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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

FGFR2 overexpression and compromised survival in diffuse-type gastric cancer in a large central European cohort.

The significance of fibroblast growth factor receptor 2 (FGFR2) in gastric cancer (GC) has been studied predominantly in Asian patient cohorts. Data on White patients are scarce. Here, we aimed to independently validate the expression and putative tumor biological significance of FGFR2 in a large non-Asian GC cohort. Immunohistochemistry (IHC) was performed on large-area tissue sections from 493 patients with GC and evaluated using the HScore. GCs with moderate and strong FGFR2 expression were studied for Fgfr2 amplification using chromogenic in situ hybridization (CISH). Median overall survival was determined using the Kaplan-Meier method. The majority [240 (99.1%)] of FGFR2-positive GCs showed a variable combination of staining intensities with marked intratumoral heterogeneity, including weak [198 (40.2%) cases], moderate [145 (29.4%)], and strong [108 (21.9%)] staining in diverse combinations. 250 (50.9%) GCs expressed no FGFR2. Fgfr2 gene amplification was found in 40% of selected cases with high protein expression and was also heterogeneous at the cell level. FGFR2 protein expression did not correlate with patient survival in the entire cohort However, using different cutoff values, a negative correlation between FGFR2-expression and patient outcome was found for diffuse-type GC. FGFR2 expression was associated with a lower tumor grade and intestinal phenotype (p≤0.0001). FGFR2-positive diffuse-type GCs classify a small subset of patients with a poor tumor specific survival (5.29±1.3 vs. 14.67±1.9 months; p = 0.004).

Neoadjuvant/Perioperative Treatment Affects Spatial Distribution and Densities of Tumor Associated Neutrophils and CD8+ Lymphocytes in Gastric Cancer.

Tumor associated neutrophils (TANs) and cytotoxic T cells (CTLs) are part of the tumor microenvironment of gastric cancer (GC). We explored their tumor biological significance in neoadjuvantly/perioperatively treated GC. Immunostaining was performed on whole tissue sections of 173 GCs, using antibodies directed against myeloperoxidase (MPO) and CD8. Stained specimens were digitalized, and the densities of TANs and CTLs were assessed separately in the mucosa, tumor surface, tumor center, invasion front, and tumor scar. The densities were correlated with clinicopathological patient characteristics. Compared with a historical cohort of 449 treatment naive GCs, the TAN density in the invasion front was significantly lower in neoadjuvantly/perioperatively treated GCs. TAN density in the tumor center and invasion front correlated with tumor regression. TAN density also correlated with CTL density in the tumor center and invasion front. A high density of CTL in the tumor center correlated with an improved overall survival and tumor specific survival. We show that neoadjuvant/perioperative (radio-) chemotherapy impacts on the immune microenvironment of GC, while also depending on sex. The density of TANs in neoadjuvantly/perioperatively treated GCs differed from findings made in a treatment naive GC cohort.

Multiscale heterogeneity in gastric adenocarcinoma evolution is an obstacle to precision medicine.

BACKGROUND: Cancer is a somatic evolutionary disease and adenocarcinomas of the stomach and gastroesophageal junction (GC) may serve as a two-dimensional model of cancer expansion, in which tumor subclones are not evenly mixed during tumor progression but rather spatially separated and diversified. We hypothesize that precision medicine efforts are compromised when clinical decisions are based on a single-sample analysis, which ignores the mechanisms of cancer evolution and resulting intratumoral heterogeneity. Using multiregional whole-exome sequencing, we investigated the effect of somatic evolution on intratumoral heterogeneity aiming to shed light on the evolutionary biology of GC. METHODS: The study comprised a prospective discovery cohort of 9 and a validation cohort of 463 GCs. Multiregional whole-exome sequencing was performed using samples form 45 primary tumors and 3 lymph node metastases (range 3-10 tumor samples/patient) of the discovery cohort. RESULTS: In total, the discovery cohort harbored 16,537 non-synonymous mutations. Intratumoral heterogeneity of somatic mutations and copy number variants were present in all tumors of the discovery cohort. Of the non-synonymous mutations, 53-91% were not present in each patient's sample; 399 genes harbored 2-4 different non-synonymous mutations in the same patient; 175 genes showed copy number variations, the majority being heterogeneous, including CD274 (PD-L1). Multi-sample tree-based analyses provided evidence for branched evolution being most complex in a microsatellite instable GC. The analysis of the mode of evolution showed a high degree of heterogeneity in deviation from neutrality within each tumor. We found evidence of parallel evolution and evolutionary trajectories: different mutations of SMAD4 aligned with different subclones and were found only in TP53 mutant GCs. CONCLUSIONS: Neutral and non-neutral somatic evolution shape the mutational landscape in GC along its lateral expansions. It leads to complex spatial intratumoral heterogeneity, where lymph node metastases may stem from different areas of the primary tumor, synchronously. Our findings may have profound effects on future patient management. They illustrate the risk of mis-interpreting tumor genetics based on single-sample analysis and open new avenues for an evolutionary classification of GC, i.e., the discovery of distinct evolutionary trajectories which can be utilized for precision medicine.

Insulin Receptor in Pancreatic Cancer-Crown Witness in Cross Examination.

BACKGROUND: The proximity of pancreatic cancer (PDAC) to the physiological source of the growth promoting hormone insulin might be exploited by this highly malignant cancer entity. We investigated if (I) PDACs express the insulin receptor (IR) in cancer cells and cancer vasculature, (II) if IR correlates with clinicopathological patient characteristics, including survival, and hence is involved in PDAC biology, (III) if IR is already expressed in precursor lesions, if (IV) the IGF1 receptor (IGF1R) is associated with clinicopathological patient characteristics and survival and (V) is linked to IR expression. METHODS: 160 PDAC samples were examined for IR and IGF1R expression by immunohistochemistry. A modified HistoScore was correlated with clinicopathological characteristics and survival. RESULTS: IR overexpression was already observed in pancreatic intraepithelial neoplasia. Furthermore, it was more frequently observed in advanced disease and associated with distant metastasis, UICC stage, lymphatic invasion and an increased lymph node ratio, but without impacting survival in the end. IGF1R expression was not associated with clinicopathological parameters or survival, in contrast to former paradigms. CONCLUSIONS: We hypothesize that the close proximity to the pancreatic islets might be advantageous for cancer growth at first, but it experiences self-limitation due to surgical removal or local destruction following accelerated cancer growth.

Complement 9 in amyloid deposits.

Amyloidosis is a disease group caused by pathological aggregation and deposition of peptides in diverse tissue sites. Apart from the fibril protein, amyloid deposits frequently enclose non-fibrillar constituents. In routine diagnostics, we noticed the presence of complement 9 (C9) in amyloid. Based on this observation, we systematically explored the occurrence of C9 in amyloid. Apolipoprotein E (apoE), caspase 3 and complement 3 (C3) served as controls. From the Amyloid Registry Kiel, we retrieved 118 formalin-fixed and paraffin-embedded tissue samples, including eight different amyloid- and 18 different tissue types. The expression patterns were assessed immunohistochemically in relation to amyloid deposits. A literature search on proteomic data was performed. Amyloid deposits stained for C9 and apoE in 117 (99.2%) and 112 of 118 (94.9%) cases, respectively. A homogeneous immunostaining of the entire amyloid deposits was found in 75.4% (C9) and 61.9% (apoE) of the cases. Caspase 3 and C3 were present only in 22 (19.3%) of 114 and 20 (36%) of 55 assessable cases, respectively. Caspase 3 and C3 immunostaining rarely covered substantial areas of the amyloid deposits. The literature search on proteomic data confirmed the frequent detection of apoE and the occurrence of C9 and C3 in amyloid deposits. No data were found regarding caspase 3. Our findings demonstrate the ubiquitous, spatial and specific enrichment of C9 in amyloid deposits irrespective of amyloid-, organ- or tissue type. Our findings lend support to the hypothesis that amyloidosis might activate the complement cascade, which could lead to the formation of the membrane attack complex and cell death.

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.

p53 immunostaining cannot be used to predict TP53 mutations in gastric cancer: results from a large Central European cohort.

Four molecular subgroups of gastric cancer (GC) have been proposed, ie, Epstein-Barr virus (EBV)-positive, microsatellite instable, chromosomal instable (CIN), and genomically stable GC. Based on the complex relationship between chromosomal instability and TP53 mutational status, we hypothesized that the typical clinicopathological characteristics caused by chromosomal instability are correlated with the p53 expression that is detected by immunohistochemistry. Four hundred sixty-seven whole-tissue sections of patients with therapy-naive GC were stained with anti-p53 antibody. The histoscore and staining pattern were analyzed for each slide. Different algorithms of immunohistochemistry evaluation were formed and correlated with clinicopathological characteristics. The algorithms were validated by assessing the mutational status of TP53 in 111 cases. Four hundred forty-two GCs were p53 positive, and 25 were negative, including 414 GCs with a homogeneous pattern and 53 GCs with a heterogeneous staining pattern. There was no correlation with overall or tumor-specific survival. In comparison with clinicopathological characteristics, the algorithm high versus low showed correlations with microsatellite instability, hepatocyte growth factor receptor (MET), and TP53 mutational status. The algorithm Q1/Q4 versus Q2/Q3 appeared to be correlated with the phenotype as per the Laurén classification, microsatellite instability, EBV status, and p53 expression pattern. The algorithm <90% = 0 and <50% = 3+ versus ≥90% = 0 or ≥50% = 3+ showed correlations with the EBV status, microsatellite instability, grading, and p53 expression pattern. The algorithm homogeneous versus heterogeneous did not correlate with any clinicopathological characteristic. Our results showed that the immunohistochemistry of p53, TP53 mutational status, and CIN subtype were connected. However, different algorithms for p53 immunohistochemical evaluation cannot be used to predict TP53 mutations in CIN GCs in individual cases.

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.

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.