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.

Prevalence of MMTV-like sequences in breast cancer samples in Romanian patients-there is a geographic difference compared to the Western world.

BACKGROUND: Breast cancer, although the most frequently diagnosed malignant tumor in humans, has a less clear etiology compared to other frequent cancer types. Mouse-mammary tumor virus (MMTV) is involved in breast cancer in mice and dogs and might play a role in the etiology of some breast cancers in humans, since an MMTV-like sequence was identified in 20-40% of breast cancer samples in Western Europe, USA, Australia and some other parts of the world. The purpose of our study was to identify MMTV-like DNA sequences in breast tissue samples from breast cancer patients who underwent curative surgery in our regional academic center in Romania, EU. METHODS: We selected 75 patients with non-metastatic breast cancer treated surgically with curative intent, who did not undergo any neoadjuvant treatment. Out of these patients, 50 underwent radical lumpectomy and 25 modified radical mastectomy. Based on previous reports in the literature we searched using PCR the MMTV-like DNA env sequence in the breast cancer tissue and normal breast tissue obtained from the same patients. RESULTS: None of the examined samples was positive for MMTV-like target sequences on PCR. CONCLUSIONS: We could not prove that MMTV plays a role in the etiology of breast cancer in our patient group. This finding is similar to those from publications of other geographically related research groups.

Spectrum of High-Risk Mutations among Breast Cancer Patients Referred for Multigene Panel Testing in a Romanian Population.

(1) Background: Multigene panel testing for Hereditary Breast and Ovarian Cancer (HBOC) using next generation sequencing (NGS) is becoming a standard in medical care. There are insufficient genetic studies reported on breast cancer (BC) patients from Romania and most of them are focused only on BRCA 1/2 genes (Breast cancer 1/2). (2) Methods: NGS was performed in 255 consecutive cases of BC referred for management in our clinic between 2015-2019. (3) Results: From the 171 mutations identified, 85 were in the high-penetrance BC susceptibility genes category, 72 were pathogenic genes, and 13 genes were in the (variants of uncertain significance) VUS genes category. Almost half of the mutations were in the BRCA 1 gene. The most frequent BRCA1 variant was c.3607C>T (14 cases), followed by c.5266dupC (11 cases). Regarding BRCA-2 mutations we identified c.9371A>T (nine cases), followed by c.8755-1G>A in three cases, and we diagnosed VUS mutations in three cases. We also identified six pathogenic variants in the PALB2 gene and two pathogenic variants in (tumor protein P 53) TP53. (4) Conclusions: The majority of pathogenic mutations in the Romanian population with BC were in the BRCA 1/ 2 genes, followed by PALB2 (partner and localizer of BRCA2) and TP53, while in the CDH1 (cadherin 1) and STK11 (Serine/Threonine-Protein Kinase) genes we only identified VUS mutations.

Revisiting the Implications of Positive Germline Testing Results Using Multi-gene Panels in Breast Cancer Patients.

BACKGROUND/AIM: The use of multi-gene panels for germline testing in breast cancer enables the estimation of cancer risk and guides risk-reducing management options. The aim of this study was to present data that demonstrate the different levels of actionability for multi-gene panels used in genetic testing of breast cancer patients and their family members. MATERIALS AND METHODS: We performed an analysis in our clinical database to identify breast cancer patients undergoing genetic testing. We reviewed positive results in respect of risk estimation and management, cascade family testing, secondary findings and information for treatment decision-making. RESULTS: A total of 415 positive test reports were identified with 57.1%, 18.1%, 10.8% and 13.5% of individuals having pathogenic/likely pathogenic variants in high, moderate, low and with insufficient evidence for breast cancer risk genes, respectively. Six point seven percent of individuals were double heterozygotes. CONCLUSION: Germline findings in 92% of individuals are linked to evidence-based treatment information and risk estimates for predisposition to breast and/or other cancer types. The use of germline findings for treatment decision making expands the indication of genetic testing to include individuals that could benefit from targeted treatments.

T4 retroperitoneal liposarcoma. Challenges of big size sarcomas surgical treatment.

Our article presents some of the challenges of the surgical treatment of T4 (>15 cm) retroperitoneal liposarcomas (up to 65∕56∕30 cm, 25.5 kg) series of cases treated by the Department of Surgical Oncology, Prof. Dr. Ion Chiricuta Oncology Institute, Cluj-Napoca (IOCN), Romania, with illustrations, insisting on important blood vessels and nerves dissection and preservation and discussions of strategies with references to important articles from the last 10 years specialty literature. Challenges do not come only from intraoperative difficulties but also from establishing the right attitude from the extent of resection and oncological safety point of view, the role of the pathologist being very important because histological subtype and completeness of the resections are the most important prognostic factors for such tumors. Despite all today available aids in decision making, like nomograms or high-resolution imagery, sometimes this decision is to be taken intraoperative based on surgeon's expertise and skills. That is why is strongly advised that such cases to be treated in high-volume specialized tertiary centers of surgical oncology.

Endoscopic Ultrasound Elastography in the Assessment of Rectal Tumors: How Well Does It Work in Clinical Practice?

Endorectal ultrasound applications in the evaluation of rectal tumors could be a useful tool in achieving proper staging of rectal cancer. The purpose of this study was to compare the efficacy of rectal tumor staging by flexible endoscopic ultrasound (EUS) with real-time elastography (RTE) using the gold standard post-surgery histological analysis of the resected tissue as the control. The second aim of our research was to establish cutoff values for the EUS-RTE strain ratio corresponding to stages by independently comparing the stiffness values obtained with histology and EUS-RTE staging in order to minimize observation bias. We evaluated the records of 130 patients with a rectal tumor confirmed by biopsy. EUS was used in 70 patients, EUS-RTE-in the other 60. We found no statistically significant differences in staging accuracy when comparing EUS to EUS-RTE. Through a correspondence method between staging assessment and the EUS-RTE stain ratio, we identified cutoff intervals for T2, T3, and T4 staging that were nonoverlapping and proved to be statistically significant in terms of EUS-RTE values (significantly different ascending values from one interval to the other). We found that EUS-RTE offers slightly better, although not statistically significant sensitivity and specificity for T and N stage predictions compared to 2D EUS. Our results showed that EUS-RTE offers slightly higher sensitivity and specificity compared to EUS. Reliable cutoff intervals were found for strain rate elastography, previously available only for shear wave elastography (SWE) which is currently unavailable on any EUS system. Thus, these commonly available EUS-RTE systems can serve as a complementary tool in the staging of rectal tumors.

Lipidomic Signatures for Colorectal Cancer Diagnosis and Progression Using UPLC-QTOF-ESI+MS.

Metabolomics coupled with bioinformatics may identify relevant biomolecules such as putative biomarkers of specific metabolic pathways related to colorectal diagnosis, classification and prognosis. This study performed an integrated metabolomic profiling of blood serum from 25 colorectal cancer (CRC) cases previously classified (Stage I to IV) compared with 16 controls (disease-free, non-CRC patients), using high-performance liquid chromatography and mass spectrometry (UPLC-QTOF-ESI+ MS). More than 400 metabolites were separated and identified, then all data were processed by the advanced Metaboanalyst 5.0 online software, using multi- and univariate analysis, including specificity/sensitivity relationships (area under the curve (AUC) values), enrichment and pathway analysis, identifying the specific pathways affected by cancer progression in the different stages. Several sub-classes of lipids including phosphatidylglycerols (phosphatidylcholines (PCs), phosphatidylethanolamines (PEs) and PAs), fatty acids and sterol esters as well as ceramides confirmed the "lipogenic phenotype" specific to CRC development, namely the upregulated lipogenesis associated with tumor progression. Both multivariate and univariate bioinformatics confirmed the relevance of some putative lipid biomarkers to be responsible for the altered metabolic pathways in colorectal cancer.

Clinical Utility of Functional RNA Analysis for the Reclassification of Splicing Gene Variants in Hereditary Cancer.

BACKGROUND: Classification of splicing variants (SVs) in genes associated with hereditary cancer is often challenging. The aim of this study was to investigate the occurrence of SVs in hereditary cancer genes and the clinical utility of RNA analysis. MATERIAL AND METHODS: 1518 individuals were tested for cancer predisposition, using a Next Generation Sequencing (NGS) panel of 36 genes. Splicing variant analysis was performed using RT-PCR and Sanger Sequencing. RESULTS: In total, 34 different SVs were identified, 53% of which were classified as pathogenic or likely pathogenic. The remaining 16 variants were initially classified as Variant of Uncertain Significance (VUS). RNA analysis was performed for 3 novel variants. CONCLUSION: The RNA analysis assisted in the reclassification of 20% of splicing variants from VUS to pathogenic. RNA analysis is essential in the case of uncharacterized splicing variants, for proper classification and personalized management of these patients.

Malignant phyllodes tumors of the breast associating malignancy of both mesenchymal and epithelial components (invasive or in situ ductal carcinoma).

Phyllodes tumors of the breast are biphasic tumors consisting from an epithelial component and a mesenchymal component. Usually, the mesenchymal component of the tumor is the one who dictates the malignancy of the biphasic proliferation. Presence of the malignancy of the both, epithelial [under the form of invasive carcinoma or ductal carcinoma in situ (DCIS)] and mesenchymal components is very rare. Most of the data available from the literature refers to single case presentations. This paper presents the experience of Prof. Dr. Ion Chiricuta Oncological Institute (IOCN), Cluj-Napoca, Romania, with the malignant phyllodes tumors with both epithelial and mesenchymal components showing malignancy. Over two decades (1999-2018), four cases of malignant phyllodes tumors with concomitant epithelial and mesenchymal malignancy were found and presented as a case series. Two out of four cases were malignant phyllodes tumors harboring invasive breast carcinomas (one case with associated DCIS and one case of pure invasive carcinoma) and two cases were malignant phyllodes tumors with the epithelial component showing DCIS. Average follow-up period was 67 months (from 39 to 132 months) with a disease-free survival of 58 months.

The role of imaging techniques in the diagnosis, staging and choice of therapeutic conduct in pregnancy associated breast cancer.

Breast cancer diagnosed during pregnancy is at increasing incidence due to the increased frequency of obesity, the postponement of the first pregnancy to later decades of life and the advances of diagnostic techniques. Clinical and imaging diagnosis is difficult during gestation due to adaptive changes of the maternal organism , the mammary glads in particular. Furthermore, the therapeutic approach is limited both by the possible side effects on the fetus and by the skepticism of the couple over these therapeutic regimens.The present paper aims to review the main diagnostic steps to confirm pregnancy associated breast cancer, as well as the therapeutic possibilities during this period, related to the potential adverse effects concerning pregnancy.

The influence of reproductive factors on breast cancer risk in women with pathogenic mutations.

PURPOSE: To assess the influence of reproductive factors in the occurrence of breast cancer in women, taking into account the presence/absence of genetic predisposing mutations. METHODS: 100 patients with breast cancer were included. The genetic testing was conducted through a multigene panel. Reproductive characteristics were noted for all patients: age of menarche, age of the patient at first full term pregnancy, number of pregnancies, number of full-term pregnancies, breastfeeding interval, number of abortions, and menopausal status at the time of diagnosis. The patients were divided into three groups according to their mutations: BRCA1, positive for mutations other than BRCA1 and negative. RESULTS: The risk of breast cancer was not influenced by the number of abortions, parity, age at first pregnancy, age at menarche and menopausal status, or by oral contraceptive use in carriers of pathogenic mutations group in the BRCA1 group. The present study has demonstrated the protective effect of breastfeeding only in patients without genetic risk (p=0.0344). In contrast, breastfeeding did not influence breast cancer occurrence in BRCA1 mutation carriers' group (p=0.2321). CONCLUSIONS: Breastfeeding represents a protective mechanism only in patients without genetic breast cancer predisposing mutations. Environmental and reproductive factors can impact the risk and the age of onset of breast cancer in patients carrying pathogenic mutations, but the mechanisms of action are not fully understood.

Analysis of hereditary cancer syndromes by using a panel of genes: novel and multiple pathogenic mutations.

BACKGROUND: Hereditary cancer predisposition syndromes are responsible for approximately 5-10% of all diagnosed cancer cases. In the past, single-gene analysis of specific high risk genes was used for the determination of the genetic cause of cancer heritability in certain families. The application of Next Generation Sequencing (NGS) technology has facilitated multigene panel analysis and is widely used in clinical practice, for the identification of individuals with cancer predisposing gene variants. The purpose of this study was to investigate the extent and nature of variants in genes implicated in hereditary cancer predisposition in individuals referred for testing in our laboratory. METHODS: In total, 1197 individuals from Greece, Romania and Turkey were referred to our laboratory for genetic testing in the past 4 years. The majority of referrals included individuals with personal of family history of breast and/or ovarian cancer. The analysis of genes involved in hereditary cancer predisposition was performed using a NGS approach. Genomic DNA was enriched for targeted regions of 36 genes and sequencing was carried out using the Illumina NGS technology. The presence of large genomic rearrangements (LGRs) was investigated by computational analysis and Multiplex Ligation-dependent Probe Amplification (MLPA). RESULTS: A pathogenic variant was identified in 264 of 1197 individuals (22.1%) analyzed while a variant of uncertain significance (VUS) was identified in 34.8% of cases. Clinically significant variants were identified in 29 of the 36 genes analyzed. Concerning the mutation distribution among individuals with positive findings, 43.6% were located in the BRCA1/2 genes whereas 21.6, 19.9, and 15.0% in other high, moderate and low risk genes respectively. Notably, 25 of the 264 positive individuals (9.5%) carried clinically significant variants in two different genes and 6.1% had a LGR. CONCLUSIONS: In our cohort, analysis of all the genes in the panel allowed the identification of 4.3 and 8.1% additional pathogenic variants in other high or moderate/low risk genes, respectively, enabling personalized management decisions for these individuals and supporting the clinical significance of multigene panel analysis in hereditary cancer predisposition.

The decrease of some serum free amino acids can predict breast cancer diagnosis and progression.

This study was targeted on a metabolomic approach to compare the blood serum free amino acid profiles and concentration of confirmed breast cancer (stages I-III) patients to healthy controls in order to establish reliable biomarkers of early detection and prediction of breast cancer. The ultra-high-performance liquid chromatography coupled with mass spectrometry using positive ionization electrospray was applied for the picoline-derivatized serum free amino acids using the EZ:faastTM kit. Multivariate statistical analysis principal component analysis, partial least squares discrimination analysis and univariate analysis were applied in order to discriminate between patient groups and putative amino acid biomarkers for breast cancer. A significant decrease of amino acid concentrations between the breast cancer group and the control group was positively correlated with breast cancer progression. Arginine, Alanine, Isoleucine, Tyrosine and Tryptophan were identified as being good potential discriminants (AUROC ≥0.85) and suitable candidates to diagnose and predict the breast cancer progression.

Blue nevus-like melanoma of the uterine cervix. Case report and review of the literature.

We present the clinical and pathological aspects of a patient diagnosed with a very rare tumor, a blue nevus-like melanoma of the uterine cervix. The patient turned to our Service for a second opinion regarding a cervical polyp causing vaginal bleeding, polyp which has been excised in another Hospital and interpreted initially as a pleomorphic sarcoma. In the presentation, we emphasize upon the stages of solving a difficult diagnosis, pathological description and treatment of these rare, aggressive tumors with poor prognosis, which represent the fundamental precondition in order to formulate the best therapeutic strategy.

A malignant phyllodes tumor with liposarcomatous differentiation case with 3-year follow-up.

Phyllodes tumors (PTs) are a group of rarely breast tumors of fibro-epithelial origin, counting for about 1% of the breast malignancies divided, based on histological features, in benign, borderline and malignant neoplasms, arising most of them in women in their 40's. Among this complex group of tumors, the liposarcomatous differentiation is an even more rare lesion, counting for about 0.3% of all primary sarcomas of the breast. This article presents a case of a 48-year-old woman with a breast malignant PT with liposarcomatous differentiation, diagnosed by guided core biopsy, treated by excision and subsequent simple mastectomy followed by radiotherapy, with a 3-year follow-up.

Prevalence of deleterious mutations among patients with breast cancer referred for multigene panel testing in a Romanian population.

AIM: Multigene panel testing for Hereditary Breast and Ovarian Cancer (HBOC) using next generation sequencing is becoming more common in medical care.We report our experience regarding deleterious mutations of high and moderate-risk breast cancer genes (BRCA1/2, TP53, STK11, CDH1, PTEN, PALB2, CHEK2, ATM), as well as more recently identified cancer genes, many of which have increased risk but less well-defined penetrance. METHODS: Genetic testing was performed in 130 consecutive cases with breast cancer referred to our clinic for surgical evaluation and who met the 2016 National Comprehensive Cancer Network (NCCN) criteria for genetic testing. RESULTS: 82 patients had pathogenic/likely pathogenic mutations and VUS mutations, and 48 were negative; 36 of the pathogenic mutations were in the high-risk genes and 16 were in the moderate risk genes and only 5 cases in the intermediary risk group.From the VUS mutation group 21 cases were in the intermediary risk group, 9 cases were in the moderate risk group and only 7 cases in high risk group.The most frequent BRCA1 variant was c.3607C>T (7 cases) followed by c.5266dupC and c.4035delA (each in 4 cases). Regarding BRCA-2 mutations we identified c.9371A>T and c.8755-1G>A in 6 cases and we diagnosed VUS mutations in 3 cases. CONCLUSION: Our study identified 2 mutations in the BRCA1 gene that are less common in the Romanian population, c.3607C>T and c.4035delA. Both variants had particular molecular phenotypes, c.3607C>T variant respecting the triple negative pattern of BRCA1 breast cancer while c.4035delA were Luminal B HER positive.