Germline Co-deletion of CDKN2A and CDKN2B Genes in Pleomorphic Xanthoastrocytoma: Case Report.

BACKGROUND/AIM: Gliomas are highly heterogeneous malignancies originating from diverse cell types within the brain. Although their precise etiology is frequently unknown, risk factors, such as chemical exposure, radiation, and specific uncommon genetic disorders have been identified. Diagnosis typically entails imaging tests, such as magnetic resonance imaging and computed tomography, complemented by a biopsy for confirmation, which may be further validated through genetic testing. CASE REPORT: Next-generation sequencing technology revealed germline co-deletion deletion of cyclin-dependent kinase inhibitor 2 A and B genes (CDKN2A and CDKN2B) in a patient diagnosed with pleomorphic xanthoastrocytoma based on the tumor's molecular characteristics. Following this result, we performed focused genetic analysis with use of multiplex ligation-dependent probe amplification technology for the mother that revealed the same co-deletion. Moreover, due to the father's neuroendocrine pancreatic cancer, application of the NGS technology detected a pathogenic variant in the BRCA1-interacting helicase 1 (BRIP1) gene. Comprehensive multi-gene testing conducted within the familial context, marked by a varied spectrum of cancer type, revealed a constellation of genetic predispositions. CONCLUSION: This case study underscores the critical importance of molecular testing for tumor characterization and highlights the pivotal role of genetic testing in facilitating early intervention and screening for at-risk family members. Furthermore, the identification of germline co-deletions in cancer lays the foundation for the development of targeted therapeutic strategies aimed at restoring normal cellular regulation and improving patient management.

Microsatellite Instability Is Insufficiently Used as a Biomarker for Lynch Syndrome Testing in Clinical Practice.

PURPOSE: The pan-cancer presence of microsatellite instability (MSI)-positive tumors demonstrates its clinical utility as an agnostic biomarker for identifying immunotherapy-eligible patients. Additionally, MSI is a hallmark of Lynch syndrome (LS), the most prevalent cancer susceptibility syndrome among patients with colorectal and endometrial cancer. Therefore, MSI-high results should inform germline genetic testing for cancer-predisposing genes. However, in clinical practice, such analysis is frequently disregarded. METHODS: A next-generation sequencing (NGS)-based technique was used for MSI analysis in 4,553 patients with various tumor types. Upon request, somatic BRAF gene analysis was conducted. In addition, hereditary testing of cancer-associated genes was performed in MSI-high cases using a capture-based NGS protocol. MLH1 promoter methylation analysis was conducted retrospectively in patients with colorectal and endometrial cancer to further investigate the origin of MSI at the tumor level. RESULTS: The MSI positivity rate for the entire cohort was 5.27%. Endometrial, gastric, colorectal, urinary tract, and prostate cancers showed the highest proportion of MSI-high cases (15.69%, 8.54%, 7.40%, 4.55%, and 3.19%, respectively). A minority of 45 patients (22.73%) among the MSI-high cases underwent germline testing to determine whether the mismatch repair pathway deficiency was inherited. 24.44% of those who performed the genetic test carried a pathogenic variant in an LS-associated gene. Three MSI-high individuals had non-LS gene alterations, including BRCA1, BRCA2, and CDKN2A pathogenic variants, indicating the presence of non-LS-associated gene alterations among MSI-high patients. CONCLUSION: Although MSI analysis is routinely performed in clinical practice, as many as 77% of MSI-high patients do not undergo LS genetic testing, despite international guidelines strongly recommending it. BRAF and MLH1 methylation analysis could shed light on the somatic origin of MSI in 42.50% of the MSI-high patients; however, MLH1 analysis is barely ever requested in clinical practice.

Only 32.3% of Breast Cancer Families with Pathogenic Variants in Cancer Genes Utilized Cascade Genetic Testing.

BACKGROUND: Hereditary cancer predisposition syndromes are responsible for approximately 5-10% of all diagnosed cancer cases. In order to identify individuals at risk in a cost-efficient manner, family members of individuals carrying pathogenic alterations are tested only for the specific variant that was identified in their carrier relative. The purpose of this study was to investigate the clinical use and implementation of cascade family testing (CFT) in families of breast cancer patients with pathogenic/likely pathogenic variants (PVs/LPVs) in cancer-related predisposition genes. METHODS: Germline sequencing was carried out with NGS technology using a 52-gene panel, and cascade testing was performed by Sanger sequencing or MLPA. RESULTS: In a cohort of 1785 breast cancer patients (families), 20.3% were found to have PVs/LPVs. Specifically, 52.2%, 25.1%, and 22.7% of patients had positive findings in high-, intermediate-, and low-penetrance breast cancer susceptibility genes, respectively. Although CFT was recommended to all families, only 117 families (32.3%) agreed to proceed with genetic testing. Among the first-degree relatives who underwent CFT, 70.3% were female, and 108 of 121 (89.3%) were cancer free. Additionally, 42.7%, 36.7%, and 20.6% were offspring, siblings, and parents of the subject, respectively. Our data suggest that CFT was mostly undertaken (104/117, 88.8%) in families with positive findings in high-risk genes. CONCLUSIONS: Cascade family testing can be a powerful tool for primary cancer prevention by identifying at-risk family members. It is of utmost importance to implement genetic counseling approaches leading to increased awareness and communication of genetic testing results.

Copy Number Variations (CNVs) Account for 10.8% of Pathogenic Variants in Patients Referred for Hereditary Cancer Testing.

BACKGROUND/AIM: Germline copy number variation (CNV) is a type of genetic variant that predisposes significantly to inherited cancers. Today, next-generation sequencing (NGS) technologies have contributed to multi gene panel analysis in clinical practice. MATERIALS AND METHODS: A total of 2,163 patients were screened for cancer susceptibility, using a solution-based capture method. A panel of 52 genes was used for targeted NGS. The capture-based approach enables computational analysis of CNVs from NGS data. We studied the performance of the CNV module of the commercial software suite SeqPilot (JSI Medical Systems) and of the non-commercial tool panelcn.MOPS. Additionally, we tested the performance of digital multiplex ligation-dependent probe amplification (digitalMLPA). RESULTS: Pathogenic/likely pathogenic variants (P/LP) were identified in 464 samples (21.5%). CNV accounts for 10.8% (50/464) of pathogenic variants, referring to deletion/duplication of one or more exons of a gene. In patients with breast and ovarian cancer, CNVs accounted for 10.2% and 6.8% of pathogenic variants, respectively. In colorectal cancer patients, CNV accounted for 28.6% of pathogenic/likely pathogenic variants. CONCLUSION: In silico CNV detection tools provide a viable and cost-effective method to identify CNVs from NGS experiments. CNVs constitute a substantial percentage of P/LP variants, since they represent up to one of every ten P/LP findings identified by NGS multigene analysis; therefore, their evaluation is highly recommended to improve the diagnostic yield of hereditary cancer analysis.

Autoimmune pancreatitis associated with retroperitoneal fibrosis mimicking cholangiocarcinoma.

Autoimmune pancreatitis (AIP) is a rare systematic autoimmune disease that causes chronic pancreatitis. Type 1-AIP (IgG4-related disease) may involve other organs as well. In this report we are presenting a case of a 74-year-old man with obstructive abdominal pain jaundice, mild and a history of retroperitoneal fibrosis and hydronephrosis. Labs were remarkable for hyperbilirubinemia, high serum IgG4 levels, mildly elevated CA 19-9, elevated rheumatoid factor and new onset diabetes. MRI revealed pancreatic enlargement, dilated intrahepatic bile ducts and stricture of the distal common bile duct concerning for cholangiocarcinoma. EUS-FNA biopsy was negative for malignancy but showed findings of pancreatitis. The diagnosis of type 1-AIP was made and the patient was treated with steroids. After one month of treatment jaundice and MRI findings resolved. It is important to include AIP in the differential diagnosis of pancreatic conditions causing obstructive jaundice, especially in the presence of other autoimmune conditions like retroperitoneal fibrosis.

Uveal Melanoma: GNAQ and GNA11 Mutations in a Greek Population.

BACKGROUND/AIM: Uveal melanoma is the most common primary adult intraocular malignancy. It is known to have a strong metastatic potential, fatal for the vast majority of patients. In recent years, meticulous cytogenetic and molecular profiling has led to precise prognostication, that unfortunately is not matched by advancements in adjuvant therapies. G Protein subunits alpha Q (GNAQ) and alpha 11 (GNA11) are two of the major driver genes that contribute to the development of uveal melanoma. Understanding their prognostic significance can allow tailored management and facilitate their use in the on-going quest of targeted uveal melanoma therapies. MATERIALS AND METHODS: Formalin-fixed, paraffin-embedded specimens were obtained from 47 patients of Greek origin, with uveal melanoma. GNAQ and GNA11 genes were screened for mutations in exons 4 and 5, by polymerase chain reaction and Sanger sequencing. RESULTS: The overall mutation frequency of GNAQ/GNA11 genes was 42.4%. A novel mutation c.625_626delinsGC was identified in GNA11. No correlation was observed between the mutation status and metastasis occurrence or overall survival time of patients. CONCLUSION: Mutations in GNAQ and GNA11 genes in this Greek population present frequencies that qualify them as potential targets for customized therapy.

Tumor heterogeneity revealed by KRAS, BRAF, and PIK3CA pyrosequencing: KRAS and PIK3CA intratumor mutation profile differences and their therapeutic implications.

Current clinical problems in colorectal cancer (CRC) diagnostics and therapeutics include the disease complexity, tumor heterogeneity, and resistance to targeted therapeutics. In the present study, we examined 171 CRC adenocarcinomas from Greek patients undergoing surgery for CRC to determine the frequency of KRAS, BRAF, and PIK3CA point mutations from different areas of tumors in heterogeneous specimens. Ninety two out of 171 (53.8%) patients were found to bear a KRAS mutation in codons 12/13. Of the 126 mutations found, 57.9% (73/126) were c.38G>A mutations (p.G13D) and 22.2% (28/126) were c.35G>T (p.G12V). Remarkably, RAS mutations in both codons 12 and 13 were recorded in the same tumor by pyrosequencing. Moreover, differences in KRAS mutations between tumor center and periphery revealed tumor heterogeneity in 50.7% of the specimens. BRAF c.1799T>A (V600E) mutations were moderately detected in 4/171 (2.3%) specimens, whereas most PIK3CA mutations were revealed by pyrosequencing 6/171 (3.5%). Remarkable tumor heterogeneity is revealed, where double mutations of KRAS in the same tumor and different KRAS mutation status between tumor core and margin are detected with high frequency. It is expected that these findings will have a major impact in cancer diagnosis and personalized therapies.

Multiplex PCR-based detection of circulating tumor cells in lung cancer patients using CK19, PTHrP, and LUNX specific primers.

INTRODUCTION: The aim of this study was to develop a multiplex polymerase chain reaction (PCR)-based method for detection of circulating tumor cells in peripheral blood of lung cancer (LC) patients. PATIENTS AND METHODS: Peripheral blood was collected from 71 healthy donors and 125 LC patients at different pathological stages. Samples were analyzed using multiplex PCR, and specific primers for CK19, PTHrP, and LUNX mRNA. The sensitivity of our method was set at 10 LC cells (A549 cells) in 3 mL of peripheral blood of healthy donors using spiking experiments. RESULTS: The detection rates in LC patients for CK19, PTHrP, and LUNX were 45.6%, 64.8%, and 28%, and in healthy individuals were 7%, 7%, and 5.6%, respectively. Overall, our method produced 77.8% positive detections for at least 1 molecular marker. Twenty-eight (22.2%) were negative for expression of all markers, 39 (31.2%) were positive for expression of 1 marker, 42 (33.6%) were positive for expression of 2 markers, and 17 (13.6%) were positive for expression of all 3 markers. Detection of CK19 mRNA expression positively correlated with LC stage and distant metastases. PTHrP mRNA detection correlated positively with LC stage, presence of bone metastasis, and squamous cell carcinoma, and LUNX mRNA detection correlated with lymph node involvement. Combined detection of 2 or 3 markers was significantly correlated with metastatic disease, and negative detection of all 3 molecular markers was correlated with early stage nonmetastatic disease. CONCLUSION: Multiple PCR-based detection of CK19, PTHrP, and LUNX mRNA expression provides useful information for disease stage and dissemination in LC patients.

Laparoscopic adrenalectomy in patients with subclinical Cushing syndrome.

BACKGROUND: Subclinical Cushing syndrome in patients with adrenal incidentalomas has been associated with an increased prevalence of the metabolic syndrome and cardiovascular risk. The management of these patients, be it conservative or surgical, is still debated, but there is accumulating evidence that surgery is best and that laparoscopic adrenalectomy, when possible, is the most preferred procedure. Here we present the short- and long-term results of laparoscopic adrenalectomy for subclinical Cushing syndrome and determine the effect of this procedure on components of the metabolic syndrome. METHODS: Twenty-nine patients, 8 men and 21 women with adrenal incidentalomas and subclinical Cushing syndrome who underwent laparoscopic adrenalectomy, were studied retrospectively. They had undergone postoperative follow-up for improvement or worsening of their arterial blood pressure, body weight, and fasting glucose level for a mean period of 77 months. RESULTS: Preoperatively, 17 patients (58.6 %) had arterial hypertension, 14 (48.3 %) had a body mass index exceeding 27 kg/m(2), and 12 (41.4 %) had diabetes mellitus. Postoperatively, a decrease in mean arterial pressure was found in 12 patients (70.6 %), a decrease in body mass index in 6 patients (42.9 %), and an improvement in glycemic control in 5 patients (41.7 %). CONCLUSIONS: Laparoscopic adrenalectomy is beneficial in many patients with subclinical Cushing syndrome because it reduces arterial blood pressure, body weight, and fasting glucose levels. Prospective randomized studies are needed to compare laparoscopic adrenalectomy with a conservative approach and to confirm these results.

Pancreatic leak related hemorrhage following pancreaticoduodenectomy. A case series.

CONTEXT: Delayed arterial hemorrhage, secondary to pancreaticojejunal leakage, is an infrequent complication (2-4%) of pancreaticoduodenectomy but it carries a high mortality rate with more than half of the patients dying from overwhelming sepsis and/or bleeding. Its ideal management remains unclear. CASE REPORTS: We hereby present our experience with respect to the presentation and management of this severe post-pancreaticoduodenectomy complication which occurred in 3/149 patients (2.1%) operated on between 1996 and 2008 in our department and we review the role of endoscopy, interventional radiology and surgery in its management. CONCLUSIONS: The severity of the underlying sepsis and the prompt identification of the sentinel bleed determine surgical and angiographic intervention and define the outcome in the treatment of a pancreatic leak-related hemorrhage. Endoscopy has no role in this setting.

TRAIL receptor upregulation and the implication of KRAS/BRAF mutations in human colon cancer tumors.

TRAIL raises hopes as a promising anti-tumor agent due to its selectivity toward cancer cells. Higher expression of its pro-death receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5) attenuates higher sensitivity to TRAIL-induced apoptosis, and represents a marker for better cancer prognosis and treatment. Since receptor availability can be analogous to ligand efficacy, we performed RT-PCR analysis of DR4 and DR5 in 51 colon cancer biopsy specimens and respective normal mucosa, while 11 of these tumors were determined immunohistochemically for protein expression. Transcriptional analysis showed that DR4 and DR5 were significantly upregulated in 37 and 47% of the tumor samples respectively, while both DR4 and DR5 were coinstantaneously upregulated in 31% of the samples analyzed. Positive transcriptional regulation of DRs was recorded as early as Dukes' A stage. Furthermore, protein expression analysis yielded results comparable to DR4 and DR5 increased mRNA levels. Possible contributing events to DR upregulation involve presence of frequent oncogenic mutations in the MAPK pathway, and was investigated by direct sequencing in all 51 tumors. Samples (6/8) hosting either a KRAS(G12V) or BRAF(V600E) mutation, significantly amplified the upregulated expression of DR4 and DR5, showing strong inter-relation between overexpression and presence of oncogenic KRAS/ BRAF mutations. In the light of recent data concerning TRAIL receptor distribution, we contribute further by presenting DR5 as the most frequently upregulated DR in colon cancer. Furthermore, oncogenic mutations may directly or indirectly enhance DR expression, potentially sensitizing these tumors to TRAIL-based therapies.