Identification of Rare EIF3E::RSPO2 Fusion in Recurrent and Aggressive Urachal Adenocarcinoma.

INTRODUCTION: Urachal cancer (UC) is a rare genitourinary malignancy arising from the urachus, an embryonic remnant of the placental allantois. Its diagnosis remains ambiguous with late-stage cancer detection and represents a highly aggressive disease. Due to its rarity, there is no clear consensus on molecular signatures and appropriate clinical management of UC. CASE REPORT: We report a 45-year-old man with recurrent urachal adenocarcinoma (UA) treated with cystectomies, chemotherapy, and radiotherapy. The patient initially presented with hematuria and abdominal pain. Imaging revealed a nodular mass arising from the superior wall of the urinary bladder and extending to the urachus. Biopsy results suggested moderately differentiated UA with muscle layer involvement. The tumor recurred after 20 months, following which, another partial cystectomy was performed. Repeat progression was noted indicating highly aggressive disease. Targeted next-generation sequencing revealed the presence of EIF3E::RSPO2 fusion, along with BRAF and TP53 mutations, and EGFR gene amplification. This is the first case reporting the presence of this fusion in UA. Palliative medication and radiotherapy were administered to manage the disease. CONCLUSION: Current treatment modality of surgery may be effective in the early stages of recurrent UA; however, a standard chemotherapy and radiotherapy regimen is yet to be determined for advanced stages. The detection of the rare EIF3E::RSPO2 fusion warrants further studies on the significance of this variant as a possible therapeutic target for improved clinical management.

EGFR analysis on scrapings from cytology smears in lung carcinoma, an effective alternative to testing on trucut biopsies.

BACKGROUND: Lung carcinomas are a leading cause of cancer morbidity and mortality. Many cases present at an advanced stage of disease where definitive treatment by surgical resection is not feasible. Molecular testing using materials derived from minimally invasive procedures aid in targeted therapy with least iatrogenic burden to the patient. METHODS: Cases diagnosed as non-small cell lung carcinoma (NSCLC) on cytology were included in the study. Scrapings from the smears with adequate tumor cell load were submitted for molecular testing. The DNA was extracted and quantified. Mutations in exons 18, 19, 20, and 21 of the EGFR gene were detected using Sanger sequencing. DNA quantity and EGFR mutation status on equal number of consecutive trucut biopsy specimens were also analyzed. RESULTS: Seventy cases of NSCLC tested for EGFR mutation had a median DNA concentration of 40.2 ng/µl and 31% cases showed mutation. Majority of mutations (14/21, 66.66%) were identified in exon 19. Among 70 trucut biopsy samples, DNA concentration was 41.42 ng/µl and 30% cases showed mutation. No significant difference was seen in DNA quantity and EGFR mutation between cytology smears and trucut biopsies. CONCLUSION: EGFR testing on cytology smears provides adequate DNA yield with minimal invasiveness and is equally effective as biopsies. Testing on samples like pleural effusion allows for concomitant diagnosis, staging, and molecular testing in one procedure. Tests done on the smears rather than on cell block or trucut biopsies ensures superior quality DNA from the tumor cells as they are unexposed to cross linking formalin fixative.

Somatic mutation detection efficiency in EGFR: a comparison between high resolution melting analysis and Sanger sequencing.

BACKGROUND: High resolution melting curve analysis is a cost-effective rapid screening method for detection of somatic gene mutation. The performance characteristics of this technique has been explored previously, however, analytical parameters such as limit of detection of mutant allele fraction and total concentration of DNA, have not been addressed. The current study focuses on comparing the mutation detection efficiency of High-Resolution Melt Analysis (HRM) with Sanger Sequencing in somatic mutations of the EGFR gene in non-small cell lung cancer. METHODS: The minor allele fraction of somatic mutations was titrated against total DNA concentration using Sanger sequencing and HRM to determine the limit of detection. The mutant and wildtype allele fractions were validated by multiplex allele-specific real-time PCR. Somatic mutation detection efficiency, for exons 19 & 21 of the EGFR gene, was compared in 116 formalin fixed paraffin embedded tumor tissues, after screening 275 tumor tissues by Sanger sequencing. RESULTS: The limit of detection of minor allele fraction of exon 19 mutation was 1% with sequencing, and 0.25% with HRM, whereas for exon 21 mutation, 0.25% MAF was detected using both methods. Multiplex allele-specific real-time PCR revealed that the wildtype DNA did not impede the amplification of mutant allele in mixed DNA assays. All mutation positive samples detected by Sanger sequencing, were also detected by HRM. About 28% cases in exon 19 and 40% in exon 21, detected as mutated in HRM, were not detected by sequencing. Overall, sensitivity and specificity of HRM were found to be 100 and 67% respectively, and the negative predictive value was 100%, while positive predictive value was 80%. CONCLUSION: The comparative series study suggests that HRM is a modest initial screening test for somatic mutation detection of EGFR, which must further be confirmed by Sanger sequencing. With the modification of annealing temperature of initial PCR, the limit of detection of Sanger sequencing can be improved.

Cerebrospinal Fluid Hyaluronan and Neurofibromatosis Type 2.

Neurofibromatosis type 2 (NF-2) is associated with mainly three types of recurrent benign tumors restricted to the central nervous system: schwannoma, meningioma and ependymoma. The absence of the protein NF2/Merlin causes an uninterrupted cell proliferation cascade originating from an abnormal interaction between an extracellular mucopolysaccharide, hyaluronan (HA), and schwann cell surface CD44 receptor, which has been identified as one of the central causative factors for schwannoma. Most tumors in NF-2 have a predilection to originate from either arachnoid cap cells or schwann cells of the cisternal portion of nerve rootlets that share a continuous exposure to cerebrospinal fluid (CSF). We hypothesize that the CSF HA may play a role in tumorigenesis in NF-2. In a prospective analysis over a period of one year, the levels of medium to low molecular weight HA (LMW HA) was estimated in the CSF of three subjects with central schwannomas and compared against that of age-sex matched controls, using Cetyltrimethylammonium bromide coupled turbidimetric assay and found to be seventeen-fold higher in the schwannoma subjects compared to the controls. HA was observed to be actively secreted by cultured schwannoma cells isolated from tumor tissues commensurate with their proliferation rate. On cell viability index analysis to compare the cell proliferation of astrocytoma cells with LMW HA vs. oligomeric HA (OHA), we found a decrease in cell proliferation of up to 30% with OHA. The study provides initial evidence that CSF HA may have a central role in the tumorigenesis of schwannoma in NF-2.

Metabolism and mechanisms of action of hyaluronan in human biology.

Hyaluronan is a ubiquitous high-molecular weight polymer of repeated disaccharides of glucuronic acid and N-acetylglucosamine. It is a membrane-bound, viscous material extruded into the extracellular matrix after being synthesized in the cytoplasm by hyaluronan synthases complex and a regulated degradation by a group of enzymes called hyaluronidases. Hyaluronan has varied biological roles on many vital organismal functions, such as cellular and tissue development, migration and repair after injury or inflammation and cancer genesis. Hyaluronan in the tissue microenvironment is regulated by its concentration as well as the chain length of the polysaccharide. Many functions of hyaluronan are mediated by specific receptors at the cellular level, though its general physiochemical properties facilitate and coordinate many organ functions as well as in development. These fundamental characteristics of hyaluronan are reviewed, focusing on human biological context.

Genotype-phenotype analysis of von Hippel-Lindau syndrome in fifteen Indian families.

The general prevalence of the familial multi-organ tumor disorder, von Hippel-Lindau syndrome (VHL), was estimated to be 1 in 25-40,000 in western studies two decades back. Few studies were done in Indian sub-continent, amidst a surge in clinical reports on VHL specific manifestations. The syndrome is correlated with mutations of the gene VHL (located in Chr 3p25.3). We aimed to conduct a prospective case series describing phenotypic and genotypic characteristics in Indian population. The VHL-specific clinical and radiological features were collected from patients and family members. Genotypic changes such as deletion/duplication or point mutation in the VHL locus were identified using sequencing and MLPA. Thirty-one subjects, from fifteen families with diagnosed VHL, were included in the study. Multicystic pancreas was found in 71% (22/31), CNS hemangioblastoma in 68% (21/31), renal cell carcinoma and retinal angiomas in 23% (7/31) each, pheochromocytoma in 9.7% (3/31) of the population and endolymphatic sac tumor in one subject. Four families (9 subjects) had full length deletion of VHL, three families (4 subjects) had a deletion of exon 3, eight families (18 subjects) had different exonic, splice-site and intronic point mutations and one subject had a de novo in-frame indel in exon 1. Multicystic pancreas and CNS hemangioblastomas were the most common manifestations in our population. The phenotypic expression patterns in terms of tumorigenesis, tissue tropism and penetrance in comparison to the genotypic features were found to be different from previous correlative studies.