Ring Finger 43 Hot-spot Frameshift Mutation G659V in Colorectal Cancer Patients: Report from a Tertiary Cancer Care Hospital in North India.

Background: The Ring Finger 43 (RNF43) is a tumor suppressor gene that negatively regulates the Wnt/ß-catenin signaling. The p.G659fs is a recurrent RNF43 C-terminal truncating variant frequent in colorectal cancer (CRC) patients. We aimed to identify this hotspot variant in CRC patients and assessed the relationship between the mutation, clinical characteristics, and tumor ß-catenin localization. Materials and Methods: Formalin-fixed, paraffin-embedded tissue samples of upfront, surgically resected, sporadic colorectal adenocarcinoma cases were selected. The p.G659fs mutation was determined by capillary sequencing with sequence-specific primers. Tissue microarray and immunohistochemistry were employed to analyze nuclear ß-catenin expression and the expression of mismatch repair (MMR) proteins, respectively. In addition, clinical details were retrieved from the hospital medical records and data were analyzed. Results: The RNF43 p.G659fs mutation was observed in 8% of CRC patients. In total, 25% of tumors showed a loss of immunostaining for one or more MMR proteins and 14.6% of tumors showed positive nuclear ß-catenin staining. The p.G659fs variant was significantly enriched in MMR-deficient tumors (P = 0.04). Importantly, no correlation was observed between the variant and nuclear ß-catenin localization (P = 0.48), indicating a Wnt-independent role of this variant in CRC tumors. Conclusions: To the best of our knowledge, this is the first study from North India to show the involvement of RNF43 p.G659fs variant in CRC patients. The mutation correlated with MMR protein deficiency and seems to be conferring tumorigenicity independent of the Wnt pathway.

Expression of glucose transporter 1 (SLC2A1) - Clinicopathological associations and survival in an Indian cohort of colorectal cancer patients.

Background: Glucose transporter 1 (GLUT1) facilitates the uptake of glucose in malignant cells. We investigated GLUT1 transcript expression in colorectal cancer (CRC) tumors and explored its relationship to clinicopathological features, diabetes condition, and patient survival. Materials and Methods: The expression of GLUT1 was determined using fluorescent probe-based quantitative real-time polymerase chain reaction assay of tumor tissue and corresponding normal mucosa from 180 archived formalin-fixed, paraffin-embedded tissue blocks of ninety upfront surgically resected colorectal adenocarcinoma cases. Clinical information was collected from the hospital medical records and statistical analyses were performed. Results: Compared to normal mucosa tissue, the GLUT1 expression was significantly elevated in CRC tumor tissue (0.024 ± 0.056 vs. 0.004 ± 0.005; P < 0.0001). The expression was significantly more in poorly differentiated tumors than well/moderately differentiated tumors (P = 0.024) and in patients with liver metastasis (P = 0.013). The high GLUT1 expression correlated with advanced tumor stage (P = 0.003), liver metastasis (P = 0.003), poor tumor differentiation (P = 0.02), and death (P = 0.001). In univariate Cox regression analysis for survival, high GLUT1 expression, presence of any comorbidity, diabetic condition, advanced or metastatic stage, and liver metastasis were significant risk factors for death. CRC patients with high GLUT1 expression showed worse survival outcomes than those with low GLUT1 expression (P = 0.001). Furthermore, the high GLUT1/diabetes (+) patients had an inferior survival outcome than the patients with low GLUT1/diabetes (+) condition. Conclusions: GLUT1 is significantly upregulated in colorectal adenocarcinoma. The expression correlated with poor tumor histology, higher stage, hepatic metastases, and adverse survival in the study cohort.

Lung Cancer in Non-Smokers: Clinicopathological and Survival Differences from Smokers.

Background Lung cancer in non-smokers is a clinically distinct entity based on unique epidemiology, clinicopathology, genetics, treatment response, and outcome. Data from Indian centres are scarce. The objective of this study was to compare the frequency, clinical characteristics, driver mutations, and survival of non-smoking and smoking lung cancer patients treated at a tertiary cancer centre in North India. Methodology Two years of data on 724 consecutive lung cancer patients were assessed. Clinical, demographics, smoking history, and EGFR and ALK mutation test results were collected. Descriptive and inferential statistics were applied. Survival analysis was performed using the Kaplan-Meier method. Results Non-smokers comprised 40.9% of the study sample. Non-smokers were more likely than smokers to experience disease onset at a younger age (P = 0.004) and metastasis (P < 0.001). The tumor histology showed significant differences (P < 0.001), with non-smokers more likely to be diagnosed with adenocarcinoma (77.4%), while squamous and small cell histologies were commonly found among smokers (37.6% and 13.8%, respectively). The EGFR mutation and ALK rearrangement rates in the cohort were 23.3% and 10.1%, respectively, and were more frequent in non-smoking patients. Overall, 10-year survival was 7%, with a significantly better survival rate of non-smokers than smokers (median survival time of 15.13 vs 10.17 months; P = 0.012). Conclusions About four out of 10 patients diagnosed with lung cancer at our centre were non-smokers. They were more often young, diagnosed at an advanced stage, with predominantly adenocarcinoma histology, and had a threefold higher frequency of EGFR mutations than smokers. In our cohort, non-smokers appear to be a targetable group with better survival than smokers.

Plasma biopsy by Tag-sequencing: an acceptable alternative to tumor tissue profiling in non-small-cell lung cancer.

Tag-sequencing is a modified next-generation sequencing (NGS) approach wherein targeted regions are tagged with unique molecular identifiers enabling error-free detection of rare genomic alterations. We aimed to perform this high- fidelity sequencing to identify actionable variants from the plasma of lung cancer patients. Targeted sequencing was performed from plasma-derived cell-free nucleic acid in twenty-one advanced, treatment naïve, non-small-cell lung cancer (NSCLC) patients. Clinically significant genetic alterations were compared with matched tumor NGS profile for each patient (patient-level), and separately for each alteration (variant-level). Cross-platform validation was done for EGFR and KRAS mutations (real-time PCR) and ALK1 rearrangement (immunohistochemistry). Forty-seven alterations (26 in plasma and 21 in tumor tissue) were detected in 19/21 tested cases. Overall-concordance between the two assays was 94.87% (κ of 0.71, 95% CI: 0.54-0.89). Patient-level and genic-concordance was 57.1% (12/21 cases) and 67.86%, respectively. Almost perfect agreement was reached for detecting actionable EGFR mutations and ALK1 rearrangement (κ of 0.89 and κ of 1, respectively), which was confirmed by single-gene testing. Substantial agreement between the assays makes Tag-sequencing a viable option for identifying multibiomarkers from the plasma of advanced NSCLC patients in special circumstances where tissue has depleted/tumor is inaccessible/high risk of biopsy due to existing comorbidities.

Rare epidermal growth factor receptor gene alterations in non-small cell lung cancer patients, tyrosine kinase inhibitor response and outcome analysis.

BACKGROUND: The predictive value of rare epidermal growth factor receptor gene (EGFR) mutations for non-small cell lung carcinoma (NSCLC) patients remain elusive. We evaluated the distribution, clinicopathological association, tyrosine kinase inhibitor (TKI) response, and outcome of NSCLC patients carrying uncommon EGFR aberrations in comparison to classical EGFR mutations. METHODS: Treatment naïve, advanced NSCLC cases tested by Next-Generation sequencing (NGS) method between 2015 and 2020 were included. The objective response rate (ORR), disease control rate (DCR), progression-free survival (PFS), and overall survival (OS) were analyzed. RESULTS: A total of 237 tumor samples were sequenced. Among the sixty-nine (29%) EGFR mutated cases, 41 (59.4%) harbored classical mutation (37.7% Del19, 21.7% p.L858R). Non-classical aberrations included missense mutations in exon 18/20/21 (15.9%), EGFR amplification (8.7%), exon 20 insertions (7.2%), EGFR Variant III (4.3%), exon 18 indel (2.9%), exon 21 missense (2.9%) and exon 19 missense mutation (1.4%). These occurred as complex mutations in 16% of cases. Oral TKI was administered in 66.7% cases. The patients harboring non-classical variants had a lower ORR and DCR (23.1% and 61.5%) than those carrying a common mutation (57.6% and 84.8%). Collectively, compared to the patients with common EGFR mutations, the uncommon group showed early disease progression and had shorter overall survival. CONCLUSION: NGS testing has the capacity to reveal a diverse spectrum of uncommon EGFR variants. Our study can contribute to the database of uncommon EGFR mutations with a positive influence on evidence-based care of advanced lung cancer patients with EGFR mutations.

COVID-19 mortality in cancer patients: a report from a tertiary cancer centre in India.

BACKGROUND: Cancer patients, especially those receiving cytotoxic therapy, are assumed to have a higher probability of death from COVID-19. We have conducted this study to identify the Case Fatality Rate (CFR) in cancer patients with COVID-19 and have explored the relationship of various clinical factors to mortality in our patient cohort. METHODS: All confirmed cancer cases presented to the hospital from June 8 to August 20, 2020, and developed symptoms/radiological features suspicious of COVID-19 were tested by Real-time polymerase chain reaction assay and/or cartridge-based nucleic acid amplification test from a combination of naso-oropharyngeal swab for SARS-CoV-2. Clinical data, treatment details, and outcomes were assessed from the medical records. RESULTS: Of the total 3,101 cancer patients admitted to the hospital, 1,088 patients were tested and 186 patients were positive for SARS-CoV-2. The CFR in the cohort was 27/186 (14.52%). Univariate analysis showed that the risk of death was significantly associated with the presence of any comorbidity (OR: 2.68; (95% CI [1.13-6.32]); P = 0.025), multiple comorbidities (OR: 3.01; (95% CI [1.02-9.07]); P = 0.047 for multiple vs. single), and the severity of COVID-19 presentation (OR: 27.48; (95% CI [5.34-141.49]); P < 0.001 for severe vs. not severe symptoms). Among all comorbidities, diabetes (OR: 3.31; (95% CI [1.35-8.09]); P = 0.009) and cardiovascular diseases (OR: 3.77; (95% CI [1.02-13.91]); P = 0.046) were significant risk factors for death. Anticancer treatments including chemotherapy, surgery, radiotherapy, targeted therapy, and immunotherapy administered within a month before the onset of COVID-19 symptoms had no significant effect on mortality. CONCLUSION: To the best of our knowledge, this is the first study from India reporting the CFR, clinical associations, and risk factors for mortality in SARS-CoV-2 infected cancer patients. Our study shows that the frequency of COVID-19 in cancer patients is high. Recent anticancer therapies are not associated with mortality. Pre-existing comorbidities, especially diabetes, multiple comorbidities, and severe symptoms at presentation are significantly linked with COVID-19 related death in the cohort.

Clinical characteristics and survival profile of young versus old colorectal cancer patients at a tertiary cancer center in North India over a period of 5 years.

BACKGROUND: Colorectal cancer (CRC) is mostly considered a disease of the elderly. But the rate is increasing among young adults and is associated with different clinical patterns. The objective was to study the frequency of CRC in young patients and compare the clinicopathological profile and survival with the older cohort. METHODS: Five-year (2012-2016) data of the 912 consecutive CRC cases treated at the center were analyzed. Clinical and histopathological characteristics were compared in young (≤40) and older (>40) patients. Descriptive statistics were used for data presentation. Categorical data were compared by the Chi-square test; survival analyses were performed by Kaplan-Meier method. RESULTS: In total, 231 (25.3%) and 681 (74.7%) cases were in the young and older age groups, respectively. Male predominance was noted. Young patients presented predominantly in stage III (46%). Majority of the young patients harbored left-sided tumors (75.8% vs 63.7% in old patients, P = 0.001) and rectum was the favored site in young patients (53.7% vs 37%; P < 0.001). Poorly differentiated adenocarcinoma was more common in the young age group (46.88% vs 24.16% in old patients, P < 0.001), also signet-ring cell morphology occurred more often in young patients (11.7% vs 4%, P < 0.001). Survival was inferior in the patients presenting at an advanced stage or with adverse histology or poor tumor grade. However, stage-specific survival showed no significant difference between both groups. CONCLUSION: This study shows that though young CRC patients present with higher stage, aggressive morphology, and predominantly rectal localization, the overall survival and stage-specific survival did not differ significantly from the older patients.

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

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.

Germline BRCA1 and BRCA2 deleterious mutations and variants of unknown clinical significance associated with breast/ovarian cancer: a report from North India.

BACKGROUND: The spectrum of BRCA mutations that predispose to development of breast/ovarian cancer in Indian population remains unexplored. We report incidence and various types of pathogenic, likely pathogenic and variants of unknown significance (VUS) mutations in BRCA1 and BRCA2 genes observed at a tertiary cancer center in North India. MATERIALS AND METHODS: A total of 206 unrelated breast and/or ovarian cancer patients, who met the National Comprehensive Cancer Network (NCCN) guidelines for genetic testing, were screened for germline BRCA1/BRCA 2 mutations on high-throughput sequencing platform; large genomic rearrangements were assessed by multiple ligation probe assay. Mutations were mined in mutational databases, PubMed, and discerned into classes. Furthermore, the clinicopathological correlation of BRCA mutation status with prognostic markers in breast cancer and tumor histology in ovarian cancer was performed. RESULTS: In total, 45/206 and 17/206 cases showed positivity for BRCA1 and BRCA2 mutations, respectively, whereas 1/206 was positive for a mutation in both the genes. Altogether, 33 distinct BRCA1 mutations were observed, among which 27 were deleterious (12 frameshifts, 8 nonsense, 1 missense, 3 splice-site variants, 2 big deletions and 1 large duplication) and 6 were VUS. Five novel BRCA1 mutations (c.541G>T, c.1681delT, c.2295delG, c.4915C>T and exon 23 deletion) were identified. Seven mutations (c.2214_2215insT, c.2295delG, c.3607C>T,c.4158_4162delCTCTC, c.4571C>A, splicesite_3 (C>T) and exon 21-23 duplication) occurred more than once, whereas 16 distinct BRCA2 mutations were noted - 9 were lethal (6 frameshifts, 2 nonsense and 1 big deletion) and 7 VUS. One unique pathogenic BRCA2 mutation (c.932_933insT) was recognized. Two mutations (c.9976A>T and c.10089A>G) recurred twice. No significant difference in hormone receptor status was observed among BRCA1 carriers, BRCA2 carriers and noncarriers. CONCLUSION: We have documented various pathogenic and VUS mutations in BRCA1 and BRCA2 genes observed in the cohort. Six novel mutations were identified. The knowledge shared would assist genetic testing in enabling more focused site-specific screening for mutations in biological relatives.

6-Shogaol Inhibits Breast Cancer Cells and Stem Cell-Like Spheroids by Modulation of Notch Signaling Pathway and Induction of Autophagic Cell Death.

Cancer stem cells (CSCs) pose a serious obstacle to cancer therapy as they can be responsible for poor prognosis and tumour relapse. In this study, we have investigated inhibitory activity of the ginger-derived compound 6-shogaol against breast cancer cells both in monolayer and in cancer-stem cell-like spheroid culture. The spheroids were generated from adherent breast cancer cells. 6-shogaol was effective in killing both breast cancer monolayer cells and spheroids at doses that were not toxic to noncancerous cells. The percentages of CD44+CD24-/low cells and the secondary sphere content were reduced drastically upon treatment with 6-shogaol confirming its action on CSCs. Treatment with 6-shogaol caused cytoplasmic vacuole formation and cleavage of microtubule associated protein Light Chain3 (LC3) in both monolayer and spheroid culture indicating that it induced autophagy. Kinetic analysis of the LC3 expression and a combination treatment with chloroquine revealed that the autophagic flux instigated cell death in 6-shogaol treated breast cancer cells in contrast to the autophagy inhibitor chloroquine. Furthermore, 6-shogaol-induced cell death got suppressed in the presence of chloroquine and a very low level of apoptosis was exhibited even after prolonged treatment of the compound, suggesting that autophagy is the major mode of cell death induced by 6-shogaol in breast cancer cells. 6-shogaol reduced the expression levels of Cleaved Notch1 and its target proteins Hes1 and Cyclin D1 in spheroids, and the reduction was further pronounced in the presence of a γ-secretase inhibitor. Secondary sphere formation in the presence of the inhibitor was also further reduced by 6-shogaol. Together, these results indicate that the inhibitory action of 6-shogaol on spheroid growth and sustainability is conferred through γ-secretase mediated down-regulation of Notch signaling. The efficacy of 6-shogaol in monolayer and cancer stem cell-like spheroids raise hope for its therapeutic benefit in breast cancer treatment.

Diaminothiazoles evade multidrug resistance in cancer cells and xenograft tumour models and develop transient specific resistance: understanding the basis of broad-spectrum versus specific resistance.

Acquired drug resistance poses a challenge in cancer therapy. Drug efflux is the most common mechanism of resistance displayed by hydrophobic drugs beyond a certain size. However, target specific changes and imbalance between the pro- and anti-apoptotic proteins are also found quite often in many tumours. A number of small antimitotic agents show high potential for multidrug resistant tumours, mainly because they are able to evade the efflux pumps. However, these compounds are also likely to suffer from resistance upon prolonged treatment. Thus, it is important to find out agents that are sensitive to resistant tumours and to know the resistance mechanisms against small molecules so that proper combinations can be planned. In this report, we have studied the efficiency of diaminothiazoles, a novel class of tubulin targeting potential anticancer compounds of small size, in multidrug resistant cancer. Studies in model cell lines raised against taxol and the lead diaminothiazole, DAT1 [4-amino-5-benzoyl-2-(4-methoxy phenyl amino) thiazole], and the xenograft tumours derived from them, show that diaminothiazoles are highly promising against multidrug resistant cancers. They were able to overcome the expression of efflux protein MDR1 and certain tubulin isotypes, could sensitize improper apoptotic machinery and ablated checkpoint proteins Bub1 and Mad2. Further, we have found that the resistance against microtubule binding compounds with higher size is broad-spectrum and emerges due to multiple factors including overexpression of transmembrane pumps. However, resistance against small molecules is transient, specific and is contributed by target specific changes and variations in apoptotic factors.

Upregulation of DR5 receptor by the diaminothiazole DAT1 [4-amino-5-benzoyl-2-(4-methoxy phenyl amino) thiazole] triggers an independent extrinsic pathway of apoptosis in colon cancer cells with compromised pro and antiapoptotic proteins.

Mitochondria mediated signalling is the more common way of apoptosis induction exhibited by many chemotherapeutic agents in cancer cells. Death receptor mediated signalling for apoptosis in many cells also requires further amplification from the mitochondrial pathway activation through tBid. Thus the potential of most chemotherapeutic agents in tumours with intrinsic apoptosis resistance due to changes in molecules involved in the mitochondrial pathway is limited. Diaminothiazoles were shown earlier to bind to tubulin thereby exhibiting cytotoxicity towards different cancer cells. We observed that the lead diaminothiazole, DAT1 [4-amino-5-benzoyl-2-(4-methoxy phenyl amino) thiazole] could induce apoptosis in the colon cancer cell line HCT116 by both pathways. However, in contrast to many other chemotherapeutic agents, DAT1 triggered apoptosis where the intrinsic pathway was blocked by changing the pro and antiapoptotic proteins. An independent extrinsic pathway activation triggered by the upregulation of DR5 receptor accounted for that. The induction of DR5 occurred in the transcriptional level and the essential role of DR5 was confirmed by the fact that siRNA downregulation of DR5 significantly reduced DAT1 induced apoptosis. HCT116 cells were earlier shown to have a type II response for apoptosis induction where extrinsic pathway was connected to the intrinsic pathway via the mediator protein tBid. Our finding thus indicates that the signalling events in the manifestation of apoptosis depend not only on the cancer cell type, but also on the inducer. Our results also place diaminothiazoles in a promising position in the treatment of tumours with compromised apoptotic factors.