Experimental and Molecular Pathology

Validation of a next generation sequencing assay for BRCA1, BRCA2, CHEK2 and PALB2 genetic testing

BRCA1, BRCA2, CHEK2 and PALB2 genes are associated with hereditary breast and ovarian cancer syndrome. Genetic testing of these genes is of increasing importance to guide therapeutic and management decisions. In this study, we evaluated the performance of a next generation sequencing (NGS) assay for the complete analysis of BRCA1, BRCA2, CHEK2 and PALB2 genes using Agilent's SureMASTR BRCA Screen that enabled the detection of single nucleotide variants (SNVs), small insertions/deletions (indels) and copy number variations (CNVs) in a single-tube PCR based library preparation. The results showed 100% sensitivity and specificity on a set of 52 known samples from de-identified patients and external quality assessment program. A concordance rate of 87.5% was achieved in the comparison of variant classification with the external laboratories. The high accuracy of the assay supports the use of SureMASTR BRCA Screen in clinical diagnostic laboratories (SureMASTR BRCA Screen is for research use only, not for use in diagnostic procedures).

Roles of a TMPO-AS1/microRNA-200c/TMEFF2 ceRNA network in the malignant behaviors and 5-FU resistance of ovarian cancer cells

Competing endogenous RNA (ceRNA) networks consisted of long non-coding RNA (lncRNA), microRNA (miRNA) and mRNAs have aroused great interests recently. The current study aims to probe the mechanisms of lncRNA TMPO-AS1 in ovarian cancer (OC) development. A 5-fluorouracil (5-FU)-resistant subline of OC SKOV3 cells was developed, and differentially expressed lncRNAs in OC tissues and SKOV3 cells were analyzed. The miRNAs, genes and signaling pathways interacted with TMPO-AS1 were predicted and validated. TMPO-AS1 and the validated miRNA were inhibited to analyze their roles in malignant behaviors and 5-FU resistance of OC cells. In vivo studies were performed by inducing xenograft tumors in nude mice. Consequently, TMPO-AS1 was highly expressed in OC tissues and SKOV3 cells. TMPO-AS1 regulated transmembrane protein with epidermal growth factor and two follistatin motifs 2 (TMEFF2) through sponging miR-200c in OC cells, during which the PI3K/Akt signaling pathway was activated. Silenced TMPO-AS1 and over-expressed miR-200c inhibited epithelial-mesenchymal transition (EMT), invasion, migration and 5-FU resistance of OC cells. This study demonstrated that silencing of TMPO-AS1 might attenuate OC progression through inhibiting the invasion, metastasis and drug resistance of OC cells via the miR-200c/TMEFF2 network and the disruption of the PI3K/Akt signaling pathway.

miRNA profile in ovarian cancer

Ovarian cancer is a gynecological cancer with high mortality and a heterogeneous nature which complicates its early detection and primary prevention. Numerous studies have evaluated expression profile microRNAs (miRNAs) in tissue and serum samples of ovarian cancer patients to find appropriate biomarkers for this malignancy. Functional experiments also verified the oncogenic or suppressor effects of a number of miRNAs. miRNAs exert their role through degradation or inhibition of translation of the target mRNA. Through this regulatory function, they modulate numerous cellular processes which are ultimately associated with carcinogenesis. A number of miRNAs including miR-135a-3p, miR-200c, miR-216a and miR-340 regulate epithelial-mesenchymal transition program thus modulate invasiveness of ovarian cancer cell. Others have been shown to regulate some fundamental pathways in carcinogenesis such as mTOR and PI3K/AKT pathways. Such vast area of function of miRNAs in ovarian cancer has suggested them as putative therapeutic options for future years. In this review, we summarize the recent findings regarding the role of miRNAs in ovarian cancer pathogenesis, their application as biomarkers and the future perspectives of this research area.

Assessing pathogenicity of mismatch repair variants of uncertain significance by molecular tumor analysis

Functional analyses are the main method to classify mismatch repair (MMR) gene variants of uncertain significance (VUSs). However, the pathogenicity remains unclear for many variants because of conflicting results between clinical, molecular, and functional data. In this study, we evaluated whether whole exome sequencing (WES) could add another layer of evidence to elucidate the pathogenicity of MMR variants with conflicting interpretations. WES was performed on formalin-fixed paraffin-embedded tumor tissue of eight patients with a constitutional MMR VUS (seven families), including eight colorectal and two endometrial carcinomas and one ovarian carcinoma. Cell-free CIMRA assays were performed to assign Odds of Pathogenicity to these VUSs. In four families, seven tumors showed MMR deficiency-associated mutational signatures, supporting the pathogenicity of the VUS. Moreover, somatic (second) MMR hits identified in the WES data were found to explain MMR staining patterns when the MMR staining was discordant with the reported germline MMR gene variant. In conclusion, WES did not significantly reclassify VUS in these cases but clarified some phenotypic aspects such as age of onset and explanations in case of discordant MMR stainings.

Accurate detection of copy number aberrations in FFPE samples using the mFAST-SeqS approach

Shallow whole genome sequencing (Shallow-seq) is used to determine the copy number aberrations (CNA) in tissue samples and circulating tumor DNA. However, costs of NGS and challenges of small biopsies ask for an alternative to the untargeted NGS approaches. The mFAST-SeqS approach, relying on LINE-1 repeat amplification, showed a good correlation with Shallow-seq to detect CNA in blood samples. In the present study, we evaluated whether mFAST-SeqS is suitable to assess CNA in small formalin-fixed paraffin-embedded (FFPE) tissue specimens, using vulva and anal HPV-related lesions. Seventy-two FFPE samples, including 36 control samples (19 vulva;17 anal) for threshold setting and 36 samples (24 vulva; 12 anal) for clinical evaluation, were analyzed by mFAST-SeqS. CNA in vulva and anal lesions were determined by calculating genome-wide and chromosome arm-specific z-scores in comparison with the respective control samples. Sixteen samples were also analyzed with the conventional Shallow-seq approach. Genome-wide z-scores increased with the severity of disease, with highest values being found in cancers. In vulva samples median and inter quartile ranges [IQR] were 1[0-2] in normal tissues (n = 4), 3[1-7] in premalignant lesions (n = 9) and 21[13-48] in cancers (n = 10). In anal samples, median [IQR] were 0[0-1] in normal tissues (n = 4), 14[6-38] in premalignant lesions (n = 4) and 18[9-31] in cancers (n = 4). At threshold 4, all controls were CNA negative, while 8/13 premalignant lesions and 12/14 cancers were CNA positive. CNA captured by mFAST-SeqS were mostly also found by Shallow-seq. mFAST-SeqS is easy to perform, requires less DNA and less sequencing reads reducing costs, thereby providing a good alternative for Shallow-seq to determine CNA in small FFPE samples.

Expression of free fatty acid receptor 2 in normal and neoplastic tissues

FANCD2 expression affects platinum response and further characteristics of high grade serous ovarian cancer in cells with different genetic backgrounds

High-grade serous ovarian cancer (HGSOC) is the most prevalent subtype of ovarian cancer and demonstrates 5-year survival of just 40%. One of the major causes of mortality is the development of tumour resistance to platinum-based chemotherapy, which can be modulated by dysregulation of DNA damage repair pathways. We therefore investigated the contribution of the DNA interstrand crosslink repair protein FANCD2 to chemosensitivity in HGSOC. Increased FANCD2 protein expression was observed in some cell line models of platinum resistant HGSOC compared with paired platinum sensitive models. Knockdown of FANCD2 in some cell lines, including the platinum resistant PEO4, led to increased carboplatin sensitivity. Investigation into mechanisms of FANCD2 regulation showed that increased FANCD2 expression in platinum resistant cells coincides with increased expression of mTOR. Treatment with mTOR inhibitors resulted in FANCD2 depletion, suggesting that mTOR can mediate platinum sensitivity via regulation of FANCD2. Tumours from a cohort of HGSOC patients showed varied nuclear and cytoplasmic FANCD2 expression, however this was not significantly associated with clinical characteristics. Knockout of FANCD2 was associated with increased cell migration, which may represent a non-canonical function of cytoplasmic FANCD2. We conclude that upregulation of FANCD2, possibly mediated by mTOR, is a potential mechanism of chemoresistance in HGSOC and modulation of FANCD2 expression can influence platinum sensitivity and other tumour cell characteristics.

Analysis of BRCA1 and BRCA2 alternative splicing in predisposition to ovarian cancer

The mRNA splicing is regulated on multiple levels, resulting in the proper distribution of genes' transcripts in each cell and maintaining cell homeostasis. At the same time, the expression of alternative transcripts can change in response to underlying genetic variants, often missed during routine diagnostics. The main aim of this study was to define the frequency of aberrant splicing in BRCA1 and BRCA2 genes in blood RNA extracted from ovarian cancer patients who were previously found negative for the presence of pathogenic alterations in the 25 most commonly analysed ovarian cancer genes, including BRCA1 and BRCA2. Frequency and spectrum of splicing alterations in BRCA1 and BRCA2 genes were analysed in blood RNA from 101 ovarian cancer patients and healthy controls (80 healthy women) using PCR followed by gel electrophoresis and Sanger sequencing. The expression of splicing events was examined using RT-qPCR. We did not identify any novel, potentially pathogenic splicing alterations. Nevertheless, we detected six naturally occurring transcripts, named BRCA1ΔE9-10, BRCA1ΔE11, BRCA1ΔE11q, and BRCA2ΔE3, BRCA2ΔE12 and BRCA2ΔE17-18 of which three (BRCA1ΔE11q, BRCA1ΔE11 and BRCA2ΔE3) were significantly higher expressed in the ovarian cancer cohort than in healthy controls (p ≤ 0.0001). This observation indicates that the upregulation of selected naturally occurring transcripts can be stimulated by non-genetic mechanisms and be a potential systemic response to disease progression and/or treatment. However, this hypothesis requires further examination.

Targeted molecular profiling of epithelial ovarian cancer from Italian BRCA wild-type patients with a BRCA and PARP pathways gene panel

Ovarian cancer (OC) is the fifth most common type of cancer in women and the fourth most common cause of cancer death in women. Identification of pathogenic variants in OC tissues has an important clinical significance for therapeutic and prevention purposes. This study aims to evaluate the mutational profile of a patient cohort, negative for BRCA1/2 germinal variants and Mismatch Repair defects, using next-generation sequencing (NGS) approach on DNA from formalin-fixed paraffin-embedded samples. We used a custom NGS panel, targeting 34 cancer-related genes, mainly of the BRCA and PARP pathways, and analyzed NGS data to identify somatic and germline variants in Italian patients affected by primary epithelial ovarian cancer. We analyzed 75 epithelial ovarian cancer tissues and identified 54 pathogenic variants and 56 variants of unknown significance. TP53 was characterized by the highest mutational rate, occurring in 55% of tested epithelial ovarian cancers (EOCs). Interestingly, a subset of 8 EOCs showed pathogenic variants of homologous recombination pathway, which could be sensitive to PARP-inhibitor therapies. Germline analysis of actionable genes revealed 4 patients carrier of pathogenic germline variants respectively of RAD51C (2 patients), RAD51D, and PALB2. Molecular profiling of EOCs using our custom NGS panel has enabled the detection of both somatic and germline variants, allowing the selection of patients suitable for targeted therapies, and the identification of high-risk OC families that can benefit from genetic counseling and testing.

Targeted next generation sequencing panel for HPV genotyping in cervical cancer

Cervical cancer are generally caused by a persistent infection with the oncogenic virus, HPV. Patients with HPV integration are more prone to develop cervical cancer than patients without integration. In this proof-of-concept study, we aimed to develop a sensitive method based on targeted amplicon based NGS for early and precise detection of high-risk HPV-genotypes that are highly associated with the development of cervical cancer. Furthermore, we aimed to investigate if amplicon based NGS allowed for HPV genotyping in cervical lesions and whether it could detect HPV integration. The cohort included a group of CIN3

Down-regulation of lncRNA PCGEM1 inhibits cervical carcinoma by modulating the miR-642a-5p/LGMN axis

LncRNA PCGEM1 (PCGEM1) has been reported to exert essential effects on the development and progress of various tumors, while the detailed effects and possible mechanisms of PCGEM1 in cervical carcinoma remain unknown. In the present study, PCGEM1 was over-expressed in cervical carcinoma cells as evidenced by real-time quantitative polymerase chain reaction (RT-qPCR) assay. Knockdown of PCGEM1 significantly repressed proliferation, migration, and invasion, while induced G1 arrest in cervical carcinoma cells. In addition, PCGEM1 was predicted to target miR-642a-5p by bioinformatics software, which was further confirmed by luciferase reporter assay. Besides, RT-qPCR assay indicated that miR-642a-5p expression was decreased in cervical carcinoma cells and knockdown of PCGEM1 could accelerate miR-642a-5p expression. Moreover, inhibition of miR-642a-5p partly abolished the functions of PCGEM1 knockdown on proliferation, cell cycle, migration and invasion of cervical carcinoma cells. Furthermore, miR-642a-5p could bind to the 3'-UTR of LGMN, which was over-expressed in the cervical carcinoma cells. Suppression of LGMN partly restored the functions of miR-642a-5p inhibitor on proliferation, cell cycle distribution, migration and invasion in the cervical carcinoma cells treated with the PCGEM1 shRNA. Taken together, our data indicated that knockdown of PCGEM1 inhibited proliferation, migration and invasion in cervical carcinoma by modulating the miR-642a-5p/ LGMN axis.

Alternations in mitochondrial genome in carcinogenesis of HPV positive cervix

It is well known that mitochondrial dysfunctions are involved in tumorigenesis. A special interest of scientists is mitochondrial ND1 gene (mtND1). Recently detected mutations in the mtND1 can disrupt the normal activity of complex I and affect oxidative phosphorylation, thus leading to increase reactive oxygen species production. This study was undertaken to determine the alternations in the mtND1 and evaluate their association with development of precancerous lesions and cervical cancer. In the study 29 cervical cancer, 28 low grade squamous intraepithelial lesion (L-SIL) and 30 high grade squamous intraepithelial lesion (H-SIL) HPV positive fragments tissue were screened for the presence of mtND1 mutations. Our study showed that mutations in the mtND1 gene were detected in patients with precancerous stage, as well as cervical cancer. We have identified 12 point mutations in 116 analyzed precancerous and cancer samples HPV positive. Most detected missense mutations were previously described, except one (p. M156K) with Grantham value 95. The lower expression of mRNA ND1 was detected in cervical cancer cases and in all samples in which mtND1 mutations were identified. Our analyses revealed that level of ROS production was higher in cervical cancer tissues and all cases characterized by mtND1 mutations. We hypothesize that mutations in MT-ND1 observed in H-SIL and cancer could activate cervical carcinogenesis by increased ROS production.

RETRACTED: Knockdown of long non-coding RNA CCAT1 suppresses proliferation and EMT of human cervical cancer cell lines by down-regulating Runx2

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Editor-in-Chief. Following the concerns raised about the background pattern of the Western Blots from Figures 3A and 3C, the corresponding author has contacted the journal to request the retraction of the article. Given the comments of Dr Elisabeth Bik regarding this article “This paper belongs to a set of over 400 papers (as per February 2020) that share very similar Western blots with tadpole-like shaped bands, the same background pattern, and striking similarities in title structures, paper layout, bar graph design, and - in a subset - flow cytometry panels”, the journal requested the authors to provide the raw data. However, the authors were not able to fulfil this request and therefore the Editor-in-Chief decided to retract the article.

MiR-1258 promotes the apoptosis of cervical cancer cells by regulating the E2F1/P53 signaling pathway

Cervical cancer is the most common malignant tumor in gynaecology with high mortality. MiRNA has been reported to regulate cell biological processes in cervical cancer. This study aimed to explore the expression of miR-1258 and role of miR-1258 by targeting E2F1 in cervical cancer cells. The expression of miR-1258 and E2F1 in cervical cancer cells and transfection effects was determined by RT-qPCR analysis. The expression of E2F1, MMP2, MMP7, MMP9, Bcl2, Bax, cleaved caspase3, caspase3, KI67, p-AKT, cyclinD1, CDK2, P53 and AKT in cervical cancer cells was detected by western blot analysis. The proliferation, invasion, migration and apoptosis were respectively analyzed by CCK-8 assay, transwell assay, wound healing assay and flow cytometry analysis. E2F1 was a potential target of miR-1258, which demonstrated by a dual-luciferase reporter assay. miR-1258 expression was decreased while E2F1 expression was increased in cervical cancer cells. MiR-1258 overexpression could down-regulate the E2F1 expression. Overexpression of miR-1258 inhibited the proliferation, invasion and migration and promoted the apoptosis of cervical cancer cells by AKT and P53 signal pathway. And, Overexpression of miR-1258 also suppressed the tumor growth by AKT and P53 signal pathway. Overexpression of E2F1 reduced the inhibition effects of miR-1258 in cervical cancer. Taken together, miR-1258 overexpression exerts its inhibition effects on the proliferation, invasion and migration and promotion effects on the apoptosis of cervical cancer cells by targeting the E2F1, which might provide new ideas for clinical treatment of cervical cancer.

IGFBP-2 in cervical cancer development

Increased expression of insulin-like growth factor binding protein 2, IGFBP-2, is associated with many cancers, though its role in cervical cancer is unclear. The aim of this study was to investigate the expression of IGFBP-2 protein and the transcriptomics profile of genes involved in the IGF signaling pathway during cervical cancer development. Immunohistochemical expression of IGFBP-2 protein was semi-quantitatively assessed in tissue microarrays containing 9 normal cervix, 10 low grade cervical intraepithelial neoplasia (LGCIN), 10 high grade cervical intraepithelial neoplasia (HGCIN) and 42 squamous cell carcinoma (SCC) cases. The gene expression profiles of IGFBP-2, IGF-1, IGF-1R, PTEN, MDM2, AKT1 and TP53 were determined in three cervical tissue samples each from normal cervix, human papillomavirus (HPV)-infected LGCIN, HGCIN and SCC, using Human Transcriptome Array 2.0. IGFBP-2 protein was highly expressed in the cytoplasm of SCC cells compared to normal cervix (p = .013). The expression was not significantly associated with CIN grade or SCC stage. Transcriptomics profiling demonstrated upregulation of IGFBP-2 and TP53 in HGCIN and SCC compared to normal cervix. IGF-1, IGF-1R and PTEN genes were downregulated in all histological groups. IGF-1 gene was significantly downregulated in SCC (p = .031), while PTEN gene was significantly downregulated in HGCIN (p = .012), compared to normal cervix. MDM2 and AKT1 genes were downregulated in LGCIN and HGCIN, while upregulated in SCC. In cervical carcinogenesis, IGFBP-2 appears to play an oncogenic role, probably through an IGF-independent mechanism.

The inhibitory effect and safety of GANT61 on HeLa cells in nude mice

The Hedgehog (Hh) pathway effector Gli1 plays an important role in cervical cancer, and GANT61 is an Hh signaling inhibitor. In this study, we aimed to investigate the inhibitory effect of GANT61 on cervical cancer and to study its safety in nude mice. We used in vivo experiments to assess the effect of GANT61 on the growth of cervical cancer HeLa cells, and we measured the WBC, HGB, PLT, ALT, AST and Cre levels in nude mice. Next, we examined the organ and tumor morphology and distant metastasis by HE staining. We used immunohistochemistry to monitor the expression levels of Gli1, FoxM1, Ki-67, cyclinD1, E-cadherin, vimentin, survivin, caspase-3 and CD34+. Western blotting and RT-RCR were used to measure Gli1 expression. GANT61 inhibited the growth and metastasis of HeLa cervical cancer cells upon their transplantation into nude mice, and we preliminarily propose that GANT61 is safe for nude mice. These findings suggest that GANT61 could be used as a Hedgehog inhibitor to inhibit EMT and proliferation and to promote apoptosis via Gli1 downregulation.

The role of NFKB1/NFKBIA genetic variants in HPV infection: A cross-sectional cohort study

Human Papillomavirus (HPV) is the most frequent etiological agent sexually transmitted. In the context of the immune response, NF-kB pathway plays an important role controlling the expression of several genes essential to cellular activity and structural and/or functional changes in components of this pathway can promote the development of several tumors. Thus, the study purpose was to evaluate the influence of NFKB1 rs28362491 and NFKBIA rs696 genetic variants on HPV infection and cervical lesions development. In this study 334 patients were recruited, of whom 48.8% (n = 163) were HPV infected, and considered our case group. HPV-DNA was detected by polymerase chain reaction (PCR) and the genetic variants were assessed in blood cells and tumor tissues paraffin embedded samples through restriction fragment length polymorphism analysis. Among women who were recruited for this study who were infected, 37.4% presented precursor lesions and 16.8% were diagnosed with cervical cancer (CC). The present study did not observe significant effects of the interaction between such genetic variants on HPV infection, nor on the development of lesions and progression to CC. Further studies will be important to investigate if under some circumstance the NFKB1 rs28362491 and NFKBIA rs696 genetic variants influence the progression of HPV-associated lesions.

Prognostic and clinicopathological significance of TMEFF2, SMOC-2, and SOX17 expression in endometrial carcinoma

Background there is a need for novel biomarkers and targeting therapies for predicting Endometrial carcinoma (EC) progression and recurrence. TMEFF2 is a gene that was found to play a role in EMT. SMOC-2 is expressed in embryogenesis and it was identified as a recent stem cell-related gene that has a role in cancer progression. SRY-box 17 (SOX17) is a member of the SRY-related HMG-box (SOX) family of transcription factors. Dysregulation or downregulation of SOX17 expression was found in many cancer tissues. In the present study, we aimed to assess the tissue protein expressions of TMEFF2, SMOC-2, and SOX17 in EC using immunohistochemistry to evaluate their clinicopathological values and prognostic roles in EC patients. This is prospective cohort study included 120 patients with EC. Sections from 120 paraffin blocks were retrieved and stained with TMEFF2, SMOC-2, and SOX17 using immunohistochemistry, the expression of markers in all tissue samples was assessed, analyzed and correlation of pathological parameters with the levels of expression was done. All patients were followed up till death or till the last known alive data for about 50 months (range from 25 to 60). TMEFF2, SMOC-2 expression was correlated with the presence of lymph node metastases (p = 0.023), distant metastasis (p = 0.039) recurrence of the tumor after successful therapy, overall survival, and disease-free survival (p < 0.001). SOX17 positive expression was positively correlated with low grade (p = 0.019), absence of lymph node metastasis (p = 0.001), absence of distant metastasis (p = 0.013), low stage (p = 0.03), and its negative expression was positively correlated with recurrence of the tumor after successful therapy, overall survival and disease-free survival (p = 0.001). In conclusion, we demonstrated that both TMEFF2 and SMOC-2 were highly expressed in EC and were associated with a shortened survival rate, dismal outcome, and poor prognosis in EC patients. While SOX17 expression was related to a favorable outcome and its down-regulation was associated with dismal EC patient's survival.