Gingivo-buccal oral squamous cell carcinoma (OSCC-GB), an anatomical and clinical subtype of head and neck squamous cell carcinoma (HNSCC), is prevalent in regions where tobacco-chewing is common. Exome sequencing (n=50) and recurrence testing (n=60) reveals that some significantly and frequently altered genes are specific to OSCC-GB (USP9X, MLL4, ARID2, UNC13C and TRPM3), while some others are shared with HNSCC (for example, TP53, FAT1, CASP8, HRAS and NOTCH1). We also find new genes with recurrent amplifications (for example, DROSHA, YAP1) or homozygous deletions (for example, DDX3X) in OSCC-GB. We find a high proportion of C>G transversions among tobacco users with high numbers of mutations. Many pathways that are enriched for genomic alterations are specific to OSCC-GB. Our work reveals molecular subtypes with distinctive mutational profiles such as patients predominantly harbouring mutations in CASP8 with or without mutations in FAT1. Mean duration of disease-free survival is significantly elevated in some molecular subgroups. These findings open new avenues for biological characterization and exploration of therapies.

Lipocalin 2 is a siderophore‐binding protein that regulates iron homeostasis. Lipocalin 2 expression is elevated in multiple tumor types; however, the mechanisms that drive tumor progression upon Lipocalin 2 expression remain unclear. When Lipocalin 2 is over‐expressed, it leads to resistance to 5‐fluorouracil in colon cancer cell lines in vitro and in vivo by inhibiting ferroptosis. Lipocalin 2 inhibits ferroptosis by decreasing intracellular iron levels and stimulating the expression of glutathione peroxidase4 and a component of the cysteine glutamate antiporter, xCT. The increase in xCT levels is dependent on increased levels of ETS1 in Lipocalin 2 over‐expressing cells. Inhibiting Lipocalin 2 function with a monoclonal antibody leads to a decrease in chemo‐resistance and transformation in vitro, and a decrease in tumor progression and chemo‐resistance in xenograft mouse models. Lipocalin 2 and xCT levels exhibit a positive correlation in human tumor samples suggesting that the pathway we have identified in cell lines is operative in human tumor samples. These results indicate that Lipocalin 2 is a potential therapeutic target and that the monoclonal antibody described in our study can serve as the basis for a potential therapeutic in patients who do not respond to chemotherapy.

For oral cancers, screening and monitoring of high-risk populations can aid in early diagnosis and improve overall outcomes. Of the new methods, approaches based on exfoliative cytology are more practical for mass screening and monitoring of high-risk populations. Raman spectroscopy and exfoliative cytology for cervical cancers has shown promise in differentiating normal and abnormal samples. In this study, feasibility of Raman oral exfoliative cytology along with cytopathology for oral cancer diagnosis was evaluated on 70 specimens. Exfoliated cells were obtained from 15 healthy volunteers (HV), 15 healthy tobacco users (HT), and 20 contralateral or disease control (DC) and 20 tumor (T) sites of oral-cancer patients. Pap staining was carried out post Raman spectral acquisition. Spectral findings demonstrate that with increase in severity of pathology from HV to T, higher DNA and changes in secondary structure of proteins were encountered. Owing to heterogeneity in cellular samples, two different approaches-point-spectra and patient-wise were evaluated for data analysis. PCA and PC-LDA using both approaches indicate that HV and HT are distinct from cancer groups DC and T.Misclassifications were also observed between HT and DC. These findings also correlate with cytopathological findings. Less misclassifications and higher classification efficiency was observed for patient-wise approach. Large-scale validation study needs to be undertaken for evaluating utility of Raman oral exfoliative cytology for screening of oral cancers using patientwise approach.The incidence of oral cancer worldwide is around 300000 new cases every year 1 . Tobacco abuse (smoking and smokeless) is the most common etiological factor for oral cancer 2 . The overall 5year survival rate of around 50% is mainly attributed to delayed diagnosis and recurrence. These rates are less than occult cancers like breast, colorectal, cervix 3 . Early detection of oral cancer and its curable precursors remains the best way to ensure patient survival and improved quality of life 4, 5 . South Asian countries like India are major contributors to the global cancer burden, due to rampant tobacco habits. In fact, it is estimated that over 90% of the global smokeless tobacco burden is in South Asia-around 100 million people use smokeless tobacco in India and Pakistan alone 6, 7 . Due to a known dose-response relationship between tobacco consumption and development of oral cancer, chronic tobacco abusers are at high risk for development of oral cancer 8 . Thus, screening and monitoring of these high-risk populations, along with the general population is crucial.The current standard screening procedure for oral cancers is visual inspection followed by biopsy and histopathology of suspicious lesions. These currently employed methods have shown limited potential in detecting precancerous or early cancerous lesions 9 . In fact, visual examination was useful as a method of screening for oral cancer only in high risk cases like chronic smokers or alcoholics 10 . The susp...

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 heterodimerize 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 Afatninb 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.

Oral cancer is associated with high rates of recurrence, attributable to field cancerization. Early detection of advanced field changes that can potentially progress to carcinoma can facilitate timely intervention and can lead to improved prognosis. Previous in vivo studies have successfully detected advanced field effects in oral cancers. Raman exfoliative cytology has previously shown to differentiate normal, oral precancer and cancers. The present study explores Raman-exfoliative-cytology-based detection of field effects. Exfoliated cells were collected from tumor (n = 16) and contralateral-normal appearing mucosa (n = 16) of oral cancer patients, and healthy tobacco habitués (n = 20). After spectral acquisition, specimens were Papstained for cytological evaluation. Data analysis, by Principal Component Analysis and Principal Component-Linear Discriminant Analysis, indicate several spectralmisclassifications between contralateral normal and tumor, which were investigated and correlated with spectral, cytological and clinical outcomes. A qualitative analysis by grouping patients with number of misclassifications with tumor (Group 1: 0, Group 2: 1 and Group 3: >1) was explored. Group 3 with highest misclassifications showed spectral and cytological similarity to tumor groupone patient was a case of early inoperable residual disease, despite clear margins on histopathology.Thus, these misclassifications could be indicative of cancer field changes, and can prospectively help to identify patients susceptible to recurrences. K E Y W O R D Sfield cancerization, oral cancers, pap staining, Raman exfoliative cytology, Raman spectroscopy

Background: Poor-responsiveness of tumors to radiotherapy is a major clinical problem. Owing to the dynamic nature of the epigenome, the identification and targeting of potential epigenetic modifiers may be helpful to curb radio-resistance. This requires a detailed exploration of the epigenetic changes that occur during the acquirement of radio-resistance. Such an understanding can be applied for effective utilization of treatment adjuncts to enhance the efficacy of radiotherapy and reduce the incidence of tumor recurrence. Results: This study explored the epigenetic alterations that occur during the acquirement of radio-resistance. Sequential irradiation of MCF7 breast cancer cell line up to 20 Gy generated a radio-resistant model. Micrococcal nuclease digestion demonstrated the presence of compact chromatin architecture coupled with decreased levels of histone PTMs H3K9ac, H3K27 ac, and H3S10pK14ac in the G 0 /G 1 and mitotic cell cycle phases of the radio-resistant cells. Further investigation revealed that the radio-resistant population possessed high HDAC and low HAT activity, thus making them suitable candidates for HDAC inhibitor-based radio-sensitization. Treatment of radio-resistant cells with HDAC inhibitor valproic acid led to the retention of γH2AX and decreased H3S10p after irradiation. Additionally, an analysis of 38 human patient samples obtained from 8 different tumor types showed variable tumor HDAC activity, thus demonstrating inter-tumoral epigenetic heterogeneity in a patient population. Conclusion: The study revealed that an imbalance of HAT and HDAC activities led to the loss of site-specific histone acetylation and chromatin compaction as breast cancer cells acquired radio-resistance. Due to variation in the tumor HDAC activity among patients, our report suggests performing a prior assessment of the tumor epigenome to maximize the benefit of HDAC inhibitor-based radio-sensitization.

Oral premalignant lesions (OPLs) such as leukoplakia, erythroplakia, and oral submucous fibrosis, often precede oral cancer. Screening and management of these premalignant conditions can improve prognosis. Raman spectroscopy has previously demonstrated potential in the diagnosis of oral premalignant conditions (in vivo), detected viral infection, and identified cancer in both oral and cervical exfoliated cells (ex vivo). The potential of Raman exfoliative cytology (REC) in identifying premalignant conditions was investigated. Oral exfoliated samples were collected from healthy volunteers (n=20), healthy volunteers with tobacco habits (n=20), and oral premalignant conditions (n=27, OPL) using Cytobrush. Spectra were acquired using Raman microprobe. Spectral acquisition parameters were: λex: 785 nm, laser power: 40 mW, acquisition time: 15 s, and average: 3. Postspectral acquisition, cell pellet was subjected to Pap staining. Multivariate analysis was carried out using principal component analysis and principal component-linear discriminant analysis using both spectra- and patient-wise approaches in three- and two-group models. OPLs could be identified with ∼77% (spectra-wise) and ∼70% (patient-wise) sensitivity in the three-group model while with 86% (spectra-wise) and 83% (patient-wise) in the two-group model. Use of histopathologically confirmed premalignant cases and better sampling devices may help in development of improved standard models and also enhance the sensitivity of the method. Future longitudinal studies can help validate potential of REC in screening and monitoring high-risk populations and prognosis prediction of premalignant lesions.

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