|Year : 2017 | Volume
| Issue : 1 | Page : 38-42
|Immunogenetics of chronic lymphocytic leukemia
Nikhil Patkar1, Nikhil Rabade1, Pratibha Amare Kadam2, Falguni Mishra1, Aditi Muranjan1, Prashant Tembhare1, Shruti Chaudhary1, Swapnali Joshi1, Hasmukh Jain3, Uma Dangi3, Bhausaheb Bagal3, Navin Khattry3, Hari Menon3, Sumeet Gujral1, Manju Sengar3, PG Subramanian1
1 Hematopathology Laboratory, Tata Memorial Centre, Parel, Mumbai, Maharashtra, India
2 Department of Cancer Cytogenetics, Tata Memorial Centre, Parel, Mumbai, Maharashtra, India
3 Department of Medical Oncology, Tata Memorial Centre, Parel, Mumbai, Maharashtra, India
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|Date of Web Publication||14-Feb-2017|
| Abstract|| |
Introduction: Cytogenetic aberrations as well as presence of IGVH mutations are the underlying reason for clinical heterogeneity in Chronic Lymphocytic Leukemia (CLL). The presence of IGVH mutations as well as the predominant gene usage shows geographical variations. However, there is no study from India addressing immunogenetics of CLL. In a first Indian study we document the immunogenetics of CLL in a large tertiary hospital. Methods: We analyzed IGVH mutation status, VH gene usage, cytogenetic abnormalities using FISH, immunophenotyping data and correlated them with standard clinical variables in 84 patients of CLL. Results: Advanced Rai stage (Stage 3/4) was seen in 45% of our patients, where as 13q deletion was the commonest clonal cytogenetic abnormality detected in 48.4% of the cases. IGVH unmutated cases (55.2%) showed higher proportion expressing CD38 and CD49d, a preferential usage for VH1 and VH3 families (55.2%), presentation at an advanced Rai stage (52.8%) as well as more frequent presence of p53 deletions. As compared to the IGVH mutated cases greater proportion of IGVH unmutated patients (70%) required treatment. However, there was no significant difference in the time to treatment between mutated and unmutated cases which can be attributed to relatively short median follow up of 10 months. Conclusion: To summarize, we have seen a higher proportion of IGVH unmutated patients in our cohort (55.2%). The commonly used VH genes in the Indian population are IGVH 2-5, IGVH 1-2 and IGVH 1-69. Longer clinical follow up and a larger cohort is necessary to confirm the prognostic value of IGVH mutation analysis in Indian Patients with CLL.
Keywords: Chronic lymphocytic leukemia, immunogenetics, prognosis, VH gene usage
|How to cite this article:|
Patkar N, Rabade N, Kadam PA, Mishra F, Muranjan A, Tembhare P, Chaudhary S, Joshi S, Jain H, Dangi U, Bagal B, Khattry N, Menon H, Gujral S, Sengar M, Subramanian P G. Immunogenetics of chronic lymphocytic leukemia. Indian J Pathol Microbiol 2017;60:38-42
|How to cite this URL:|
Patkar N, Rabade N, Kadam PA, Mishra F, Muranjan A, Tembhare P, Chaudhary S, Joshi S, Jain H, Dangi U, Bagal B, Khattry N, Menon H, Gujral S, Sengar M, Subramanian P G. Immunogenetics of chronic lymphocytic leukemia. Indian J Pathol Microbiol [serial online] 2017 [cited 2017 Jul 23];60:38-42. Available from: http://www.ijpmonline.org/text.asp?2017/60/1/38/200051
Nikhil Patkar, Nikhil Rabade
| Introduction|| |
Chronic lymphocytic leukemia (CLL) is a common type of mature B lymphoid neoplasm among the B cell chronic lymphoproliferative disorders. The last few years have witnessed a rapid change in the concepts related to pathobiology of CLL. What was thought initially to be an indolent disease of immune incompetent naïve B-lymphocytes is today recognized to be a clinically and biologically heterogeneous disease. This heterogeneity stems from the fact that both intrinsic, as well as extrinsic factors, drive lymphomagenesis in CLL.
Structural abnormalities in the leukemic cells as evidenced by an abnormal karyotype are important prognostic factors in deciding disease outcome. The common structural abnormalities; del 11q22-23, del17p, and del6q are predictive of a poorer outcome whereas patients harboring isolated deletion 13q deletion are associated with a favorable response.,
The B-cell receptor (BCR) is one of the defining units of a B lymphocyte and contains the immunoglobulin (IG) molecule as a key component. In CLL cells, alterations in the BCR form a foundation on which CLL immunogenetics is based upon., It is known that in addition to abnormal karyotype, CLL with IGVH mutations has a favorable prognosis as compared to its nonmutated counterpart.,,,,,,, A restricted and biased usage of the VH genes has been reported in CLL as well as pointing toward a role of specific antigens that drive lymphomagenesis. From a clinical perspective, identifying the gene usage is relevant as certain subgroups of the IGVH genes are associated with poor outcome (e.g., IGVH 3–21) whereas others were associated with good outcome.
Numerous studies from India have looked at “clinicopathological” profiles of CLL. Typically, these are retrospective studies investigating the immunophenotype and lymph node or bone marrow pathology.,,, Very few papers have studied the clinical presentation and genetic variables that influence CLL in India. One of the very few papers is by Nair et al., who analyzed the presence of trisomy 12 in CLL in Tata Memorial Hospital. In their study, patients with CLL presented with higher tumor burden in the form of higher blood counts, higher percentage of trisomy 12 in high-risk groups as well as lower median age. These preliminary observations were seconded by Agrawal et al., who also saw CLL at an advanced clinical stage when compared to published data. Both studies hinted that perhaps Indian CLL has different, more aggressive disease biology, however, definitive conclusions could not be drawn. The most surprising data came from Gunawardana et al., in the UK, who evaluated the influence of ethnicity on outcome in CLL. In their study, patients with Southern Asian origin, being managed in one UK region presented at younger age, at an advanced clinical stage and required treatment earlier as compared to their Western counterparts. This finding was statistically significant even when accounting for IGVH hypermutations. This study, however, investigated only 28 South Asian CLL patients. A natural extension of these three studies would be to address the question “Is Indian CLL biologically different?”
In this manuscript, we have evaluated the baseline frequencies of somatic hypermutations in CLL, the predominant VH gene usage, and correlated them with cytogenetics, immunophenotyping, and clinical data. This is the first study from India that addresses the immunogenetics of CLL.
| Methods|| |
Consecutive patients of CLL who were referred for diagnosis or treatment at Tata Memorial Hospital from 2013 to 2014 were accrued in the study.
Diagnosis of chronic lymphocytic leukemia
All patients who had unexplained lymphocytosis were diagnosed as per WHO 2008 criteria. Flow cytometric panels evaluated for lymphoma based on the morphology. This test was performed on peripheral blood or a bone marrow aspirate. In some cases, the diagnosis of CLL was made using two eight-color tubes (pre-2014). Subsequently, this assay evolved to a two-tube nine-color assay; on a Navios (Beckman Coulter) flow cytometer, meant to diagnose chronic lymphoproliferative disorders. CD19 was used as a common tracking marker (CD19 gating) in all cases once a B cell abnormality was suspected.
The first two tubes of this assay (antibodies and fluorochrome conjugates) are demonstrated in [Table 1]. Analysis of the.lmd files was done using Kaluza analysis software (Beckman Coulter, Seattle, USA) (version 1.3). The panel of antibodies with fluorochrome conjugates used is shown in [Table 1].
Cytogenetic abnormalities in chronic lymphocytic leukemia
Fluorescence in situ hybridization (FISH) was done on the interphase cells obtained from the submitted sample (bone marrow aspirate/peripheral blood). The panel of probes was as follows: LSI D13S319 (13q14.3)/LSI 13q34, LSI ATM (11q22.3)/CEP11, LSI TP53 ( 17p13.1)/CEP17, CEP 12, LSI 6q21/SE6, and LSI break apart IgH translocation probe (Vysis Abbott Molecular, Netherlands). Processing for FISH was done as per the manufacturer's recommendations.
IGVH mutations and gene usage
Amplification of VH genes was done using a mixture of leader specific VH (VH 1–6) primers and a consensus JH primer  using genomic DNA or cDNA as starting template. The amplified clonal product was bidirectionally sequenced on an ABI3500 genetic analyzer. The consensus sequence was aligned to IMGT/VQuest database to identify the VH gene usage as well as detect the presence of somatic hypermutations. A cutoff of 98% similarity to the germline VH segment was used to define the mutational status of the clonal B cells.,,,,,,
All patients were diagnosed and followed up in Tata Memorial Hospital. Patients were risk stratified according to the Rai staging system. The decision to treat was made by the treating physician on the patient's symptoms.
Statistical analysis was done on SPSS 21(IBM SPSS Statistics, IBM, USA). The clinical significance between IGVH mutated and unmutated groups was calculated using the time to treatment as a prognostic parameter.
| Results|| |
Patients and clinical variables
We studied 85 cases of CLL with a median age of 59 (range 36–82 years) which included 61 males and 24 females diagnosed over a period of 2 years. Among these, three were CLL-prolymphocytic leukemia (PLL). Based on the Rai classification (n = 62), 15 were stage 4, 13 stage 3, 15 stage 2, 16 stage 1, and 3 patients were stage 0.
Treatment was reserved for symptomatic and advanced disease only as per discretion of treating physician. 38.4% of patients were kept on observation only. The remaining was treated by chemotherapy with 6 cycles of bendamustine and rituximab (50%) or chlorambucil (nonaffording patients) either as single agent or with addition of steroids.
Majority of the cases coexpressed CD5/CD23 (96.7%) and CD43/CD200 (97.1%). Only 2 cases were positive for CD43 but negative for CD200, of which one had high-grade transformation. Twenty-one (26.9%) out of 78 cases showed CD38 expression. As CD49d was added later to the diagnostic panel, it was included in 16 cases only and 62.5% (10 out of 16) showed >30% positivity. Seven out of these 10 (70%) also showed CD38 expression. Studies have shown that the zeta-associated protein of 70 kD (ZAP-70) is aberrantly expressed in a subset of CLL and serves as a surrogate marker for the IgH mutational status. However, polymerase chain reaction amplification followed by Sanger sequencing is the gold standard for the determination of IgH mutation status. In addition, ZAP-70 expression by flow cytometry is relatively weak in the vast majority of cases, represented by relatively small shifts above the baseline threshold set by the autofluorescence control. Thus, compared with the expression of most of the other markers commonly used to diagnose CLL, detection of ZAP-70 is overall not a robust assay. Hence, due to lack of a robust assay and to avoid redundancy in our study, ZAP 70 expression was not included in the study.
Cytogenetic abnormalities in chronic lymphocytic leukemia
Cytogenetic data, detailed in [Table 2] and [Table 3], obtained from patient records, were available in 66 cases. 13q deletion was the most common clonal cytogenetic abnormality detected in 48.4% of the cases. Multiple (>2) abnormalities were seen in 17 (25.8%) cases.
|Table 2: Cytogenetic abnormalities and VH gene usage in chronic lymphocytic leukemia|
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IGVH mutations and gene usage
Details of IGVH status, gene usage, and correlation with cytogenetics are seen in [Table 2] and [Table 3]. We found a higher proportion of unmutated cases (55.2%). VH1 and VH3 were the most frequently used VH gene families. The most commonly used VH genes were IGVH 2–5 (11.7%), IGVH 1–2 and IGVH 1–69 (9.4%), IGVH 3–30, IGVH 3–33, IGVH 4–59, and IGVH 4–34 (5.8% each) which accounted for 54.1% (46 of 85) cases.
Differences in clinical and biological variables between mutated and unmutated groups are seen in [Table 3]. Over 50% (52.8%) of the unmutated cases presented as Rai stage 3 or 4 as compared to 34.7% of the mutated cases.
The cytogenetic abnormalities were evenly distributed among the mutated and unmutated cases except p53 deletion which was more common in the unmutated group. Approximately 70% (26 out of 37) of the unmutated cases required treatment as compared to 50% (14 out of 28) of the mutated ones (P = 0.09).
Follow-up and survival
Follow-up data were available in 78 of the 85 cases. The median follow-up duration was 10 months. There was no significant difference in the time to treatment from the date of diagnosis and overall survival between mutated and unmutated cases (P = 0.69). All the four cases that subsequently transformed into high-grade lymphomas were IGVH unmutated. In addition, two patients had concurrent infiltrating lobular carcinoma and metastatic infiltrating duct carcinoma breast. These patients remain under routine follow-up on a 6 monthly basis. Patients are monitored based on laboratory tests, including routine hemograms. We are in the process of accruing more patients for further genomic studies. Such studies include next generation sequencing for detection of somatic mutations, SNP/array CGH for detection of copy number alterations as well as digital karyotyping. Such data that would incorporate immunophenotyping, IGVH mutations as well as somatic mutations in the future will be then correlated with clinical outcome for prognostication and risk-adapted therapy.
| Discussion|| |
Matutes et al. first proposed a scoring system for the diagnosis of CLL based on the expression level of CD5, CD23, FMC7, smIg, and CD22 (use of CD22 instead of CD79b were suggested by Moreau et al.)., CD200 has been reported as a marker to distinguish CLL from mantle cell lymphomas and CD43+CD200 coexpression is strongly associated with CLL. We could reliably diagnose CLL based on CD200 expression and the coexpression of CD43 and CD200 was seen in 97.1% (69 out of 71) of the cases.
Damle et al. first found a significant correlation between CD38 expression and lack of IGVH mutations. We also found that a higher number of unmutated cases (37.7%) expressed CD38 as compared to the mutated ones (12.1%). Recently, CD49d has been reported as “the strongest independent flow cytometry based prognostic indicator in CLL.” This novel marker was overexpressed in 81.8% of the unmutated cases, however, the number of cases analyzed is very small (n = 16). More numbers will be required to confirm these findings.
Clonal cytogenetic abnormalities may be seen in over 80% of the CLLs. 13q deletion is the most common favorable cytogenetic abnormality in CLL, was detected in up to 48.8% of our patients. It is more frequent in IGVH mutated cases. Although the frequency of 13q deletion in our study was similar to what has been reported in literature, we did not find any significant correlation with mutated or unmutated status. p53 deletion was seen more frequently in unmutated cases (16.3%) as has been previously reported. The presence of p53 deletion is known to be frequently associated with chemo refractoriness, IGVH unmutated status, and overall a poor prognostic indicator., In our study, 12.1% of the cases showed evidence of p53 deletion which is similar to that reported in literature. The association of trisomy 12 with unmutated IGVH status, as reported in literature, was not found in our study.
Damle et al. first used the presence of somatic hypermutations to divide CLL into two groups. They found that close to 50% of the CLL patients had these mutations. Their findings were subsequently confirmed by Hamblin et al. (54.8%), Fais et al. (51.6%), Oscier et al. (60%), and Shanafelt et al. (56%), all of whom found that more than 50% of their patients showed somatic hypermutations. On the other hand, Thunberg et al. (55.1%), Tobin et al. (58%), Ritgen et al. (~65%), and Krober et al. (56%) found a higher proportion of unmutated cases. However, this data represent the Western population. This paper is the first series from India, which describes the immunogenetics of CLL. Although the numbers are small, we describe here a comparable proportion of IGVH unmutated cases as compared to what has been published. In our series, 46.7% of all the patients presented in the later stages of the disease, i.e., Rai stage 3 or 4. As has been published, majority of these cases were from the IGVH unmutated group. A study by Gunawardana et al. compared the characteristics of CLL in the Western population with that of people of South Asian origin. They found that both these populations showed a higher proportion of mutated patients. Studies by Chen et al. from China and Koiso et al. from Japan also showed a higher proportion of mutated cases (69.2% and 58.1%, respectively)., Our data showed a bias toward unmutated status, with 55.2% of the cases falling in this group.
Frequent usage of VH3 and VH4 families has been reported in Asian studies., Although the frequency of VH3 (30.6%) and VH4 (21.2%) usage was high in our study, VH1 (31.8%) was the most common, which has not been reported in any of the aforementioned studies.
With respect to individual VH gene usage, most of the previous studies report a biased usage of IGVH 1–69, IGVH 4–34, IGVH 3–30, and IGVH 3–21.,,, IGVH 3–23 has been reported to be more common in Asians.,, However, our data shows a higher frequency of use of IGVH 2–5 (11.7%), IGVH 1–2, and IGVH 1–69 (9.4%, respectively). Interestingly, the frequency of IGVH 1–69 usage was lower in both studies from Asian populations (~2.3%) as compared to Western studies as well as our findings., The overall frequency of individual VH gene usage in our study was similar to that reported by Ghia et al. except for the higher usage of IGVH 2–5. IGVH 2–5 and IGVH 1–2 did not show any preference to either the mutated or the unmutated group. A significant tendency of biased IGVH 1–69 usage in unmutated cases has been previously established and an association of IGVH 4–34 with the mutated status as seen in our study has also been reported.,, We also found a tendency of IGVH 4–33 and IGVH 3–66 usage to occur in IGVH mutated cases, which has not been previously described.
The mutated and unmutated cases showed no significant difference in the time interval between diagnosis and initiation of treatment. The overall survival between the two groups also showed no statistical significance; however, the duration of follow-up is relatively short in our study. With regard to survival, the association of lack of IGVH mutation with an inferior outcome has already been established.,,
To summarize, in a first of its kind study from India, we looked at the IGVH mutation status and individual IGVH gene usage in 85 cases of CLL and found that higher proportion of unmutated cases and preferential usage of IGVH 2–5, IGVH 1–2, and IGVH 1–69. In this regard, the immunogenetics of Indian CLL differs from that in the Western world. However, we found no difference in the outcome of these two groups of patients. This could be because of limited numbers and shorter median follow-up.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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P G Subramanian
727, Hematopathology Laboratory, 7th Floor, Annexe Building, Tata Memorial Hospital, Parel, Mumbai - 400 012, Maharashtra
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2], [Table 3]