Rates of severe complications in patients undergoing colorectal surgery for deep endometriosis-a retrospective multicenter observational study.

INTRODUCTION Surgical experience and hospital procedure volumes have been associated with the risk of severe complications in expert centers for endometriosis in France. However, little is known about other certified units in Central European countries. MATERIAL AND METHODS This retrospective observational study included 937 women who underwent surgery for colorectal endometriosis between January 2018 and January 2020 in 19 participating expert centers for endometriosis. All women underwent complete excision of colorectal endometriosis by rectal shaving, discoid or segmental resection. Postoperative severe complications were defined as grades III-IV of the Clavien-Dindo classification system including anastomotic leakage, fistula, pelvic abscess and hematoma. Surgical outcomes of centers performing less than 40 (group 1), 40-59 (group 2) and ≥60 procedures (group 3) over a period of 2 years were compared. RESULTS The overall complication rate of grade III and IV complications was 5.1% (48/937), with rates of anastomotic leakage, fistula formation, abscess and hemorrhage in segmental resection, discoid resection and rectal shaving, respectively, as follows: anastomotic leakage 3.6% (14/387), 1.4% (3/222), 0.6% (2/328); fistula formation 1.6% (6/387), 0.5% (1/222), 0.9%; (3/328); abscess 0.5% (2/387), 0% (0/222) and 0.6% (2/328); hemorrhage 2.1% (8/387), 0.9% (2/222) and 1.5% (5/328). Higher overall complication rates were observed for segmental resection (30/387, 7.8%) than for discoid (6/222, 2.7%, P = 0.015) or shaving procedures (12/328, 3.7%, P = 0.089). No significant correlation was observed between the number of procedures performed and overall complication rates (rSpearman  = -0.115; P = 0.639) with a high variability of complications in low-volume centers (group 1). However, an intergroup comparison revealed a significantly lower overall severe complication rate in group 3 than in group 2 (2.9% vs 6.9%; P = 0.017) without significant differences between other groups. CONCLUSIONS A high variability in complication rates does exist in centers with a low volume of activity. Major complications may decrease with an increase in the volume of activity but this effect cannot be generally applied to all institutions and settings

Selective Induction of Cell Death in Melanoma Cell Lines through Targeting of Mcl-1 and A1

Melanoma is an often fatal form of skin cancer which is remarkably resistant against radio- and chemotherapy. Even new strategies that target RAS/RAF signaling and display unprecedented efficacy are characterized by resistance mechanisms. The targeting of survival pathways would be an attractive alternative strategy, if tumor-specific cell death can be achieved. Bcl-2 proteins play a central role in regulating survival of tumor cells. In this study, we systematically investigated the relevance of antiapoptotic Bcl-2 proteins, i.e., Bcl-2, Bcl-xL, Bcl-w, Mcl-1, and A1, in melanoma cell lines and non-malignant cells using RNAi. We found that melanoma cells required the presence of specific antiapoptotic Bcl-2 proteins: Inhibition of Mcl-1 and A1 strongly induced cell death in some melanoma cell lines, whereas non-malignant cells, i.e., primary human fibroblasts or keratinocytes were not affected. This specific sensitivity of melanoma cells was further enhanced by the combined inhibition of Mcl-1 and A1 and resulted in 60% to 80% cell death in all melanoma cell lines tested. This treatment was successfully combined with chemotherapy, which killed a substantial proportion of cells that survived Mcl-1 and A1 inhibition. Together, these results identify antiapoptotic proteins on which specifically melanoma cells rely on and, thus, provide a basis for the development of new Bcl-2 protein-targeting therapies

Expression of antiapoptotic Bcl-2 proteins in non-malignant skin cells and melanoma cell lines.

<p>(A) Expression of Bcl-2, Bcl-xL, Bcl-w, and Mcl-1 protein in primary human fibroblasts, keratinocytes, and melanocytes was analyzed by immunoblotting. For each cell type, 2 donors are shown. (B) Expression of Bcl-2, Bcl-xL, Bcl-w and Mcl-1 protein in melanocytes isolated from 4 donors. (C) Expression of Bcl-2, Bcl-xL, Bcl-w and Mcl-1 protein in melanoma cell lines of different progression levels. Blot is representative for 2 independent experiments. (D) Levels of A1 mRNA were measured by quantitative RT-PCR. Mean +/− SD of 3 donors is shown for non-malignant cells, mean +/− SD of 3 cell passages is shown for melanoma cell lines. β-actin served as loading control in immunoblots. The same protein sample of melanocytes from donor #2 was loaded on all gels as a reference to allow the comparison of expression levels among the immunoblots shown in A, B, and C. RGP: radial-growth phase (non-invasive); VGP: vertical growth phase (invasive).</p

Chemotherapy adds to cell death induced by Mcl-1 and A1 inhibition.

<p>(A) 1205Lu melanoma cells were treated with the indicated siRNAs together with 1 µM 5-fluorouracil (5-FU) or not (no treatment) and analyzed on day 3 after transfection. Left panel: Cell viability was determined. Viability of control siRNA-treated cells without 5-FU treatment was set to 100%. Right panel: Cell death analysis measured by FACS after staining with Annexin V and propidium iodide. (B) Analysis of cell viability (left panel) or cell death (right panel) of 1205Lu cells on day 6 treated as described in A. (C) Analysis of cell viability (left panel) or cell death (right panel) of primary human fibroblasts on day 6 treated as described in A. If not otherwise indicated, asterisks represent significant increase in cell death compared to control siRNA-treated cells. Mean +/− SD of 3 independent experiments is shown in A, B, and C. AN, Annexin V; PI, propidium iodide.</p

Combined inhibition of Mcl-1 and A1 results in efficient induction of apoptosis in 1205Lu melanoma cells.

<p>(A) 1205Lu melanoma cells were transfected with A1- or Mcl-1-specific siRNAs or control siRNAs as indicated. Cell death was assessed 72 hours after transfection. Mean +/− SD of 3 independent experiments is shown. Asterisks represent significant increase in cell death compared to single inhibition. (B) 1205Lu melanoma cells were transfected with different siRNA sequences targeting A1 or Mcl-1 (A1b, Mcl-1b). Analysis was carried out as described in A. Asterisks represent significant increase in cell death compared to single inhibition. Also, cell death induction by A1b, Mcl-1b, and A1b/Mcl-1b versus control was significant (not indicated in the figure). (C) Caspase activation of 1205Lu cells treated as described in A was assessed by immunoblotting with antibodies detecting the proform as well as the active subunits. Caspase 9 (upper panel) and caspase 3 (lower panel) were analyzed 48 hours after transfection. (D) 1205Lu cells treated with siRNAs as described in A with or without the pan-caspase inhibitor zVAD-FMK (100 µM). Cell death was measured 72 hours after transfection. Mean +/− SD of 3 independent experiments is shown. Asterisks represent significant decrease in cell death of zVAD-FMK-treated cells compared to cells not treated with zVAD-FMK. AN, Annexin V; PI, propidium iodide.</p

Representative light microscopy images of 1205Lu melanoma cells (A) or fibroblasts (B) 6 days after transfection of the indicated siRNAs alone (upper panel) or together with 1 µM 5-FU (lower panel).

<p>Scale bar = 0.5 mm.</p

Combined inhibition of Mcl-1 and A1 results in efficient induction of cell death in a panel of melanoma cell lines, but not in keratinocytes.

<p>(A) Primary human keratinocytes (upper left panel), non-invasive melanoma cells (WM3211), invasive (WM793), and metastatic melanoma cells (WM1232, WM239A, WM1158) were transfected with the siRNAs as indicated. Cell death was determined 72 hours after transfection. Mean +/− SD of 3 (keratinocytes, WM1232, WM239A, WM1158) or 5 (WM3211 and WM793) independent experiments is shown. (B) A1 mRNA was measured by quantitative RT-PCR in indicated cells treated with A1-specific- or control siRNA for 48 hours. Mean +/− SD of 3 independent experiments is shown. Asterisks represent significant increase in cell death compared to control siRNA-treated cells. AN, Annexin V; PI, propidium iodide.</p