Osimertinib for lung cancer cells harboring low-frequency EGFR T790M mutation.

Osimertinib, a third-generation EGFR tyrosine kinase inhibitor, shows significant benefit among patients with EGFR T790M mutation at disease progression. We analyzed the whole exome sequence of 48 samples obtained from 16 lung cancer patients with a longitudinal follow-up: treatment-naïve-baseline primary tumors positive for EGFR activating-mutations, paired re-biopsies upon disease progression but negative for EGFR T790M mutation based on qPCR, and their matched normal blood samples. Our Next generation sequencing (NGS) analysis identified an additional set of 25% re-biopsy samples to harbor EGFR T790M mutation occurring at a low-allele frequency of 5% or less, undetectable by conventional qPCR-based assays. Notably, the clinical utility of osimertinib among patients harboring low-allele frequency of EGFR T790M in tissue biopsy upon disease progression remains less explored. We established erlotinib-resistant PC-9R cells and twenty single-cell sub-clones from erlotinib-sensitive lung cancer PC-9 cells using in vitro drug-escalation protocol. NGS and allele-specific PCR confirmed the low-allele frequency of EGFR T790M present at 5% with a 100-fold higher resistance to erlotinib in the PC-9R cells and its sub-clones. Additionally, luciferase tagged PC-9, and PC-9R cells were orthotopically injected through the intercostal muscle into NOD-SCID mice. The orthotopic lung tumors formed were observed by non-invasive bioluminescence imaging. Consistent with in vitro data, osimertinib, but not erlotinib, caused tumor regression in mice injected with PC-9R cells, while both osimertinib and erlotinib inhibited tumors in mice injected with PC-9 cells. Taken together, our findings could extend the benefit of osimertinib treatment to patients with low EGFR T790M mutation allele frequency on disease progression.

TMC-SNPdb 2.0: an ethnic-specific database of Indian germline variants.

Cancer is a somatic disease. The lack of Indian-specific reference germline variation resources limits the ability to identify true cancer-associated somatic variants among Indian cancer patients. We integrate two recent studies, the GenomeAsia 100K and the Genomics for Public Health in India (IndiGen) program, describing genome sequence variations across 598 and 1029 healthy individuals of Indian origin, respectively, along with the unique variants generated from our in-house 173 normal germline samples derived from cancer patients to generate the Tata Memorial Centre-SNP database (TMC-SNPdb) 2.0. To show its utility, GATK/Mutect2-based somatic variant calling was performed on 224 in-house tumor samples to demonstrate a reduction in false-positive somatic variants. In addition to the ethnic-specific variants from GenomeAsia 100K and IndiGenomes databases, 305 132 unique variants generated from 173 in-house normal germline samples derived from cancer patients of Indian origin constitute the Indian specific, TMC-SNPdb 2.0. Of 305 132 unique variants, 11.13% were found in the coding region with missense variants (31.3%) as the most predominant category. Among the non-coding variations, intronic variants (49%) were the highest contributors. The non-synonymous to synonymous SNP ratio was observed to be 1.9, consistent with the previous version of TMC-SNPdb and literature. Using TMC SNPdb 2.0, we analyzed a whole-exome sequence from 224 in-house tumor samples (180 paired and 44 orphans). We show an average depletion of 3.44% variants per paired tumor and significantly higher depletion (P-value < 0.001) for orphan tumors (4.21%), demonstrating the utility of the rare, unique variants found in the ethnic-specific variant datasets in reducing the false-positive somatic mutations. TMC-SNPdb 2.0 is the most exhaustive open-source reference database of germline variants occurring across 1800 Indian individuals to analyze cancer genomes and other genetic disorders. The database and toolkit package is available for download at the following: Database URL  http://www.actrec.gov.in/pi-webpages/AmitDutt/TMCSNPdb2/TMCSNPdb2.html.

Fusobacterium nucleatum is associated with inflammation and poor survival in early-stage HPV-negative tongue cancer.

Persistent pathogen infection is a known cause of malignancy, although with sparse systematic evaluation across tumor types. We present a comprehensive landscape of 1060 infectious pathogens across 239 whole exomes and 1168 transcriptomes of breast, lung, gallbladder, cervical, colorectal, and head and neck tumors. We identify known cancer-associated pathogens consistent with the literature. In addition, we identify a significant prevalence of Fusobacterium in head and neck tumors, comparable to colorectal tumors. The Fusobacterium-high subgroup of head and neck tumors occurs mutually exclusive to human papillomavirus, and is characterized by overexpression of miRNAs associated with inflammation, elevated innate immune cell fraction and nodal metastases. We validate the association of Fusobacterium with the inflammatory markers IL1B, IL6 and IL8, miRNAs hsa-mir-451a, hsa-mir-675 and hsa-mir-486-1, and MMP10 in the tongue tumor samples. A higher burden of Fusobacterium is also associated with poor survival, nodal metastases and extracapsular spread in tongue tumors defining a distinct subgroup of head and neck cancer.

IPD 2.0: To derive insights from an evolving SARS-CoV-2 genome.

BACKGROUND: Rapid analysis of SARS-CoV-2 genomic data plays a crucial role in surveillance and adoption of measures in controlling spread of Covid-19. Fast, inclusive and adaptive methods are required for the heterogenous SARS-CoV-2 sequence data generated at an unprecedented rate. RESULTS: We present an updated version of the SARS-CoV-2 analysis module of our automated computational pipeline, Infectious Pathogen Detector (IPD) 2.0, to perform genomic analysis to understand the variability and dynamics of the virus. It adopts the recent clade nomenclature and demonstrates the clade prediction accuracy of 92.8%. IPD 2.0 also contains a SARS-CoV-2 updater module, allowing automatic upgrading of the variant database using genome sequences from GISAID. As a proof of principle, analyzing 208,911 SARS-CoV-2 genome sequences, we generate an extensive database of 2.58 million sample-wise variants. A comparative account of lineage-specific mutations in the newer SARS-CoV-2 strains emerging in the UK, South Africa and Brazil and data reported from India identify overlapping and lineages specific acquired mutations suggesting a repetitive convergent and adaptive evolution. CONCLUSIONS: A novel and dynamic feature of the SARS-CoV-2 module of IPD 2.0 makes it a contemporary tool to analyze the diverse and growing genomic strains of the virus and serve as a vital tool to help facilitate rapid genomic surveillance in a population to identify variants involved in breakthrough infections. IPD 2.0 is freely available from http://www.actrec.gov.in/pi-webpages/AmitDutt/IPD/IPD.html and the web-application is available at http://ipd.actrec.gov.in/ipdweb/ .

Weekly osimertinib dosing prevents EGFR mutant tumor cells destined to home mouse lungs.

The recently conducted ADAURA trial concludes daily dosing of adjuvant osimertinib, a third-generation EGFR tyrosine kinase inhibitor (TKI), improves disease-free survival with stage IB/II/IIIA EGFR -mutated non-small cell lung cancer patients in comparison to placebo. We have developed a preclinical orthotopic mouse model, using luciferase tagged lung adenocarcinoma cells harboring EGFR TKI sensitive exon 19 deletion to model and extend trial implications comparing a weekly vs daily dosing outcome of osimertinib to a first-generation TKI- erlotinib. We find that 100% of mice in both the groups receiving osimertinib daily or weekly before injection of cells show a complete absence of homing of cells in mice's lungs from day three until day 18 post-injection of cells. On the other hand, 25% and 75% of mice receiving erlotinib daily and weekly before injecting cells show homing of cells to the lungs. The tumors observed in the lungs, when dissected at day 30, confirmed the colonization of the injected cells homing to the organ. Thus, our study establishes the efficacy of pretreatment with osimertinib in reducing tumor cells' homing to mouse lungs in an in vivo mouse model.

Molecular characterization of lung squamous cell carcinoma tumors reveals therapeutically relevant alterations.

INTRODUCTION: Unlike lung adenocarcinoma patients, there is no FDA-approved targeted-therapy likely to benefit lung squamous cell carcinoma patients. MATERIALS AND METHODS: We performed survival analyses of lung squamous cell carcinoma patients harboring therapeutically relevant alterations identified by whole exome sequencing and mass spectrometry-based validation across 430 lung squamous tumors. RESULTS: We report a mean of 11.6 mutations/Mb with a characteristic smoking signature along with mutations in TP53 (65%), CDKN2A (20%), NFE2L2 (20%), FAT1 (15%), KMT2C (15%), LRP1B (15%), FGFR1 (14%), PTEN (10%) and PREX2 (5%) among lung squamous cell carcinoma patients of Indian descent. In addition, therapeutically relevant EGFR mutations occur in 5.8% patients, significantly higher than as reported among Caucasians. In overall, our data suggests 13.5% lung squamous patients harboring druggable mutations have lower median overall survival, and 19% patients with a mutation in at least one gene, known to be associated with cancer, result in significantly shorter median overall survival compared to those without mutations. CONCLUSIONS: We present the first comprehensive landscape of genetic alterations underlying Indian lung squamous cell carcinoma patients and identify EGFR, PIK3CA, KRAS and FGFR1 as potentially important therapeutic and prognostic target.

Raman spectroscopy-based detection of RNA viruses in saliva: A preliminary report.

Several non-invasive Raman spectroscopy-based assays have been reported for rapid and sensitive detection of pathogens. We developed a novel statistical model for the detection of RNA viruses in saliva, based on an unbiased selection of a set of 65 Raman spectral features that mostly attribute to the RNA moieties, with a prediction accuracy of 91.6% (92.5% sensitivity and 88.8% specificity). Furthermore, to minimize variability and automate the downstream analysis of the Raman spectra, we developed a GUI-based analytical tool "RNA Virus Detector (RVD)." This conceptual framework to detect RNA viruses in saliva could form the basis for field application of Raman Spectroscopy in managing viral outbreaks, such as the ongoing COVID-19 pandemic. (http://www.actrec.gov.in/pi-webpages/AmitDutt/RVD/RVD.html).

ERBB2 and KRAS alterations mediate response to EGFR inhibitors in early stage gallbladder cancer.

The uncommonness of gallbladder cancer in the developed world has contributed to the generally poor understanding of the disease. Our integrated analysis of whole exome sequencing, copy number alterations, immunohistochemical, and phospho-proteome array profiling indicates ERBB2 alterations in 40% early-stage rare gallbladder tumors, among an ethnically distinct population not studied before, that occurs through overexpression in 24% (n = 25) and recurrent mutations in 14% tumors (n = 44); along with co-occurring KRAS mutation in 7% tumors (n = 44). We demonstrate that ERBB2 heterodimerizes with EGFR to constitutively activate the ErbB signaling pathway in gallbladder cells. Consistent with this, treatment with ERBB2-specific, EGFR-specific shRNA or with a covalent EGFR family inhibitor Afatinib inhibits tumor-associated characteristics of the gallbladder cancer cells. Furthermore, we observe an in vivo reduction in tumor size of gallbladder xenografts in response to Afatinib is paralleled by a reduction in the amounts of phospho-ERK, in tumors harboring KRAS (G13D) mutation but not in KRAS (G12V) mutation, supporting an essential role of the ErbB pathway. In overall, besides implicating ERBB2 as an important therapeutic target under neo-adjuvant or adjuvant settings, we present the first evidence that the presence of KRAS mutations may preclude gallbladder cancer patients to respond to anti-EGFR treatment, similar to a clinical algorithm commonly practiced to opt for anti-EGFR treatment in colorectal cancer.

Genomic characterization of tobacco/nut chewing HPV-negative early stage tongue tumors identify MMP10 asa candidate to predict metastases.

OBJECTIVES: Nodal metastases status among early stage tongue squamous cell cancer patients plays a decisive role in the choice of treatment, wherein about 70% patients can be spared from surgery with an accurate prediction of negative pathological lymph node status. This underscores an unmet need for prognostic biomarkers to stratify the patients who are likely to develop metastases. MATERIALS AND METHODS: We performed high throughput sequencing of fifty four samples derived from HPV negative early stage tongue cancer patients habitual of chewing betel nuts, areca nuts, lime or tobacco using whole exome (n=47) and transcriptome (n=17) sequencing that were analyzed using in-house computational tools. Additionally, gene expression meta-analyses were carried out for 253 tongue cancer samples. The candidate genes were validated using qPCR and immuno-histochemical analysis in an extended set of 50 early primary tongue cancer samples. RESULTS AND CONCLUSION: Somatic analysis revealed a classical tobacco mutational signature C:G>A:T transversion in 53% patients that were mutated in TP53, NOTCH1, CDKN2A, HRAS, USP6, PIK3CA, CASP8, FAT1, APC, and JAK1. Similarly, significant gains at genomic locus 11q13.3 (CCND1, FGF19, ORAOV1, FADD), 5p15.33 (SHANK2, MMP16, TERT), and 8q24.3 (BOP1); and, losses at 5q22.2 (APC), 6q25.3 (GTF2H2) and 5q13.2 (SMN1) were observed in these samples. Furthermore, an integrated gene-expression analysis of 253 tongue tumors suggested an upregulation of metastases-related pathways and over-expression of MMP10 in 48% tumors that may be crucial to predict nodal metastases in early tongue cancer patients. In overall, we present the first descriptive portrait of somatic alterations underlying the genome of tobacco/nut chewing HPV-negative early tongue cancer, and identify MMP10 asa potential prognostic biomarker to stratify those likely to develop metastases.

Air gasification of rice husk in bubbling fluidized bed reactor with bed heating by conventional charcoal.

An experimental study of air gasification of rice husk was conducted in a bench-scale fluidized bed gasifier (FBG) having 210 mm diameter and 1600 mm height. Heating of sand bed material was performed using conventional charcoal fuel. Different operating conditions like bed temperature, feeding rate and equivalence ratio (ER) varied in the range of 750-850 °C, 25-31.3 kg/h, and 0.3-0.38, respectively. Flow rate of air was kept constant (37 m(3)/h) during FBG experiments. The carbon conversion efficiencies (CCE), cold gas efficiency, and thermal efficiency were evaluated, where maximum CCE was found as 91%. By increasing ER, the carbon conversion efficiency was decreased. Drastic reduction in electric consumption for initial heating of gasifier bed with charcoal compared to ceramic heater was ∼45%. Hence rice husk is found as a potential candidate to use directly (without any processing) in FBG as an alternative renewable energy source from agricultural field.