International Validation of the Immunoscore Biopsy in Patients With Rectal Cancer Managed by a Watch-and-Wait Strategy.

PURPOSE: No biomarker capable of improving selection and monitoring of patients with rectal cancer managed by watch-and-wait (W&W) strategy is currently available. Prognostic performance of the Immunoscore biopsy (ISB) was recently suggested in a preliminary study. METHODS: This international validation study included 249 patients with clinical complete response (cCR) managed by W&W strategy. Intratumoral CD3+ and CD8+ T cells were quantified on pretreatment rectal biopsies by digital pathology and converted to ISB. The primary end point was time to recurrence (TTR; the time from the end of neoadjuvant treatment to the date of local regrowth or distant metastasis). Associations between ISB and outcomes were analyzed by stratified Cox regression adjusted for confounders. Immune status of tumor-draining lymph nodes (n = 161) of 17 additional patients treated by neoadjuvant chemoradiotherapy and surgery was investigated by 3'RNA-Seq and immunofluorescence. RESULTS: Recurrence-free rates at 5 years were 91.3% (82.4%-100.0%), 62.5% (53.2%-73.3%), and 53.1% (42.4%-66.5%) with ISB High, ISB Intermediate, and ISB Low, respectively (hazard ratio [HR; Low v High], 6.51; 95% CI, 1.99 to 21.28; log-rank P = .0004). ISB was also significantly associated with disease-free survival (log-rank P = .0002), and predicted both local regrowth and distant metastasis. In multivariate analysis, ISB was independent of patient age, sex, tumor location, cT stage (T, primary tumor; c, clinical), cN stage (N, regional lymph node; c, clinical), and was the strongest predictor for TTR (HR [ISB High v Low], 6.93; 95% CI, 2.08 to 23.15; P = .0017). The addition of ISB to a clinical-based model significantly improved the prediction of recurrence. Finally, B-cell proliferation and memory in draining lymph nodes was evidenced in the draining lymph nodes of patients with cCR. CONCLUSION: The ISB is validated as a biomarker to predict both local regrowth and distant metastasis, with a gradual scaling of the risk of pejorative outcome.

Evaluation of circulating tumor cells in colorectal cancer using flow cytometry.

OBJECTIVE: We aimed to evaluate the prognostic value of circulating tumor cells (CTCs) and the impact of intraoperative tumor manipulation on CTCs in colorectal cancer (CRC) patients. METHODS: We performed a prospective study on 40 patients with CRC stages I to IV who received curative surgery using the no-touch technique. Flow cytometry was used to identify CTCs in peripheral blood samples (4 mL/sample) collected at two surgical moments: skin incision (T1) and after surgical resection (T2). A threshold of ≥4 CTCs/4 mL blood was established for considering patients CTC positive. RESULTS: In the univariate analysis, CTC evaluation at T2 was correlated with female sex, vascular invasion, tumor localization in the colon and metastatic lymph nodes. In the multivariate analysis, only female sex and colon cancer maintained statistical significance. At a medium follow-up of 15 months (1-25 months), the mortality rate was 10% (n = 4), with no significant differences between the overall survival of T1 or T2 CTC-positive and CTC-negative patients. CONCLUSIONS: Flow cytometry is a feasible CTC identification technique in CRC, and although surgical manipulation has no influence on CTC numbers, CTCs may serve as a prognostic and predictive factor.

Uterine Epithelioid Trophoblastic Tumor in a 44-Year-Old Woman: A Diagnostic Dilemma.

BACKGROUND: Epithelioid trophoblastic tumor (ETT) is a rare and newly defined disease, which most commonly occurs in women of reproductive age and can be a sequela of any gestational event. ETT can be present in both intrauterine and extrauterine sites. CASE REPORT: A woman of reproductive age, without specific comorbidities and with a single pregnancy and natural childbirth eight years ago, was diagnosed initially with poorly differentiated pleomorphic leiomyosarcoma on the hemostatic uterine curettage. CONCLUSION: Our case highlights that ETT presents a diagnostic challenge due to its rarity and histologic resemblance to other pathologies. Misdiagnosis delays effective treatment and affects survival. To date, only 8 cases of ETT of the uterus without previous gestational event and normal human chorionic gonadotropin (ß-HCG) levels in a 60-year literature survey have been reported.

A Diagnostic Biopsy-Adapted Immunoscore Predicts Response to Neoadjuvant Treatment and Selects Patients with Rectal Cancer Eligible for a Watch-and-Wait Strategy.

PURPOSE: No biomarker to personalize treatment in locally advanced rectal cancer (LARC) is currently available. We assessed in LARC whether a diagnostic biopsy-adapted immunoscore (ISB) could predict response to neoadjuvant treatment (nT) and better define patients eligible to an organ preservation strategy ("Watch-and-Wait"). EXPERIMENTAL DESIGN: Biopsies from two independent cohorts (n 1 = 131, n 2 = 118) of patients with LARC treated with nT followed by radical surgery were immunostained for CD3+ and CD8+ T cells and quantified by digital pathology to determine ISB. The expression of immune-related genes post-nT was investigated (n = 64 patients). Results were correlated with response to nT and disease-free survival (DFS). The ISB prognostic performance was further assessed in a multicentric cohort (n = 73 patients) treated by Watch-and-Wait. RESULTS: ISB positively correlated with the degree of histologic response (P < 0.001) and gene expression levels for Th1 orientation and cytotoxic immune response, post-nT (P = 0.006). ISB high identified patients at lower risk of relapse or death compared with ISB low [HR, 0.21; 95% confidence interval (CI), 0.06-0.78; P = 0.009]. Prognostic performance of ISB for DFS was confirmed in a validation cohort. ISB was an independent parameter, more informative than pre- (P < 0.001) and post-nT (P < 0.05) imaging to predict DFS. ISB combined with imaging post-nT discriminated very good responders that could benefit from organ preservation strategy. In the "Watch-and-Wait" cohort (n = 73), no relapse was observed in patients with ISB high (23.3%). CONCLUSIONS: ISB predicts response to nT and survival in patients with LARC treated by surgery. Its usefulness in the selection of patients eligible for a Watch-and-Wait strategy is strongly suggested.

Analytical validation of the Immunoscore and its associated prognostic value in patients with colon cancer.

BACKGROUND: New and fully validated tests need to be brought into clinical practice to improve the estimation of recurrence risk in patients with colon cancer. The aim of this study was to assess the analytical performances of the Immunoscore (IS) and show its contribution to prognosis prediction. METHODS: Immunohistochemical staining of CD3+ and CD8+ T cells on adjacent sections of colon cancer tissues were quantified in the core of the tumor and its invasive margin with dedicated IS modules integrated into digital pathology software. Staining intensity across samples collected between 1989 and 2016 (n=595) was measured. The accuracy of the IS workflow was established by comparing optical and automatic counts. Analytical precision of the IS was evaluated within individual tumor block on distant sections and between eligible blocks. The IS interlaboratory reproducibility (n=100) and overall assay precision were assessed (n=3). Contribution of the IS to prediction of recurrence based on clinical and molecular parameters was determined (n=538). RESULTS: Optical and automatic counts for CD3+ or CD8+ were strongly correlated (r=0.94, p<0.001 and r=0.92, p<0.001, respectively). CD3 and CD8 staining intensities were not altered by the age of the tumor block over a period of 30 years. Neither the position of tested tissue sections within a tumor block nor the selection of the tissue blocks affected the IS. Reproducibility of the IS was not affected by multiple variables (eg, antibody lots, DAB revelation kits, immunohistochemistry automates and operators). Interassay repeatability of the IS was 100% and interlaboratory reproducibility between two testing centers was 93%. Finally, in a case series of patients with stage II-III colon cancer, the relative proportion of variance for time to recurrence was greatest for the IS (53% of prognostic variability) in a model that included IS, T-stage, microsatellite instability status and total number of lymph nodes. CONCLUSION: IS is a robust and validated clinical assay leveraging immune scoring to predict recurrence risk of patient with localized colon cancer. The strong and independent prognostic value of IS should pave the way for it use in clinical practice.

The Influence of Neoadjuvant Treatment on the Number of Lymph Nodes on the Surgical Specimen in Mid and Low Rectal Cancer - A Retrospective Single-Centre Study.

Introduction: In this study, we aim to identify the impact of neoadjuvant radiation treatment upon the number of harvested and positive lymph nodes in the surgical specimen; in addition, we tried to identify the impact of chemotherapy in association with radiotherapy on said structures. Patients and methods: In the study we included patients treated for rectal cancer within a single oncologic surgical Unit serving the north-eastern part of Romania, over a period of 5 and a half years, between May 2013 and April 2018. Firstly, we compared pathologic lymph node status to pretherapeutic staging. Secondly, we compared lymph node values in relation to the treatment scheme. Results: There was a total of 498 patients treated radically through open surgery for low and mid rectal cancer. We saw a decrease in N staging in 218 cases, 65 remaining stationary and 10 increasing their lymph node staging on the surgical specimen. We identified significant differences between the total number of lymph nodes (17.4 vs 24.2, p 0.001), the number of positive lymph nodes (1.4 vs 3.4, p 0.001) and the ratio between positive and total lymph nodes (0.08 vs 0.14, p 0.001) in patients with and without neoadjuvant treatment respectively. However, there was no statistical difference between patients with and without chemotherapy associated to radiotherapy in the neoadjuvant treatment plan (p=0.539, p=0.58, p=0.575). Conclusion: This study shows there are significant variations according to the application of neoadjuvant treatment, between the numbers of positive and total lymph nodes, as well as the positive/total lymph node ratio.

Hindgut and Midgut Neuroendocrine Tumors - Therapeutic Approach.

Introduction: Neuroendocrine tumors of the gastro-entero-pancreatic system have a variety of components, clinical manifestations and prognostic indices according to their anatomical site. Therefore, their diagnostic and management strategies differ a great deal. Prognosis concerning NETs can be poor due to the degree of differentiation, early metastasizing and the high degree of invasiveness. Material and Methods: For the present study, the patient files were evaluated and the parameters of interest were followed. Results: Over the course of 6 years there were 37 patients diagnosed with and treated for NETs, regardless of primary tumor site. There were 9 patients with NETs of the primite mid- and hindgut thusly: 5 cases with colorectal NETs and 4 cases of small bowel NETs. 6 patients benefited from radical surgical treatment, 2 cases with palliative procedures and only one patient with tumor biopsy. The tumors were evaluated according to the 2010 WHO classification based on the number of mitoses and the Ki67 proliferation index. Adjuvant treatment was adapted according to staging and histopathological parameters. Conclusions: Despite recent progress in managing NETs, there are still many controversial aspects regarding the management of these cases, mainly about timing the right sequence of therapy.

Pelvic exenteration, a surgical treatment option for locally advanced, primary and recurrent neoplasia.

Pelvic exenteration (PE) is an extensive surgical procedure for locally advanced primary neoplasia (LAPN) or recurrent neoplasia (RN) that consists in the en bloc removal of the pelvic organs (rectum, internal genital organs and bladder) associated with pelvic lymph nodes. PE is classified into anterior, posterior and total, supra or infralevatorian approaches. Our aim was to evaluate the surgical procedure and the resection margins in correlation with postoperative complications and morbidity rates after PE in patients treated in a single surgical unit. The study group comprised patients diagnosed with different malignancies, surgically treated by using PE procedure, during 2012-2018. The cohort included 121 cases with LAPN (n=98, 80.99%) and RN (n=23, 19%), mostly female (n=114, 94.21%), with a mean age of 61.16 (33-85) years. LAPN had predominantly digestive (n=48, 49.98%) and gynecological (n=28, 28.57%) origins, while the majority of RN cases were cervical cancers (n=9, 39.13%). The univariate analysis showed that the gynecological origin of the tumor (p=0.02), urinary stoma (p=0.02) and posterior PE (PPE) (p=0.004) were significant prognostic factors for postoperative complications. After performing the multivariate analysis, only the gynecological origin (p=0.02) of the tumor and PPE (p=0.03) remained determining factors for postoperative complications. PE is a disabling surgical procedure associated with high postoperative mortality and morbidity, although it is often the only solution for advanced cases. The judicious selection of patients who can benefit from such extensive surgery is compulsory. Our study suggests that the gynecological origin of the tumor and PPE are key factors in postoperative complications.

Factors influencing the pathological quality of the surgical specimen in rectal cancer - a retrospective single-centre study.

AIM: The pathologist's role in the multidisciplinary treatment of rectal cancer is to evaluate and stage the tumor according to the latest standards, as well as indicate the quality of the surgical act. This study aims to evaluate circumferential and distal resection margins as well as quality of mesorectal resection and correlate them with different clinical, pathological and therapeutic factors. PATIENTS, MATERIALS AND METHODS: Four hundred ninety-eight patients treated radically for mid and low rectal cancer within one Clinic of Oncological Surgery in Iasi, Romania, were included in this study. RESULTS: The distal resection margin showed significant correlations with the type of surgical intervention, chemotherapy in the neoadjuvant treatment plan and pathological node staging. The circumferential resection margin depended mostly on pathological node staging and the length of the interval between neoadjuvant treatment and surgery. Finally, the aspect of the mesorectum varied according to neoadjuvant treatment and the type of surgical intervention performed. CONCLUSIONS: The study reached its aim in providing important data for the expected outcome of the specimen after curative treatment for rectal cancer.

Pathological aspects underlying pancreatogenous hyperinsulinemic hypoglycemia--report of three cases.

Pancreatogenous hyperinsulinemic hypoglycemia (PHH) is a rare disorder determined by an abnormally high secretion of insulin in the pancreas, in the absence of other medical or pharmacological factors. Either ß-cell tumors (insulinomas) or ß-cell hyperplasia (nesidioblastosis) can determine this pathology. Most publications on insulinomas or nesidioblastosis approached these subjects from a clinical point of view. This paper aims to analyze pathological aspects underlying pancreatogenous hyperinsulinemic hypoglycemia. We present two cases of insulinomas with unusual pancreatic localization and size, one of them showing amyloid deposits in the stroma. In both cases, immunohistochemistry confirmed the clinical and imagistic supposition. The third reported case refers to a 57-year-old patient with nesidioblastosis with isolated disposition of endocrine cells and areas of focal organization, both morphological aspects being extremely rare in adults. Although clinical and laboratory data are usually identical in the two forms of PHH, histopathological and immunohistochemical diagnosis is essential in differentiating insulinomas from nesidioblastosis, as the surgical management is different: enucleation for insulinomas and total or subtotal pancreatectomy for nesidioblastosis.

Prognostic and predictive values of the immunoscore in patients with rectal cancer.

PURPOSE: To determine whether the tumor immune infiltrate, as recently evaluated with the Immunoscore methodology, could be a useful prognostic marker in patients with rectal cancers. EXPERIMENTAL DESIGN: The influence of the immune infiltrate on patient's outcome was investigated in patients with or without preoperative chemoradiation therapy (pCRT). The density of total (CD3(+)) and cytotoxic (CD8(+)) T lymphocytes was evaluated by immunohistochemistry and quantified by a dedicated image analysis software in surgical specimens of patients with rectal cancer (n = 111) who did not receive pCRT and in tumor biopsies performed before pCRT from additional 55 patients. The results were correlated with tumor recurrence, patient's survival, and response to pCRT. RESULTS: The densities of CD3(+) and CD8(+) lymphocytes and the associated Immunoscore (from I0 to I4) were significantly correlated with differences in disease-free and overall survival (HR, 1.81 and 1.72, respectively; all P < 0.005). Cox multivariate analysis supports the advantage of the Immunoscore compared with the tumor-node-metastasis (TNM) staging in predicting recurrence and survival (all P < 0.001). Lymph node ratio added information in a prognostic model (all P < 0.05). In addition, high infiltration of CD3(+) and CD8(+) lymphocytes in tumor biopsies was associated with downstaging of the tumor after pCRT (CD3(+) cells; Fisher exact test P = 0.01). CONCLUSIONS: The Immunoscore could be a useful prognostic marker in patients with rectal cancer treated by primary surgery. The determination of the immune infiltrate in biopsies before treatment could be a valuable information for the prediction of response to pCRT.

Expression of bmi-1 protein in cervical, breast and ovarian cancer.

UNLABELLED: B-cell-specific Moloney murine leukemia virus integration site 1 (Bmi-l) is a member of polycomb group, which participates in axial patterning, hematopoiesis, cell cycle regulation, and senescence. Overexpression of Bmi-1 has been reported in various human cancers and proved to be associated with poor survival. DISSUCION: Bmi-I is expressed by various tumors and therefore may contribute to malignant transformation. Bmi-I not only can lead mammary epithelial cells to senescence and immortalization, but also plays a key role in breast cancer. A significant correlation was observed between Bmi-1 expression and axillary lymph node metastases in lymph-ductal breast cancer. Bmi-1 is expressed in cervical cancer and correlated with a poorer prognosis, suggesting that this protein participates in the development and progress of cervical cancer. Regarding ovarian cancer, the results of several immunohistochemical studies revealed overexpression of Bmi-1, especially in poorly differentiated ovarian carcinoma. There is a strong correlation between histological grade, clinical stage and its expression. CONCLUSIONS: Human genes of polycomb group correlated with various hematological and epithelial cancers identify new mechanisms of malignant transformation and pave the way for developing new cancer treatments and identify new diagnostic markers. Bmi-1 and its expression in tissues taken from patients with cervical, breast and ovarian cancer could be a marker for diagnosis and prognosis, and not least a potential target of antitumor therapy.