KRAS and MAPK1 Gene Amplification in Type II Ovarian Carcinomas

In this study, we examined the clinical significance of KRAS and MAPK1 amplification and assessed whether these amplified genes were potential therapeutic targets in type II ovarian carcinoma. Using fluorescence in situ hybridization, immunohistochemistry, and retrospectively collected clinical data, KRAS and MAPK1 amplifications were identified in 9 (13.2%) and 5 (7.4%) of 68 type II ovarian carcinoma tissue samples, respectively. Interestingly, co-amplification of KRAS and MAPK1 seemed to be absent in the type II ovarian carcinomas tested, except one case. Active phospho-ERK1/2 was identified in 26 (38.2%) out of 68 type II ovarian carcinomas and did not correlate with KRAS or MAPK1 amplification. There was no significant relationship between KRAS amplification and overall or progression-free survival in patients with type II ovarian carcinoma. However, patients with MAPK1 amplification had significantly poorer progression-free survival than patients without MAPK1 amplification. Moreover, type II ovarian carcinoma cells with concomitant KRAS amplification and mutation exhibited dramatic growth reduction following treatment with the MEK inhibitor PD0325901. These findings indicate that KRAS/MAPK1 amplification is critical for the growth of a subset of type II ovarian carcinomas. Additionally, RAS/RAF/MEK/ERK pathway-targeted therapy may benefit selected patients with type II ovarian carcinoma harboring KRAS/MAPK1 amplifications.     

The G-Protein-Coupled Estrogen Receptor (GPER/GPR30) in Ovarian Granulosa Cell Tumors

Ovarian granulosa cell tumors (GCTs) are thought to arise from cells of the ovarian follicle and comprise a rare entity of ovarian masses. We recently identified the G-protein-coupled estrogen receptor (GPER/GPR30) to be present in granulosa cells, to be regulated by gonadotropins in epithelial ovarian cancer and to be differentially expressed throughout folliculogenesis. Thus, supposing a possible role of GPER in GCTs, this study aimed to analyze GPER in GCTs. GPER immunoreactivity in GCTs (n = 26; n (primary diagnosis) = 15, n (recurrence) = 11) was studied and correlated with the main clinicopathological variables. Positive GPER staining was identified in 53.8% (14/26) of GCTs and there was no significant relation of GPER with tumor size or lymph node status. Those cases presenting with strong GPER intensity at primary diagnosis showed a significant reduced overall survival (p = 0.002). Due to the fact that GPER is regulated by estrogens, as well as gonadotropins, GPER may also be affected by endocrine therapies applied to GCT patients. Moreover, with our data supposing GPER to be associated with GCT prognosis, GPER might be considered as a possible confounder when assessing the efficacy of hormone-based therapeutic approaches in GCTs.     

High Concordance of Genomic Profiles between Primary and Metastatic Colorectal Cancer

The comparison of the genetic profiles between primary and metastatic colorectal cancer (CRC) is needed to enable the discovery of useful therapeutic targets against metastatic CRCs. We performed the targeted next generation sequencing assay of 170 cancer-associated genes for 142 metastatic CRCs, including 95 pairs of primary and metastatic CRCs, to reveal their genomic characteristics and to assess the genetic heterogeneity. The most frequently mutated gene in primary and metastatic CRCs was APC (71% vs. 65%), TP53 (54% vs. 57%), KRAS (45% vs. 44%), PIK3CA (16% vs. 19%), SMAD4 (15% vs. 14%) and FBXW7 (11% vs. 11%). The concordance in the top six frequently mutated genes was 85%, on average. The overall mutation frequencies were consistent with two sets of public data (TCGA and MSKCC). To the author’s knowledge, this is the first study to compare the genetic profiles of our cohort with that of the metastatic CRCs from MSKCC. Comparative sequencing analysis between primary and metastatic CRCs revealed a high degree of genetic concordance in the current clinically actionable genes. Therefore, the genetic investigation of archived primary tumor samples with the challenges of obtaining an adequate sample from metastatic sites appears to be sufficient for the application of cancer precision medicine in the metastatic setting.     

If Virchow and Ehrlich Had Dreamt Together: What the Future Holds for KRAS-Mutant Lung Cancer

Non-small-cell lung cancer (NSCLC) with Kirsten rat sarcoma (KRAS) mutations has notoriously challenged oncologists and researchers for three notable reasons: (1) the historical assumption that KRAS is “undruggable”, (2) the disease heterogeneity and (3) the shaping of the tumor microenvironment by KRAS downstream effector functions. Better insights into KRAS structural biochemistry allowed researchers to develop direct KRAS(G12C) inhibitors, which have shown early signs of clinical activity in NSCLC patients and have recently led to an FDA breakthrough designation for AMG-510. Following the approval of immune checkpoint inhibitors for PDL1-positive NSCLC, this could fuel yet another major paradigm shift in the treatment of advanced lung cancer. Here, we review advances in our understanding of the biology of direct KRAS inhibition and project future opportunities and challenges of dual KRAS and immune checkpoint inhibition. This strategy is supported by preclinical models which show that KRAS(G12C) inhibitors can turn some immunologically “cold” tumors into “hot” ones and therefore could benefit patients whose tumors harbor subtype-defining STK11/LKB1 co-mutations. Forty years after the discovery of KRAS as a transforming oncogene, we are on the verge of approval of the first KRAS-targeted drug combinations, thus therapeutically unifying Paul Ehrlich’s century-old “magic bullet” vision with Rudolf Virchow’s cancer inflammation theory.     

Sanger Sequencing for BRCA1 c.68_69del,BRCA1 c.5266dup and BRCA2 c.5946del Mutation Screen on Pap Smear Cytology Samples

Three sets of polymerase chain reaction (PCR) primers were designed for heminested PCR amplification of the target DNA fragments in the human genome which include the site of BRCA1 c.68_69del, BRCA1 c.5266dup and BRCA2 c.5946del respectively, to prepare the templates for direct Sanger sequencing screen of these three founder mutations. With a robust PCR mixture, crude proteinase K digestate of the fixed cervicovaginal cells in the liquid-based Papanicolaou (Pap) cytology specimens can be used as the sample for target DNA amplification without pre-PCR DNA extraction, purification and quantitation. The post-PCR products can be used directly as the sequencing templates without further purification or quantitation. By simplifying the frontend procedures for template preparation, the cost for screening these three founder mutations can be reduced to about US $200 per test when performed in conjunction with human papillomavirus (HPV) assays now routinely ordered for cervical cancer prevention. With this projected price structure, selective patients in a high-risk population can be tested and each provided with a set of DNA sequencing electropherograms to document the absence or presence of these founder mutations in her genome to help assess inherited susceptibility to breast and ovarian cancer in this era of precision molecular personalized medicine.     

Pro-Inflammatory Microenvironment Modulates the Transfer of Mutated TP53 Mediated by Tumor Exosomes

Exosomes released from tumor cells are instrumental in shaping the local tumor microenvironment to allow cancer progression. Recently, it has been shown that tumor exosomes carry large fragments of dsDNA, which may reflect the mutational status of parental cells. Although it has been described that a stressful microenvironment can influence exosomal cargo, the effects on DNA packing and its transfer into recipient cells have yet to be investigated. Here, we report that exosomes derived from SW480 (human colorectal adenocarcinoma cell line) cells can carry dsDNA fragments containing the entire coding sequence of both TP53 and KRAS genes, harboring the SW480-related TP53 c.818G > A and KRAS c.35G > T typical mutations. We also report the following: that cell stimulation with lipopolysaccharides (LPS) promotes the selective packaging of the TP53 gene, but not the KRAS gene; that exosomes secreted by SW480 cells efficiently transfer the mutated sequences into normal CCD841-CoN colon epithelial and THLE-2 hepatic cells; that this mechanism is more efficient when the cells had been previously incubated with pro-inflammatory cytokines; that the TP53 gene appears actively transcribed in both recipient cells; and that mutated mRNA levels are not influenced by cytokine treatment. Our data strongly suggest that pro-inflammatory stimulation promotes the horizontal transfer of an oncogene by exosomes, although this remains a rare event. Further studies are needed to assess the impact of the oncogenic transfer by exosomes in malignant transformation and its role in tumor progression.     

BTG1 Expression Correlates with the Pathogenesis and Progression of Ovarian Carcinomas

BTG (B-cell translocation gene) can inhibit cell proliferation, metastasis, and angiogenesis and regulate cell cycle progression and differentiation in a variety of cell types. We aimed to clarify the role of BTG1 in ovarian carcinogenesis and progression. A BTG1-expressing plasmid was transfected into ovarian carcinoma cells and their phenotypes and related proteins were examined. BTG1 mRNA expression was detected in ovarian normal tissue (n = 17), ovarian benign tumors (n = 12), and ovarian carcinoma (n = 64) using real-time RT-PCR. Ectopic BTG1 expression resulted in lower growth rate, high cisplatin sensitivity, G₁ arrest, apoptosis, and decreased migration and invasion. Phosphoinositide 3-kinase, protein kinase B, Bcl-xL, survivin, vascular endothelial growth factor, and matrix metalloproteinase-2 mRNA and protein expression was reduced in transfectants as compared to control cells. There was higher expression of BTG1 mRNA in normal tissue than in carcinoma tissue (p = 0.001) and in benign tumors than in carcinoma tissue (p = 0.027). BTG1 mRNA expression in International Federation of Gynecology and Obstetrics (FIGO) stage I/II ovarian carcinomas was higher than that in FIGO stage III/IV ovarian carcinomas (p = 0.038). Altered BTG1 expression might play a role in the pathogenesis and progression of ovarian carcinoma by modulating proliferation, migration, invasion, the cell cycle, and apoptosis.     

The Involvement of RhoA and Wnt-5a in the Tumorigenesis and Progression of Ovarian Epithelial Carcinoma

Background

Ras homolog gene family member A (RhoA) is involved in Wnt-5a–induced migration of gastric and breast cancer cells. We investigated the roles of RhoA and Wnt-5a in ovarian carcinoma.

Methods

RhoA and Wnt-5a mRNA and protein expression in normal fallopian tube epithelium, benign tumors, primary ovarian carcinomas, and metastatic omentum were quantified. RhoA or Wnt-5a was knocked down in OVCAR3 ovarian carcinoma cells using siRNAs and cell phenotype and expression of relevant molecules were assayed.

Results

RhoA and Wnt-5a mRNA and protein expression were found to be significantly higher in metastatic omentum than in ovarian carcinomas, benign tumors, and normal fallopian tube epithelium (p < 0.05), and positively associated with differentiation and FIGO staging (stage I/II vs. stage III/IV) in ovarian carcinoma (p < 0.05). RhoA and Wnt-5a expression were positively correlated in ovarian carcinoma (p = 0.001, R² = 0.1669). RhoA or Wnt-5a knockdown downregulated RhoA and Wnt-5a expression; reduced cell proliferation; promoted G₁ arrest and apoptosis; suppressed lamellipodia formation, cell migration, and invasion; and reduced PI3K, Akt, p70S6k, Bcl-xL, survivin, and VEGF mRNA or protein expression.

Conclusions

This is the first demonstration that RhoA and Wnt-5a are associated with ovarian carcinogenesis and apoptosis inhibition; there might be positive correlation between RhoA and Wnt-5a expression. RhoA is a potential tumorigenesis, differentiation, and progression biomarker in ovarian carcinoma.     

Amplification Target ADRM1: Role as an Oncogene and Therapeutic Target for Ovarian Cancer

Approximately 25,000 ovarian cancers are diagnosed in the U.S. annually, and 75% are in the advanced stage and largely incurable. There is critical need for early detection tools and novel treatments. Proteasomal ubiquitin receptor ADRM1 is a protein that is encoded by the ADRM1 gene. Recently, we showed that among 20q13-amplified genes in ovarian cancer, ADRM1 overexpression was the most highly correlated with amplification and was significantly upregulated with respect to stage, recurrence, and metastasis. Its overexpression correlated significantly with shorter time to recurrence and overall survival. Array-CGH and microarray expression of ovarian cancer cell lines provided evidence consistent with primary tumor data that ADRM1 is a 20q13 amplification target. Herein, we confirm the ADRM1 amplicon in a second ovarian cancer cohort and define a minimally amplified region of 262 KB encompassing seven genes. Additionally, using RNAi knock-down of ADRM1 in naturally amplified cell line OAW42 and overexpression of ADRM1 via transfection in ES2, we show that (1) ADRM1 overexpression increases proliferation, migration, and growth in soft agar, and (2) knock-down of ADRM1 results in apoptosis. Proteomic analysis of cells with ADRM1 knock-down reveals dysregulation of proteins including CDK-activating kinase assembly factor MAT1. Taken together, the results indicate that amplified ADRM1 is involved in cell proliferation, migration and survival in ovarian cancer cells, supporting a role as an oncogene and novel therapeutic target for ovarian cancer.     

2021 Update on Diagnostic Markers and Translocation in Salivary Gland Tumors

Salivary gland tumors are a rare tumor entity within malignant tumors of all tissues. The most common are malignant mucoepidermoid carcinoma, adenoid cystic carcinoma, and acinic cell carcinoma. Pleomorphic adenoma is the most recurrent form of benign salivary gland tumor. Due to their low incidence rates and complex histological patterns, they are difficult to diagnose accurately. Malignant tumors of the salivary glands are challenging in terms of differentiation because of their variability in histochemistry and translocations. Therefore, the primary goal of the study was to review the current literature to identify the recent developments in histochemical diagnostics and translocations for differentiating salivary gland tumors.     

The Changes of Angiogenesis and Immune Cell Infiltration in the Intra- and Peri-Tumoral Melanoma Microenvironment

Malignant melanoma (MM) urgently needs identification of new markers with better predictive value than currently-used clinical and histological parameters. Cancer cells stimulate the formation of a specialized tumor microenvironment, which reciprocally affects uncontrolled proliferation and migration. However, this microenvironment is heterogeneous with different sub-compartments defined by their access to oxygen and nutrients. This study evaluated microvascular density (MVD), CD3⁺ lymphocytes (TILs) and FOXP3⁺ T-regulatory lymphocytes (Tregs) on formalin-fixed paraffin-embedded tissue sections using light microscopy. We analyzed 82 malignant melanomas, divided according to the AJCC TNM classification into four groups—pT1 (35), pT2 (17), pT3 (18) and pT4 (12)—and 25 benign pigmented nevi. All parameters were measured in both the central areas of tumors (C) and at their periphery (P). A marked increase in all parameters was found in melanomas compared to nevi (p = 0.0001). There was a positive correlation between MVD, TILs, FOXP3⁺ Tregs and the vertical growth phase. The results show that MVD, TILs and FOXP3⁺ Tregs substantially influence cutaneous melanoma microenvironment. We found significant topographic differences of the parameters between central areas of tumors and their boundaries.     

Study on the Expression and Clinical Significances of Lewis y Antigen and Integrin αv, β3 in Epithelial Ovarian Tumors

Objective

To detect the expression and clinical significances of Lewis y antigen and integrin αv, β3 in epithelial ovarian tumors, and to explore the expression correlation between Lewis y antigen and integrin αv, β3.

Methods

Immunohistochemical staining was performed in 95 cases of epithelial ovarian cancer, 37 cases of borderline tumors, 20 cases of benign tumors, and 20 cases of normal ovarian tissue, for the detection of Lewis y antigen and integrin αv, β3 expressions, and to analyze the relationship between Lewis y antigen and integrin, and the relationship between clinical and pathological parameters of ovarian cancer. In addition, immunofluorescence double labeling was utilized to detect the expression correlation between Lewis y antigen and integrin αv, β3 in ovarian cancer.

Results

In epithelial ovarian tumors, the expression rate of Lewis y antigen was 81.05%, significantly higher than that of borderline (51.53%) (P < 0.05) and benign (25%) (P < 0.01) tumors, and normal ovarian tissues (0) (P < 0.01). The expression rate of integrin αv, β3 in malignant epithelial ovarian tumors was 78.95% and 82.11%, respectively, significantly higher than that of the borderline (45.94%, 40.54%) (both P < 0.05), benign group (10.00%, 15.00%) (both P < 0.01) and normal ovary group (5%, 15%) (both P < 0.01).

Conclusions

Lewis y and integrins αv, β3 are relevant to pelvic and abdominal diffusion and metastasis of ovarian cancer cells, suggesting that these two molecules mediate a boosting function for tumor metastasis.     

Prognostic and Predictive Roles of KRAS Mutation in Colorectal Cancer

The RAS gene family is among the most studied and best characterized of the known cancer-related genes. Of the three human ras isoforms, KRAS is the most frequently altered gene, with mutations occurring in 17%–25% of all cancers. In particular, approximately 30%–40% of colon cancers harbor a KRAS mutation. KRAS mutations in colon cancers have been associated with poorer survival and increased tumor aggressiveness. Additionally, KRAS mutations in colorectal cancer lead to resistance to select treatment strategies. In this review we examine the history of KRAS, its prognostic value in patients with colorectal cancer, and evidence supporting its predictive value in determining appropriate therapies for patients with colorectal cancer.     

KRAS Promoter G-Quadruplexes from Sequences of Different Length: A Physicochemical Study

DNA G-quadruplexes (G4s) form in relevant genomic regions and intervene in several biological processes, including the modulation of oncogenes expression, and are potential anticancer drug targets. The human KRAS proto-oncogene promoter region contains guanine-rich sequences able to fold into G4 structures. Here, by using circular dichroism and differential scanning calorimetry as complementary physicochemical methodologies, we compared the thermodynamic stability of the G4s formed by a shorter and a longer version of the KRAS promoter sequence, namely 5′-AGGGCGGTGTGGGAATAGGGAA-3′ (KRAS 22RT) and 5′-AGGGCGGTGTGGGAAGAGGGAAGAGGGGGAGG-3′ (KRAS 32R). Our results show that the unfolding mechanism of KRAS 32R is more complex than that of KRAS 22RT. The different thermodynamic stability is discussed based on the recently determined NMR structures. The binding properties of TMPyP4 and BRACO-19, two well-known G4-targeting anticancer compounds, to the KRAS G4s were also investigated. The present physicochemical study aims to help in choosing the best G4 target for potential anticancer drugs.     

Claudins Overexpression in Ovarian Cancer: Potential Targets for Clostridium Perfringens Enterotoxin (CPE) Based Diagnosis and Therapy

Claudins are a family of tight junction proteins regulating paracellular permeability and cell polarity with different patterns of expression in benign and malignant human tissues. There are approximately 27 members of the claudin family identified to date with varying cell and tissue-specific expression. Claudins-3, -4 and -7 represent the most highly differentially expressed claudins in ovarian cancer. While their exact role in ovarian tumors is still being elucidated, these proteins are thought to be critical for ovarian cancer cell invasion/dissemination and resistance to chemotherapy. Claudin-3 and claudin-4 are the natural receptors for the Clostridium perfringens enterotoxin (CPE), a potent cytolytic toxin. These surface proteins may therefore represent attractive targets for the detection and treatment of chemotherapy-resistant ovarian cancer and other aggressive solid tumors overexpressing claudin-3 and -4 using CPE-based theranostic agents.     

Differentiated Epithelial- and Mesenchymal-Like Phenotypes in Subcutaneous Mouse Xenografts Using Diffusion Weighted-Magnetic Resonance Imaging

Epithelial-mesenchymal transition (EMT) is important for tumor metastasis. Detection of EMT protein expression and observation of morphological changes are commonly used to identify EMT. Diffusion-weighted magnetic resonance imaging (DW-MRI) and measuring apparent diffusion coefficient (ADC) values are noninvasive techniques for characterizing tumor microenvironments. We investigated the difference in ADC values between epithelial- and mesenchymal-like subcutaneous mouse xenografted tumors using DW-MRI. Epithelial-like MM189 PB-Klf4 and BL322 PB-Klf4 cells were generated from tumor suppressive Kruppel-like factor 4 (Klf4)-expressing mesenchymal-like MM189 and BL322 cells. The ADC values of xenografted tumors from epithelial-like MM189 PB-Klf4 and BL322 PB-Klf4 were significantly lower than those from their mesenchymal-like counterparts (p < 0.05 and p < 0.01, respectively). Our results suggested that DW-MRI is a potential tool for observing mesenchymal- or epithelial-like characteristics of subcutaneous xenografted tumors.     

Highlights on the Role of KRAS Mutations in Reshaping the Microenvironment of Pancreatic Adenocarcinoma

The most frequent mutated oncogene family in the history of human cancer is the RAS gene family, including NRAS, HRAS, and, most importantly, KRAS. A hallmark of pancreatic cancer, recalcitrant cancer with a very low survival rate, is the prevalence of oncogenic mutations in the KRAS gene. Due to this fact, studying the function of KRAS and the impact of its mutations on the tumor microenvironment (TME) is a priority for understanding pancreatic cancer progression and designing novel therapeutic strategies for the treatment of the dismal disease. Despite some recent enlightening studies, there is still a wide gap in our knowledge regarding the impact of KRAS mutations on different components of the pancreatic TME. In this review, we will present an updated summary of mutant KRAS role in the initiation, progression, and modulation of the TME of pancreatic ductal adenocarcinoma (PDAC). This review will highlight the intriguing link between diabetes mellitus and PDAC, as well as vitamin D as an adjuvant effective therapy via TME modulation of PDAC. We will also discuss different ongoing clinical trials that use KRAS oncogene signaling network as therapeutic targets.     

The Association of Ovarian Teratoma and Anti-N-Methyl-D-Aspartate Receptor Encephalitis: An Updated Integrative Review

Ovarian teratomas are by far the most common ovarian germ cell tumor. Most teratomas are benign unless a somatic transformation occurs. The designation of teratoma refers to a neoplasm that differentiates toward somatic-type cell populations. Recent research shows a striking association between ovarian teratomas and anti-N-methyl-D-aspartate receptor (anti-NMDAR) encephalitis, a rare and understudied paraneoplastic neurological syndrome (PNS). Among teratomas, mature teratomas are thought to have a greater relevance with those neurological impairments. PNS is described as a neurologic deficit triggered by an underlying remote tumor, whereas anti-NMDAR encephalitis is characterized by a complex neuropsychiatric syndrome and the presence of autoantibodies in cerebral spinal fluid against the GluN1 subunit of the NMDAR. This review aims to summarize recent reports on the association between anti-NMDAR encephalitis and ovarian teratoma. In particular, the molecular pathway of pathogenesis and the updated mechanism and disease models would be discussed. We hope to provide an in-depth review of this issue and, therefore, to better understand its epidemiology, diagnostic approach, and treatment strategies.