Nikhil Patkar

Standardizing minimal residual disease by flow cytometry for precursor B lineage acute lymphoblastic leukemia in a developing country

In addition to standard risk criteria at diagnosis, minimal residual disease (MRD) following initiation of therapy is a well‐recognized risk factor to predict relapse. Literature from developing countries addressing therapeutic or laboratory practices related to MRD, is largely lacking. In a first paper from India, we describe our experience in establishing a flow cytometry‐based MRD assay for precursor B lineage ALL (BCP‐ALL) with emphasis on the assay standardization and cost.

MYD88 mutant lymphoplasmacytic lymphoma/Waldenström macroglobulinemia has distinct clinical and pathological features as compared to its mutation negative counterpart

Abstract In a first series from India, we report 32 cases of lymphoplasmacytic lymphoma/Waldenström macroglobulinemia (LPL/WM) over 7 years. Here, we analyzed 32 patients with LPL/WM for MYD88 L265P mutation and correlated mutation staus with hematological and biochemical parameters and also with the International Prognostic Scoring System (ISSWM) and treatment response. Twenty-seven out of 32 cases of LPL/WM (84.3%) harbored the MYD88 L265P mutation. MYD88 wild-type WM was associated with a lower number of tumor cells (p < 0.01) and older age (p = 0.02) and a lower ISSWM score at presentation (p = 0.03) as compared to mutated LPL/WM. On evaluation of response (n = 23), 44.4% of patients with MYD88 mutated LPL/WM had progressive disease, whereas no patient in the MYD88 unmutated group changed their baseline status. We confirm the high frequency of MYD88 mutations in LPL/WM. Although the number of MYD88 wild-type cases was limited, our data indicate that MYD88 may represent an adverse prognostic marker for LPL/WM.

Evaluation of new markers for minimal residual disease monitoring in B-cell precursor acute lymphoblastic leukemia: CD73 and CD86 are the most relevant new markers to increase the efficacy of MRD 2016; 00B: 000–000

Multiparametric flow cytometry (MFC) is a popular technique for minimal residual disease (MRD) analysis. However, its applicability is still limited to 90% of B‐cell precursor acute lymphoblastic leukemia (BCPALL) due to two major issues, i.e. a proportion of cases do not express adequate leukemia associated immunophenotype (LAIPs) with currently used markers and drug‐induced antigen modulation. Hence, the incorporation of additional reliable markers is required for the further improvement of MFC‐based MRD evaluation. We studied the utility of new markers in improvising MFC‐based MRD detection in BCPALL.

A novel and easy FxCycle™ violet based flow cytometric method for simultaneous assessment of DNA ploidy and six-color immunophenotyping

Abnormal DNA ploidy is a valuable prognostic factor in many neoplasms, especially in hematological neoplasms like B‐cell acute lymphoblastic leukemia (B‐ALL) and multiple myeloma (MM). Current methods of flow‐cytometric (FC) DNA‐ploidy evaluation are either technically difficult or limited to three‐ to four‐color immunophenotyping and hence, challenging to evaluate DNA‐ploidy in minute tumor population with background rich of its normal counterpart cells and other hematopoietic cells. We standardized a novel sensitive and easy method of simultaneous evaluation of six‐ to seven‐color immunophenotyping and DNA‐ploidy using a dye–FxCycle Violet (FCV). Linearity, resolution, and coefficient of variation (CV) for FCV were studied using chicken erythrocyte nuclei. Ploidy results of FCV were compared with Propidium iodide (PI) in 20 samples and intra‐assay variation for FCV was studied. Using this six‐color immunophenotyping & FCV‐protocol DNA‐ploidy was determined in bone‐marrow samples from 124 B‐ALL & 50 MM patients. Dilution experiment was also conducted to determine the sensitivity in detection of aneuploidy in minute tumor population. FCV revealed high linearity and resolution in 450/50 channel. On comparison with PI, CV of Go/G1‐peak with FCV (mean‐CV 4.1%) was slightly higher than PI (mean‐CV 2.9%) but had complete agreement in ploidy results. Dilution experiment showed that aneuploidy could be accurately detected up to the limit of 0.01% tumor cells. Intra‐assay variation was very low with CV of 0.005%. In B‐ALL, hypodiploidy was noted in 4%, hyperdiploidy in 24%, near‐hyperdiploidy in 13% and remaining 59% were diploid. In MM, hypodiploidy was in 2%, hyperdiploidy in 58%, near‐hyperdiploidy in 8% and remaining 30% were diploid. FCV‐based DNA‐ploidy method is a sensitive and easy method for simultaneous evaluation of six‐color immunophenotyping and DNA analysis. It is useful in DNA‐ploidy evaluation of minute tumor population in cases like minimal residual disease and MM precursor conditions.

Clinicopathological features of hepatosplenic T cell lymphoma: a single centre experience from India

Abstract In a first series from India, we report 9 cases of hepatosplenic T cell lymphoma (HSTCL) seen in 23 months accounting for 4.2% of all mature T-non-Hodgkin lymphomas (NHLs) in our institution. All patients presented with organomegaly, cytopenias and had evidence of bone marrow involvement. The tumor cells had a blastic (55%) morphology with predominantly intrasinusoidal (33.3%) or intrasinusoidal with an additional interstitial component (33.3%). On flow cytometry, the classical phenotype (CD3+, CD7+, CD4−, CD8−, CD5−, CD56+/−) was seen only in 4 patients. Unusual variations included CD45 (overexpression), CD7 (dim expression), CD3 (overexpression, heterogeneous and dim), CD2 (overexpression), CD5 (heterogeneous), CD8 (heterogeneous or dim or overexpression) and aberrant expression of CD19. Fluoresvent in situ hybridisation (FISH) and karyotyping was abnormal in 5 out of 7 patients evaluated. All of the 5 cases showed abnormalities in chromosome 7 (ring chromosome or isochromosome 7q). Five patients died of disease and related complications in a span of 1–3 months after diagnosis whereas 4 were alive at their last follow up out of which 2 had documented a relapse. In our series, HSTCL was characterized by typical clinical and variable immunophenotypic features and a dismal clinical outcome.

An integrated genomic profile that includes copy number alterations is highly predictive of minimal residual disease status in childhood precursor B-lineage acute lymphoblastic leukemia

Introduction: Copy number alterations (CNA) have been described in childhood precursor B-lineage acute lymphoblastic leukemia (B-ALL) which in conjunction with chromosomal abnormalities drive leukemogenesis. There is no consensus on the clinical incorporation of CNA in B-ALL. An integrated genomic classification (IGC) has been proposed which includes CNA and cytogenetics. Methods: We correlated this IGC with immunophenotypic minimal residual disease (MRD) as well as other standard criteria for 245 patients of B-ALL such as National Cancer Institute (NCI) risk, D+8 prednisolone response, cytogenetics, and ploidy status. Results: MRD was detectable in 81 patients (33.1%). The most common abnormalities were seen in CDKN2A/B (25.7%) followed by PAX5(20%), ETV6(16.7%), IKZF1(15.5%), Rb1(5.3%), BTG (3.3%), EBF1(2.0%), and PAR1(0.8%). On integrating CNA into the IGC, 170 patients (69.4%) were classified into good genomic risk (GEN-GR) whereas 75 (30.6%) belonged to the poor genomic risk (GEN-PR) category. The IGC showed a significant correlation with MRD and NCI risk. The presence of CNA predicted MRD clearance in intermediate cytogenetics group. Conclusion: These data seem to indicate that in addition to cytogenetics, CNA should be incorporated into routine clinical testing and risk algorithms for B-ALL. The IGC is of prognostic relevance and offers an additional avenue for prognostication and risk-adapted therapy.

Characteristics of BCR-ABL kinase domain mutations in chronic myeloid leukemia from India: not just missense mutations but insertions and deletions are also associated with TKI resistance

Abstract We document the characteristics of BCR-ABL kinase domain mutations (KDM) in the largest study from India comprising of 385 patients and demonstrate that more than half (51.9%) of these patients have detectable abnormalities in the KD both in adult and in pediatric chronic myelogenous leukemia (CML). These comprise singly occurring missense mutations (25.5%), polyclonal/compound point mutations (4.9%), and insertions/deletions (29.6%). Missense mutations were most commonly seen in the imatinib-binding region followed by the P-loop. The commonest mutation in our dataset was T315I. Other common missense mutations were Y253H, M244V, and F317L. A high prevalence of BCR-ABL exon7 deletion (p.R362fs*) was also seen (25.5% of the entire cohort), whereas the 35bpintron-derived insertion/truncation mutation detected in 12 patients. In the pediatric age group, 58.8% of patients harbored missense mutations, polyclonal/compound mutations as well as insertions and deletions. We detected 11 novel mutations (seven missense mutations and four insertions/deletions).

Molecular basis of Wiskott–Aldrich syndrome in patients from India

To the Editor: Wiskott–Aldrich syndrome (WAS) [OMIM: 301000] is an X-linked immunodeficiency disease characterized by thrombocytopenia and small platelets, eczema, recurrent infections, with an increased risk for autoimmunity and malignancy (1, 2). The gene responsible for this syndrome, WAS, comprises 12 exons and approximately 1.8 kb in length (3). WAS encodes a 502-amino-acid protein (WASp) that is expressed selectively in hematopoietic stem cell– derived lineages and is involved in cell signaling and cytoskeleton reorganization (4). A milder allelic variant caused by WAS gene mutation leads to X-linked thrombocytopenia (XLT), a congenital disorder characterized by thrombocytopenia and small platelets but, in general, without the other complications of WAS (5). So far, ~369mutations have been reported in WAS gene (http://www.hgmd.cf.ac.uk/ ac/gene.php?gene=WAS). Detection of additional mutations in this gene is important for the precise genetic diagnosis in families affected by this disorder as well as for studying the molecular basis of this disease (6). We report here for the first time the WAS gene mutations identified in patients with WAS/XLT from India and their genotype–phenotype correlations. Ten patients from eight families were evaluated at the Department of Haematology, Christian Medical College, Vellore, with clinical features suggestive of WAS on written, informed consent. All patients underwent hematological and biochemical evaluation (Table 1). Blood was collected in citrated buffer and in ethylenediamine tetra-acetic acid (EDTA) from the probands and, whenever available, from family members. Similar samples were also collected from healthy controls. Blood smears were stained using modified Giemsa–Wright stain (Beckman, Duarte, CA, USA) and evaluated for morphology. Platelet count and the mean platelet volume (MPV) were estimated in a cell counter (Coulter LH 755; Beckman Coulter). A mononuclear cell suspension in phosphate-buffered saline (PBS) was isolated from heparinized blood by Ficoll gradient centrifugation. The cells were fixed (Fix and Perm Medium; Invitrogen, Carlsbad, CA, USA) and permeabilized (Fix and Perm medium B; Invitrogen) for intracellular staining and incubated with phycoerythrin (PE)-conjugated antibody against WASP DI (SantaCruz Biotechnology, SantaCruz, CA, USA) and WAS B9 (Santa Cruz Biotechnology) with appropriate IgG1 and IgG2a controls (BD Pharmingen, San Jose, CA, USA). After processing, cells were analyzed for intracellular bound florescence in a flow cytometer (BD FACS Calibur, Manifield, MA, USA). The patient WASP expression was compared to normal controls processed at the same time, and the data are expressed as percentage of normal WASP-positive cells (7). Genomic DNA from EDTA anti-coagulated blood was isolated by standard phenol–chloroform method. The human WAS gene exonic and flanking intronic regions were amplified by twelve pairs of primers as described previously (8). Nucleotide changes in the amplified fragments were screened by a conformation-sensitive gel electrophoresis (CSGE) and DNA sequencing strategy (9). Samples displaying abnormal CSGE patterns were sequenced by the Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Warrington, UK) on an ABI 3130 genetic analyzer (PE Applied Biosystems, Foster City, CA, USA). All the novel mutations identified were confirmed as unique to the patients by screening 100 normal control alleles. The normal controls were recruited based on a written informed consent. Mutations at or near the splice junction consensus sequences were analyzed by ‘Splice Site Prediction program’ (http://www.fruitfly.org/seq_tools/splice.html) to predict changes in RNA splicing. The clinical features, and hematological and molecular genetic data of all the 10 patients are detailed in Table 1. Diagnosis was based on low platelet count (9000–95 000/ mm), presence of small platelets (mean platelet volume – 6.2 ± 1.5) and reduced levels of WASp by flow cytometry. Of these, two were sibling pairs (WAS-9/WAS-10; WAS15/WAS-35). The median age at first clinical symptoms of these patients was 2.5 yr (range 1–12 yr). Six of them had a family history of bleeding. To differentiate between classical WAS and XLT in these patients, the disease was scored from 1 to 5 based on the presence of thrombocytopenia, small platelets, eczema, immunodeficiency, infections, autoimmunity or malignancy and congenital neutropenia as described previously (10). In patients who were lost to follow-up, we could not comprehensively analyze the biochemical or genetic data, and this was a limitation to our study. Genotypic analysis revealed mutations in eight of ten patients (Table 1). Among them, three had nonsense mutations, two splice site variations, two deletions, and one missense mutation. Two of these eight mutations were novel. These included a single ‘T-nucleotide’ deletion (c.108delT),

Evaluation of CD229 as a new alternative plasma cell gating marker in the flow cytometric immunophenotyping of monoclonal gammopathies: CD229, A NEW RELIABLE PLASMA CELL GATING MARKER

Current flow‐cytometric plasma cell (PC) gating is based on CD138, CD38, and CD45 expression. CD138 is known for variable expression and loss during storage and processing. Introduction of anti‐CD38 and anti‐CD138 monoclonal‐antibody therapies has limited the use of these markers during follow‐up. Hence, additional reliable PC‐gating markers are required. Recently, CD229 has been claimed as an alternative PC‐gating marker. However, these studies are limited to a small cohort of samples. We evaluated the utility of CD229 as a new PC‐gating marker in routine laboratory practice.

Immunogenetics of chronic lymphocytic leukemia

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.