Multitarget Fluorescence In Situ Hybridization Diagnostic Applications in Tumors.

Multitarget fluorescence in situ hybridization (mFISH) is a technique that allows the detection of multiple target sequences on the same sample using spectrally distinct fluorophore labels. The mFISH approach is currently a useful assay in the oncologic field for the detection of predictive, prognostic, and diagnostic biomarkers. In this chapter, we summarize the application of mFISH in the identification of target genetic aberrations in formalin-fixed, paraffin-embedded (FFPE) tissue samples of several tumor types. We discuss the mFISH protocols in FFPE samples, the innovative multitarget probes used, and the critical issues related to their interpretation.

Pelvic floor and sexual function 3 years after hysterectomy - A prospective cohort study.

INTRODUCTION: Long term effects after hysterectomy, such as a worsening of pelvic floor and sexual function, have been studied with diverse results. Therefore, we investigated the long-term effects of hysterectomy for benign indication on pelvic floor and sexual function as well as differences in outcome depending on mode of hysterectomy. MATERIAL AND METHODS: In a prospective clinical cohort study, we included 260 women scheduled for hysterectomy who answered validated questionnaires; pelvic floor impact questionnaire (PFIQ-7), pelvic floor distress inventory (PFDI-20) and female sexual function index (FSFI). Participants were followed up to 3 years after surgery. Nonparametric statistics and mixed effect models were used in analyses of the data. RESULTS: After exclusions, 242 women remained in the study, with a response rate at the 3-year follow-up of 154/242 (63.6%) for all questionnaires. There was an improvement of pelvic floor function with a mean score of PFIQ-7 at baseline of 42.5 (SD 51.7) and at 3 years 22.7 (SD 49.4), (p < 0.001) and mean score of PFDI-20 at baseline was 69.6 (SD 51.1) and at 3 years 56.2 (SD 54.6), (p = 0.001). A deterioration of sexual function was seen among the sexually active women after 3 years with a mean score of FSFI at baseline 25.2 (SD 6.6) and after 3 years 21.6 (SD 10.1), (p < 0.001). However, this was not consistent with the unaltered sexual function for the whole cohort. No difference in pelvic floor or sexual function was detected when comparing robotic assisted laparoscopic hysterectomy, laparoscopic hysterectomy and abdominal hysterectomy. CONCLUSIONS: Three years after surgery robotic assisted laparoscopic hysterectomy, total laparoscopic hysterectomy and abdominal hysterectomy improve pelvic floor function to the same extent. Among the sexually active women, a decline of sexual function was seen after 3 years, not consistent with the entire cohort and independent of surgical methods. Whether this is a trend associated with aging or menopausal transition remains to be studied.

To Get the Most out of the Least: BRAF Molecular Evaluation in Melanoma Metastases on Cell Suspension from Fine Needle Aspiration Cytology Needle Rinses.

INTRODUCTION: Cytological samples from cutaneous melanoma (CM) metastases may be the only biomaterial available for diagnostic and predictive purpose in the clinical practice. BRAF evaluation from cytological samples actually implies the loss of one or more diagnostic smears, or the execution of one or more passes to obtain dedicated cytological samples. We tested BRAF molecular evaluation in CM metastases on cell suspension obtained from fine needle aspiration cytology (FNAC) needle rinses. METHODS: Forty-two patients with lymph node enlargements and a previous CM were enrolled. Patients were submitted to FNAC, and direct smears and cell-block were prepared for diagnostic purpose. The needle was carefully flushed in a vial containing 350 µL of nuclease-free water and a cell suspension was obtained for BRAF molecular evaluation. Molecular evaluation was also performed on histological samples for statistics. RESULTS: The series included 35 CM metastases and 7 reactive lymphadenopathies. Three cases resulted inadequate and adequacy was 92.9%. BRAF V600E/Ec mutations were found in 7 out of 32 (21.9%) CM metastases cases. BRAF mutations other than V600E/Ec were found in 2 out of 32 (6.25%) cases. Sensitivity, specificity, positive predictive value, and negative predictive value resulted 100%. CONCLUSION: BRAF molecular evaluation in CM metastases on cell suspension obtained from FNAC needle rinses is a time-sparing and accurate technique allowing to spare biomaterial in the clinical setting.

Effects of hysterectomy on pelvic floor function and sexual function-A prospective cohort study.

INTRODUCTION: Hysterectomy is one of the most common major surgical procedures in women. The effects of hysterectomy on pelvic floor and sexual function are uncertain. Our objective was to investigate the effects of hysterectomy for benign indications on pelvic floor and sexual function and to compare different modes of surgery. MATERIAL AND METHODS: We performed a prospective clinical cohort study. In all, 260 women scheduled for hysterectomy answered validated questionnaires (Pelvic Floor Impact Questionnaire, Pelvic Floor Distress Inventory and Female Sexual Function Index). Participants were followed 6 months and 1 year after surgery. Data were analyzed using nonparametric statistics and mixed effect models. RESULTS: Women with subtotal hysterectomy, vaginal hysterectomy, laparoscopic assisted vaginal hysterectomy, and previous prolapse/incontinence surgery were excluded from further analysis, leaving the remaining cohort to 242 patients. The response rate at 6 months and 1 year follow-up was 180/242 (74.3%) and 169/242 (69.8%), respectively. There was an improvement of pelvic floor function at both follow-ups; mean score of Pelvic Floor Impact Questionnaire at baseline was 42.5 (51.7), at 6 months 19.9 (42.2) and at 1 year 23.7 (50.3) (p < 0.001). The mean score of Pelvic Floor Distress Inventory at baseline was 69.6 (51.1), at 6 months 49 (43.2) and at 1 year 49 (43.2) (p < 0.001). There was an improvement of sexual function after 6 months (mean score of Female Sexual Function Index at baseline 17.9 [SD 11.7] and at 6 months 21.0 [SD 11.7]) (p < 0.001). There was no difference in pelvic floor or sexual function when comparing surgical techniques. CONCLUSIONS: Robotic assisted laparoscopic hysterectomy, laparoscopic hysterectomy and abdominal hysterectomy improve pelvic floor function to the same extent at 6 months and 1 year after surgery. There was an overall improvement of sexual function 6 months after hysterectomy, but this did not persist after 1 year.

Microsatellite Instability: From the Implementation of the Detection to a Prognostic and Predictive Role in Cancers.

Microsatellite instability (MSI) has been identified in several tumors arising from either germline or somatic aberration. The presence of MSI in cancer predicts the sensitivity to immune checkpoint inhibitors (ICIs), particularly PD1/PD-L1 inhibitors. To date, the predictive role of MSI is currently used in the selection of colorectal cancer patients for immunotherapy; moreover, the expansion of clinical trials into other cancer types may elucidate the predictive value of MSI for non-colorectal tumors. In clinical practice, several assays are used for MSI testing, including immunohistochemistry (IHC), polymerase chain reaction (PCR) and next-generation sequencing (NGS). In this review, we provide an overview of MSI in various cancer types, highlighting its potential predictive/prognostic role and the clinical trials performed. Finally, we focus on the comparison data between the different assays used to detect MSI in clinical practice.

Microsatellite Status Detection in Gastrointestinal Cancers: PCR/NGS Is Mandatory in Negative/Patchy MMR Immunohistochemistry.

BACKGROUND: Microsatellite instability (MSI) is a predictive biomarker for immune checkpoint inhibitors. The main goal was to investigate the discordance between IHC and PCR/NGS for MSI testing in gastrointestinal cancers. METHODS: Two series were analyzed through IHC for mismatch-repair-system proteins (MMRP) and PCR, with one series of 444 colorectal cancers (CRC) and the other of 176 gastric cancers (GC). All cases with discordant results between IHC and PCR were analyzed by NGS. IHC staining was evaluated as follows: proficient MMR (pMMR), with all MMR positive; deficient MMR (dMMR), with the loss of one heterodimer; and cases with the loss/patchy expression of one MMR (lo-paMMR). Cases with instability in at least two markers by PCR were MSI-high (MSI-H) and with instability in one marker, MSI-low (MSI-L). Cases without instability were evaluated as microsatellite-stable (MSS). RESULTS: In the CRC cohort, 15 out of 444 cases were dMMR and 46 lo-paMMR. Among the 15 dMMR, 13 were MSI-H and 2 MSS. Among the 46 lo-paMMR, 13 were MSI-H and 33 were MSS. In the GC cohort, 13 out of 176 cases were dMMR and 6 cases lo-paMMR. Among the 13 dMMR, 12 were MSI-H and only 1 was MSS. All six lo-paMMR cases were MSS. All NGS results were in agreement with PCR. CONCLUSIONS: In clinical practice, MMR-IHC could be used as a screening test and additional molecular analysis is mandatory exclusively in cases carrying loss/patchy MMR-IHC.

An Integrated In Silico, In Vitro and Tumor Tissues Study Identified Selenoprotein S (SELENOS) and Valosin-Containing Protein (VCP/p97) as Novel Potential Associated Prognostic Biomarkers in Triple Negative Breast Cancer.

BACKGROUND: Triple negative breast cancer (TNBC) is a heterogeneous group of tumors with early relapse, poor overall survival, and lack of effective treatments. Hence, new prognostic biomarkers and therapeutic targets are needed. METHODS: The expression profile of all twenty-five human selenoproteins was analyzed in TNBC by a systematic approach.In silicoanalysis was performed on publicly available mRNA expression datasets (Cancer Cell Line Encyclopedia, CCLE and Library of Integrated Network-based Cellular Signatures, LINCS). Reverse transcription quantitative PCR analysis evaluated selenoprotein mRNA expression in TNBC versus non-TNBC and normal breast cells, and in TNBC tissues versus normal counterparts. Immunohistochemistry was employed to study selenoproteins in TNBC tissues. STRING and Cytoscape tools were used for functional and network analysis. RESULTS: GPX1, GPX4, SELENOS, TXNRD1 and TXNRD3 were specifically overexpressed in TNBC cells, tissues and CCLE/LINCS datasets. Network analysis demonstrated that SELENOS-binding valosin-containing protein (VCP/p97) played a critical hub role in the TNBCselenoproteins sub-network, being directly associated with SELENOS expression. The combined overexpression of SELENOS and VCP/p97 correlated with advanced stages and poor prognosis in TNBC tissues and the TCGA dataset. CONCLUSION: Combined evaluation of SELENOS and VCP/p97 might represent a novel potential prognostic signature and a therapeutic target to be exploited in TNBC.