Dushyant Kumar

Outcome of HER2 Testing by FISH applying ASCO/CAP 2007 and 2013 guideline in IHC equivocal group of breast cancer: Experience at tertiary cancer care centre

Background and Objectives: HER2 testing guideline of ASCO/CAP for interpretation and reporting has recently been revised. The study is aimed to measure the impact of 2013 CAP guideline on equivocal HER2 test outcome (immunohistochemistry [IHC] 2+) when tested by fluorescent in situ hybridization (FISH). The study also aims at finding the frequency of polysomy and monosomy of chromosome 17. Materials and Methods: Specimens were collected in Rajiv Gandhi Cancer Institute and Research Centre, New Delhi, India. IHC was performed in every case, and FISH was performed in IHC2+ cases. Results: In final analysis includes 557 subjects on the basis of CAP guideline 2007 and CAP guideline 2013. One hundred ninety-two subjects (34.4%) were HER2 amplified according to CAP scoring 2007, and 246 subjects (44%) according to 2013 CAP scoring. Conclusions: FISH results were evaluated (IHC2 + interpreted according to CAP 2007 guideline) with both 2007 and 2013 ASCO/CAP scoring criteria, we identified significantly more HER2 positive cases as compared to cases evaluated using the 2007 criteria (P < 0.05). We also found that in breast carcinoma, HER2 status in the presence of polysomy 17 may vary with the scoring criteria used. Evaluation of FISH result using 2013 ASCO/CAP criteria means that more patients with breast cancer may be appropriate for targeted treatment with trastuzumab, potentially improving their outcome.

MET Amplification and Response to MET Inhibitors in Stage IV Lung Adenocarcinoma

Background: Non-small-cell lung cancers with MET amplification may respond to c-MET inhibitors. Methods: We examined lung adenocarcinoma patients for mutations and amplification status of epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), ROS, MET. The clinical characteristics of patients with MET amplification and their responses to MET inhibitor therapy were studied. Results: Of the 76 patients analyzed, 5 were positive for c-MET gene amplification and 4 cases showed an intermediate result. For 12 patients who were EGFR positive, a c-MET analysis on secondary biopsy tissue was performed following disease progression. All 5 c-MET-positive patients were men. The age range in the study was 34-83 years. 4 of the 5 patients were started on crizotinib. 2 of these cases were positive following tyrosine kinase inhibitor therapy. 3 patients showed a response. 1 patient showed no response and was later found to have a concurrent T790M mutation. Conclusions: There are 2 categories of MET gene amplification in lung cancer patients, de novo and that secondary to TKI therapy. These patients can benefit from MET inhibitor therapy. Dual mechanisms of resistance, EGFR T790M mutation and c-MET amplification after TKI therapy, may suggest a poor prognosis.

Detection of false positive mutations in BRCA gene by next generation sequencing.

BRCA1 and BRCA2 genes are implicated in 20–25% of hereditary breast and ovarian cancers. New age sequencing platforms have revolutionized massively parallel sequencing in clinical practice by providing cost effective, rapid, and sensitive sequencing. This study critically evaluates the false positives in multiplex panels and suggests the need for careful analysis. We employed multiplex PCR based BRCA1 and BRCA2 community Panel with ion torrent PGM machine for evaluation of these mutations. Out of all 41samples analyzed for BRCA1 and BRCA2 five were found with 950_951 insA(Asn319fs) at Chr13:32906565 position and one sample with 1032_1033 insA(Asn346fs) at Chr13:32906647, both being frame-shift mutations in BRCA2 gene. 950_951 insA(Asn319fs) mutation is reported as pathogenic allele in NCBI dbSNP. On examination of IGV for all these samples, it was seen that both mutations had ‘A’ nucleotide insertion at 950, and 1032 position in exon 10 of BRCA2 gene. Sanger Sequencing did not confirm these insertions. Next-generation sequencing shows great promise by allowing rapid mutational analysis of multiple genes in human cancer but our results indicate the need for careful sequence analysis to avoid false positive results.

Quantification of DNA Extracted from Formalin Fixed Paraffin-Embeded Tissue Comparison of Three Techniques: Effect on PCR Efficiency

INTRODUCTIONnMutation detection from Formalin Fixed Paraffin-Embedding (FFPE) tissue in molecular lab became a necessary tool for defining potential targeted drug. Accurate quantification of DNA extracted from FFPE tissue is necessary for downstream applications like Polymerase Chain Reaction (PCR), sequencing etc.nnnAIMnTo check and define which method for FFPE DNA quantification is suitable for downstream processes.nnnMATERIALS AND METHODSnIn this experimental experience study Biorad Smartspec Plus spectrophotomery, Qubit Fluorometer, and Qiagen Rotorgene qPCR was used to compare 20 FFPE DNA quantification in Rajiv Gandhi Cancer Institute and Research Centre, in 2015 and quantified amount of DNA used for PCR reaction.nnnRESULTSnThe average concentration of DNA extracted from FFPE tissue measured using the spectrophotometer was much higher than the concentration measured using the Qubit Fluorometer and qPCR.nnnCONCLUSIONnResults varied depending upon the technique used. A fluorometric analysis may be more suitable for quantification of DNA samples extracted from FFPE tissue compared with spectrophotometric analysis. But qPCR is the best technique because it details DNA quantity along with quality of amplifiable DNA from FFPE tissue.

Evaluating New Markers for Minimal Residual Disease Analysis by Flow Cytometry in Precursor B Lymphoblastic Leukemia

Minimal residual disease is currently the most powerful prognostic indicator in Precursor B lymphoblastic leukemia. Multiparameter flow cytometry is the most commonly used modality. Seventy three B ALL cases and 15 normal marrows were evaluated for expression patterns of leukemia markers (CD38, CD58, CD73) in all 73 cases and CD66c, CD86 and CD123 in 23 cases. CD73 was aberrantly expressed in 90.41% cases and CD86 in 60.87% B ALL cases. Thus addition of these markers in MRD panels can increase the sensitivity of the assay.

Novel e8a2 BCR/ABL Fusion Transcript in Case of a Myeloproliferative Neoplasm

Main objective of this work was to confirm the occurrence of rare BCR-ABL fusion variant involving the a2 region of the ABL gene and e8 of BCR gene in a patient of Myeloproliferative neoplasm positive for t(9;22) translocation but negative for common major and minor breakpoint cluster regions. A patient with elevated white blood cell count was subjected to classical cytogenetics, FISH as well as RT-PCR testing using commercial kits as well as published primers and in house testing protocol. The translocation event in chromosome 9 and 22 could be successfully detected. BCR/ABL dual color, dual fusion probe generated a classical balanced translocation scenario within the nucleus. In RT-PCR we found an unexpectedly large amplification band around 1700xa0bp, which is consistent with e8a2 transcript. Nine metaphases showed 46,XY,t(9;22)(q34;q11.2)[9] by cytogenetic. A rare e8a2 break point in the BCR-ABL gene in myeloproliferative neoplasm disease detected in India. It also emphasizes the utility of cytogenetic and FISH for primary diagnosis of any neoplasm in blood. Our Patient detected rare BCR-ABL fusion variant e8a2 was on imatinib 400xa0mg since last 3xa0months. After 3xa0months fluorescent in situ analysis and reverse transcriptase pcr analysis showed negative results.

Clinico-pathological Features of PIK3CA Mutation in HER2-Positive Breast Cancer of Indian Population

PIK3CA pathway is one of the important signaling pathways in cells, which is involved in cell proliferation, cell survival, motility, and growth. Mutation in PIK3CA gene negatively effects to anti-HER2 therapy in breast cancer patients. PIK3CA gene of HER2-positive breast cancers associated with reduced sensitivity to neoadjuvant therapy. In this study, we assessed the frequency of PIK3CA mutations and influence of PIK3CA mutations on patient survival in a series of HER2-positive breast cancer patients. PIK3CA mutations were assessed by pyrosequencing and next generation sequencing in 107 HER2-positive breast cancer patients of a tertiary Cancer Centre of India from Jan 2012 to Jun 2013 with minimum follow-up of 3xa0years. We found PIK3CA mutations in 26 tumors (24.2%) of which 5 were in exon 9, 20 were in exon 20, and 1 was in both exon 9 and 20. In exon 9, the mutation c.1634A>G was found in 4 cases and mutation c.1636C>A was found in 2 cases. In exon 20, the mutation c.3140A>G was found in 15 cases and c.3140A>T was found in 6 cases. The outcome between PIK3CA mutated versus PIK3CA wild type was significant showing p value 0.014. Overall survival of mutation and treatment with herceptin, mutation with other chemotherapy treatment in both early breast cancer (EBC), and locally advanced breast cancer (LABC) showed significant p value 0.037 and 0.044 respectively. In conclusion, we identified 24.2% somatic mutation of PIK3CA in HER2-positive breast cancer patients. PIK3CA mutation is significantly associated with ER-positive tumors. The frequency and distribution pattern reported in this study is similar to the global report. Overall survival of PIK3CA mutation is slightly lower but in patients who received herceptin with PIK3CA mutation showed better clinical outcome.

Benefits of Pre-harvest Peripheral Blood CD34 Counts Guided Single Dose Therapy with PLERIXAFOR in Autologous Hematopoietic Stem Cell Transplantation: A Retrospective Study at a Tertiary Care Institute in India

Peripheral blood is a convenient source of stem cells for hematopoietic stem cell transplantation. However, in autologous transplants, the harvest failure rates are high because of inadequate mobilization using G-CSF alone. Plerixafor is a potent mobilizer when used with G-CSF. However, its routine use is limited by high cost. This is a retrospective study done at a tertiary care oncology centre in India. All the harvest records were analyzed between Jan 2015 and Nov 2017. May 2016 onwards pre-harvest peripheral blood CD34 count was done in all cases of autologous transplants on day 4 of G-CSF therapy and they were given a single dose of Plerixafor if counts wereu2009<u200920 cell per cumm. The results were compared amongst various groups. A total of 321 cases were analyzed. 172/321 were allogenic transplant cases of which 5% (nu2009=u20097) failed to achieve a target live stem cell dose ofu2009>u20092 million per kg of the recipient. The overall failure rate in autologous group (nu2009=u2009149) was 27% (nu2009=u200941) (pu2009≤u20090.001 auto vs. allo). The failure rate was higher (36%, nu2009=u200928/77) when no intervention with Plerixafor was done. The overall failure rate in the group treated with pre-harvest 34 count based single dose therapy of Plerixafor was 18% (nu2009=u200913/72, pu2009=u20090.01). However, within this intervention group, the patients who had pre-harvest peripheral blood CD34 above the desired cutoff had a higher failure rate of 21% (pu2009=u20090.13). Pre-harvest CD34 count based intervention with Plerixafor help optimizing the cost.

Diagnostic Dilemma in Ambiguous Lineage Acute Leukemia: A Case Report

Acute leukemias with ambiguous lineage encompass those leukemias that show no clear evidence of differentiation along a single lineage [1]. They are rare and account for less than 4% of all cases of acute leukemias. They include acute undifferentiated leukemia (AUL) and mixed phenotype acute leukemia (MPAL) with bilineage and biphenotypic blast subpopulations. Co-expression of myeloid and B-lymphoid antigens is most common in MPAL, followed by co-expression of myeloid and T-lymphoid antigens, accounting for 66–70% and 23–24% of MPALs, respectively [2]. Co-expression of Band T-lineage-associated antigens is even rarer; overall there are too few cases to make any specific statements about clinical features, genetic lesions or prognosis of such patients. In assigning B lineage to a case of T cell leukaemia, CD79a and CD10 should not be considered as evidence of B-cell differentiation. Multiparameter flow cytometry is the method of choice for recognizing MPAL, with only few cases requiring immunohistochemical staining for characterization of blasts. Even when there are not two distinctly separable populations, most cases of MPAL show heterogeneity of expression of some antigens [3]. We present a challenging case of B/T type MPAL with equivocal expression of lineage-specific antigens on flowcytometry. A previously healthy 42 year old male presented to the OPD with flu-like symptoms. A complete blood count (CBC) revealed pancytopenia with a white blood cell count of 1.9 9 10/L, hemoglobin of 8.3 g/dl, platelet count of 30 9 10/L and a differential count of N: 08, L: 31, M: 03, Blast: 58. Bone marrow aspiration and biopsy was performed and the samples were submitted for morphologic, flowcytometric, cytochemical, immunohistochemical, cytogenetic and molecular analysis. Bone marrow aspirate smears showed near-total replacement of normal hematopoietic components by a heterogenous population of small to medium sized blasts with round to slightly indented nuclei, condensed chromatin, inconspicuous nucleoli and scant cytoplasm. Blasts were negative for myeloperoxidase on cytochemical staining. Flowcytometry was performed on FACS CANTO-II (BD Biosciences) instrument using stain, lyse and wash technique. On flowcytometric analysis, blasts gated in the dim to intermediate CD45 region were positive for CD34, CD38, CD33, CD7 with heterogenous expression of CD19 (49%, moderate intensity; when compared to normal positive control in the sample), cytoCD79a (42.6%, moderate intensity) and cytoCD3 (14%, dim to moderate) [Fig. 1]. They were negative for all other markers including CD10, CD20, CD73, CD13, HLA-DR, CD14, CD117, CD64, CD5, CD2, CD4, CD8, CD3, CD56, cytoMPO, cytoCD22, CD15, CD11c, TDT, CD41a and CD1a. As clear-cut lineage determination was not possible due to absence of strong expression of lineage-specific antigens such as cytoCD3, CD19 and cytoCD79a, immunohistochemistry was done on bone marrow biopsy which showed diffuse Disclaimer: The identity of the patient is not disclosed here in this case.

Prevalence and Clinical Significance of FLT3 and NPM1 Mutations in Acute Myeloid Leukaemia Patients of Assam, India

Acute Myeloid Leukaemia (AML) is one of the common forms of haematological malignancy in adults. We analysed the prevalence and clinical significance of FMS-like tyrosine kinase 3 (FLT3) and Nucleophosmin 1 (NPM1) mutations in AML patients of North East India. Co-prevalence and clinical significance of three recurrent chromosomal translocations namely t(15; 17), t(8; 21), t(16; 16) and expression of epidermal growth factor receptor (EGFR), flow markers were also documented and co-related with disease progress. We analysed bone marrow aspirates or peripheral blood samples from 165 newly diagnosed AML patients. All clinical samples were analysed by Real Time PCR and DNA sequencing based assays. NPM1 was the most frequently detected mutation in the study population (46/165xa0=xa027.90%, 95% CI 20.75–35.05). FLT3 mutations were detected in 27/165 (16.40%, 95% CI 10.45–22.35) patients with internal tandem duplication (FLT3-ITD) in 24/165 (14.60%, 95% CI 8.91–20.29) and FLT3-D835 in 3/165 (1.80%, 95% CI 0–4.13) patients. NPM1 mutations were associated with a higher complete remission rate and longer overall survival (Pxa0<xa00.01) compared to FLT3-ITD whereas FLT3-ITD showed adverse impact with poor survival rate (Pxa0<xa00.01), leukocytosis (Pxa0<xa00.01) and a packed bone marrow. EGFR expression was more in patients with NPM1 mutation compared to FLT3 mutation (Pxa0=xa00.09). Patients with FLT3 and NPM1 mutations uniformly expressed CD13 and CD33 whereas CD34 was associated with poor prognosis (Pxa0≤xa00.01) in patients with NPM1 mutation. FLT3-ITD was associated with inferior overall survival. However the clinical significance of FLT3-D835 was not clear due to small number of samples. NPM1 mutation showed better prognosis with increased response to treatment in the absence of FLT3-ITD.