Sensitivity and specificity of loss of heterozygosity analysis for the classification of rare germline variants in BRCA1/2: results of the observational AGO-TR1 study (NCT02222883).

Variant-specific loss of heterozygosity (LOH) analyses may be useful to classify BRCA1/2 germline variants of unknown significance (VUS). The sensitivity and specificity of this approach, however, remains unknown. We performed comparative next-generation sequencing analyses of the BRCA1/2 genes using blood-derived and tumour-derived DNA of 488 patients with ovarian cancer enrolled in the observational AGO-TR1 trial (NCT02222883). Overall, 94 pathogenic, 90 benign and 24 VUS were identified in the germline. A significantly increased variant fraction (VF) of a germline variant in the tumour indicates loss of the wild-type allele; a decreased VF indicates loss of the variant allele. We demonstrate that significantly increased VFs predict pathogenicity with high sensitivity (0.84, 95% CI 0.77 to 0.91), poor specificity (0.63, 95% CI 0.53 to 0.73) and poor positive predictive value (PPV; 0.71, 95% CI 0.62 to 0.79). Significantly decreased VFs predict benignity with low sensitivity (0.26, 95% CI 0.17 to 0.35), high specificity (1.0, 95% CI 0.96 to 1.00) and PPV (1.0, 95% CI 0.85 to 1.00). Variant classification based on significantly increased VFs results in an unacceptable proportion of false-positive results. A significantly decreased VF in the tumour may be exploited as a reliable predictor for benignity, with no false-negative result observed. When applying the latter approach, VUS identified in four patients can now be considered benign. Trial registration number NCT02222883.

Optimized PD-L1 scoring of gastric cancer.

BACKGROUND: PD-1/PD-L1-Immunotherapy has been approved for gastric carcinoma. PD-L1 assessment by immunohistochemistry is the principle biomarker. Are biopsies able to map the actual PD-L1 status of the entire tumor? METHODS: Whole tumor slides of 56 gastric carcinoma were analyzed to determine the distribution of PD-L1 positive cells in the entire tumor areas. Tissue micro arrays with four cores of the tumor surface, which represents the endoscopically accessible biopsy zone, were built from the same tumors. The PD-L1 CPS value was determined separately for each core. Preoperative diagnostic biopsies were available for 22 of the tumors. PD-L1 prevalence, sensitivity and specificity were analyzed using the whole tumor slides as reference scores. Molecular subtyping was performed and related to the PD-L1 status. RESULTS: 27.3% of cases were PD-L1 negative (CPS < 1), 43.6% showed low PD-L1 expression (CPS ≥ 1 to < 5), 12.7% moderate (CPS ≥ 5 to < 10) and 16.4% strong expression (CPS ≥ 10). The biopsies showed best test characteristics if four surface biopsies were analyzed combined, i.e., the CPS was calculated across all four biopsies. The prevalence showed a distribution similar to the resection specimens, sensitivity was 0.73 and specificity 1.0. Using fewer surface biopsies decreased sensitivity and specificity and caused false-negative classifications. Compared to the TMAs, the preoperative biopsies showed reduced sensitivity (0.412). CONCLUSIONS: This is the first comprehensive study to optimize PD-L1 assessment in gastric cancer using endoscopically available tissue. The obtained PD-L1 prevalence is consistent with data of current clinical studies. Calculation of the test characteristics shows that surface biopsies can be indicative of the true PD-L1 status based on the resection specimen. However, an adequate number of biopsies is required. In this study, n = 4 biopsies yielded best results.

Sex-specific prognostic effect of CD66b-positive tumor-infiltrating neutrophils (TANs) in gastric and esophageal adenocarcinoma.

BACKGROUND: Tumor-associated neutrophils (TANs) have recently been identified as a relevant component of the tumor microenvironment (TME) in solid tumors. Within the TME TANs mediate either tumor-promoting or tumor-inhibiting activities. So far, their prognostic relevance remains to be determined. This study aims to evaluate the prognostic relevance of TANs in different molecular subtypes of gastric and esophageal adenocarcinoma. METHODS: We analyzed a total of 1118 Caucasian patients divided into gastric adenocarcinoma (n = 458) and esophageal adenocarcinoma cohort (n = 660) of primarily resected and neoadjuvant-treated individuals. The amount of CD66b + TANs in the tumor stroma was determined using quantitative image analysis and correlated to both molecular, as well as clinical data. RESULTS: An accumulation of TANs in the tumor stroma of gastric carcinomas was associated to a significant favorable prognosis (p = 0.026). A subgroup analysis showed that this effect was primarily related to the molecular chromosomal instable subtype (CIN) of gastric carcinomas (p = 0.010). This was only observed in female patients (p = 0.014) but not in male patients (p = 0.315). The same sex-specific effect could be confirmed in adenocarcinomas of the esophagus (p = 0.027), as well as in female individuals after receiving neoadjuvant therapy (p = 0.034). CONCLUSIONS: Together, we show a sex-specific prognostic effect of TANs in gastric cancer within a Caucasian cohort. For the first time, we showed that this sex-specific prognostic effect of TANs can also be seen in esophageal cancer.

Amplification of KRAS and its heterogeneity in non-Asian gastric adenocarcinomas.

BACKGROUND: Gastric cancer is one of the deadliest cancer entities worldwide. While surgery is the only curative treatment option in early tumors, for locally advanced and metastatic patients further therapeutic targets are needed. Several studies not only reported mutations but also amplifications of the KRAS locus in different cancer entities. More recently, KRAS amplification was discussed as a new therapeutic target. Little is known about the (prognostic) relevance and (heterogenic) distribution of KRAS amplification in gastric adenocarcinomas, especially in Non-Asian patients. METHODS: Amplification of the KRAS locus and corresponding protein expression was analyzed in 582 gastric adenocarcinomas employing fluorescence in-situ hybridization (FISH) and immunohistochemistry. Amplification status was correlated with clinico-pathological features, clinical outcome and molecular tumor data including a correlation to the TCGA subtypes of gastric carcinoma. RESULTS: KRAS amplification was detected in 27 out of 470 analysable tumors (5.7%) and correlated with protein expression of KRAS in all amplified tumors. Within the KRAS amplified gastric tumors 14/27 (51.9%) showed a heterogeneous distribution with also KRAS non-amplified tumor parts. According to TCGA 24 tumors (88.8%) were related to chromosomal instable tumors (CIN). The survival analysis of the entire patient cohort did not show any difference in overall survival in dependence on the KRAS status. However, a significant survival difference with a worse outcome for patients with KRAS amplified tumors was identified when analysing patients without neoadjuvant pre-treatment. CONCLUSIONS: We confirm the unfavorable prognosis of KRAS amplified tumors reported by other studies in (Asian) patient groups, at least in patients without neoadjuvant pre-treatment. Within KRAS amplified tumors we revealed intratumoral heterogeneity that may define a (more aggressive) tumor cell population which is more frequently observed in patients with lymph node metastases. Despite the heterogeneous distribution of KRAS amplified tumor clones, KRAS amplified locally advanced or metastasized gastric adenocarcinomas represent a therapeutically highly relevant tumor subgroup.

Deleterious somatic variants in 473 consecutive individuals with ovarian cancer: results of the observational AGO-TR1 study (NCT02222883).

BACKGROUND: For individuals with ovarian cancer (OC), therapy options mainly depend on BRCA1/2 germline status. What is the prevalence of deleterious somatic variants, that is, does genetic tumour testing identify subgroups of individuals who also might benefit from targeted therapy? METHODS: Paired analysis of tumour-derived versus blood-derived DNA to determine the prevalence of deleterious somatic variants in OC predisposition genes (ATM, BRCA1/2, BRIP1, MSH2/6, PALB2, RAD51C/D and TP53) and the PIK3CA and PTEN genes in individuals with OC (AGO-TR1 study, NCT02222883). Results were complemented by BRCA1, PALB2 and RAD51C promoter methylation analyses and stratified by histological subtype; 473 individuals were included. RESULTS: The combined analyses revealed that deleterious germline variants in established OC predisposition genes (all: 125/473, 26.4%; BRCA1/2: 97/473, 20.5%), deleterious somatic variants in established OC predisposition genes excluding TP53 (all: 39/473, 8.2%; BRCA1/2: 30/473, 6.3%) and promoter methylation (all: 67/473, 14.2%; BRCA1: 57/473, 12.1%; RAD51C: 10/473, 2.1%; PALB2: 0/473) were mutually exclusive, with a few exceptions. The same holds true for deleterious somatic PIK3CA and/or PTEN variants (33/473, 7.0%) found to be enriched in endometrioid and clear cell OC (16/35, 45.7%); 84.3 % of the deleterious single-nucleotide/indel germline variants in established OC predisposition genes showed significantly higher variant fractions (VFs) in the tumour-derived versus blood-derived DNA, indicating a loss of the wild-type alleles. CONCLUSION: Tumour sequencing of the BRCA1, BRCA2, PIK3CA and PTEN genes along with BRCA1 and RAD51C promoter methylation analyses identified large subgroups of germline mutation-negative individuals who may be addressed in interventional studies using PARP or PI3K/AKT/mTOR inhibitors. TRIAL REGISTRATION NUMBER: NCT02222883.

Tumour area infiltration and cell count in endoscopic biopsies of therapy-naive upper GI tract carcinomas by QuPath analysis: implications for predictive biomarker testing.

Guidelines regulate how many (tumour-bearing) tissue particles should be sampled during gastric cancer biopsy to obtain representative results in predictive biomarker testing. Little is known about how well these guidelines are applied, how the number of tissue particles correlates with the actual tumour-infiltrated area and how many absolute tumour cells are captured. The study included endoscopic biopsies of untreated carcinomas of the upper gastrointestinal (GI)-tract during the 2016-2020 review period. Archival (H&E)-stained histological sections were digitised and the tumour areas were manually annotated. The tumour-bearing tissue area and absolute carcinoma cell count per case were determined by image analysis and compared with a reference primary surgical specimen. Biopsies from 253 patients were analysed. The following mean values were determined: (a) tumour tissue particle number: 6.5 (range: 1-25, standard deviation (SD) = 3.33), (b) number of tumour-bearing tissue particles: 4.7 (range: 1-20, SD = 2.80), (c) tumour-infiltrated area: 7.5 mm2 (range: 0.18-59.46 mm2, SD = 6.67 mm2), (d) absolute tumour cell count: 13,492 (range: 193-92,834, SD = 14,185) and (e) tumour cell count in a primary surgical specimen (tumour size: 6.7 cm): 105,200,176. The guideline-recommended tissue particle count of 10 was not achieved in 208 patients (82.2%) and the required tumour-bearing tissue particle count of 5 was not achieved in 133 patients (52.6%). Tissue particle count, tumour-infiltrated area and tumour cell count were only weakly correlated. Most cases featured an infiltrated area ≥ 4.5 mm2 (156, 61.7%). Cases with more tissue particles showed only a moderate increase in infiltrated area and tumour cells compared to cases with fewer particles. Biopsies are often used to determine predictive biomarkers, particularly Her2/neu and PD-L1. Diagnostic standards to ensure representative material have been suggested in guidelines to reduce false-negative predictions. However, the real-world practice seems to substantially deviate from recommended standards. To the best of our knowledge, this is the first systematic study describing the relationships between endoscopic tissue fragment number, actual infiltrated tumour area and carcinoma cell number. The data question the tissue particle number as a quality assessment parameter. We advocate histopathological reports indicating on which basis statements on therapy-relevant biomarkers were made. Digital pathology has the potential to objectively quantify the tissue for documentation, quality assessment and future clinical studies.

Microsatellite instability and sex differences in resectable gastric cancer - A pooled analysis of three European cohorts.

OBJECTIVE: Biological sex differences in cancer are increasingly acknowledged. Here, we examined these differences in clinicopathological characteristics and survival in microsatellite instability (MSI)-high and microsatellite stable (MSS) gastric cancer (GC). DESIGN: We analysed MSI status by polymerase chain reaction (PCR) and/or mismatch repair (MMR) status by immunohistochemistry in a pooled analysis of individual patient data from one retrospective cohort from Cologne, and the randomised phase III clinical trials D1/D2 and CRITICS. All patients had resectable adenocarcinoma of the stomach and/or gastro-oesophageal junction. Patients were treated with either surgery only or perioperative chemo(radio)therapy. RESULTS: MSI and/or MMR analyses on 1307 tumours resulted in 1192 (91.2%) MSS and/or MMR proficient (MMRP) [median age, 65 years; 759 males (63.7%); 619 treated with surgery only (51.9%)], and 115 (8.8%) MSI-high [median age, 69 years; 67 males (58.3%); 76 treated with surgery only (66.1%)] GC cases. Males had shorter overall survival (OS) than female MSI-high GC (5-year OS 34.7% vs. 69.7%; hazard ratio (HR) 2.68, 95%CI 1.60 to 4.49; p < 0.001). Females with MSI-high had longer OS than those with MSS/MMRP GC (HR 0.61, 95%CI 0.41 to 0.92; p = 0.02). Males with MSI-high did not have longer OS than those with MSS/MMRP GC (HR 1.26, 95%CI 0.94 to 1.69; p = 0.12). CONCLUSIONS: MSI-high GC males had a significantly worse prognosis compared to their female counterparts in three independent cohorts. In addition, the favourable prognostic value of MSI was only seen in females and not in males. These observations emphasise the need to consider sex differences in prognosis and treatment effects in oncology. CLINICAL TRIAL REGISTRATION: The CRITICS trial is registered at ClinicalTrials.gov, number NCT00407186; EudraCT, number 2006-004130-32; and CKTO, 2006-02.

First description of hematogenously metastasized sclerosing epithelioid fibrosarcoma arising in the uterine cervix.

•First Case of hematogenously metastasized sclerosing epithelioid fibrosarcoma arising primarily in the cervix uteri.•Tumor cells were strongly and diffusely positive for MUC4.•Tumor showed a rare EWSR1-CREB3L2 gene fusion.

Occurrence of High Microsatellite-Instability/Mismatch Repair Deficiency in Nearly 2,000 Human Adenocarcinomas of the Gastrointestinal Tract, Pancreas, and Bile Ducts: A Study From a Large German Comprehensive Cancer Center.

INTRODUCTION: Knowledge of the high microsatellite-instability (MSI-H)/mismatch repair deficiency (MMRd) status is of increasing interest for personalized neoadjuvant or adjuvant therapy planning. Only a few studies are available on MSI-H distribution in the Northern European Caucasian patient population. In this study, we focused on a large cohort of tumors of the upper gastrointestinal tract. MATERIALS AND METHODS: Surgical material from a total of 1,965 patients was analyzed for MSI-H/MMRd status (including 1,267 carcinomas of the esophagus or stomach). All tumors were analyzed with an internationally recommended immunohistochemical panel consisting of four antibodies (MLH1, MSH2, PMS2, and MSH6). The results were molecularly objectified. RESULTS: Adenocarcinomas with MSI-H/MMRd were detected with the following distribution: esophagus (1.4%), stomach (8.3%), small intestine (18.2%), large intestine (8.5%), intrahepatic bile ducts (1.9%), and pancreas (0%). In case of gastric tumors with MSI-H/MMRd, neoadjuvant therapy did not influence the prognosis of patients (p = 0.94). Within all tumor entities with MSI-H/MMRd, patients with a UICC stage 4 were also represented. In this advanced stage, 11.7% of patients with MSS tumors were diagnosed compared to 0.5% of patients with MSI-H tumors relative to the entire tumor collective. DISCUSSION: In this study, the proportion of MSI-H/MMRd tumors in the stomach is smaller than would have been expected in knowledge of the data published by TCGA or AGRC. Negative prognostic effects regarding MSI-H status and neoadjuvant therapy as described by the MAGIC study group were not seen in our cohort. The extent to which the MSI-H/MMRd status should be known for neoadjuvant therapy planning must be clarified in prospective studies in the future. At present, there is no convincing data to dispense the neoadjuvant therapy for gastric carcinoma. Due to the very convincing, positive data regarding the response rates of MSI-H tumors to treatment with PD1/PD-L1 inhibitors, every metastatic carcinoma of the gastrointestinal tract should be tested for its MSI-H status.