaw AA, Voytek TM, Haja J, Wilbur DC. Distinguishing small cell

carcinoma from non-small cell carcinoma of the lung. Correlating cytologic

features and performance in the College of American Pathologists non-

gynecologic cytology program. Arch Pathol Lab Med 2005;129:619–23.

17. Saad RS, Silverman JF. Respiratory cytology: differential diagnosis and pitfalls.

Diagn Cytopathol 2010;38:297–307.

18. Cagle PT, Kovach M, Ramzay I. Causes of false results in transthoracic fine

needle lung aspirates. Acta Cytol 1993;37:16–20.

19. Crapanzano JP, Zakowski MF. Diagnostic dilemmas in pulmonary cytology.

Cancer (Cancer Cytopathol) 2001;93:364–75.

20. Crapanzano JP, Saqi A. Pitfalls in pulmonary cytopathology. Diagn Cytopathol

2011;39:144–54.

21. Lawther RE, Graham AN, McCluggage WG, et al. Pulmonary infarct

cytologically mimicking adenocarcinoma of the lung. Ann Thorac Surg

2002;73:1964–5.

22. Naryshkin S, Young NA. Respiratory cytology: a review of non-neoplastic

mimics of malignancy. Diagn Cytopathol 1993;9:89–97.

23. Policarpio-Nicolas ML, Wick MR. False-positive interpretations in respiratory

cytopathology: exemplary cases and literature review. Diagn Cytopathol

2008;36:13–9.

24. Butnor KJ. Avoiding underdiagnosis, overdiagnosis, and misdiagnosis of lung

carcinoma. Arch Pathol Lab Med 2008;132:111.

25. Auger M, Moriarty AT, Laucirica R, et al. Granulomatous inflammation – an

underestimated cause of false-positive diagnoses in lung fine-needle aspirates:

observations from the CAP nongynecologic cytopathology interlaboratory

comparison program. Arch Pathol Lab Med 2010;134:1793–6.

26. Hughes JH, Young NA, Wilbur DC, et al. Fine-needle aspiration of pulmonary

hamartoma: a common source of false-positive diagnoses in the College of

American Pathologists Interlaboratory Comparison Program in Non-

Gynecologic Cytology. Arch Pathol Lab Med 2005;129:19–22.

27. Cameron SE, Andrade RS, Pambuccian SE. Endobronchial ultrasound-guided

transbronchial needle aspiration cytology: a state of the art review.


Cyclin D1–Positive Plasma Cell Leukemia with Lymphoplasmacytic Morphology andt(11;14)(q13;q32): Case Report and Brief

Literature Review

Irfan K. Khan, MD, Fariborz Rashid-Kolvear, PhD, Adnan Mansoor, MD, and Jay L. Patel, MD, are members of the Departmentof Pathology and Laboratory Medicine, University of Calgary, in Calgary, Alberta. Correspondence may be directed to Jay L.Patel at [email protected]. This article has been peer reviewed.Competing interests: None declared

Irfan K. Khan, MD, Fariborz Rashid-Kolvear, PhD, Adnan Mansoor, MD, Jay L. Patel, MD

ABSTRACTA 67-year-old man presented with back pain and a peripheral leukocytosis consisting

predominantly of lymphoplasmacytoid cells that were difficult to classify by morphology.

Atypical plasma cells predominated in the bone marrow. Immunohistochemistry and flow

cytometry confirmed CD138-positive, κ light chain–restricted plasma cells with strong nuclear

positivity for cyclin D1. Cytogenetic studies revealed a t(11;14)(q13;q32) translocation. Plasma

cell leukemia with lymphoplasmacytic morphology, cyclin D1 up-regulation, and

t(11;14)(q13;q32) is a distinct presentation of plasma cell myeloma.

RÉSUMÉ Voici le cas d’un homme de 67 ans affligé de dorsalgie présentant une leucocytose périphérique

caractérisée par la prédominance de cellules de type lymphoplasmocytoïde difficiles à classer

selon la morphologie. Les plasmocytes atypiques sont abondants dans la moelle osseuse. Les

résultats de l’immunohistochimie et de la cytométrie en flux confirment la nature des cellules,

soit des plasmocytes à chaînes légères avec restriction kappa exprimant CD138 et forte présence

de cycline D1 dans le noyau. Les épreuves cytogénétiques mettent en évidence une translocation

t(11;14)(q13;q32). La leucémie à plasmocytes caractérisée par une morphologie

lymphoplasmocytaire, une régulation haussière de la cycline D1 et la translocation

t(11;14)(q13;q32) constitue une présentation particulière du myélome plasmocytaire.

ORIGINAL ARTICLE

We report an unusual case of cyclin D1–positive plasma

cell leukemia with lymphoplasmacytic morphology

in a 67-year-old man who presented clinically with back pain.

Imaging studies demonstrated marrow replacement in the

axial skeleton with destruction of the L2 vertebral body and

a soft tissue mass extending from the vertebral body into the

spinal cord and causing compression of the cauda equina.

Serum chemistry studies revealed hypercalcemia and renal

failure. Complete blood count showed macrocytic anemia

(hemoglobin, 81 g/L; mean corpuscular volume, 120 fL),

thrombocytopenia (platelets, 69 × 109/L), and leukocytosis

(white blood cells, 27.2 × 109/L). Leukocytes consisted

predominantly of plasmacytoid lymphocytes and mature

plasma cells (24.4 × 109/L, 90%, based on a 200-cell

differential count), but these were difficult to classify by

morphology alone and were initially miscategorized as

“blasts” (Figure 1). This error led to diagnostic confusion and

raised a broad differential diagnosis including multiple

myeloma, lymphoma, and acute leukemia. Bone marrow

examination was performed, and smears of the aspirate


CYCLIN D1–POSITIVE PLASMA CELL LEUKEMIA

showed a marked predominance of plasma cells (75% of

nucleated cells) with atypical features, including small size,

scant basophilic cytoplasm, and variably conspicuous

nucleoli. The core biopsy sections showed effacement by

sheets of lymphoplasmacytoid cells (Figure 2).

Immunohistochemical and flow cytometry studies

confirmed CD45-negative, CD38- and CD138-positive,

cytoplasmic κ light chain–restricted plasma cells with strong

diffuse nuclear positivity for cyclin D1 (Figure 3). Surface

light chain expression was absent, and CD20, PAX-5, and

CD56 were not expressed. Mature B cells expressed polytypic

surface light chains. Cytogenetic and fluorescence in situ

hybridization studies revealed t(11;14)(q13;q32). A final

diagnosis of plasma cell leukemia with cyclin D1 expression

and t(11;14)(q13;q32) was rendered.

DiscussionPlasma cell leukemia with cyclin D1 positivity and

t(11;14)(q13;q32) is a well-recognized (although rare)

pathological entity and is associated with small lymphocyte-

like or lymphoplasmacytic morphology and a poor

prognosis.1,2 The incidence of t(11;14)(q13;q32) in plasma

cell myeloma is approximately 5% and is associated with a

favourable prognosis when other high-risk clinical features

(e.g., leukemic presentation) and adverse prognostic genetic

abnormalities – such as t(4;14)(p16;q32) or

t(14;16)(q32;q23) – are absent.3 The incidence of t(11;14)

translocations in plasma cell leukemia has been reported to

be much higher (19.5–71.0%).1

We report an instance of plasma cell myeloma presenting in

the peripheral blood as plasma cell leukemia, which resulted

in initial diagnostic confusion, although the presence of

destructive bone lesions provided an important clue in this

case. However, mature B-cell lymphoma (subtypes of which

express cyclin D1 to varying degrees) could reasonably be

considered in the differential diagnosis.4 Nuclear cyclin D1

expression is characteristic of mantle cell lymphoma but is

also commonly observed in hairy cell leukemia and rarely in

diffuse large B-cell lymphoma. Aberrant expression of the

mature B-cell antigens CD19 and/or CD20 on neoplastic

Figure 1. Peripheral blood smear showing atypical cells withlymphoplasmacytoid features. (May-Grünwald Giemsa)

Figure 2. A, Bone marrow aspirate smear with atypicallymphoplasmacytic cells as seen in peripheral blood. B, Bonemarrow core biopsy section showing effacement by sheets oflymphoplasmacytoid cells. (A, Wright; B, hematoxylin and eosin)


KHAN ET AL.

plasma cells could add to the difficulty in differentiation from

lymphoma.5 The differential diagnosis, however, is usually

easily resolvable with clinical correlation and bone marrow

examination, including appropriate immunophenotyping

studies. Plasma cell neoplasms, in contrast to B-cell

lymphoma, are characterized by a lack of CD45 expression

and by expression of CD38 and CD138 with cytoplasmic

light-chain restriction.6 Aberrant CD56 expression, though

sometimes helpful in the diagnosis of plasma cell neoplasms,

is often notably absent in the setting of plasma cell leukemia.7

References1. Fernandez de Larrea C, Kyle RA, Durie BG, et al. Plasma cell leukemia:

consensus statement on diagnostic requirements, response criteria and

treatment recommendations by the International Myeloma Working Group.

Leukemia 2013;27:780–91.

2. Albarracin F, Fonseca R. Plasma cell leukemia. Blood Rev 2011;25:107–12.

3. Sawyer JR. The prognostic significance of cytogenetics and molecular profiling

in multiple myeloma. Cancer Genet 2011;204:3–12.

4. Gladkikh A, Potashnikova D, Korneva E, et al. Cyclin D1 expression in B-cell

lymphomas. Exp Hematol 2010;38:1047–57.

5. Seegmiller AC, Xu Y, McKenna RW, Karandikar NJ. Immunophenotypic

differentiation between neoplastic plasma cells in mature B-cell lymphoma vs

plasma cell myeloma. Am J Clin Pathol 2007;127:176–81.

6. Heerema-McKenney A, Waldron J, Hughes S, et al. Clinical,

immunophenotypic, and genetic characterization of small lymphocyte-like

plasma cell myeloma: a potential mimic of mature B-cell lymphoma. Am J Clin

Pathol 2010;133:265–70.

7. Pellat-Deceunynck C, Barille S, Jego G, et al. The absence of CD56 (NCAM)

on malignant plasma cells is a hallmark of plasma cell leukemia and of a special

subset of multiple myeloma. Leukemia 1998;12:1977–82.

Figure 3. Immunohistochemical studies of bone marrow core biopsy specimens. A, CD138. B, cyclin D1. C, κ. D, λ. (Immunoperoxidase)


Medulloblastoma – An Unusual Variant ofPrimitive Neuroectodermal Tumour Arising in a

Mature Teratoma of Testis: Case Report

Michelle R. Downes, MB, MRCSI, MD, FRCPC, and Theo van der Kwast, MD, PhD, are members of the Department of Labo-ratory Medicine and Pathobiology, University Health Network, in Toronto, Ontario. Correspondence may be directed [email protected]. This article has been peer reviewed.Competing interests: None declared

Michelle R. Downes, MB, MRCSI, MD, FRCPC, Theo van der Kwast, MD, PhD

ABSTRACTPrimitive neuroectodermal tumours (PNETs) are one of the most common non–germ cell

malignancies to arise in a testicular germ cell tumour and represent malignant transformation

along mesodermal lines. In this article, the authors report on the first case in the literature in

which a central PNET resembling a medulloblastoma has been identified as a malignant

component in a primary testicular germ cell tumour.

RÉSUMÉ Les tumeurs primitives neuro ectodermiques (TPNE) sont parmi les tumeurs malignes non

germinales les plus fréquentes à survenir dans une tumeur testiculaire germinale et représentent

une transformation maligne au niveau du mésoderme. Dans cet article, les auteurs discutent

du premier cas documenté dans lequel une TPNE centrale ressemblant à un médulloblastome

a été identifiée comme étant une composante maligne dans une tumeur testiculaire germinale

primaire.

ORIGINAL ARTICLE

Primitive neuroectodermal tumours (PNETs) are one of

the most common non–germ cell malignancies to arise

in a testicular germ cell tumour and represent malignant

transformation along mesodermal lines. They are associated

with increased numbers of relapses and decreased overall

survival.1 The majority of gonadal PNETs do not resemble

peripheral PNET/Ewing’s sarcoma and morphologically are

closer to central PNET (cPNET).2We report on the first case

in the literature in which a cPNET resembling a

medulloblastoma has been identified as a malignant

component in a primary testicular germ cell tumour.

Case ReportA 26-year-old man, previously healthy, presented with

swelling of his left testicle. Clinical examination and imaging

confirmed a suspicious mass, and he underwent a left radical

Figure. 1. Mature teratoma (arrow) and adjacent primitiveneuroectodermal tumour with features of medulloblastoma.(Hematoxylin and eosin)


DOWNES AND VAN DER KWAST

orchiectomy. There was no personal or family history of

cryptorchidism or testicular malignancy. Preoperatively,

serum concentrations of bio-markers were AFP 1 µg/L

(normal, <6.0 µg/L), β-HCG <1 IU/L (normal, up to

2.0 IU/L), and LDH 373 U/L (normal, 125–220 U/L).

The orchiectomy specimen weighed 87.9 g and measured

7.0 × 6.0 × 4.0 cm. The parenchyma was replaced by a tan-

white circumscribed mass, 6.0 × 6.0 × 4.0 cm, with

hemorrhagic and cystic foci. The tunicae albuginea and

vaginalis were grossly uninvolved.

Microscopically, there was complete replacement of

parenchyma by sheets of monomorphic basophilic cells with

admixed pale areas and concentration of the basophilic cells

in a sleeve-like pattern around vessels. The nuclei were round

and hyperchromatic without nucleoli. Mitotic figures were

easily identified. The paler areas showed cells with dark, round

uniform nuclei embedded in a fine fibrillary matrix. There

was focal gland formation, and the glands were lined by

mucinous columnar epithelium consistent with a mature

teratoma (Figure 1).

Immunohistochemical studies demonstrated intense

cytoplasmic expression of synaptophysin and CD56. AE1/AE3

showed a paranuclear dot-like positivity. GFAP, CD45, CD99,

WT-1, and S100 were not expressed. The morphology and

immunohistochemical profile was consistent with a cPNET

showing features of medulloblastoma, nodular desmoplastic

subtype (Figure 2). There was invasion of hilar fat and rete

testis without vascular space invasion; the spermatic cord

margin was negative (stage pT1).

DiscussionThe finding of PNET in germ cell tumours is not uncommon.

PNET is distinguished from “immature teratoma” based on

the criterion of overgrowth of primitive neuroectodermal

tissue such that the majority of a low-power (4×) objective

field is occupied.3 The term PNET initially was used to

describe embryonal appearing tumours in the central nervous

system (CNS) and included entities such as cerebral

neuroblastoma, ependymoblastoma, pineoblastoma,

medulloblastoma, medulloepithelioma, atypical teratoid/

rhabdoid tumour, and rhabdomyosarcoma. Medulloblastoma

is the main cPNET in childhood and is a heterogeneous entity

with four subtypes designated by the World Health

Organization: large cell variant, anaplastic type, nodular

desmoplastic type, and medulloblastoma with extensive

nodularity.4 The nodular desmoplastic subtype is

characterized by pale “islands” of neurocytic cells surrounded

by densely packed, small to medium-sized, highly proliferative

cells that represent zones of neuronal maturation.

Medulloepithelioma has been reported as being the most

frequent cPNET morphology in testicular germ cell

malignancies, despite being relatively uncommon as a CNS

Figure 2. Medulloblastoma (A) with neurocytic stroma and small, round, blue cell component (B). (Hematoxylin and eosin)

A B


MEDULLOBLASTOMA – AN UNUSUAL VARIANT OF PRIMITIVE NEUROECTODERMAL TUMOUR

PNET. A previous study of 14 PNETs (12 metastatic, 2 primary

testicular) classified 9 tumours as medulloepithelioma, 3 as

medulloblastoma, 1 as small cell embryonal tumour, and 1 as

a neuroblastic tumour. The medulloblastoma cases were all

identified in metastatic sites.2 In another study,

medulloblastoma was not identified in the testis or the

metastatic sites in the 29 PNET cases reviewed.1 Occasional

case reports of testicular PNETs similar to peripheral

PNET/Ewing’s sarcoma are noted in the literature,5,6 but it is

recognized that testicular PNETs usually resemble pediatric-

type CNS embryonal neoplasms and lack the characteristic

rearrangement of chromosome 22.2

The diagnosis of cPNET in a teratoma is straightforward

using a combination of light microscopy features and

immunohistochemistry. We present the first report of a

cPNET showing features of medulloblastoma in a primary

testicular germ cell tumour.

References1. Michael H, Hull MT, Ulbright TM, et al. Primitive neuroectodermal tumors

arising in testicular germ cell neoplasms. Am J Surg Pathol 1997;21:896–904.

2. Ulbright TM, Hattab EM, Zhang S, et al. Primitive neuroectodermal tumors in

patients with testicular germ cell tumors usually resemble pediatric-type central

nervous system embryonal neoplasms and lack chromosome 22 rearrangements.

Mod Pathol 2010;23:972–80.

3. Ulbright TM, Loehrer PJ, Roth LM, et al. The development of non-germ cell

malignancies within germ cell tumors. A clinicopathologic study of 11 cases.

Cancer 1984;54:1824–33.

4. Giangaspero F, Eberhart CG, Haapasalo H, et al., eds. WHO Classification of

Tumours of the Central Nervous System. Lyon, France: International Agency

for Research on Cancer; 2007:132–40.

5. Heikaus S, Schaefer KL, Eucker J, et al. Primary peripheral primitive

neuroectodermal tumor/Ewing’s tumor of the testis in a 46 year old man –

differential diagnosis and review of the literature. Hum Pathol 2009;40:893–97.

6. Gupta P, Dhingra KK, Singhal S, et al. Primary primitive neuroectodermal

tumour (PNET) of the testis: an unsuspected diagnosis. Pathology 2010;42:179–

81.

65th Annual Meeting of the Canadian Assocation of Pathologists

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