Medical and Pediatric Oncology 12:4-8 (1984)

Cell Markers in Lymphoma Syndrome Leukemia in Children: A Pilot Study

Elaine Morgan, MD

Children with acute lymphocytic leukemia negative. In two patients E-rosettes could not (ALL) who have a “lymphoma syndrome” be accurately determined because of a low (LySLk) defined by the presence of at least percentage of lymphoblasts in the samples three of the following criteria: a) Hg > 10 g/ studied. Follow-up data on CALLA-positive and dl, b) lymph nodes > 3 cm, c) spleen below CALLA-negative patients revealed 414 CALLA- umbilicus, d) liver below umbilicus, and e) positive patients with no evidence of disease mediastinal mass, appear to represent a (NED) at 22+ to 45+ months from diagnosis subgroup of ALL. These children have a poor and 2/16 CALLA-negative patients NED at 19+ prognosis for survival when treated with and 57+ months. Thus it appears that the standard chemotherapy for ALL. We per- majority of children with LySLk have lympho- formed a retrospective review of 21 patients blasts which are CALLA negative. Patients who at Children’s Memorial Hospital with LySLk meet clinical criteria for LySLk but whose sur- diagnosed from Jan 24,1977 to July 8,1981 and face markers are E negative, CALLA positive of the surface markers on their leukemic cells may have a better prognosis and may repre- at diagnosis. Surface markers identified in- sent a separate subgroup of patients with ALL cluded E-rosettes (E), surface immunoglobulin and, therefore, should be given therapy ap- (Slg), and common ALL antigen (CALLA). Four propriate for their prognostic classification by patients were CALLA positive and E-rosette more standard criteria, such as white blood negative; nine patients were E-negative, CALLA count, age, and sex. negative; six patients were E positive, CALLA

Key words: lymphoma syndrome, At

I N T RO D U CT I0 N

Acute lymphocytic leukemia (ALL) in children can be subcategorized into several clinical groups based on vari- ables that include the initial total leukocyte count, age, and sex [1,2]. This type of subclassification has been used to establish the prognostic significance of each of these variables, and subsequently as a means for assign- ing more specifically appropriate treatment regimens for these patients. One clinical subgroup of ALL, which has been called “lymphoma syndrome leukemia” (LySLk), has been noted to have an extremely poor prognosis for survival [3,4]. Patients in this group are characterized by the presence of massive organomegaly and lymphade- nopathy, mediastinal mass, relatively high hemoglobin values at presentation, and frequently, high peripheral white blood cell counts, E-rosette-positive lymphoblasts, older age, and male sex [3,4].

We performed a retrospective analysis of patients at Children’s Memorial Hospital in whom a diagnosis of ALL was made during the time period from Jan 1, 1977 to July 31, 1981 and who were felt to have LySLk. In each of these patients leukemia cell surface markers were

, cell surface markers, CALLA, leukemia

determined, and these findings were correlated with the response of the patients to therapy.

MATERIALS AND METHODS

Leukemia cell surface markers that were performed in all of the patients included sheep cell E-rosettes, cell surface immunoglobulin, Ia-like antigen, and ALL anti- gen (CALLA; Greaves antigen) [5].

Ia-like and CALLA antigens were identified by an immunofluorescent assay with antisera prepared in Dr. C.C.S. Hsu’s laboratory at Northwestern Memorial Hos- pital and previously documented to be specific for these antigens [6,7]. Patients were considered E-rosette posi- tive or antigen positive for any specific antigen if more than 30% of mononuclear cells obtained by Ficoll-Hy- paque gradient cell separation were positive.

Our clinical criteria for the diagnosis of LySLk, based on the definition established by the Children’s Cancer

From the Department of Pediatrics, Northwestern University Medical School, Children’s Memorial Hospital, Chicago.

Address reprint requests to Elaine Morgan, MD, Children’s Memo- rial Hospital, 2300 Children’s Plaza, Chicago, IL 60614.

El 1984 Alan R. Liss, Inc.

Cell Markers in Lymphoma Syndrome Leukemia 5

negative; the cells of the remaining 17 (18%) of the children were CALLA negative. The cells of all patients were Surface Immunoglobulin (SIg) negative. Among the patients with CALLA-negative cells, results of E-rosette tests were negative in nine and positive in six. In two patients, E-rosetting could not be accurately determined because of a relatively low percentage of lymphoblasts in the sample studies (< 35 a), with consequent contami- nation by normal peripheral T-lymphocytes yielding un- interpretable results. The leukemic cells of the four patients that were CALLA positive were also Ia positive. The majority of patients with CALLA-negative cells also demonstrated absence of the Ia antigen (12 of 17 pa- tients). However, la antigen was present on the cells of three of nine E-negative patients and one of two E-? patients, and could not be accurately determined on the cells of one of six E-positive patients.

The presenting clinical characteristics of the CALLA- positive and CALLA-negative were quite similar (Table I). Ten of 17 CALLA-negative and two of four CALLA- positive patients were male. Proportionately greater numbers (1 1 of 17) of the CALLA-negative patients than of the CALLA-positive patients (one of four) were in an unfavorable age range (< 2 yr or > 10 yr). However, two of the four CALLA-positive and seven of the 17 CALLA-negative patients had peripheral white blood cell counts > 50,000 cm.

Fifteen of the CALLA-negative patients and none of the CALLA-positive patients have suffered an adverse clinical event (Table 11). There were three induction deaths-two from infection and one from hyperkalemia. There were two remission deaths-one from pancreatitis secondary to L-asparaginase and one from disseminated varicella. There were eight bone marrow relapses at intervals from 7 to 27 months from diagnosis with a median of 16 months; there was one central nervous sytem relapse followed by bone marrow relapse and one primary testicular relapse in a patient who has remained in bone marrow remission for 12 + months following his testicular relapse while receiving an intensified chemo- therapeutic regimen.

Six of the 21 lymphoma syndrome patients are cur- rently alive in first remission form 19 + to 57 + months. All four of the CALLA-positive patients remain in first remission. Two of the CALLA-negative patients remain in first remission 57+ and 19+ months from diagnosis. These two patients are both girls, ages 3 % and 1 ‘/2 yr, respectively. The first patient had a low total white blood count but was E-rosette positive and was treated with “standard therapy.” The other patient had a high total white blood count, was E-rosette negative, and is receiv- ing intensive antileukemia therapy (BFM).

Life table analysis revealed disease-free survival of these two groups of patients to be significantly different

Group (CCG), included: a) a diagnosis of ALL with > 25% lymphoblasts in the bone marrow, and b) the pres- ence of three or more of the following clinical features: massive hepatomegaly (below the umbilicus), massive splenomegaly (below the umbilicus), massive lymphade- nopathy (lymph nodes > 3 cm in diameter), mediastinal mass, and hemoglobin level greater than 10 g/dl [3, unpublished data]. This definition is strictly clinical based on statistical analysis of survival in earlier CCG studies [ 3 , unpublished data] and includes both T-cell and non- T-cell disease patients. Patients with < 25% lympho- blasts were not included here. They have been arbitrarily considered to have non-Hodgkin lymphoma by CCG and have been treated on protocols designed for lymphoma rather than leukemia.

A diagnosis of ALL was established in 170 patients at Children’s Memorial Hospital during the study period. Of these, 21 patients fulfilled our criteria for LySLk. These 21 patients were evaluated for their clinical find- ings at the time of diagnosis, leukemia cell surface mark- ers, occurrence of adverse clinical events, and survival in first remission.

The patients were treated with one of several treatment programs. The majority of patients were given chemo- therapy that was considered standard for the treatment of ALL, consisting of vincristine (VCR), prednisone (PDN), L-asparaginase (L-asp), and intrathecal (IT) methotrexate (MTX) for induction, CNS prophylaxis with cranial ir- radiation and IT MTX, and maintenance with 6-mercap- topurine (6MP), MTX, VCR, and PDN. Approximately one third of the patients (one CALLA- positive patient and five CALLA-negative patients) re- ceived additive or cyclic doses of cyclophosphamide, cytosine arabinoside, doxorubicin, adriamycin, and mod- erate-dose methotrexate infusions according to the pro- tocol regimens (CCG protocol 162 and 163) in use at the time of their diagnosis [unpublished data]. Two of the CALLA-negative and one of the CALLA-positive patients whose treatment was initiated toward the end of this study, received an intensive antileukemic regimen mod- eled on the Berlin-Frankfort-Munster program (BFM) [8]. Three of the CALLA-negative patients were treated with an intensive multidrug protocol modeled on the LSA2L2 program designed by the Memorial Sloan-Ket- tering Cancer Center [4]. Decisions to treat patients ac- cording to these regimens were made by the individual investigators caring for the patients.

RESULTS

The clinical characteristics of our patients and the results of cell surface marker studies are included in Table I. Cell marker studies revealed that the leukemia cells of four patients were CALLA positive and E-rosette

6 Morgan

TABLE I. CALLA Positive*

Hemoglobin Clinical findings WBC Patient Agelsex x 10"Icmm gmldl nodes/L-Slmass Markers -

JBd MB" LBd MK" CALLA Negative CB AB C M" DS JL

AC ES MR RG HN CWa TS VS KW CB RC

QH

4/M I I/F 5/F 2/M

6/F 8/M 2/F 9/M 4/M 3/M

12/M 51F 1 %/M 5/M

13/M 1 %IF

1 month/F 12lF 6lF 7/M

8.2 141.2

5.6 60.4

14.4

7.9 2 10

25 89 1 296

120 32 38 16

550 218

16 163 39.4

5.3

7/M 5.7 - *Med, mediastinal mass;

L S , hepatosplenomegaly ; Y, present; N , not present.

aPatient alive in first remission.

TABLE 11. Occurrence of First Adverse Events - No. of patients Time

Event CALLA-/CALLA+ postdiagnosis

Induction death 310 2, 2 , 4 , wk Toxic death 110 3 months Infectious death 1 10 15 months

-

Bone marrow relapse 810 7, 7, 12, 12, 20 ,20 ,21 , 27 months

CNS relapse 1 10 6 months Testicular relapse 1 I0 12 months

(P' = 0.04 by Gehan-Wilcox on two-tailed test) [9] (Fig. 1). For this analysis patients who died during induction or during treatment prior to relapse were withdrawn alive. Even if one eliminates the five CALLA-negative patients who died from toxicity or during induction, disease-free survival of the remaining 12 CALLA-nega- tive patients is significantly different from the CALLA- positive patients (two of 12 vs four of four, P = 0.008 by Fisher Exact test). Furthermore, exclusion of E-posi- tive patients from the analysis shows a similar survival advantage of CALLA-positive patients compared to E-

12.3 13.4 6.4 9.3

Y /Y /Kidney Y/Y/N NIYlMed Y/Y/Med

12.5 Y/N/Med 14.0 NIYIN 9.4 Y/Y/Med 7.1 Y/Y/ Med 8.5 YlY/ Med 6.9 N/Y/Med

13.4 Y/N/Med 8.0 Y/Y/ Med 9.7 N/Y/ Med

11.3 Y/N/Med 13.4 Y/ N/ Med 7.0 YIYIMed

16.0 YIYIKidney 6.9 N/Y/ Med

10.3 YINIMed 10.0 Y/N/Med 13.1 YINIMed

E- SIg- Ia+ E- SIg- Ia+ E- SIg- Ia+ E- SIg- Ia+

E+ SIg- Ia- E+ SIg- Ia- E+ SIg- la- E+ SIg- Ia- E+ SIg- Ia- E+ SIg- Ia'! E- SIg- Iaf E- SIg- Ia+ E- SIg- Ia+ E- SIg- Ia- E- SIg- Ia- E- SIg- Ia- E- SIg- Ia- E- SIg- Ia- E- SIg- Ia- E? SIg- Ia+ E'' SIg- la-

negative patients (four of four vs one of nine; P = 0.007 by Fisher Exact test).

Although strict multivariate analysis was not per- formed, the group of patients surviving in first remission was compared to the nonsurvivors using the Fisher Exact test for the statistical significance of well-known prog- nostic factors. The presence of E-positive cells (P = 0.28), total white blood count over 50,000/cmm (P = 0.34), unfavorable age (< 2 yr or > 10 yr) (P = 0.39), and male sex (P = 0.15) were found to be nonsignificant. The absence of CALLA (P = 0.0025) however, was a significant prognostic factor in these patients.

DISCUSSION

The overall survival of our lymphoma syndrome pa- tients has been very poor (six of 21 currently alive in first remission) but it is similar to other series of such patients. This supports the segregation of this arbitrarily defined clinical subgroup of leukemia patients. The ma- jority of our patients with LySLk were CALLA negative (81%). However, the subgroup of patients who are CALLA positive appear in this small series to have a better prognosis for disease-free survival (four of four

PERCENT

100

90 -

00 -

70 -

6 0 -

50 -

40 -

30 -

20 -

10 -

0

Cell Markers in Lymphoma Syndrome Leukemia

CALLA + N = 414

' : : --- H-1- - - - - --:

4

: * 1

b

t CALLA - N = 2/17

8 :

l ' l ' l ' l ' l ' l ' l ' l ' l ' i ' i

7

P = 0.04

MONTHS FROM DIAGNOSIS

Fig. 1. Disease-free survival in "lymphoma syndrome" leukemia.

alive in first remission versus two of 17 CALLA-negative patients alive in first remission). CALLA-negative and CALLA-positive LySLk patients are clinically indistin- guishable at the time of diagnosis. Thus, their better prognosis does not appear to be related to other well- known prognostic signs such as total white blood cell count, age or sex. Furthermore, it is not the simple absence of E-rosette-positive lymphoblasts in CALLA- positive patients which is influencing prognosis, inas- much as nine of the 17 CALLA-negative patients were also E-rosette negative and only one of these children remains alive.

The influence of treatment in our patients was also evaluated. The additional pulses of chemotherapeutic agents added to the basic maintenance program of 6MP, MTX, VCR, and PDN were shown to be no more effi- cacious in obtaining a long-term continuous remission

than standard maintenance therapy in children with ALL [unpublished data]. Thus, only the more intensive thera- pies (BFM and LSA2L2) could possibly have influenced survival in our patients. Only one of the CALLA-positive patients is receiving such an intensified program while five of the CALLA-negative patients have been treated with one of these two programs. Even if one excludes these patients from analysis, survival still appears to be different in the two groups (three of three CALLA-posi- tive vs one of 12 CALLA-negative patients alive in first remission). It is, therefore, unlikely that differences in therapy in our patients have significantly influenced outcome.

At the present time, because of the previously noted poor prognosis of children with LySLk, several groups are treating these children with very intensive chemother- apy programs regardless of the presence or absence of

8 Morgan

other known prognostic signs (ie, total white blood count, age, sex) [l-31. Such therapy, or perhaps even therapy which has been designed for patients with lymphoma r.ather than ALL, in general appears to be warranted be- cause of the heretofore dismal outlook for these children. Preliminary results suggest that such intensified therapy may indeed improve the outlook in this group of patients [81.

Unfortunately, our study was performed on a very small number of patients. However, these results suggest that there is a subset of patients with clinical LySLk who are CALLA positive. This subset, although clinically in- distinguishable from other LySLk patients, appears to have a better prognosis even when treated with standard antileukemia therapy. Therefore, if our results can be supported by large-scale studies, perhaps such patients should not automatically be assigned to receive intensive therapy or anti-lymphoma therapy, but should instead be treated with antileukemia therapy which is appropriate to their prognostic classification by other more standard criteria such as white blood cell count, age, and sex.

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ance of the predictive value of prognostic variables in childhood

acute lymphoblastic leukemia. A report from Children’s Cancer Study Group. Cancer 48:370-376, 1981.

2. Heideman RL, Falletta JM, Mukhopadhyay N, Fernbach DJ: Lymphocytic leukemia in children: Prognostic significant of clini- cal and laboratory findings at time of diagnosis. J Pediatr 92:540- 545, 1978.

3. Hammond D, Sather H, Honour RC, Nesbit ME, Miller D, Siege1 S, Coccia P: The relative importance of clinical and biological predictors of outcome in the treatment of acute lymphocytic leu- kemia. Cancer Treat Rep (submitted).

4. Duque, L, Wollner N, Miller DR: LSA2L2 protocol treatment of non-Hodgkin’s lymphoma (NHL) in children with partial and extensive bone marrow replacement. Proc Am Assoc Cancer Res 20:113, 1979.

5. Greaves MF, Brown G: Antisera to acute lymphoblastic leukemia cells. Clin Immunol Immunopathol4:67-84, 1975.

6 . Morgan ER, Hsu CCS: Prognostic significance of the acute lym- phoblastic leukemia (ALL) cell-associated antigen in children with null-cell ALL. Am J Ped Hem Onc 2:99-102, 1980.

7. Hsu CCS, Morgan ER: Detection of B-lymphocyte (B-cell)-asso- ciated antigens on human leukemic lymphocytes. Masking of membrane antigens. Am J Clin Pathol 70:741-747, 1978.

8. Henze G, Langermann JH, Schellong G, Riehm H: Therapy re- sults of the BFM studies in childhood acute lymphoblastic leuke- mia. 12th Annual Meeting of the International Society of Pediatric Oncology, Abstract 1980, p 42.

9. Gross A, Clark V: Survival Distributions: Reliability Applications in the Biomedical Sciences. New York: John Wiley and Sons, 1975, 243-249.