Trisomy 12 in chronic lymphocytic leukemia—geographical variation

Leukemia Research

PERGAMON

Leukemia Research 22 (1998) 313-317

Trisomy 12 in chronic lymphocytic leukemia-geographical variation Chandrika N. Nair *, Anuradha Chougule, Subodh Dhond, Rajat Goyal, Purvish M. Parikh, Swati Pai, Darshana Telang, Suresh H. Advani Tata Memorial Hospital, Dr. Ernerst Barges Road, Pare1 Mumbai-12, India Received 12 June 1997; accepted 7 October 1997

Abstiact Incidence of trisomy 12 was studied in 60 casesof chronic lymphocytic leukemia (CLL) with chromosome 12 specific a-satellite DNA probe by fluorescence in situ hybridization (FISH). Trisomy 12 was observed in 37 (61.8%) patients. Cells with trisomy 12 were detected in a varying proportion, ranging from > 2% to 86%. Patients with trisomy 12 were predominantly observed with total white blood cell (WBC) count > 80 x lo9 l- ’ (P < 0.001). In addition, the percentage of trisomy 12 positive lymphocytes correlated with the high WBC counts. Trisomy 12 was observed equally in typical and atypical CLL. 90% of our patients were in the intermediate and high risk groups. It was seen that there was significantly higher percentage of trisomy 12 positive lymphocytes ( > 10%) in the high risk groups (P < 0.05). A higher incidence of FMC7 positivity in atypical CLL was seen in our study. However, there was no significant relationship found between trisomy 12 positivity and expression of either FMC7 or CD23 in our cases. It appears that the CLL that we see at our centre is at a different phase of evolution and perhaps biologically different compared to the CLL seen in the West. 0 1998 Elsevier Science Ltd. All rights reserved. Keywords: Trisomy

12; Chronic lymphocytic

leukemia; Immunophenotypes;

1. Introduction

Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder usually involving a distinct subset of recirculating B-cells which accumulate in the peripheral blood, lymph nodes, spleen, liver and other organs [l]. Important prognostic features include stage of the disease,chromosomal abnormalities, pattern of marrow involvement, intensity of surface immunoglobulin and doubling time [2-61. Chromosomal abnormalities commonly seenare trisomy 12 and structural changes in 14, the former being the more frequent [7,8]. Since CLL lymphocytes have low frequency of mitosis, fluorescence in situ hybridization (FISH) using chromosome 12 a-satellite DNA probe has evolved as a highly sensitive technique for detection of trisomy 12 [9- 131. Abbreviations: CLL, chronic lymphocytic leukemia; DIG, digoxigenin; DNA, deoxyribonucleic acid; FISH, fluorescence in situ hybridization; WBC, white blood cell. *Corresponding author. Tel.: + 91 22 4146750; fax: + 91 22 4146937; e-mail: [email protected] 0145-2126/98/$19.00 0 1998 Elsevier Science Ltd. All rights reserved. PII: SO145-2126(97)00169-O

Geographical

variation

Although B-CLL appears to be an accumulation of clonal B-lymphocytes with CD5 receptors, it has been reported to be morphologically, immunologically and clinically heterogeneous in nature [ 13- 181. Geographical data also suggest that the incidence of CLL is low in Asian population as compared to the West [19]. The present project was undertaken to evaluate the clinical and biological features of CLL in our population.

2. Materials

and methods

2.1. Patients From September 1995 to December 1996, 60 patients with CLL were newly diagnosed on the basis of clinical features, cell morphology and immunological markers. The clinical diagnosis of CLL was based on lymphocytosis > 10 x 10’ I-- ’ with or without generalised lymphadenopathy and hepatosplenomegaly. The morphology of small lymphocyte with clumped nuclear

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chromatin, regular outline and no visible nucleolus, along with > 20% cells showing surface marker expression of CD19, weak surface immunoglobulin and light chain restriction to kappa or lambda are used as criteria for the diagnosis of B-CLL [20]. The staging was performed according to Rai’s staging system [l] and morphological subtypes were identified as described by Vallespi et al. [17]. The risk group assignment was done as per the report of Montserrat [19]. 2.2. Immunophenotyping Peripheral blood mononuclear cells were isolated by density gradient centrifugation on Ficoll-Hypaque. Cells were analysed by direct immunofluorescence with monoclonal antibody against CD5 (Dako), CD23 (Immunotech), FMC7 (Immunotech) along with CD19 (Dako), kappa and lambda (Zymed) and immunoglobulin heavy chain (Zymed). 2.3. Fluorescence in situ hybridisation FISH was performed with a DIG labelled chromosome 12 specific cc-satellite DNA probe (Oncor) for the molecular detection of trisomy 12 in the interphase cells. Hybridization was done according to the manufacturer’s protocol. In all the cases, 500 cells were examined for well-delineated fluorescent spots. The test samples were considered unanalysable if there was < 90% hybridization and by corollary if > 10% cells had zero signal. 2.4. Control Peripheral blood samples were obtained from 10 normal people between the ages of 40 and 60 years.

Research 22 (1998) 313-317 Table 1

Sr. no.

0 1 2 3 4 5 6 7 8 9 10

In each of the controls, 500 cells were analysed for 0, 1, 2, 3 and 4 hybridization signals. The mean value for each of the above was 0, 0.36, 99.2, 0.42 and 0%. Detection of two chromosomes in over 99% of the normal population indicated a high quality of hybridization. The percentage trisomy observed was 0.42% f 0.22 (mean f 1 S.D.) and the upper limit for trisomy in the control group was 1.08%. Considering this data, a very safe control level for presence of true trisomy was taken as 2% (Table 1). Out of 60 patients, 53 were males and the median age was 55 years (range: 28-80). The total WBC count varied from 6 x lo9 1-l to 786 x lo9 1-l. The mean WBC count in our group of patients was 87.5 x lo9 1-r + 137 (mean f SD.). Typical CLL was diagnosed in 39 patients while 21 were found to have atypical CLL. Only six patients belonged to low risk group. 90%

-

1

2

3

4

3 1 2 5 ~ 2 3 2 -

495 499 496 492 496 498 495 498 491 497

2 2 2 3 4 2 2 1 3

-

0.4 0.4 0.4 0.6 0.8 0.0 0.4 0.4 0.2 0.6

of our patients were in the intermediate and high risk group (Rai stage, I-IV) (Table 2). 2.6. Trisomy by FISH in patient 37 patients with CLL showed presence of trisomy 12. The range of trisomy 12 positive cells were 2-86.8%. The positive samples were divided into two groups: Group I: lymphocytes Group II: lymphocytes

The with The with

low positivity group with 2-10% trisomy 12. high positivity group with > 10% trisomy 12.

The incidence of > 10% trisomy 12 positive cells was significantly higher in high risk groups (P < 0.05) (Table 3). It was found that when the WBC counts Table 2

Clinical

2.5. Trisomy by FISH in control population

% of trisomy

No. of signals

No. (%)

features

1. Age

<40 >40<60 >60

4 (6.6) 33 (55.0) 23 (38.0)

2. Sex

Males Females

53 (88.3) 7 (11.6)

3. Total WBC count

<10x lo9 I-’ >10x1091-‘-50x1091-’ >50 x lo9 1-‘-80x lo9 1-l >8Ox lo9 1-l

4 23 11 22

4. Morphology

Typical Atypical

39 (65.0) 21 (35.0)

5. Stage

Risk group Low Intermediate

Rai’s stage 0 I II

High

III IV

(6.6) (38.3) (18.3) (36.6)

6 (10) 5 23 28 14 12 26

(8.3) (38.3) (46.6) (23.3) (20.0) (46.3)

C.N. Nair et al. /Leukemia

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Research 22 (1998) 313-317

Table 3 CLL stage and incidence of trisomy 12 Rai’s stage

Total cases

< 2% negative

0

I II III IV

6 5 23 14 12

4 3 9 2 5

2 2 8 4 5

0 0 6 8 2

Total

60

23

21

16

were > 80 x lo9 l- ’ the incidence of trisomy positivity was significantly high (P < 0.001) (Table 4). Similarly a significant co-relation was found between WBC counts and the number of trisomy positive lymphocytes (P < 0.02). There was no significant difference observed in the incidence of trisomy 12 among the typical and atypical CLL cases (Table 5). Immunophenotyping with CD5, CD23 and FMC were carried out in 50% of our patients. CD5 was positive in 31 out of 34 cases (91%) and CD23 in 23 out of 30 cases (76.6%). Only nine out of 27 cases (33.3%) were positive for FMC7 (Table 6). The FMC7 positivity (7/9) had significant correlation with the atypical morphology of CLL (P = 0.01) FMC7 and CD23 expression did not show any relation with trisomy 12 (Tables 7 and 8). 3. Discussion CLL is rare at a young age and the incidence increaseswith age. The Western reported median age of patients with CLL at diagnosis is z 65 years [19-211. However, in our study, the median age at presentation was 55 years, a decade earlier in contrast to the Western countries. As reported in most series, the male predominance (53.7) was seen in our study as well. In Western countries 70% of patients with CLL are diagnosed at an asymptomatic phase or at a low risk stage (stage 0 of Rai’s staging system) [19], while 90% of our patients with CLL had intermediate or high risk stage (Stage I-IV). A low incidence of CLL is reported in Asian population [21]. During 1996, among the 842 cases of adult leukemias, 144 cases were diagnosed as CLL. The incidence of CLL was 17.1% in our center. Table 4 Correlation between white cell count and trisomy 12 WBC count

Total cases

2- 10% low positive

Cytogenetic analysis in CLL has shown high frequency of trisomy 12 as a sole abnormality or associated with other abnormalities such as del6q, dell3q, 14q + etc. [22,23]. The origin of the third copy of chromosome 12 is unknown. In addition, trisomy 12 has been detected in benign ovarian tumors and hence is not specific for B-CLL [24]. Proto oncogene K-ras-2 located on chromosome 12 is reported to be amplified in high grade ovarian tumor and it is suggested that probably there is increased gene dosage of an altered or activated putative oncogene [25]. However the role of this proto oncogene is not yet known in CLL. Though a very low incidence of trisomy 12 as compared to other chromosomal anomalies like 1lq and 6p deletion have been reported by Emilia et al., in B-CLL the role of trisomy 12 in the determination of prognosis, the residual disease and the progression is well documented [26-291. FISH has been shown as a simple and more sensitive method for determination of trisomy 12 [l 1- 131. The reported incidence of trisomy 12 by FISH in CLL varies from 11.5 to 42.6% [ll-13,29-321. However we have seen higher incidence of trisomy 12 (61.6%) with 26.6% of casesshowing > 10% of cells showing trisomy 12. We consider that, as our cases present to us at the advanced disease status and with high count, the trisomy 12 clone has already become evident. This is shown by the fact that varying proportion of trisomy 12 + ve cells (ranging from 2 to 86%) are seen. The occurrence of trisomy 12 has been reported to be significantly higher in atypical CLL as compared to the typical CLL [30]. Our data failed to demonstrate a significant relationship between trisomy 12 and the morphological sub-types, which is probably because of Table 5 CLL morphology and incidence of trisomy 12

Trisomy 12 Percentage of trisomy +ve cells Positive

<80x log I-’ >8Ox 109 I-’

38 22

> 10% high positive

Morphological type

Negative

>2-10%

> 10%

< 2%

13 8

5 11

20 3

Negative ( < 2%) Low +ve (2-10%) High +ve (>lO%)

Typical

Atypical

18 13 8

5 8 8

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C.N. Nair et al. /Leukemia

Table 6 CDS, CD23 and FMC7 in CLL Monoclonal

antibody

CDS CD23 FMC7

Table 8 Correlation

No. of cases tested

34 30 27

between FMC7, morphology

Total no. Typical CLL Atypical CLL Trisomy 12 positivity

between CD23, morphology

Positive cases No.

%I

31 23 9

91 76.6 33.3

the small number that we have studied. However, if one looks at the high positivity ( > 10% + 12) group, S/16 and 8/21 are positive in atypical and typical morphology group, respectively. It appears that a greater number of trisomy 12 positive cells are seen frequently in atypical morphology group. However, statistical difference is not seen, probably due to the small numbers studied. There is no significant difference in the incidence of trisomy 12 in various risk groups in the present study. But a greater number ( > 10%) of trisomy positive cells was seen in the intermediate and high risk groups. Though CD5 and CD23 are constant markers in CLL, there are reports when CD5 and CD23 have not always been expressed [13,16]. It is reported that 668% of CLL are negative for these markers, In our diagnostic criteria we had not included CD5 and CD23 markers and we have been able to perform these markers only on 50% of our patients when these monoclonals became available to us. To avoid the possibility of reluctance to admit CD5, CD23 negative casesas CLL and to support our present data we have done, additionally, all three markers (CD5, CD23 and FMC7) in another set of 32 patients. On analysing these 32 CLL cases, where both CD5 and CD23 were positive, 19 cases showed presence of trisomy 12 ( > 2% trisomy + cells). Among this group of 32 cases FMC7 was positive in 12. This data also shows a high incidence of trisomy 12 and FMC7 positivity. We consider that the trisomy 12 is a late event and is seen frequently in our casesbecause the patients come late to us. Heterogeneity in the immunological markers in CLL has been described by various workers [33-361. Heterogenous expression of CD5, CD23 and FMC7 was seen in our patients as well. Prognostic significance of these markers are reported in an extensive study by Geisler et al. Table 7 Correlation

Research 22 (1998) 313-317

Total no. Typical CLL Atypical CLL Trisomy 12 positivity

and trisomy

CD23 positive

CD23 negative

23 13 10 14

I 5 2 3

[35]. An association of FMC7 with atypical morphology has also been reported in various studies [31,35]. Higher incidence of FMC7 positivity in atypical CLL was seen in our study as well. However there was no significant relationship found between trisomy 12 positivity and expression of either FMC7 or CD23 in our cases. We have observed a high incidence of trisomy 12 in our patients and a correlation with the WBC count and stage at initial presentation. However, a follow up study to evaluate the prognostic significance of trisomy 12 is yet to be done. Trisomy 12 is considered as an event occuring in an established leukemic clone in CLL and data is emerging that this abnormality is associated with progression [29,31,34]. Both from the biological and clinical point of view, B-CLL seemsto be heterogenous even within a given clinical stage, morphological sub-type and immunological markers. We see CLL with younger agemedian age 55 (range 28-80) with high count mean 87.5 x lo9 1-l and with high risk stage as compared to the Western countries. As the WBC count > 80000 cumm - ‘, correlated with increased incidence of trisomy 12 in our patients, it appears that the presence of trisomy is an evidence of tumour load and advanced diseasestatus. Hence, we feel that the CLL caseswe see is a ‘geographical variation: representing a different phase of evaluation’. Further follow-up will help us to understand the impact of trisomy 12, atypia in morphology and expression of FMC7 on the clinical course in CLL. Acknowledgements

We wish to thank ‘The Lady Tata Trust’ Bombay for supporting this study and Mr. Ajay Kumar for the satistical analysis. Also we thank Mr. Sushi1 S. Sarkar for secretarial assistance.

and trisomy

FMC7 positive

FMC7 negative

9 2 7 6

18 14 4 9

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