Modifiable (Sleeping Pattern and Stress) and Non-Modifiable Risk Factors Associated with Breast Cancer: A Matched Case-Control Study in Delhi, India.

BACKGROUND: An utmost increase of breast cancer burden during the last several decades was reported in Asian countries. Findings from literature confirm that risk factors of breast cancers can be modifiable and non-modifiable in nature. OBJECTIVE: The present study is designed to identify specific modifiable and non-modifiable risk factors associated with breast cancer. METHODS: A matched case-control study was conducted considering 187 cases as women diagnosed with breast cancer and 187 hospital-controls as women without having breast cancer visiting the hospital. Other than standard risk factors, stress is measured using Perceived Stress Scale (PSS) and stress is measured using Pittsburgh Sleep Quality Index (PSQI). Several modifiable and non-modifiable risk factors were assessed using conditional logistic regression to find out significant association with breast cancer. RESULTS: Regular multi-vitamin uptake (OR = 3.38; 95%CI = 1.69 - 6.77; p-value = 0.001), poor sleep (OR = 11.29; 95%CI = 4.36 - 29.25; p-value < 0.001), irregular sleep (OR = 34.11; 95%CI = 10.03 - 115.92; p-value < 0.001) and severe stress (OR = 6.74; 95%CI = 3.06 - 14.81; p-value < 0.001) were found to be the highest odds ratio among all modifiable risk factor of breast cancer. Also, age at first childbirth less than 30 years (OR = 0.44; 95%CI = 0.25 - 0.78; p-value = 0.005) was found protective against breast cancer. CONCLUSION: In our study, stress, sleeping pattern, and regular multi-vitamin uptake were found to be significant modifiable risk factors of breast cancer. None of the non-modifiable risk factors were found to be significantly associated with the risk of breast cancer.

Non-amplification genetic alterations of HER2 gene in non-small cell lung carcinoma.

AIMS: The present study investigated the incidence and spectrum of human epidermal growth factor receptor 2 (HER2) mutations, associated clinicopathological characteristics and the co-occurrence of HER2 gene amplification in the HER2 gene mutated cases in non-small cell lung cancer (NSCLC). METHODS: All patients with advanced lung adenocarcinoma (LUAD) who underwent broad genomic profiling by next generation sequencing (NGS) from 2015 to 2019 were included in the study. HER2 gene amplification was checked in all the HER2 gene mutated cases. Tumour tissues of all the mutated cases were examined by fluorescent in situ hybridisation (FISH). RESULTS: Fifty-four (37.2%) out of the 145 cases harboured tier 1 driver mutations comprising EGFR in 22.1%, ALK rearrangements in 7.6% cases, ROS1 rearrangements and BRAF V600E in 3.5% cases each, and NTRK fusion in 0.7% cases. Nine (6.2%) cases exhibited a significant genetic alteration in HER2 gene (tiers 2 and 3) on NGS. The most common alteration was exon 20 insertion of amino acid sequence AYVM in five cases (p.E770_A771insAYVM) followed by insertion of YVMA (p.A771_Y772insYVMA) in one case, insGSP (p.V777_G778insGSP) in one case and two missense mutations: p.G776C and p.QA795C (novel variant). The median copy number of the HER2 gene was 3.21 while on FISH, the median HER2/CEP17 ratio was 2.0. CONCLUSIONS: There is a relatively higher occurrence of HER2 exon 20 mutations as primary oncogenic driver in NSCLC especially LUAD. Our cohort has demonstrated (p.E770_A771insAYVM) as the strikingly dominant insertion mutation against the most often globally reported (p.A771_Y772insYVMA).

Biomarker testing for advanced lung cancer by next-generation sequencing; a valid method to achieve a comprehensive glimpse at mutational landscape

Background: Next-generation sequencing (NGS) based assay for finding an actionable driver in non-small-cell lung cancer is a less used modality in clinical practice. With a long list of actionable targets, limited tissue, arduous single-gene assays, the alternative of NGS for broad testing in one experiment looks attractive. We report here our experience with NGS for biomarker testing in hundred advanced lung cancer patients. Methods: Predictive biomarker testing was performed using the Ion AmpliSeq™ Cancer Hotspot Panel V2 (30 tumors) and Oncomine™ Solid Tumor DNA and Oncomine™ Solid Tumor Fusion Transcript kit (70 tumors) on IonTorrent sequencing platform. Results: One-seventeen distinct aberrations were detected across 29 genes in eighty-six tumors. The most commonly mutated genes were TP53 (43% cases), EGFR (23% cases) and KRAS (17% cases). Thirty-four patients presented an actionable genetic variant for which targeted therapy is presently available, and fifty-two cases harbored non-actionable variants with the possibility of recruitment in clinical trials. NGS results were validated by individual tests for detecting EGFR mutation, ALK1 rearrangement, ROS1 fusion, and c-MET amplification. Compared to single test, NGS exhibited good agreement for detecting EGFR mutations and ALK1 fusion (sensitivity- 88.89%, specificity- 100%, Kappa-score 0.92 and sensitivity- 80%, specificity- 100%, Kappa-score 0.88; respectively). Further, the response of patients harboring tyrosine kinase inhibitor (TKI) sensitizing EGFR mutations was assessed. The progression-free-survival of EGFR positive patients on TKI therapy, harboring a concomitant mutation in PIK3CAmTOR and/or RAS-RAF-MAPK pathway gene and/or TP53 gene was inferior to those with sole-sensitizing EGFR mutation (2 months vs. 9.5 months, P = 0.015). Conclusions: This is the first study from South Asia looking into the analytical validity of NGS and describing the mutational landscape of lung cancer patients to study the impact of co-mutations on cancer biology and treatment outcome. Our study demonstrates the clinical utility of NGS testing for identifying actionable variants and making treatment decisions in advanced lung cancer

Incidence of ROS1-Rearranged Non-Small-Cell Lung Carcinoma in India and Efficacy of Crizotinib in Lung Adenocarcinoma Patients.

BACKGROUND: The ROS1 gene is a member of the "sevenless" subfamily of tyrosine-kinase insulin-receptor genes. ROS1-fusion rearrangement causes constitutive downstream signal transduction, with an oncogenic role in non-small-cell lung carcinoma (NSCLC). Fortunately, crizotinib, an ALK1 tyrosine-kinase inhibitor, provides long-term disease control. The objective of this molecular epidemiological study was to estimate the frequency of ROS1 rearrangements and evaluate treatment outcomes with crizotinib therapy. METHODS: Patients with stage IV NSCLC adenocarcinoma histology were considered for this study. The study was conducted according to the ethical principles stated in the latest version of the Declaration of Helsinki and the applicable guidelines for good clinical practice. Clinical characteristics and treatment details were collected from patients' medical records. RESULTS: A total of 709 stage IV NSCLC adenocarcinoma patients were included in the study. There were 457 (64.46%) men and 252 (35.54%) women, with a median age of 60 years. ROS1-gene rearrangement was positive in 20 (2.82%) cases, 13 using Fluorescent In-Situ Hybridization (FISH), and two and five cases, respectively, using immunohistochemistry (IHC) and next-generation sequencing (NGS), followed by confirmation with FISH. Fourteen of the 20 patients with ROS1-gene rearrangement received crizotinib therapy, with an objective response rate of 64.28%. At a median follow-up of 6 months, the study had not achieved the end points of median progression free survival and overall survival. CONCLUSION: ROS1-gene rearrangement was present at a relatively higher frequency of 2.8% in north Indian patients with lung adenocarcinoma and was successfully targeted by crizotinib therapy. Although the only US Food and Drug Administration and Conformité Européenne approved method for testing ROS1 rearrangement is NGS, FISH alone or IHC with D4D6 antibody as initial screen with subsequent confirmation of IHC-positive cases by FISH are cost-effective methods in institutions lacking NGS facilities.

Droplet digital polymerase chain reaction offers an improvisation over conventional immunohistochemistry and fluorescent in situ hybridization for ascertaining Her2 status of breast cancer.

BACKGROUND: Droplet digital polymerase chain reaction (DDPCR) is a recent modality for detecting Her2 expression which is quantitative, cheaper, easier to standardize, and free from interobserver variation. PURPOSE: The purpose of this study is to incorporate DDPCR in the current diagnostic paradigm with clinical benefit. MATERIALS AND METHODS: Fifty-four consecutive patients were tested by immunohistochemistry (IHC), fluorescent in situ hybridization (FISH), and DDPCR. With FISH result as gold standard, receiver operating characteristic curves for DDPCR ratio were analyzed to label Her2-negative, equivocal, and positive cases as DDPCR score 1, 2, and 3, respectively. Proportion of patients labeled unequivocally as Her2 positive or negative was defined to have "clinically benefitted" from the test. Drawing parallel to inter-relationships between DDPCR, IHC, and FISH in the test cohort, four diagnostic pathways were defined - (1) initial IHC followed by FISH, (2) initial DDPCR followed by FISH, (3) initial IHC followed by DDPCR followed by FISH, and (4) initial DDPCR followed by IHC followed by FISH. RESULTS: Clinical benefit of DDPCR as an initial test in the test cohort was 57%, while it was 65% if used as a second-line test among those with an initial inconclusive IHC result. Sensitivity analysis in the simulation cohort revealed that if DDPCR cost was ≤0.6 times the cost of IHC, then a three-step pathway with DDPCR upfront would near certainly prove most cost beneficial. If DDPCR cost was >0.6 but ≤2 times the cost of IHC, then a three-step pathway with DDPCR as second-line test had a higher probability to prove most cost beneficial. If DDPCR cost was >2 times the cost of IHC, then conventional pathway had a higher probability to prove most cost-effective. CONCLUSION: Incorporating DDPCR in the current clinical diagnostic paradigm has the potential to improve its cost-effectiveness and benefit.

Predictive biomarkers in nonsmall cell carcinoma and their clinico-pathological association.

BACKGROUND: Lung cancer is the leading cause of cancer-related mortality worldwide. Genome-directed therapy is less toxic, prolongs survival and provides a better quality of life. Predictive biomarker testing, therefore, has become a standard of care in advanced lung cancers. The objective of this study was to relate clinical and pathological features, including response to targeted therapy (TT) and progression-free survival (PFS) with positive driver mutation. MATERIALS AND METHODS: Archival data of nonsmall cell carcinoma patients with Stage IV disease were retrieved. Those who tested positive for one of the four biomarkers (epidermal growth factor receptor [EGFR], anaplastic lymphoma kinase [ALK], MET, and ROS) were included. Patient demographics and clinical features were reviewed. Tumor histomorphology was correlated with oncological drivers. Treatment response, PFS, and overall survival were studied in three subcohorts of patients who received computed tomography (CT), CT followed by TT and those who received TT in the first line. RESULTS: A total of 900 patients underwent biomarker evaluation of which 288 tested positive. Frequency of the four biomarkers observed was 26.6% (229/860), 6.6% (51/775), 6.6% (5/75), and 5.1% (3/59) for EGFR, ALK, MET, and ROS-1, respectively. The median PFS for EGFR-mutated cohort was 12 months, whereas it was 21 months for ALK protein overexpressing cases. Patients treated with first-line tyrosine kinase inhibitors performed better compared to those who were switched from chemotherapy to TT or those who received chemotherapy alone (P < 0.05). CONCLUSION: Biomarker testing has improved patient outcome. Genome-directed therapy accords best PFS with an advantage of nearly 10 months over cytotoxic therapy.

The detection of primary and secondary EGFR mutations using droplet digital PCR in patients with nonsmall cell lung cancer.

BACKGROUND: We share our experience of using droplet digital polymerase chain reaction (DdPCR) in liquid biopsy specimens for detecting primary and secondary epidermal growth factor receptor (EGFR) mutations among patients with nonsmall-cell lung cancer who had tissue biopsy initially analyzed for del19, L858R and T790M. MATERIALS AND METHODS: Three groups of patients were chosen: Group 1: patients positive for EGFR mutation (del 19 or L858R) by conventional tissue biopsy that were treatment naïve, Group 2: patients positive for EGFR mutation (del 19 or L858R) by conventional tissue biopsy with acquired resistance to tyrosine kinase inhibitor (TKI) therapy, documented by radiology, and Group 3: no known EGFR mutation detected on primary tissue biopsy and treatment naive. RESULTS: One hundred and thirty-three patients were included in the study. Group 1 had 40 cases, of which 21 (52.5%) and 19 (47.5%) were positive for del19 and L858R mutations, respectively, by tissue biopsy. DdPCR detected primary mutation in all but 5 cases. DdPCR additionally found four patients to have T790M mutation. Group 2 had 73 cases and DdPCR detected T790M mutation in 39 (53.4%) cases. Liquid biopsy also picked the original primary mutation in 56/73 cases. Secondary tissue biopsy for T790M mutation status was performed in 11 patients and while it detected mutation in 2 out of 11 cases, DdPCR detected the same in 7 cases, thus providing significantly superior yield (46% difference, McNemar's test, P value 0.063). Tissue biopsy additionally detected c-MET amplification in a patient who had T790M mutation on liquid biopsy. Group 3 had 20 patients and none were falsely positive for EGFR mutation on liquid biopsy. Overall, DdPCR had a Cohen's kappa of 0.82 (standard error 0.074, 95% CI 0.68-0.97) indicating "very good agreement" with conventional tissue biopsy. CONCLUSION: DdPCR demonstrated 87.5% sensitivity and 100% specificity in detecting primary EGFR mutations in patients who were treatment naïve with overall positive and negative predictive value of 100% and 80%, respectively. DdPCR demonstrated T790M mutation postprogression on TKI therapy in 53.4% patients.

Clinical utility of RT-PCR in assessing HER 2 gene expression versus traditional IHC and FISH in breast cancer patients.

BACKGROUND: IHC and FISH are used for categorizing HER 2 status in breast cancer at the protein and DNA level, respectively. HER 2 expression at the RNA level is quantitative, cheaper, easier to standardize and free from interobserver variation. METHODS: 115 consecutive patients were tested by IHC, FISH and RT-PCR (test cohort). Assuming FISH result to be the response variable, ROC curves for RT-PCR ratio were analyzed to label HER 2 negative, equivocal and positive cases as RT-PCR score 1, 2 and 3, respectively. Inter-relationships between RT-PCR, IHC and FISH were defined. 'Clinical benefit' of a test was defined as proportion of patients labeled unequivocally as HER 2 positive or negative. Population for 1 year was simulated constraint to previous reports of HER 2 positivity and IHC category distribution by a meta-analysis of previous studies that evaluated concordance between IHC and FISH to determine HER 2 status (simulation cohort). Four diagnostic pathways in the simulation cohort were defined-(1) initial IHC, followed by FISH (conventional pathway); (2) initial RT-PCR, followed by FISH; (3) initial IHC, followed by RT-PCR and then by FISH; (4) initial RT-PCR, followed by IHC and then by FISH. The clinical benefit of IHC and RT-PCR in the four pathways was analyzed and sensitivity analysis for incremental cost-effectiveness ratio and cost-benefit comapring RT-PCR against IHC, both as first-line tests and among those with IHC score 2 as a reflex second-line test was performed by the Monte Carlo technique. FINDINGS: 115 patients comprised the study population. While none with IHC score of 0 or 1 was FISH positive for HER 2, all cases with IHC score of 3 were FISH positive. 43 cases were assigned IHC score of 2. Thus, 72 patients benefited from the initial IHC testing [clinical benefit 62.6%], with the overall concordance between IHC and FISH being 100% for those with IHC score of 0, 1 and 3 (conclusive IHC categories). For RT-PCR with 100% concordance, 15.7% (115-97 = 18) patients would have benefited from RT-PCR testing if it was used as a first-line test. If RT-PCR would have been used as a second-line test among those with IHC score 2 (n = 43), then only 6 patients would have been assigned a conclusive RT-PCR category (category 1 or 3) translating to a clinical benefit of 14% (6/43) as a second-line test. As a second-line test it had 51% probability to prove more cost-effective than the conventional pathway, provided the cost of RT-PCR was 0.4 times the cost of IHC. Also in a three-step pathway, RT-PCR upfront would have 56% probability of higher cost-benefit provided the cost of RT-PCR was 0.1 times the cost of IHC. CONCLUSION: RT-PCR results were found to be suboptimal to IHC in terms of discriminative ability and clinical benefit; thus, it is unlikely to replace IHC as a first-line test in the near future.

ROS1 rearrangement and response to crizotinib in Stage IV non-small cell lung cancer.

BACKGROUND: The frequency of ROS1 rearrangement in non-small cell lung cancers has been reported from 1.6% to 2.3%. MATERIALS AND METHODS: We examined 105 lung adenocarcinoma patients for ROS1 rearrangement which were negative for EGFR and anaplastic lymphoma kinase. Clinical characteristics of ROS1 rearranged patients and their responses to crizotinib therapy were studied. RESULTS: Of the 105 patients, three cases were positive for ROS1 rearrangement by fluorescence in situ hybridization analysis. All of them showed heterogeneous pattern. All the 3 ROS1-positive patients were females in their forties and started on crizotinib. All of them responded to treatment. One of them developed resistance after 3 months. Another one showed marked systemic response but central nervous system lesions progressed. The third case is doing well till date with inactive lesions on positron emission tomography scan. CONCLUSIONS: The frequency of ROS1 rearrangement is low in non-small cell lung carcinoma, but their diagnosis offers patients an opportunity to receive highly effective targeted therapies.