Asawari Patil

Mutational landscape of gingivo-buccal oral squamous cell carcinoma reveals new recurrently-mutated genes and molecular subgroups

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.

Quantitative Immunohistochemical Analysis Reveals Association between Sodium Iodide Symporter and Estrogen Receptor Expression in Breast Cancer

Background Human sodium iodide symporter (hNIS) gene over-expression is under active consideration worldwide as an alternative target molecule for breast cancer (BC) diagnosis and targeted radio-iodine treatment. However, the field demands better stratified analysis of endogenous hNIS expression across major BC subtypes. Therefore, we have analyzed subtype-specific variation of hNIS overexpression in breast tumor tissue samples by immunohistochemistry (IHC) and also report the development of a homogeneous, quantitative analysis method of digital IHC images. Methods hNIS expression was analyzed from 108 BC tissue samples by IHC. Sub-cellular localization of hNIS protein was analyzed by dual immunofluorescence (IF) staining method using hNIS and HER2 antibodies. An ImageJ based two-step digital analysis method was developed and applied for the bias-free analysis of the images. Results Staining of the tumor samples show 70% cases are hNIS positive indicating high incidence of hNIS positive cases in BC. More importantly, a subtype specific analysis done for the first time shows that hNIS expression is overly dominated in estrogen receptor (ER) positive cases than the receptor negative cases. Further, 56% of the ER+ve, PgR+ve, HER2-ve and 36% of ER+ve, PgR+ve, HER2+ve cases show highest intensity staining equivalent to the thyroid tissue. A significant positive correlation is also observed between hNIS and estrogen receptor expression (p = 0.0033, CI = 95%) suggesting hNIS mediated targeted radio-iodine therapy procedures may benefit both ER+ve, PgR+ve, HER2–ve as well as HER2+ve cases. Further, in a few cases, hNIS and HER2 protein localization is demonstrated by overlapping membrane co-expression. ImageJ based image analysis method shows over 70% match with manual pathological scoring method. Conclusion The study indicates a positive link between hNIS and ER expression in BC. The quantitative IHC image analysis method reported here will further help in patient stratification and potentially benefit global clinical assessment where hNIS mediated targeted 131I radio-ablative therapy is aimed.

Estrogen, progesterone and HER2 receptor expression in breast tumors of patients, and their usage of HER2-targeted therapy, in a tertiary care centre in India

BACKGROUND This study was undertaken to document the pattern of expression of estrogen (ER), progesterone (PR) and human epidermal growth factor receptor-2 (HER2) and the usage of HER2-targeted therapy in a large tertiary care hospital in India in the year 2008. MATERIALS AND METHODS The histopathology reports of all breast cancer patients registered in the hospital in 2008 were extracted from the electronic medical record system. All the cases were immunohistochemically evaluated for estrogen and progesterone receptor status (ER and PR), and c-erbB-2 protein (HER2) expression using standard immunoperoxidase method. The use of HER2-targeted therapies was evaluated by extracting relevant information from the database of the hospital pharmacy and case charts of patients enrolled in ongoing approved trials. RESULTS A total of 2001 new patients of invasive breast cancers with available pathology reports were registered in the hospital in the year 2008. ER and/or PR expression was positive in tumors of 1025 (51.2%) patients. HER2 3+ expression by immunohistochemistry (IHC) was found in 335 (16.7%) and HER2 2+ in 163 (8.1%). The triple negative phenotype was found in 596 (29.8%) patients. An estimated 441 patients were eligible to receive HER2-targeted therapy based on their HER2 status. Of these 38 (8.6%) patients received some form of HER2-targeted therapy; 20 patients (4.5%) as part of ongoing clinical trials and 18 (4.1%) as part of routine care. CONCLUSIONS The overwhelming majority of patients eligible for HER2-targeted therapy in our institution are unable to receive it because of financial constraints and limited access to health insurance. There is a higher fraction of patients with the triple negative phenotype compared to the Western population.

Genome-wide expression and copy number analysis identifies driver genes in gingivobuccal cancers.

The molecular mechanisms contributing to the development and progression of gingivobuccal complex (GBC) cancers—a sub‐site of oral cancer, comprising the buccal mucosa, the gingivobuccal sulcus, the lower gingival region, and the retromolar trigone–remain poorly understood. Identifying the GBC cancer‐related gene expression signature and the driver genes residing on the altered chromosomal regions is critical for understanding the molecular basis of its pathogenesis. Genome‐wide expression profiling of 27 GBC cancers with known chromosomal alterations was performed to reveal differentially expressed genes. Putative driver genes were identified by integrating copy number and gene expression data. A total of 315 genes were found differentially expressed (P ≤ 0.05, logFC > 2.0) of which 11 genes were validated by real‐time quantitative reverse transcriptase‐PCR (qRT‐PCR) in tumors (n = 57) and normal GBC tissues (n = 18). Overexpression of LY6K, in chromosome band 8q24.3, was validated by immunohistochemical (IHC) analysis. We found that 78.5% (2,417/3,079) of the genes located in regions of recurrent chromosomal alterations show copy number dependent expression indicating that copy number alteration has a direct effect on global gene expression. The integrative analysis revealed BIRC3 in 11q22.2 as a candidate driver gene associated with poor clinical outcome. Our study identified previously unreported differentially expressed genes in a homogeneous subtype of oral cancer and the candidate driver genes that may contribute to the development and progression of the disease.

Neoadjuvant chemotherapy followed by surgery in very locally advanced technically unresectable oral cavity cancers

BACKGROUND The median survival of technically unresectable oral-cavity cancers (T4a and T4b) with non surgical therapy is 2-12 months. We hypothesized that neoadjuvant chemotherapy (NACT) could reduce the tumour size and result in successful resection and ultimately improved outcomes. We present a retrospective analysis of consecutive patients who received NACT at our centre between January 2008 and August 2012. PATIENTS AND METHODS All patients with technically unresectable oral cancers were assessed in a multidisciplinary clinic and received 2 cycles of NACT. After 2 cycles, patients were reassessed and planned for either surgery with subsequent CTRT or nonsurgical therapy including CT-RT, RT or palliation. SPSS version 16 was used for analysis of locoregional control and overall survival (OS). Univariate and multivariate analysis was done for factors affecting the OS. RESULTS 721 patients with stage IV oral-cavity cancer received NACT. 310 patients (43%) had sufficient reduction in tumour size and underwent surgical resection. Of the remaining patients, 167 received chemoradiation, 3 radical radiation and 241 palliative treatment alone The locoregional control rate at 24 months was 20.6% for the overall cohort, 32% in patients undergoing surgery and 15% in patients undergoing non surgical treatment (p=0.0001). The median estimated OS in patients undergoing surgery was 19.6 months (95% CI, 9.59-25.21 months) and 8.16 months (95%, CI 7.57-8.76) in patients treated with non surgical treatment (p=0.0001). CONCLUSION In our analysis, NACT led to successful resection and improved overall survival in a significant proportion of technically unresectable oral-cancer patients.

Primary intraosseous myoepithelioma arising in the iliac bone and displaying trisomies of 11, 15, 17 with del (16q) and del (22q11)—A rare case report with review of literature

Mixed tumors are uncommonly observed in the musculoskeletal system, where they form a common spectrum with a myoepithelioma and a parachordoma. Herein, we present a rare case of a mixed tumor/myoepithelioma arising in the iliac bone of a young adult male who presented with swelling in his right hip. Radiological imaging disclosed a large, intraosseous, lytic, heterogenous mass with a soft tissue component. Biopsy and subsequent tumor resection showed an 18cm sized tumor involving the iliac bone and soft tissues and comprising polygonal and spindly cells, arranged in cords and aggregates, embedded in a myxohyaline stroma with osteochondroid differentiation. Tumor cells exhibited mild nuclear variation, rare mitotic figures, focal cytoplasmic clearing, and prominent squamous differentiation. On immunohistochemistry (IHC), tumor cells were diffusely positive for S100-P, EMA, CK5/6, p63, GFAP, calponin, and focally positive for CK/MNF116, but negative for Brachyury/T. Diagnosis of a myoepithelioma/mixed tumor was offered. Further, cytogenetic analysis revealed lack of EWSR1 gene rearrangement and showed clonal trisomies of 11, 15, 17 with del (16q) and del (22q11). The present case is a rare documentation of a myoepithelioma in the appendicular bones and the second such case identified in the iliac bone. IHC and cytogenetic findings supported a myoepithelial cell origin, and reinforced its relationship with a parachordoma and its distinction from mixed salivary gland tumors, a chordoma, and an extraskeletal myxoid chondrosarcoma that form its differential diagnoses.

Gross examination by the surgeon as an alternative to frozen section for assessment of adequacy of surgical margin in head and neck squamous cell carcinoma

The cost‐effectiveness of the frozen section for assessment of margin in head and neck squamous cell carcinoma (HNSCC) is still contentious. The purpose of this study was to evaluate whether gross examination of margin is an alternative to frozen section.

Keratins in oral cancer: necessity of mass spectrometry for validation of antibody based identifications.

Keratins are intermediate filament family proteins which are predominantly expressed in the epithelial cells. Most of the studies which evaluate the status of keratins in clinical samples of the oral cavity are based on the identification of their presence and localization by immunohistochemistry using monoclonal antibodies. It is very well known that many monoclonal/polyclonal antibodies show cross-reactivity with the other closely related or non-related proteins. This cross-reactivity might be the result of epitope similarity, but it is not always necessary. Therefore studies done with only antibody based techniques can mislead interpretation unless they are validated with additional techniques like mass-spectrometry. In this investigation we have evaluated the status of keratin 18 in cancer of buccal mucosa using 1DE, 2DE and western blotting with monoclonal antibody to keratin 18. The patterns emerging showed aberrant as well as differential expression of K18 in adjacent normal versus tumor tissue samples of buccal mucosa. Mass spectrometry analysis of the immunodetected spots however revealed that it is keratin 13. Thus this study emphasizes the necessity of validation of antibody based findings when dealing with proteins of a large family having similarity/homology in amino acid sequence.

Downregulation of keratin 76 expression during oral carcinogenesis of human, hamster and mouse

Background Keratins are structural marker proteins with tissue specific expression; however, recent reports indicate their involvement in cancer progression. Previous study from our lab revealed deregulation of many genes related to structural molecular integrity including KRT76. Here we evaluate the role of KRT76 downregulation in oral precancer and cancer development. Methods We evaluated KRT76 expression by qRT-PCR in normal and tumor tissues of the oral cavity. We also analyzed K76 expression by immunohistochemistry in normal, oral precancerous lesion (OPL), oral squamous cell carcinoma (OSCC) and in hamster model of oral carcinogenesis. Further, functional implication of KRT76 loss was confirmed using KRT76-knockout (KO) mice. Results We observed a strong association of reduced K76 expression with increased risk of OPL and OSCC development. The buccal epithelium of DMBA treated hamsters showed a similar trend. Oral cavity of KRT76-KO mice showed preneoplastic changes in the gingivobuccal epithelium while no pathological changes were observed in KRT76 negative tissues such as tongue. Conclusion The present study demonstrates loss of KRT76 in oral carcinogenesis. The KRT76-KO mice data underlines the potential of KRT76 being an early event although this loss is not sufficient to drive the development of oral cancers. Thus, future studies to investigate the contributing role of KRT76 in light of other tumor driving events are warranted.

Proteomic profile of keratins in cancer of the gingivo buccal complex: consolidating insights for clinical applications.

UNLABELLED Keratins play a major role in several cellular functions. Each tissue type expresses a specific set of keratins. The immense potential of keratins as diagnostic and prognostic markers for different cancers is emerging. Oral cancer is the fifteenth most common cancer worldwide. However, comprehensive information on the profile of keratins in the oral cavity is not available. Several independent reports have identified keratins using antibody based techniques which have pitfalls due to the cross reactivity of the antibodies to this set of very homologous proteins. A few recent proteomic studies have reported the identification of keratins in head and neck cancer. Majority of the studies have used tissues from the head and neck region without specifying subsites. This study reports the analysis of enriched preparations of keratins from cancer of the gingivo buccal complex (GBC) using MS, 2DE, WB, silver staining of 2DE gels and IHC. Our study reveals the absence of K4 and K13 and presence of K14, K16, and K17, in cancers of the GBC and combination of these expression patterns in the cut margins. This report also shows that K13 is glycosylated. This well characterized profile of keratins may have potential to be used in clinics. BIOLOGICAL SIGNIFICANCE In recent years the immense potential of keratins as diagnostic and prognostic markers for different cancers is emerging. However, comprehensive information on the profile of keratins in the oral cavity is not available. Several independent reports have identified keratins using only antibody based techniques which have pitfalls due to the cross reactivity of the antibodies to this set of very homologous proteins. This study reports the analysis of enriched preparations of keratins from a subsite of the oral cavity, the gingivo buccal complex (GBC) using mass spectrometry, 2DE, western blotting, silver staining of 2DE gels and IHC. The proteomic analysis shows the absence of K4 and K13 and presence of K14, K16, and K17 in cancers of the GBC and combination of these expression patterns in the cut margins. This well characterized profile of keratins from the gingivo buccal complex provides defined markers which may have potential to be used in the clinics.