Dysregulated gene subnetworks in breast invasive carcinoma reveal novel tumor suppressor genes.

Breast invasive carcinoma (BRCA) is the most malignant and leading cause of death in women. Global efforts are ongoing for improvement in early detection, prevention, and treatment. In this milieu, a comprehensive analysis of RNA-sequencing data of 1097 BRCA samples and 114 normal adjacent tissues is done to identify dysregulated genes in major molecular classes of BRCA in various clinical stages. Significantly enriched pathways in distinct molecular classes of BRCA have been identified. Pathways such as interferon signaling, tryptophan degradation, granulocyte adhesion & diapedesis, and catecholamine biosynthesis were found to be significantly enriched in Estrogen/Progesterone Receptor positive/Human Epidermal Growth Factor Receptor 2 negative, pathways such as RAR activation, adipogenesis, the role of JAK1/2 in interferon signaling, TGF-ß and STAT3 signaling intricated in Estrogen/Progesterone Receptor negative/Human Epidermal Growth Factor Receptor 2 positive and pathways as IL-1/IL-8, TNFR1/TNFR2, TWEAK, and relaxin signaling were found in triple-negative breast cancer. The dysregulated genes were clustered based on their mutation frequency which revealed nine mutated clusters, some of which were well characterized in cancer while others were less characterized. Each cluster was analyzed in detail which led to the identification of NLGN3, MAML2, TTN, SYNE1, ANK2 as candidate genes in BRCA. They are central hubs in the protein-protein-interaction network, indicating their important regulatory roles. Experimentally, the Real-Time Quantitative Reverse Transcription PCR and western blot confirmed our computational predictions in cell lines. Further, immunohistochemistry corroborated the results in ~ 100 tissue samples. We could experimentally show that the NLGN3 & ANK2 have tumor-suppressor roles in BRCA as shown by cell viability assay, transwell migration, colony forming and wound healing assay. The cell viability and migration was found to be significantly reduced in MCF7 and MDA-MB-231 cell lines in which the selected genes were over-expressed as compared to control cell lines. The wound healing assay also demonstrated a significant decrease in wound closure at 12 h and 24 h time intervals in MCF7 & MDA-MB-231 cells. These findings established the tumor suppressor roles of NLGN3 & ANK2 in BRCA. This will have important ramifications for the therapeutics discovery against BRCA.

CK7+/CK20+/CDX2+ mucinous carcinoma, arising in a mature cystic teratoma of ovary, presenting with disseminated metastasis and pseudomyxoma peritonei.

ABSTRACT: Mucinous carcinomas arising within an ovarian dermoid tumor are rare. Most of the cases reported in the literature show morphological features resembling an appendiceal mucinous neoplasm. They exhibit CK7-/CK20+ immunophenotype similar to carcinomas of the lower gastrointestinal tract. In this report, we have described a case of a well-differentiated mucinous carcinoma arising within a mature cystic teratoma. The mucinous carcinoma showed a spectrum of morphological patterns, including cystadenoma-like areas, proliferative/borderline areas, villous adenoma-like areas, and areas of invasive carcinoma. In addition, our case showed some unusual findings, namely, diffuse CK7 positivity, associated pseudomyxoma peritonei, and metastasis to lungs. These features were not demonstrated in any of the previously reported cases. Our case shows that the teratomatous mucinous neoplasm of the ovary may show CK7+/CK20+/CDX2 + immunoprofile making it immunohistochemically indistinguishable from a primary ovarian mucinous neoplasm or a metastatic mucinous carcinoma of the lower gastrointestinal tract.

Diminution of Primary Cilia in the Stromal Cells at the Tumor-stromal Interface Correlates With an Aggressive Tumor Biology in the Urothelial Carcinoma of the Urinary Bladder.

BACKGROUND AND AIMS: Primary cilia (PC) are cellular organelles that regulate the cellular homeostasis. They are the seats of many oncogenic pathways and indirectly regulate the epithelial-mesenchymal transition (EMT) and extracellular matrix, both critical for the tumor microenvironment (TME). Though there are a few studies highlighting the alteration of PC in the tumor cells of various malignancies, none depict the PC in the stromal cells in the urothelial carcinoma of the urinary bladder (UC), the stromal cells being an essential component of TME. Therefore, we intend to evaluate the PC in the stromal cells at the tumor-stromal interface in UC. METHODS: Immunohistochemistry for acetylated-α-tubulin (for PC), Ki67, E-cadherin, and SNAI1 was performed in 141 cases of UC and 5 normal controls, and primary cilium: nucleus (C:N) ratio was counted in the stromal cells at the tumor-stromal interface. The C:N ratio was correlated with various clinical and histopathological parameters. RESULTS: The C:N ratio showed significant diminution from normal control (mean=0.75) to low-grade UC (mean=0.24) ( P =0.001) to high-grade UC (mean value=0.17) ( P =0.001). There was a significant diminution of the C:N ratio from the noninvasive to invasive UC ( P =0.025). The C:N ratio did not show any correlation with EMT although negatively correlated with the Ki67 index ( r =-0.32; P =0.001), and a higher ratio showed a trend with a higher recurrence-free survival ( P =0.07). CONCLUSIONS: The diminution of the PC in the stromal cells at the tumor-stromal interface is an early event and correlates with an aggressive tumor biology of UC.

Expression of EZH2 and H3K27me3 predicts tumor biology of urothelial carcinoma.

Background: Enhancer of zeste homolog 2 (EZH2) is one of the major epigenetic modifiers involved in the transcriptional repression of target genes through trimethylation of H3K27 (lysine 27 residue of histone H3). Deregulated expression of both EZH2 and H3K27me3 has been implicated in the biological behavior and prognostic outcome of various malignancies. Aim: To assess the role of EZH2 and H3K27me3 in the carcinogenesis of urothelial carcinoma of urinary bladder. Materials and Methods: One hundred fifty consecutive urothelial carcinoma cases of urinary bladder (54.7% high-grade) were included in this study. Immunohistochemical analysis for EZH2 and H3K27me3 was performed on whole tissue sections. A multiplication score obtained by multiplying staining intensity and proportion of positively stained neoplastic cells was used for assessment. Results: EZH2 showed a significant correlation with the tumor grade and lamina propria invasion (p < 0.001). The cases with high EZH2 expression showed a significantly high proliferative index (Mean- 32.7%; p < 0.001). In contrast, negative and low expression of H3K27me3 was significantly more common in high-grade cases (p = 0.006). The expression of H3K27me3 was significantly associated with lamina propria (p = 0.01) and deep muscle invasion (p = 0.007). EZH2 showed a significantly higher expression in the high-grade invasive areas as compared to the high-grade non-invasive areas of the same tumor (p = 0.03). Conclusions: This study establishes an important role of the key epigenetic regulators EZH2 and H3K27me3 in the pathobiology of urothelial carcinomas. Strong expression of EZH2 and weak expression of H3K27me3 are associated with higher grade, proliferative index and invasive behavior.

The rhabdoid variant of adrenocortical carcinoma-Report of three cases and literature review.

Adrenocortical carcinoma (ACC) is a rare and aggressive malignancy. Extensive rhabdoid morphology in ACC has been described recently in very few cases. The proportion of rhabdoid morphology and the role of SMARCB1/ INI1 expression in these tumor cells to diagnose the specific variant is not described in the literature. We reviewed the clinicopathological features of nine cases of adrenocortical neoplasm. Out of which, three cases of ACC showed predominant rhabdoid morphology. Large discohesive cells with abundant cytoplasm containing eosinophilic inclusions, eccentric vesicular nucleus, and prominent nucleoli. INI1 immunostain was retained in all cases. We reported the rhabdoid variant of ACC, a novel entity, and its diagnostic approach from their histological mimickers. Identifying more cases of this entity will help to clearly understand the pathogenesis, biologic behaviour, and any specific molecular alterations in the future.

Margin to Depth of Invasion Ratio: A Significant Predictor of Survival in Patients With Oral Cancer.

PURPOSE: A common definition of a clear margin (≥5 mm) in oral squamous cell carcinoma (OSCC) for all stages is a subject of controversy. Studies have shown that even 1- and 2-mm margins are adequate, and few studies have identified dynamic resection margin as a criterion. We aimed to study the margin to depth of invasion ratio (MDR), margin to tumor thickness ratio (MTR), and margin to tumor size ratio (MSR) as prognostic markers for survival. Notably, to our knowledge, this is the first study to evaluate the role of MDR in OSCC. METHODS: A prospectively maintained head and neck cancer database was analyzed from January 2017 to February 2023. The MDR, MTR, and MSR were calculated for each patient. Survival outcomes were analyzed using the Cox proportional model and the Kaplan-Meier method. Akaike's information criterion (AIC) and Bayesian information criterion (BIC) were used to compare different ratio models. X-tiles software was used to identify the optimal cutoff value of MDR. RESULTS: Two hundred eighty patients in the database were assessed, of which 123 eligible patients were enrolled in the study. MDR was an independent predictor of disease-free survival (DFS) on multivariate analysis. The MDR model had the lowest values on AIC and BIC analyses. A cutoff value of 0.5 for MDR showed a significant correlation with DFS and overall survival. CONCLUSION: MDR was the best predictor of recurrence of all the three ratios studied. The minimum safe surgical margin can be calculated by multiplying the depth of invasion by 0.5. This study signifies the role of dynamic resection margin criteria on the basis of MDR in defining clear margins.

Carotid Body Tumors: Institutional Experience of 10 Cases and a Review of Literature.

Carotid body tumors are slow growing neck masses that arise from the neural crest cells at the carotid bifurcation. Majority are asymptomatic and are diagnosed incidentally. Surgical excision is accepted as the treatment of choice to reduce complications. In the present series, we report 10 cases of carotid body tumors and our institutional experience. All patients underwent radiological evaluation with an ultrasonography with Doppler, contrast enhanced computed tomography and MR angiography. 6 cases were operated by a transcervical excision. The tumor was excised in tototranscervically. One of the cases required saphenous vein graft intraoperatively due to vascular injury and also had postoperative vocal cord palsy. The rest had an uneventful recovery. Carotid body tumors although rare and seemingly indolent can cause substantial symptoms if left untreated. A prompt multi modality approach is needed for both diagnosis and treatment to avoid major complications.

Metastatic Renal Cell Carcinoma Masquerading as a Laryngeal Tumor: A Case Report.

Renal cell carcinoma (RCC) commonly metastasizes to various organs such as the lungs, liver, bones, and brain. However, isolated metastases to the head and neck region, especially the larynx, are very rare. This report presents a case of laryngeal growth that was eventually confirmed to be a metastatic deposit from an undiagnosed RCC. We report a case of a 66-year-old male who presented to the clinic with painless neck swelling and a change in voice. The scan showed a soft tissue mass in the thyroid cartilage. Histopathology of the resected laryngeal tumor confirmed metastatic clear cell carcinoma. A metastatic workup revealed a renal mass, and the patient underwent laparoscopic adrenal-sparing left cytoreductive nephrectomy. The histopathological examination established the diagnosis of clear cell RCC. Subsequently, the patient was treated with pembrolizumab and lenvatinib. Follow-up imaging showed no residual or recurrent lesions. This case highlights the rarity of laryngeal metastasis from RCC and the importance of an accurate diagnosis through advanced imaging and histopathological examination.

G-protein-coupled receptor 141 mediates breast cancer proliferation and metastasis by regulating oncogenic mediators and the p-mTOR/p53 axis.

Breast cancer morbidity is surging towards the peak in females across the globe. An inherent property of cancer cells is enhanced cell proliferation rate and migration capability, leading to deregulated cell signaling cascades. G-protein-coupled receptors (GPCRs) have recently emerged as a hot-spot target in cancer research. We identify aberrant expression of G-protein-coupled receptor 141 (GPR141) in different breast cancer subtypes that correlate with poor prognosis. However, the molecular mechanism via which GPR141 advances breast cancer remains elusive. Increased GPR141 expression enhances the migratory behavior of breast cancer, driving oncogenic pathways both in vitro and in vivo through activation of epithelial to mesenchymal transition (EMT), oncogenic mediators and regulation of p-mTOR/p53 signaling. Our study unveils a molecular mechanism for p53 downregulation and activation of p-mTOR1 and its substrates in GPR141 overexpressed cells, accelerating breast tumorigenesis. We find that an E3 ubiquitin ligase, Cullin1, partly mediates p53 degradation via proteasomal pathway. Co-immunoprecipitation result shows that the phosphorylated form of 40S ribosome protein S6 (ps6., a p-mTOR1 substrate) forms a complex with Cullin1. These findings suggest an interplay between Cullin1 and p-mTOR1 in GPR141 overexpressed cells that downregulates p53 expression, thus inducing tumor growth. GPR141 silencing restores p53 expression and attenuates p-mTOR1 signaling events, thereby impeding proliferation and migration in breast cancer cells. Our findings describe the role of GPR141 in breast cancer proliferation, and metastasis, as well as in influencing the tumor microenvironment. Modulating GPR141 expression could pave the way for a better therapeutic approach to regulating breast cancer progression and metastasis.

An Incidentally Detected Case of Atypical Bronchial Carcinoid on Stress Myocardial Perfusion SPECT/CT.

Myocardial perfusion imaging is primarily done to look for stress-induced perfusion defects in patients suspected of having coronary artery disease. However, the rotating raw images can provide significant information on the surrounding structures. The lungs lie near the heart, and any abnormality showing increased uptake can be seen on myocardial perfusion imaging. We report the case of a 52-y-old man with a history of diabetes for the previous 5 y, who presented to the cardiology outpatient department because of occasional chest pain and dyspnea. Electrocardiography and echocardiography showed no significant abnormality. The patient was referred to the nuclear medicine department for stress-induced myocardial perfusion scintigraphy. The raw 99mTc-sestamibi images showed abnormal uptake in the hilar region of the right lung, which, on subsequent investigations, was diagnosed as atypical bronchial carcinoid tumor.

Metronomic Therapy in Palliation of Oral Cancer Patients-A Home Based Approach at the End of Life.

INTRODUCTION: Despite the development of targeted therapies for the management of oral cancer patients, the cost of treatment is a concern in middle- and low-income countries. The present study assessed the feasibility of low-cost metronomic therapy as an alternative treatment modality in patients with unresectable or inoperable oral cancers. METHODOLOGY: The study was a prospective, single-arm study. Unresectable, inoperable, and metastatic lip and oral cavity cancers were started on metronomic therapy, a combination of oral methotrexate 15 mg/m2 once a week and oral celecoxib 200 mg twice daily, as palliative therapy. The primary endpoint was overall survival. The secondary endpoints were a response to metronomic therapy, compliance, and toxicity. RESULTS: From June 2018 to May 2020, 25 patients were started on metronomic therapy. The median age was 60 years. The median overall survival was 8.8 months. At eight weeks of therapy, 11 patients (44%) had a partial response, ten patients had stable disease (40%), and four patients had progressive disease (16%). The compliance with the therapy was 100%, and one patient (4%) developed grade III toxicity. CONCLUSIONS: Considering the resource constraints and cost limitations in low and middle-income countries, oral metronomic therapy in the form of methotrexate and celecoxib should be regarded as a suitable regimen in the palliative treatment of patients with unresectable, metastatic, or advanced, recurrent cancers.

Malignant gastrointestinal neuroectodermal tumor: A case-based review of literature.

Malignant gastrointestinal (GI) neuroectodermal tumor is an extremely rare entity that was first described by Zambrano et al. in 2003 as "clear cell sarcoma (CCS)-like tumor of the GI tract." It shares some of the histopathological features of CCS but lacks the immunohistochemical (IHC) reactivity for melanocytic markers. Most mesenchymal neoplasms of the GI tract belong to the category of GI stromal tumors and are characterized by the IHC expression of c-KIT. In cases, without detectable KIT receptor expression, several differential diagnoses have to be taken into consideration. In this article, we describe such a case and present a review of all the reported cases till date. We also present the current available knowledge on its pathology and molecular genetics along with the limitations in its diagnosis. Here, we report a case of a 32-year-old man with a tumor of the small bowel composed of polygonal tumor cells arranged in solid nests, alveolar pattern, and pseudopapillary and admixed with numerous osteoclast-like multinucleated giant cells. Immunohistochemically, the tumor cells strongly expressed S-100 protein only. HMB-45, melan-A, CD117, cytokeratin, desmin, smooth muscle actin, and CD-34 were absent. Ki-67 index was 15%. The diagnosis was further confirmed by fluorescence in situ hybridization (FISH) demonstrating the presence of EWSR1 (22q12) translocation. A final diagnosis of malignant gastroneuroectodermal tumor was rendered. The patient is disease-free for 20 months of postsurgery. The diagnosis of this entity should be considered in the presence of S-100-positivity and multinucleated osteoclastic giant cells and the absence of melanocytic differentiation in a tumor arising from GI tract. Further confirmation can be done by performing FISH analysis.

Second Primary Spindle Cell Carcinoma of the Tongue: A Rare Histology.

Spindle cell carcinoma (SpCC) is a rare variant of poorly differentiated squamous cell carcinoma (SCC), characterised by the presence of both squamous (carcinomatous) and spindle cell (sarcomatous) elements. Early detection and improvement in treatment for oral SCC lead to prolonged survival, thereby increasing the frequency of second primary tumours (SPTs) in the oral cavity. In this paper, we report a case of SpCC of the tongue in a 62-year-old male with a history of SCC; the right lateral border of his tongue status post-treatment completion four years ago, now presented with a polypoidal growth over the tip of his tongue for four months. An immunohistochemical study revealed features suggestive of SpCC (spindle cell pattern of cells, expression of vimentin, immunopositivity for cytokeratin (membranous), and focally positive for p40 (nuclear)). To the best of our knowledge, this is the first reported case of a spindle cell variant of SCC presenting as a second primary in an oral cancer survivor patient.

Expression of mismatch repair proteins in urothelial carcinoma of the urinary bladder.

Background: Urothelial carcinoma of the urinary bladder (UCUB) is a frequently diagnosed malignancy. Mismatch repair (MMR), a proofreading machinery of the DNA, prevents tumorigenesis. The role of MMR deficiency in UCUB in eastern Indian population is not known. Methods: Immunohistochemistry panel for MLH1, PMS2, MSH2, and MSH6 (MMR proteins) was performed on the biopsy specimens of UCUB (N=100). Results: MMR deficiency by immunohistochemistry was demonstrated in two cases (2%). One case showed deficiency of MSH2 and MSH6 and the other case showed the deficiency of all four mismatch proteins. Both cases showed high-grade invasive urothelial carcinoma by histomorphology. Conclusion: The prevalence of MMR deficiency by immunohistochemistry is 2% in eastern Indian population.

Application of the Indian Academy of Cytologists Recommendations for Reporting Serous Fluid Cytopathology in Routine Reporting of Ascitic Fluid Specimen and Assessment of the Risk of Malignancy.

Background: A five-tiered reporting system for effusion fluid cytology has been published by the Indian Academy of Cytologists (IAC). Only a single study has evaluated the applicability of this system in routine reporting. Aims: We intend to evaluate the practical utility of this system in routine reporting of ascitic fluid cytology. Materials And Methods: Nine hundred and sixty-one cases of ascitic fluid cytology were included in this study. The clinical, radiological, cytomorphological, and follow-up data of these cases were reviewed. All cases were recategorized according to the proposed IAC system, and the risk of malignancy (ROM) for each category was estimated. Results: Age of the patients ranged from 1 to 92 years, and fluid volume ranged from 10 ml to 3 l. The number of cases included in each category and their respective ROM were as follows: category 1: 41, 21.42%; category 2: 805, 14.9%; category 3: 5, 33.3%; category 4: 31, 90%; and category 5:79, 96.4%. Conclusions: The new IAC guidelines for the serous fluid is representative, informative, and could be easily applied at our institutional level. We used the recommended diagnostic categories for reclassifying the ascitic fluid samples based on their cytosmear findings and conclude that the system has enormous utility at each level starting from the collection of fluid samples till the delivery of the report.