Clinicopathological Significance of Common Genetic Alterations in Patients With Acute Promyelocytic Leukemia.

OBJECTIVE/BACKGROUND: Acute myeloid leukemia (AML) is one of the common forms of hematological malignancy and acute promyelocytic leukemia (APL) is a unique subtype of AML conferring favorable prognosis. We aimed to determine the prevalence and prognostic impact of Fms-like tyrosine kinase 3 (FLT3), nucleophosmin 1 (NPM1) mutation, epidermal growth factor receptor (EGFR), and flow marker's expression in patients with APL. METHODS: In the present study, 165 de novo APL patients were molecularly characterized for promyelocytic leukemia (PML) breakpoint and additional genetic alterations. Reverse transcriptase polymerase chain reaction (PCR) and real-time PCR assays were used to detect genetic alterations. RESULTS: PML/RARα was detected in 29/165 (17.5%) samples with breakpoint cluster region 1 (bcr1) in 17/29 (58.5%) and bcr3 in 12/29 (41.5%) samples. The prevalence of FLT3-ITD, NPM1, and EGFR were detected in 5/29 (17.5%), 11/29 (38%), and 5/29 (17.5%) patients, respectively. Patients expressing bcr-3 hybrid transcript had lower overall survival compared with bcr1 ( p = .254). White blood cell (WBC) count was significantly higher in bcr3 in comparison with bcr1 patients ( p = .002). Patients with positive EGFR expression ( p = .042) and higher WBC ( p = .002) were significantly associated with poor survival ( p < .05). CONCLUSIONS: We documented the higher prevalence of bcr1 and confirmed that the association of FLT3-ITD significantly reduced the chances of survival in APL. The mortality rate of bcr3 was comparatively higher than that of bcr1. Higher WBC count and EGFR expression were significantly associated with poor survival.

The Prognostic Impact of Epidermal Growth Factor Receptor (EGFR) in Patients with Acute Myeloid Leukaemia.

Expression of Epidermal Growth Factor Receptor (EGFR), an important proto-oncogene, regulates cell differentiation, proliferation, cell migration and survival in most of the cancer types. EGFR expression has been reported in Acute Myeloid Leukaemia (AML), however, many other reports nullified EGFR expression in AML. These contradictory data prompted us to reevaluate the expression of EGFR in AML and carry out a comparative survival analysis between EGFR expressing and non-expressing AML patients (Children and Acute Promyelocytic Leukaemia patients excluded). Bone marrow and/or peripheral blood samples were collected from 60 adult patients with AML with written informed consent. PCR, Real-Time Taqman gene expression assays were used for the detection of genetic alterations. Statistical analysis was conducted using SPSS software (IBM SPSS 20). In our study, EGFR expression was detected in 21 out of 60, in 35% (95% C.I. 23.45-48.48) AML patients. Overall survival was significantly shorter in patients with EGFR (p = < 0.01), with an average survival of 18.57 months (95% C.I. 12.42-24.73 months) compared with 31.27 months (95% C.I. 28.19-34.33 months) in patients without EGFR. EGFR expression was significantly higher in female patients compared to male (p = 0.037).This study confirms the presence of EGFR in AML and indicates that EGFR expression confers poor prognosis in AML. However, the underlying cause of this adverse prognostic effect has not been identified. Further clinical studies are warranted to determine the exact mechanism through which EGFR activity might contribute to AML progression and identify the potential therapeutic target for the reversal of resistance to conventional chemotherapeutics.

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 3 years. 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.

NPM1 Mutation Analysis in Acute Myeloid Leukemia: Comparison of Three Techniques - Sanger Sequencing, Pyrosequencing, and Real-Time Polymerase Chain Reaction.

OBJECTIVE: Nucleophosmin-1 (NPM1) mutations have prognostic importance in acute myeloid leukemia (AML) patients with intermediate-risk karyotype at diagnosis. Approximately 30% of newly diagnosed cytogenetically normal AML (CN-AML) patients harbor the NPM1 mutation in India. In this study we compared the efficiency of three molecular techniques in detecting NPM1 mutation in peripheral blood and bone marrow samples. MATERIALS AND METHODS: In a single-center cohort we analyzed 165 CN-AML bone marrow/peripheral blood samples for NPM1 mutation analysis. About 30% of the CN-AML samples revealed NPM1 mutations. For the detection, three methods were compared: Sanger sequencing, pyrosequencing, and real-time polymerase chain reaction (PCR). RESULTS: NPM1 exon 12 mutations were observed in 52 (31.51%) of all CN-AML cases. The sensitivity of Sanger sequencing, pyrosequencing, and real-time PCR was 80%, 90%, and 95%, whereas specificity was 95%, 100%, and 100%, respectively. The minimum limit of mutation detection was 20%-30% for Sanger sequencing, 1%-5% for pyrosequencing, and 0.1%-1% for real-time PCR. CONCLUSION: The sequencing method, which is the reference method, has the lowest sensitivity and is sometimes difficult to interpret. Real-time PCR is a highly sensitive method for mutation detection but is limited for specific mutation types. In our study, pyrosequencing emerged as the most suitable technique for the detection of NPM1 mutations on the basis of its easy interpretation and less time-consuming processes than Sanger sequencing.

DNMT3A (R882) mutation features and prognostic effect in acute myeloid leukemia in Coexistent with NPM1 and FLT3 mutations.

OBJECTIVE/BACKGROUND: In the absence of high-risk cytogenetic, DNMT3A (DNA Methyltransferase 3a) mutation status has an impact on outcome in the presence of FLT3 (FMS-like Tyrosine Kinase3) and/or NPM1 (Nucleophosmin). In this study, we focus on the features and effect of DNMT3A (R882) mutation in acute myeloid leukemia (AML) in the presence or absence of NPM1 and FLT3 mutations. METHODS: A total of 174 cytogenetically normal (CN)-AML cases were analyzed for NPM1, FLT3, and DNMT3A mutations. For NPM1 mutation detection, we used the pyrosequencing technique; for FLT3 mutations, polymerase chain reaction and RFLP with ECO-RV techniques were used, and for DNMT3A mutation analysis, we used Sanger sequencing and RFLP (Restriction Fragment Length Polymorphism) techniques. RESULTS: NPM1 mutation was found in 40.80%, DNMT3A in 12.06%, and FLT3 mutation was found in 16.66% of 174 CN-AML patients. We also found seven cases which were (NPM1+, FLT3+), 10 cases which were (NPM1+, DNMT3A+), and two cases were found positive for (DNMT3A+, FLT3+) mutations. Adult patients had significantly higher frequency of NPM1 mutation than children (72.22% vs. 16.66%; p = .020), whereas FLT3/ITD and DNMT3A mutation was associated with higher white blood count (p = .081). Immunophenotypically, NPM1 and DNMT3A mutations were significantly associated with the lack of CD34, whereas FLT3/ITD mutation was positively associated with the expression of CD7. We also assessed the overall survival and progression-free survival of DNMT3A mutation status among patients with CN-AML. Indeed, DNMT3A mutations within the CN-AML subset were associated with significantly shorter overall survival and progression-free survival compared to NPM1 and FLT3 mutated patients (p = .067 and p = .065, respectively). CONCLUSION: DNMT3A R882 mutation plays an important role in CN-AML patients' prognosis and clinical outcomes in the presence and absence of NPM1 and FLT3 mutations.

Prevalence Pattern of Key Polymorphisms in the Vitamin D Receptor gene among Patients of Type 2 Diabetes Mellitus in Northeast India.

AIMS: To investigate the association between Vitamin D receptor gene polymorphisms (BsmI, TaqI and FokI) and type 2 diabetes mellitus in patients in north eastern India. SETTINGS AND DESIGN: This was a case control study with 40 cases of type 2 diabetes and 20 controls. MATERIALS AND METHODS: Genomic DNA was extracted from blood and genotyped for the single nucleotide polymorphism (SNPs) of BsmI [rs1544410], TaqI [rs731236] and FokI [rs2228570] by polymerase chain reaction and gene sequencing. Genotype distribution and allelic frequencies were compared between patients and controls. Data was expressed as mean ±standard deviation. Chi square test and t test were used to compare groups. Statistical analysis was done using SAS version 9.3 software. P value of <0.05 was considered significant. RESULTS: Body weight and BMI were significantly associated with VDR polymorphisms BsmI and TaqI while BsmI was significantly associated with HbA1C. Vitamin D deficiency was significantly greater in cases than controls. The frequency of the heterozygous genotype of the BsmI polymorphism was significantly greater in type 2 diabetics than in controls. CONCLUSIONS: Vitamin D receptor polymorphisms are associated with type 2 diabetes in our population and require larger scale studies to be considered as possible risk factors or type 2 diabetes mellitus.

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 (46/165 = 27.90%, 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 (P < 0.01) compared to FLT3-ITD whereas FLT3-ITD showed adverse impact with poor survival rate (P < 0.01), leukocytosis (P < 0.01) and a packed bone marrow. EGFR expression was more in patients with NPM1 mutation compared to FLT3 mutation (P = 0.09). Patients with FLT3 and NPM1 mutations uniformly expressed CD13 and CD33 whereas CD34 was associated with poor prognosis (P ≤ 0.01) 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.

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.

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 1700 bp, 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 400 mg since last 3 months. After 3 months fluorescent in situ analysis and reverse transcriptase pcr analysis showed negative results.

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

INTRODUCTION: Mutation 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. AIM: To check and define which method for FFPE DNA quantification is suitable for downstream processes. MATERIALS AND METHODS: In 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. RESULTS: The 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. CONCLUSION: Results 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.