Intratumoral heterogeneity affects tumor regression and Ki67 proliferation index in perioperatively treated gastric carcinoma.

BACKGROUND: Intratumoral heterogeneity (ITH) is a major problem in gastric cancer (GC). We tested Ki67 and tumor regression for ITH after neoadjuvant/perioperative chemotherapy. METHODS: 429 paraffin blocks were obtained from 106 neoadjuvantly/perioperatively treated GCs (one to five blocks per case). Serial sections were stained with Masson's trichrome, antibodies directed against cytokeratin and Ki67, and finally digitalized. Tumor regression and three different Ki67 proliferation indices (PI), i.e., maximum PI (KiH), minimum PI (KiL), and the difference between KiH/KiL (KiD) were obtained per block. Statistics were performed in a block-wise (all blocks irrespective of their case-origin) and case-wise manner. RESULTS: Ki67 and tumor regression showed extensive ITH in our series (maximum ITH within a case: 31% to 85% for KiH; 4.5% to 95.6% for tumor regression). In addition, Ki67 was significantly associated with tumor regression (p < 0.001). Responders (<10% residual tumor, p = 0.016) exhibited prolonged survival. However, there was no significant survival benefit after cut-off values were increased ≥20% residual tumor mass. Ki67 remained without prognostic value. CONCLUSIONS: Digital image analysis in tumor regression evaluation might help overcome inter- and intraobserver variability and validate classification systems. Ki67 may serve as a sensitivity predictor for chemotherapy and an indicator of ITH.

Exploration of the tumor biological significance of PCLO in gastric cancer - Results from a large Central European cohort.

INTRODUCTION: A recent multiregional whole exome sequencing of 48 tumour samples from 9 gastric adenocarcinomas discovered PCLO mutations in 23 (47.9%) tumour samples. Based on that unexpected high prevalence of PCLO mutations, we hypothesized a tumour biological significance of PCLO in gastric cancer (GC). METHODS: Tumour samples (whole tissue sections) obtained from 466 patients resected for therapy-naive GC were stained with an anti-PCLO antibody. The Histoscore for tumour cells and the presence of immunostaining of stromal cells and tumour vessels was docu-mented for each case. An algorithm for PCLO immunopositivity was formed and correlat-ed with clinicopathological patient characteristics. RESULTS: 175 GCs were classified as PCLO-positive within tumour cells, and 291 as negative. Stromal cells were positive for PCLO in 106 cases and tumour vessels in 84. PCLO positive GCs more often showed an intestinal phenotype, a lower T-category and were more commonly associated with Helicobacter pylori-infection. A separate analysis of PCLO ex-pression in intestinal and diffuse type GCs, respectively, showed no significant correla-tions. Patients with PCLO negative/low tumour cells showed a shortened overall (14.0±1.4 vs. 16.0±1.8 months) and tumour specific survival (15.0±1.6 months vs. 17.9±3.6). Compar-ison of PCLO's genotype with its phenotype in 48 tumour samples obtained from nine cases showed no direct correlations with missense mutations. DISCUSSION/CONCLUSION: Our data provide evidence that PCLO is differentially expressed in GC and might delay tumour progression.

The putative pleiotropic functions of meprin ß in gastric cancer.

BACKGROUND: The gastric microbiome and inflammation play a key role in gastric cancer (GC) by regulating the immune response in a complex manner and by inflammatory events supporting carcinogenesis. Meprin ß is a zinc endopeptidase and participates in tissue homeostasis, intestinal barrier function and immunological processes. It influences local inflammatory processes, dysbiosis and the microbiome. Here, we tested the hypothesis that meprin ß is expressed in GC and of tumor biological significance. PATIENTS AND METHODS: Four hundred forty whole mount tissue sections of patients with therapy-naive GC were stained with an anti-meprin ß antibody. The histoscore and staining pattern were analyzed for each case. Following dichotomization at the median histoscore into a "low" and "high" group, the expression was correlated with numerous clinicopathological patient characteristics. RESULTS: Meprin ß was found intracellularly and at the cell membrane of GC. Cytoplasmic expression correlated with the phenotype according to Lauren, microsatellite instability and PD-L1 status. Membranous expression correlated with intestinal phenotype, mucin-1-, E-cadherin-, ß-catenin status, mucin typus, microsatellite instability, KRAS mutation and PD-L1-positivity. Patients with cytoplasmic expression of meprin ß showed a better overall and tumor-specific survival. CONCLUSIONS: Meprin ß is differentially expressed in GC and has potential tumor biological relevance. It might function as a tumor suppressor or promotor depending on histoanatomical site and context.

Deep learning-based subtyping of gastric cancer histology predicts clinical outcome: a multi-institutional retrospective study.

INTRODUCTION: The Laurén classification is widely used for Gastric Cancer (GC) histology subtyping. However, this classification is prone to interobserver variability and its prognostic value remains controversial. Deep Learning (DL)-based assessment of hematoxylin and eosin (H&E) stained slides is a potentially useful tool to provide an additional layer of clinically relevant information, but has not been systematically assessed in GC. OBJECTIVE: We aimed to train, test and externally validate a deep learning-based classifier for GC histology subtyping using routine H&E stained tissue sections from gastric adenocarcinomas and to assess its potential prognostic utility. METHODS: We trained a binary classifier on intestinal and diffuse type GC whole slide images for a subset of the TCGA cohort (N = 166) using attention-based multiple instance learning. The ground truth of 166 GC was obtained by two expert pathologists. We deployed the model on two external GC patient cohorts, one from Europe (N = 322) and one from Japan (N = 243). We assessed classification performance using the Area Under the Receiver Operating Characteristic Curve (AUROC) and prognostic value (overall, cancer specific and disease free survival) of the DL-based classifier with uni- and multivariate Cox proportional hazard models and Kaplan-Meier curves with log-rank test statistics. RESULTS: Internal validation using the TCGA GC cohort using five-fold cross-validation achieved a mean AUROC of 0.93 ± 0.07. External validation showed that the DL-based classifier can better stratify GC patients' 5-year survival compared to pathologist-based Laurén classification for all survival endpoints, despite frequently divergent model-pathologist classifications. Univariate overall survival Hazard Ratios (HRs) of pathologist-based Laurén classification (diffuse type versus intestinal type) were 1.14 (95% Confidence Interval (CI) 0.66-1.44, p-value = 0.51) and 1.23 (95% CI 0.96-1.43, p-value = 0.09) in the Japanese and European cohorts, respectively. DL-based histology classification resulted in HR of 1.46 (95% CI 1.18-1.65, p-value < 0.005) and 1.41 (95% CI 1.20-1.57, p-value < 0.005), in the Japanese and European cohorts, respectively. In diffuse type GC (as defined by the pathologist), classifying patients using the DL diffuse and intestinal classifications provided a superior survival stratification, and demonstrated statistically significant survival stratification when combined with pathologist classification for both the Asian (overall survival log-rank test p-value < 0.005, HR 1.43 (95% CI 1.05-1.66, p-value = 0.03) and European cohorts (overall survival log-rank test p-value < 0.005, HR 1.56 (95% CI 1.16-1.76, p-value < 0.005)). CONCLUSION: Our study shows that gastric adenocarcinoma subtyping using pathologist's Laurén classification as ground truth can be performed using current state of the art DL techniques. Patient survival stratification seems to be better by DL-based histology typing compared with expert pathologist histology typing. DL-based GC histology typing has potential as an aid in subtyping. Further investigations are warranted to fully understand the underlying biological mechanisms for the improved survival stratification despite apparent imperfect classification by the DL algorithm.

Gastric Carcinomas with Stromal B7-H3 Expression Have Lower Intratumoural CD8+ T Cell Density.

CD8+ T cells are the main effector cells of anti-cancer immune response that can be regulated by various costimulatory and coinhibitory molecules, including members of the B7 family. B7 homolog 3 (B7-H3) appears as a promising marker for immunotherapy; however, its significance in gastric cancer (GC) is unclear yet. We evaluated the spatial distribution of CD8+ T cells in relation to the expression of B7-H3 by double immunohistochemical staining. The level of B7-H3 intensity was scored manually (0-3) and dichotomized into B7-H3-low and B7-H3-high groups. The distribution and density of CD8+ T cells was analysed using whole slide digital imaging. B7-H3 was expressed mainly in the stromal compartment of GC (n = 73, 76% of all cases). Tumours with high expression of B7-H3 showed larger spatial differences of CD8+ T cells (86.4/mm2 in tumour centre vs. 414.9/mm2 in invasive front) when compared to B7-H3-low group (157.7/mm2 vs. 218.7/mm2, respectively) (p < 0.001). This study provides insight into the expression pattern of B7-H3 in GC of Western origin. In GCs with higher level of B7-H3 expression, CD8+ T cells were spatially suppressed in the tumour centre suggesting that B7-H3 might be involved in tumour escape mechanisms from the immune response.

Prognostic significance of tumour budding assessed in gastric carcinoma according to the criteria of the International Tumour Budding Consensus Conference.

AIMS: In this study, we aimed to independently evaluate the utility and prognostic value of tumour budding (TB) according to the International Tumour Budding Consensus Conference (ITBCC) criteria in a large and a well-characterised European gastric cancer (GC) cohort. METHODS AND RESULTS: In 456 consecutive, surgically treated GCs, TB was assessed according to the ITBCC criteria and scored as Bd0 (no buds), Bd1 (one to four buds), Bd2 (five to nine buds) or Bd3 (≥10 buds). Cases with TB present were divided into low- (Bd1/Bd2) and high-budding (Bd3) groups. The TB score was analysed in relation to the clinicopathological parameters, overall survival (OS) and tumour-specific survival (TSS); 115 (25.2%) cases had no, 104 (22.8%) had low and 237 (52.0%) had high TB. The TB score correlated significantly with sex, Laurén phenotype, pT-, pN- and M categories, histological grade, R status; and lymph node ratio, lymphatic invasion, perineural invasion and HER2-, MET- and MSI status. In both total and intestinal-type early invasive GC (n = 57 and n = 41, respectively), significant associations between the presence and extent of TB and presence of lymph node metastasis were detected. Significant differences in OS and TSS between the TB groups were found; however, TB did not retain significance in multivariate models. CONCLUSIONS: Our data show that the ITBCC criteria can be applied to GC. The data correlated significantly with the diverse clinicopathological characteristics, including patient outcome, and can help to standardise diagnostics and research into special histological features of malignant tumours in general and GC in particular.

Questioning the IGF1 receptor's assigned role in CRC - a case for rehabilitation?

BACKGROUND: The insulin-like growth factor 1 receptor (IGF1R) is suspected to be involved in colorectal carcinogenesis and has been associated with worse survival in colorectal cancer (CRC). We hypothesized that the alleged suspect might be in truth beyond any suspicion. We investigated if the expression of the IGF1R in CRC correlates with (1) clinicopathological patient characteristics, including survival, and hence is involved in colon cancer biology; (2) the expression of the IGF1R in CRC is linked to the expression of the insulin receptor (IR). METHODS: We evaluated 4497 CRC samples from 1499 patients for the expression of IGF1R in tumor cells by immunohistochemistry. Cytoplasmic (cCC-IGF1R) and membranous (mCC-IGF1R) immunostaining was evaluated by employing a modified HistoScore (HScore), which was dichotomized into low or high IGF1R expressions. The IGF1R status was correlated with clinicopathological patient characteristics, survival and the IR expression status. RESULTS: cCC-IGF1R and mCC-IGF1R (HScore> 0) were found in 85.4 and 60.8% of all CRCs. After dichotomization of the HScores, 54.9 and 48.6% were classified as cCC-IGF1R-high and mCC-IGF1R-high, respectively. IGF1R was associated with tumor localization, local tumor growth, lymphatic vessel invasion, grading, mismatch repair protein expression status and IR-expression. We found no significant association with overall or tumor-specific survival, with a tendency for an even improved overall survival for cCC-IGF1R. CONCLUSIONS: IGF1R expression is frequent and biologically relevant in CRC, but does not correlate with patient survival. The IGF1R might be beyond suspicion in CRC after all.

The expression of the insulin receptor in gastric cancer correlates with the HER2 status and may have putative therapeutic implications.

BACKGROUND: Metabolic reprogramming in gastric cancer (GC) involves not only an alteration of glucose metabolism, but also of insulin receptor (IR) expression. We investigated if (1) GCs express the IR in cancer cells (CC-IR) and vasculature (VIR), (2) IR expression is clinically relevant and may be a novel target of GC treatment. METHODS: 467 primary GCs were studied by immunohistochemistry using an IR-specific antibody. CD31-immunostaining ensured the presence of representative intratumoral microvasculature. VIR, and membranous and cytoplasmic CC-IR (mCC-IR, cCC-IR) were evaluated using a modified HistoScore (HScore) and subsequently dichotomized into low or high IR expressions. The IR status was correlated with clinico-pathological patient characteristics, including survival and HER2 status. RESULTS: VIR, mCC-IR, and cCC-IR (HScore > 0) were found in 97.0%, 87.6%, and 95.7% of all GCs. After dichotomization of the HScores, 50.7, 48.8, and 50.3% were classified as VIR-high, mCC-IR-high, and cCC-IR-high, respectively. IR was associated with the Laurén phenotype, tumor localization, local tumor growth, vascular invasion, perineural invasion, tumor budding, mucin phenotype, UICC stage, worse survival, and the HER2 status. On multivariate analysis, VIR status was an independent prognosticator of overall (p = 0.010) and tumor-specific (p = 0.006) patient survival. CONCLUSIONS: VIR and CC-IR expressions are frequent in GC, biologically significant and even correlate with the HER2 status, opening avenues for novel putative therapeutic interventions in GC.

MET in gastric cancer--discarding a 10% cutoff rule.

AIMS: We aimed to develop a putative predictive biomarker score for future hepatocyte growth factor receptor (MET)-targeted therapy of gastric cancer (GC). METHODS AND RESULTS: MET expression and MET amplification were analysed by immunohistochemistry (IHC) and chromogenic in-situ hybridization (CISH) in 470 GC patients. Immunostaining was documented with the HistoScore. The percentage area of MET-amplified tumour cell clones was assessed by virtual microscopy. The expression of MET was heterogeneous in primary and metastatic GC. Immunostaining intensity (MET-IHC 2+/3+) correlated with MET amplification and a positive MET status was defined by a combination of MET-IHC 2+ or 3+ with MET amplification, or MET-IHC 3+ without MET amplification. The prognostic significance of the MET status was independent from the percentage area of positive tumour cells (e.g. <10 versus ≥10%). MET-positive GCs were microsatellite stable and of KRAS/PIK3CA wild-type. MET-positive GCs had a very poor prognosis, with a median survival of 5.4 months and a hazard ratio of 2.126. CONCLUSIONS: A combination of immunohistochemistry and CISH is suitable to assess MET status. If MET status is used as a predictive biomarker, prospective studies should pay specific attention to adequate tissue sampling, should ignore cutoff values for tumour areas, may consider the KRAS and PIK3CA genotype as negative predictive markers and should carry out the analysis expeditiously.

Ki67--An unsuitable marker of gastric cancer prognosis unmasks intratumoral heterogeneity.

BACKGROUND AND OBJECTIVES: Due to contradictious findings of previous studies regarding Ki67's value in gastric cancer (GC), we reevaluated the expression of Ki67 in whole tissue sections (WTS) and tissue microarrays (TMAs) of GC testing the following hypotheses: does Ki67 show intratumoral heterogeneity; are TMAs representative in the determination of the Ki67 proliferation index (PI); is the Ki67 PI subject to an intralaboratory variability; and is the Ki67 PI related to clinico-pathological patient characteristics and/or prognostically relevant in GC. METHODS: Corresponding WTS and TMAs samples from 315 GCs were stained immunohistochemically. The Ki67 PI evaluated on WTS was correlated with the Ki67 PI evaluated on TMAs, sample age, clinico-pathological characteristics, and patient survival. RESULTS: The overall amount of Ki67-positive tumor cells did not depend on sample age. Three distinct, partly heterogeneous Ki67 expression patterns were observed. The mean Ki67 PI evaluated on TMAs differed on average minus 16.9% from the Ki67 PI evaluated on WTS. Ki67 in WTS correlated significantly with the Laurén phenotype and tumor grade, but not with patient survival. CONCLUSION: TMAs carry the risk of a systematic underestimation of the Ki67 PI. Ki67 has no prognostic value in GC but might be a potential indicator of intratumoral heterogeneity.

Integrins αvß3 and αvß5 as prognostic, diagnostic, and therapeutic targets in gastric cancer.

BACKGROUND: We investigated the expression of two αv integrins, αvß3 and αvß5, in gastric cancer (GC) by testing the following hypotheses: that these molecules are expressed in GC; that they are implicated in GC biology; that they help to distinguish between the two major histological subtypes of GC, according to Laurén; and that they are prognostically relevant. METHODS: Formalin-fixed and paraffin-embedded tissue samples from 482 GC samples were stained immunohistochemically using rabbit monoclonal antibodies directed against αvß3 (EM22703) and αvß5 (EM09902). Immunostaining of tumor, stroma, and endothelial cells was evaluated separately by the quantity and intensity, generating an immunoreactivity score. The immunoreactivity score of both antibodies was correlated with clinicopathology data and patient survival. RESULTS: Each integrin was expressed in at least one tumor component in all GCs. Both were expressed significantly more often in the intestinal phenotype according to Laurén. Moreover, patients who grouped as "positive" for expression of αvß3 on endothelial cells, and patients with an intestinal type GC, grouped as "negative" for expression of αvß5 on stroma cells, had significantly longer survival. The expression of αvß5 on stroma cells was confirmed to be an independent prognostic factor of intestinal-type GC. CONCLUSION: The expression of αvß3 and αvß5 in at least one tumor component in all GC samples is an interesting new result that should form a basis for further investigations; for example, regarding selective integrin antagonists and the value of αvß3 and αvß5 as putative prognostic biomarkers. Moreover, both markers might be helpful in the routine classification of GC subtypes.

Correlation between the expression of integrins in prostate cancer and clinical outcome in 1284 patients.

The aim of this study was to investigate the expression of a panel of integrins in prostate cancer in order to explore their potential for tumor biology. Formalin-fixed and paraffin-embedded tissue samples of 1284 prostate cancer patients were retrieved from the archive of the Department of Pathology. Immunostaining was done with rabbit monoclonal antibodies directed against αvß3, αvß5, αvß6, αvß8, ß3, and αv-pan. Staining results were correlated with clinicopathologic patient characteristics and patient survival. Immunostaining of tumor cells performed on whole tissue sections of 52 patients was sparse for αvß3, αvß6, and αvß8, and more prevalent for αvß5 and αv-pan. αvß5, αvß8, and αv-pan were selected for further analyses in tissue microarrays representing the entire study cohort. αvß8 staining was generally observed in peripheral nerves. αvß5 and αv-pan provided strong evidence for the differential expression of these integrins in prostate cancer. The expression was variable with regard to the histoanatomical/cytoanatomical localization, cell type, intensity of immunolabeling, and Gleason pattern. αvß5 and αv-pan are differentially expressed in prostate cancer, and the differentiation of prostate cancer seems to influence integrin expression and subcellular distribution.

Stroma AReactive Invasion Front Areas (SARIFA) predict poor survival in adenocarcinomas of the stomach and gastrooesophageal junction: a validation study.

Recently, the presence of "Stroma AReactive Invasion Front Areas" (SARIFA) has been described as a promising adverse prognostic factor in gastric cancer. However, the validity of this approach still needs to be tested. The aim of this study was to independently assess the utility of the proposed method in a well-characterised cohort of primary resected adenocarcinomas of stomach and gastrooesophageal junction (n = 392). SARIFA status was analysed on routine slides of resection specimens. Cases were divided into SARIFA-positive and negative groups and analysed in relation to clinicopathological and survival data. SARIFA positivity was found in 15.1% (n = 59) cases and was significantly associated with Lauren phenotype (p < 0.001), pT (p = 0.001), pN (p = 0.018), UICC stage (p = 0.031), tumour budding (p = 0.002), overall survival (p < 0.001) and cancer-specific survival (p < 0.001). SARIFA-positive tumours had a worse prognosis in the multivariate setting (HR = 1.847, 95% CI: 1.300-2.624, p = 0.001). SARIFA status is an independent prognostic factor in gastric cancer, in particular in locally advanced tumours.

The tumor biological significance of RNF43 and LRP1B in gastric cancer is complex and context-dependent.

Gastric cancer (GC) is the fifth most common cancer in the world with a poor prognosis. Both RNF43 and LRP1B function as tumor suppressors in the Wnt signaling pathway and have been described to be frequently mutated in GC. In this study of a large and well characterized cohort of 446 GCs we explored the significance of expression of RNF43 and LRP1B and their correlations with clinicopathological patient characteristics. Immunostaining of whole mount tissue sections was documented with the histoscore. Dichotomized at the median, we separated the cohort into a low/negative and a high/positive group of RNF43 and LRP1B expression, respectively. Apart from the entire cohort, we also examined the intestinal and diffuse type GCs separately. Regarding the entire cohort, the expression of RNF43 and LRP1B correlated significantly with the Lauren phenotype and with each other. Interestingly, differences were noted regarding RNF43 between the intestinal and diffuse type GCs. Survival analysis of the intestinal type GCs showed that RNF43 low/negative GCs tended to have a better outcome compared with RNF43 high/positive GCs [24.5 months overall survival (OS) and 25.0 months tumor-specific survival (TSS) vs. 14.1 months OS and 17.9 months TSS, respectively]. To the contrary, diffuse type GCs with RNF43 low/negative had a worse outcome compared with RNF43 high/positive GCs (12.9 months OS and 18.2 months TSS vs. 17.1 months OS and 21.5 months TSS, respectively). On multivariate analysis, RNF43 low/negative versus high/positive was an independent prognosticator of survival in diffuse type GC (hazard ratio 2.393 for OS and 2.398 for TSS). These data support the contention that the expression and biological effect of RNF43 and LRP1B in GC is context-dependent.

SMARCA4 and SMARCE1 in gastric cancer: Correlation with ARID1A, and microsatellite stability, and SMARCE1/ERBB2 co-amplification.

BACKGROUND: Recent studies have shown an association between certain subunits of the SWI/SNF complex with specific tumor characteristics in gastric cancer (GC). In an earlier study, we applied multiregional whole exome sequencing on multiple primary tumor samples and found alterations of the SWI/SNF complex in 78% of the cases. ERBB2, which encodes for Her2/neu, is a well-known predictive biomarker used to guide the treatment of GC in the palliative setting. SMARCE1, which encodes for a subunit of the SWI/SNF complex, is localized in close genetic proximity to ERBB2. AIM: As little is known about the significance of the SWI/SNF complex in GC biology and the potential relationship between ERBB2 and SMARCE1 upregulation, we examined the expression patterns of SMARCA4 and SMARCE1, two mutually exclusive catalytic ATPase subunits of the SWI/SNF complex, in a well characterized GC cohort. MATERIALS AND METHODS: The expression of SMARCA4 and SMARCE1 was studied by immunohistochemistry in connection with clinicopathological patient characteristics in a cohort of 468 GCs. Digital droplet polymerase chain reaction was performed for amplification analysis on ERBB2 and SMARCE1. RESULTS: Immunohistochemical staining of whole-mount tissue sections found a diffusely "gray scale" expression of SMARCA4 in 446 (95.2%) GCs and of SMARCE1 in 463 (98.8%) GCs. The expression of SMARCA4 and SMARCE1 correlated significantly with ARID1A, p53, and microsatellite status. No correlation was found with the patient prognosis. The amplification analysis of SMARCE1 showed amplification in 4 of 34 cases. In 3 of 34 cases, SMARCE1 was co-amplified with ERBB2. We also found a co-expression of SMARCE1 and Her2/neu in a subset of patients. CONCLUSION: While the effect of a co-amplification is currently unknown, synergistic effects of SMARCE1 and Her2/neu overexpression should be explored in future studies, holding potential for an improved treatment of GC.

MDM2 amplification is rare in gastric cancer.

The MDM2 proto-oncogene (MDM2) is a primary negative regulator of p53. The latter is frequently mutated in gastric cancer (GC). In the present study, we aimed to validate gene amplification, protein expression, and the putative tumor biological function of MDM2 in a well-characterized Western GC cohort. MDM2 amplification and protein expression were studied in a cohort of 327 GCs by fluorescence in situ hybridization (FISH) and immunohistochemistry. Gene amplification and protein expression were correlated with diverse clinicopathological patient characteristics including patient outcome. Immunohistochemically, 97 GCs (29.7%) were categorized as MDM2 positive and 230 GCs (70.3%) as negative. An amplification of MDM2 was found in 11 (3.4%) cases without evidence of intratumoral heterogeneity. Nine of these eleven (81.8%) cases showed MDM2 protein expression. MDM2 amplification correlated significantly with MDM2 protein expression (p < 0.001). On a case-by-case analysis, MDM2-amplified cases showed varied histological phenotypes and were most commonly microsatellite stable; EBV, HER2, and MET negative; and FGFR2 positive. A single case harbored both, MDM2 amplification and TP53 mutation. MDM2 amplification and MDM2 expression, respectively, did not correlate with overall or tumor-specific survival. Our targeted analysis of MDM2 in a well-characterized cohort of GC patients showed that MDM2 amplification is rare, of no specific histological phenotype, and may not be always mutually exclusive with TP53 mutations. Given the low number of cases, currently, no diagnostic or therapeutic recommendation related to MDM2 amplification can be given for GC of Western origin.

LAG3 in gastric cancer: it's complicated.

PURPOSE: Lymphocyte activation gene 3 (LAG3) is thought to contribute to T cell exhaustion within the tumor microenvironment of solid tumors. This study aimed to analyze the spatial distribution of LAG3 + cells in relation to clinicopathological and survival data in a large set of 580 primary resected and neoadjuvantly treated gastric cancers (GC). METHODS: LAG3 expression was evaluated in tumor center and invasive margin using immunohistochemistry and whole-slide digital image analysis. Cases were divided into LAG3-low and LAG3-high expression groups based on (1) median LAG3 + cell density, (2) cut-off values adapted to cancer-specific survival using Cutoff Finder application. RESULTS: Significant differences in spatial distribution of LAG3 + cells were observed in primarily resected GC, but not in neoadjuvantly treated GC. LAG3 + cell density showed evident prognostic value at following cut-offs: in primarily resected GC, 21.45 cells/mm2 in tumor center (17.9 vs. 10.1 months, p = 0.008) and 208.50 cells/mm2 in invasive margin (33.8 vs. 14.7 months, p = 0.006); and in neoadjuvantly treated GC, 12.62 cells/mm2 (27.3 vs. 13.2 months, p = 0.003) and 123.00 cells/mm2 (28.0 vs. 22.4 months, p = 0.136), respectively. Significant associations were found between LAG3 + cell distribution patterns and various clinicopathological factors in both cohorts. In neoadjuvantly treated GC, LAG3 + immune cell density was found to be an independent prognostic factor of survival (HR = 0.312, 95% CI 0.162-0.599, p < 0.001). CONCLUSION: In this study, a higher density of LAG3 + cells was associated with favorable prognosis. Current results support the need for extended analysis of LAG3. Differences in the distribution of LAG3 + cells should be considered, as they could influence clinical outcomes and treatment responses.

ATTR- and AFib amyloid - two different types of amyloid in the annular ligament of trigger finger.

INTRODUCTION: Histological examination of tissue specimens obtained during surgical treatment of trigger finger frequently encountered unclassifiable amyloid deposits in the annular ligament. We systematically explored this unknown type by a comprehensive analysis using histology, immunohistochemistry, and quantitative mass spectrometry-based proteomics. METHODS: 205 tissue specimens of annular ligaments were obtained from 172 patients. Each specimen was studied by histology and immunohistochemistry. Tissue specimens obtained from ten patients with histology proven amyloid in annular ligament were analysed by label-free quantitative proteomics. Histological and immunohistochemical findings were correlated with patient demographics. RESULTS: Amyloid was present as band like deposits along the surface of annular ligament, dot like or patchy deposits within the matrix. Immunohistochemistry identified ATTR amyloid in 92 specimens (mostly patchy in the matrix), while the band like deposits of 100 specimens remained unclassifiable. Proteomic profiles identified the unknown amyloid as most likely of fibrinogen origin. The complete cohort was re-examined by immunohistochemistry using a custom-made antibody and confirmed the presence of fibrinogen alpha-chain (FGA) in a hitherto unclassifiable type of amyloid in annular ligament. CONCLUSION: Our study shows that two different types of amyloid affect the annular ligament, ATTR amyloid and AFib amyloid, with distinct demographic patient characteristics and histomorphological deposition patterns.

Deep learning trained on lymph node status predicts outcome from gastric cancer histopathology: a retrospective multicentric study.

AIM: Gastric cancer (GC) is a tumour entity with highly variant outcomes. Lymph node metastasis is a prognostically adverse biomarker. We hypothesised that GC primary tissue contains information that is predictive of lymph node status and patient prognosis and that this information can be extracted using deep learning (DL). METHODS: Using three patient cohorts comprising 1146 patients, we trained and validated a DL system to predict lymph node status directly from haematoxylin and eosin-stained GC tissue sections. We investigated the concordance between the DL-based prediction from the primary tumour slides (aiN score) and the histopathological lymph node status (pN). Furthermore, we assessed the prognostic value of the aiN score alone and when combined with the pN status. RESULTS: The aiN score predicted the pN status reaching area under the receiver operating characteristic curves of 0.71 in the training cohort and 0.69 and 0.65 in the two test cohorts. In a multivariate Cox analysis, the aiN score was an independent predictor of patient survival with hazard ratios of 1.5 in the training cohort and of 1.3 and 2.2 in the two test cohorts. A combination of the aiN score and the pN status prognostically stratified patients by survival with p-values <0.05 in logrank tests. CONCLUSION: GC primary tumour tissue contains additional prognostic information that is accessible using the aiN score. In combination with the pN status, this can be used for personalised management of GC patients after prospective validation.

Genotypic and phenotypic characterization of side population of gastric cancer cell lines.

The side population (SP) of tumor cell lines shares characteristics with tumor stem cells. The objective of this study was to phenotypically and genotypically characterize the SP of gastric cancer cell lines. SP cells were obtained from AGS and MKN45 gastric cancer cells using Hoechst 33342 staining and fluorescence-activated cell sorting. The cells were subsequently studied morphologically at cytology and immunocytochemistry, on the transcriptional level via gene array, and in cell culture using recultivation assays. Genes differentially expressed in SP cells were evaluated at immunohistochemistry in tissue samples from 486 patients with gastric cancer. The SP cells were smaller and rounder then non-SP cells. SP cells self-renewed in recultivation experiments and differentiated into SP and non-SP cells. Recultivated SP and non-SP cells exhibited distinct phenotypes in culture insofar as cell shape and colony formation. SP cells demonstrated increased levels of the stem cell markers CD133 and Musashi-1. Transcriptional analyses demonstrated that SP cells express genes that encode for stem cell properties including FZD7, HEY1, SMO, and ADAM17. It was observed that ADAM17 and FZD7 are differentially expressed in human gastric cancer, and FZD7-positive cancers are associated with significantly shorter patient survival. In conclusion, human gastric cancer cell lines enclose a phenotypically and genotypically distinct cell population with tumor stem cell features. Phenotypic characteristics of this distinct cell population are also present in gastric cancer tissue, and correlate with patient survival.