Tumour infiltrating lymphocytes and survival after adjuvant chemotherapy in patients with gastric cancer: post-hoc analysis of the CLASSIC trial.

BACKGROUND: Only a subset of gastric cancer (GC) patients with stage II-III benefits from chemotherapy after surgery. Tumour infiltrating lymphocytes per area (TIL density) has been suggested as a potential predictive biomarker of chemotherapy benefit. METHODS: We quantified TIL density in digital images of haematoxylin-eosin (HE) stained tissue using deep learning in 307 GC patients of the Yonsei Cancer Center (YCC) (193 surgery+adjuvant chemotherapy [S + C], 114 surgery alone [S]) and 629 CLASSIC trial GC patients (325 S + C and 304 S). The relationship between TIL density, disease-free survival (DFS) and clinicopathological variables was analysed. RESULTS: YCC S patients and CLASSIC S patients with high TIL density had longer DFS than S patients with low TIL density (P = 0.007 and P = 0.013, respectively). Furthermore, CLASSIC patients with low TIL density had longer DFS if treated with S + C compared to S (P = 0.003). No significant relationship of TIL density with other clinicopathological variables was found. CONCLUSION: This is the first study to suggest TIL density automatically quantified in routine HE stained tissue sections as a novel, clinically useful biomarker to identify stage II-III GC patients deriving benefit from adjuvant chemotherapy. Validation of our results in a prospective study is warranted.

Increasing frequency of gene copy number aberrations is associated with immunosuppression and predicts poor prognosis in gastric adenocarcinoma.

BACKGROUND: Patients with Epstein-Barr virus-positive gastric cancers or those with microsatellite instability appear to have a favourable prognosis. However, the prognostic value of the chromosomal status (chromosome-stable (CS) versus chromosomal instable (CIN)) remains unclear in gastric cancer. METHODS: Gene copy number aberrations (CNAs) were determined in 16 CIN-associated genes in a retrospective study including test and validation cohorts of patients with gastric cancer. Patients were stratified into CS (no CNA), CINlow (1-2 CNAs) or CINhigh (3 or more CNAs). The relationship between chromosomal status, clinicopathological variables, and overall survival (OS) was analysed. The relationship between chromosomal status, p53 expression, and tumour infiltrating immune cells was also assessed and validated externally. RESULTS: The test and validation cohorts included 206 and 748 patients, respectively. CINlow and CINhigh were seen in 35.0 and 15.0 per cent of patients, respectively, in the test cohort, and 48.5 and 20.7 per cent in the validation cohort. Patients with CINhigh gastric cancer had the poorest OS in the test and validation cohorts. In multivariable analysis, CINlow, CINhigh and pTNM stage III-IV (P < 0.001) were independently associated with poor OS. CIN was associated with high p53 expression and low immune cell infiltration. CONCLUSION: CIN may be a potential new prognostic biomarker independent of pTNM stage in gastric cancer. Patients with gastric cancer demonstrating CIN appear to be immunosuppressed, which might represent one of the underlying mechanisms explaining the poor survival and may help guide future therapeutic decisions.

Computational Image Analysis of T-Cell Infiltrates in Resectable Gastric Cancer: Association with Survival and Molecular Subtypes.

BACKGROUND: Gastric and gastro-esophageal junction cancers (GCs) frequently recur after resection, but markers to predict recurrence risk are missing. T-cell infiltrates have been validated as prognostic markers in other cancer types, but not in GC because of methodological limitations of past studies. We aimed to define and validate the prognostic role of major T-cell subtypes in GC by objective computational quantification. METHODS: Surgically resected chemotherapy-naïve GCs were split into discovery (n = 327) and validation (n = 147) cohorts. CD8 (cytotoxic), CD45RO (memory), and FOXP3 (regulatory) T-cell densities were measured through multicolor immunofluorescence and computational image analysis. Cancer-specific survival (CSS) was assessed. All statistical tests were two-sided. RESULTS: CD45RO-cell and FOXP3-cell densities statistically significantly predicted CSS in both cohorts. Stage, CD45RO-cell, and FOXP3-cell densities were independent predictors of CSS in multivariable analysis; mismatch repair (MMR) and Epstein-Barr virus (EBV) status were not statistically significant. Combining CD45RO-cell and FOXP3-cell densities into the Stomach Cancer Immune Score showed highly statistically significant (all P ≤ .002) CSS differences (0.9 years median CSS to not reached). T-cell infiltrates were highest in EBV-positive GCs and similar in MMR-deficient and MMR-proficient GCs. CONCLUSION: The validation of CD45RO-cell and FOXP3-cell densities as prognostic markers in GC may guide personalized follow-up or (neo)adjuvant treatment strategies. Only those 20% of GCs with the highest T-cell infiltrates showed particularly good CSS, suggesting that a small subgroup of GCs is highly immunogenic. The potential for T-cell densities to predict immunotherapy responses should be assessed. The association of high FOXP3-cell densities with longer CSS warrants studies into the biology of regulatory T cells in GC.

Spatial profiling of gastric cancer patient-matched primary and locoregional metastases reveals principles of tumour dissemination.

OBJECTIVE: Endoscopic mucosal biopsies of primary gastric cancers (GCs) are used to guide diagnosis, biomarker testing and treatment. Spatial intratumoural heterogeneity (ITH) may influence biopsy-derived information. We aimed to study ITH of primary GCs and matched lymph node metastasis (LNmet). DESIGN: GC resection samples were annotated to identify primary tumour superficial (PTsup), primary tumour deep (PTdeep) and LNmet subregions. For each subregion, we determined (1) transcriptomic profiles (NanoString 'PanCancer Progression Panel', 770 genes); (2) next-generation sequencing (NGS, 225 gastrointestinal cancer-related genes); (3) DNA copy number profiles by multiplex ligation-dependent probe amplification (MLPA, 16 genes); and (4) histomorphological phenotypes. RESULTS: NanoString profiling of 64 GCs revealed no differences between PTsup1 and PTsup2, while 43% of genes were differentially expressed between PTsup versus PTdeep and 38% in PTsup versus LNmet. Only 16% of genes were differently expressed between PTdeep and LNmet. Several genes with therapeutic potential (eg IGF1, PIK3CD and TGFB1) were overexpressed in LNmet and PTdeep compared with PTsup. NGS data revealed orthogonal support of NanoString results with 40% mutations present in PTdeep and/or LNmet, but not in PTsup. Conversely, only 6% of mutations were present in PTsup and were absent in PTdeep and LNmet. MLPA demonstrated significant ITH between subregions and progressive genomic changes from PTsup to PTdeep/LNmet. CONCLUSION: In GC, regional lymph node metastases are likely to originate from deeper subregions of the primary tumour. Future clinical trials of novel targeted therapies must consider assessment of deeper subregions of the primary tumour and/or metastases as several therapeutically relevant genes are only mutated, overexpressed or amplified in these regions.

Histological intratumoral heterogeneity in pretreatment esophageal cancer biopsies predicts survival benefit from neoadjuvant chemotherapy: results from the UK MRC OE02 trial.

Despite the use of multimodal treatment, survival of esophageal cancer (EC) patients remains poor. One proposed explanation for the relatively poor response to cytotoxic chemotherapy is intratumor heterogeneity. The aim was to establish a statistical model to objectively measure intratumor heterogeneity of the proportion of tumor (IHPoT) and to use this newly developed method to measure IHPoT in the pretreatment biopsies from from EC patients recruited to the OE02 trial. A statistical mixed effect model (MEM) was established for estimating IHPoT based on variation in hematoxylin/eosin (HE) stained pretreatment biopsy pieces from the same individual in 218 OE02 trial patients (103 treated by chemotherapy and surgery (chemo+surgery); 115 patients treated by surgery alone). The relationship between IHPoT, prognosis, chemotherapy survival benefit, and clinicopathological variables was assessed. About 97 (44.5%) and 121 (55.5%) ECs showed high and low IHPoT, respectively. There was no significant difference in IHPoT between surgery (median [range], 0.1637 [0-3.17]) and chemo+surgery (median [range], 0.1692 [0-2.69]) patients (P = 0.43). Chemo+surgery patients with low IHPoT had a significantly longer survival than surgery patients (HR = 1.81, 95% CI: 1.20-2.75, P = 0.005). There was no survival difference between chemo+surgery and surgery patients with high IHPoT (HR = 1.15, 95% CI: 0.72-1.81, P = 0.566). This is the first study suggesting that IHPoT measured in the pretreatment biopsy can predict chemotherapy survival benefit in EC patients. IHPoT may represent a clinically useful biomarker for patient treatment stratification. Future studies should determine if pathologists can reliably estimate IHPoT.

Hypothesis-free deep survival learning applied to the tumour microenvironment in gastric cancer.

The biological complexity reflected in histology images requires advanced approaches for unbiased prognostication. Machine learning and particularly deep learning methods are increasingly applied in the field of digital pathology. In this study, we propose new ways to predict risk for cancer-specific death from digital images of immunohistochemically (IHC) stained tissue microarrays (TMAs). Specifically, we evaluated a cohort of 248 gastric cancer patients using convolutional neural networks (CNNs) in an end-to-end weakly supervised scheme independent of subjective pathologist input. To account for the time-to-event characteristic of the outcome data, we developed new survival models to guide the network training. In addition to the standard H&E staining, we investigated the prognostic value of a panel of immune cell markers (CD8, CD20, CD68) and a proliferation marker (Ki67). Our CNN-derived risk scores provided additional prognostic value when compared to the gold standard prognostic tool TNM stage. The CNN-derived risk scores were also shown to be superior when systematically compared to cell density measurements or a CNN score derived from binary 5-year survival classification, which ignores time-to-event. To better understand the underlying biological mechanisms, we qualitatively investigated risk heat maps for each marker which visualised the network output. We identified patterns of biological interest that were related to low risk of cancer-specific death such as the presence of B-cell predominated clusters and Ki67 positive sub-regions and showed that the corresponding risk scores had prognostic value in multivariate Cox regression analyses (Ki67&CD20 risks: hazard ratio (HR) = 1.47, 95% confidence interval (CI) = 1.15-1.89, p = 0.002; CD20&CD68 risks: HR = 1.33, 95% CI = 1.07-1.67, p = 0.009). Our study demonstrates the potential additional value that deep learning in combination with a panel of IHC markers can bring to the field of precision oncology.

Comprehensive biomarker analyses identifies HER2, EGFR, MET RNA expression and thymidylate synthase 5'UTR SNP as predictors of benefit from S-1 adjuvant chemotherapy in Japanese patients with stage II/III gastric cancer.

Purpose: A comprehensive molecular analysis was conducted to identify prognostic and predictive markers for adjuvant S-1 chemotherapy in stage II/III Japanese gastric cancer (GC) patients and to evaluate their potential suitability for alternative cytotoxic or targeted drugs. Experimental Design: We investigated genetic polymorphisms of enzymes potentially involved in 5-fluoruracil (5-FU) metabolism as well as platinum resistance, previously identified genomic subtypes potentially predicting 5-FU benefit, and mRNA expression levels of receptor tyrosine kinases and KRAS as potential treatment targets in a single institution cohort of 252 stage II/III GC patients treated with or without S-1 after D2 gastrectomy. Results: 88% and 62% GC had a potentially 5-FU sensitive phenotype by SNP analyses of TS 3'UTR, and TS 5'UTR, respectively. 24%, 46%, 40%, 5%, and 44% GC had a potentially platinum sensitive phenotype by SNP analyses of GSTP1, ERCC1 rs11615, ERCC1 rs3212986, ERCC2, and XRCC1, respectively. High HER2, EGFR, FGFR2, or MET mRNA expression was observed in 49%, 66%, 72%, and 54% GC, respectively. High HER2 expression was the only significant prognosticator (HR=3.912, 95%CI: 1.706-8.973, p=0.0005). High HER2 (p=0.031), low EGFR (p=0.124), high MET (p=0.165) RNA expression, and TS 5'UTR subtype 2R/2R, 2R/3C, or 3C (p=0.058) were significant independent predictors for S-1 resistance. Conclusions: The present study suggests that platinum-based or RTK targeted agents could be alternative treatment options for a substantial subgroup of Japanese GC patients currently treated with S-1. HER2, EGFR, MET, and TS 5'UTR SNP appear to be promising predictive markers for S-1 resistance warranting validation in an independent GC series.

DNA epigenetic signature predictive of benefit from neoadjuvant chemotherapy in oesophageal adenocarcinoma: results from the MRC OE02 trial.

BACKGROUND: DNA methylation signatures describing distinct histological subtypes of oesophageal cancer have been reported. We studied DNA methylation in samples from the MRC OE02 phase III trial, which randomised patients with resectable oesophageal cancer to surgery alone (S) or neoadjuvant chemotherapy followed by surgery (CS). AIM: The aim of the study was to identify epigenetic signatures predictive of chemotherapy benefit in patients with oesophageal adenocarcinoma (OAC) from the OE02 trial and validate the findings in an independent cohort. METHODS: DNA methylation was analysed using the Illumina GoldenGate platform on surgically resected OAC specimens from patients in the OE02 trial. Cox proportional hazard analysis was performed to select probes predictive of survival in the CS arm. Non-negative matrix factorisation was used to perform clustering and delineate DNA methylation signatures. The findings were validated in an independent cohort of patients with gastroesophageal adenocarcinoma treated with neoadjuvant chemotherapy. RESULTS: A total of 229 patients with OAC were analysed from the OE02 trial (118 in the CS arm and 111 in the S arm). There was no difference in DNA methylation status between the CS and S arms. A metagene signature was created by dichotomising samples into two clusters. In cluster 1, patients in the CS arm had significant overall survival (OS) benefit (median OS CS: 931 days vs. S: 536 days [HR: 1.54, P = 0.031]). In cluster 2, patients in the CS arm had similar (or worse) OS compared with patients in the S arm (CS: 348 days vs. S: 472 days [HR: 0.70, P = 0.1], and test of interaction was significant (p = 0.005). In the validation cohort (n = 13), there was no difference in DNA methylation status in paired pre- and post-treatment samples. When the epigenetic signature was applied, cluster 1 samples had better OS (median OS, cluster 1: 1174 days vs. cluster 2: 392 days, HR: 3.47, p = 0.059) CONCLUSIONS: This is the first and largest study of DNA methylation in patients with OAC uniformly treated in a randomised phase III trial. We identified an epigenetic signature that may serve as a predictive biomarker for chemotherapy benefit in OAC.

Correction to: KRAS status is related to histological phenotype in gastric cancer: results from a large multicentre study.

In the original publication of this article, Fig. 2 was published incorrectly. The correct Fig. 2 is given in this correction.

KRAS status is related to histological phenotype in gastric cancer: results from a large multicentre study.

BACKGROUND: Gastric cancer (GC) is histologically a very heterogeneous disease, and the temporal development of different histological phenotypes remains unclear. Recent studies in lung and ovarian cancer suggest that KRAS activation (KRASact) can influence histological phenotype. KRASact likely results from KRAS mutation (KRASmut) or KRAS amplification (KRASamp). The aim of the study was to investigate whether KRASmut and/or KRASamp are related to the histological phenotype in GC. METHODS: Digitized haematoxylin/eosin-stained slides from 1282 GC resection specimens were classified according to Japanese Gastric Cancer Association (JGCA) and the Lauren classification by at least two observers. The relationship between KRAS status, predominant histological phenotype and clinicopathological variables was assessed. RESULTS: KRASmut and KRASamp were found in 68 (5%) and 47 (7%) GCs, respectively. Within the KRASmut and KRASamp cases, the most frequent GC histological phenotype was moderately differentiated tubular 2 (tub2) type (KRASmut: n = 27, 40%; KRASamp: n = 21, 46%) or intestinal type (KRASmut: n = 41, 61%; KRASamp: n = 23, 50%). Comparing individual histological subtypes, mucinous carcinoma displayed the highest frequency of KRASmut (JGCA: n = 6, 12%, p = 0.012; Lauren: n = 6, 12%, p = 0.013), and KRASamp was more frequently found in poorly differentiated solid type (n = 12, 10%, p = 0.267) or indeterminate type (n = 12, 10%, p = 0.480) GC. 724 GCs (57%) had intratumour morphological heterogeneity. CONCLUSIONS: This is the largest GC study investigating KRAS status and histological phenotype. We identified a relationship between KRASmut and mucinous phenotype. The high level of intratumour morphological heterogeneity could reflect KRASmut heterogeneity, which may explain the failure of anti-EGFR therapy in GC.

Prognostic value of pathological lymph node status and primary tumour regression grading following neoadjuvant chemotherapy - results from the MRC OE02 oesophageal cancer trial.

AIMS: Neoadjuvant chemotherapy (NAC) remains an important therapeutic option for advanced oesophageal cancer (OC). Pathological tumour regression grade (TRG) may offer additional information by directing adjuvant treatment and/or follow-up but its clinical value remains unclear. We analysed the prognostic value of TRG and associated pathological factors in OC patients enrolled in the Medical Research Council (MRC) OE02 trial. METHODS AND RESULTS: Histopathology was reviewed in 497 resections from OE02 trial participants randomised to surgery (S group; n = 244) or NAC followed by surgery [chemotherapy plus surgery (CS) group; n = 253]. The association between TRG groups [responders (TRG1-3) versus non-responders (TRG4-5)], pathological lymph node (LN) status and overall survival (OS) was analysed. One hundred and ninety-five of 253 (77%) CS patients were classified as 'non-responders', with a significantly higher mortality risk compared to responders [hazard ratio (HR) = 1.53, 95% confidence interval (CI) = 1.05-2.24, P = 0.026]. OS was significantly better in patients without LN metastases irrespective of TRG [non-responders HR = 1.87, 95% CI = 1.33-2.63, P < 0.001 versus responders HR = 2.21, 95% CI = 1.11-4.10, P = 0.024]. In multivariate analyses, LN status was the only independent factor predictive of OS in CS patients (HR = 1.93, 95% CI = 1.42-2.62, P < 0.001). Exploratory subgroup analyses excluding radiotherapy-exposed patients (n = 48) showed similar prognostic outcomes. CONCLUSION: Lymph node status post-NAC is the most important prognostic factor in patients with resectable oesophageal cancer, irrespective of TRG. Potential clinical implications, e.g. adjuvant treatment or intensified follow-up, reinforce the importance of LN dissection for staging and prognostication.

Frequent Coamplification of Receptor Tyrosine Kinase and Downstream Signaling Genes in Japanese Primary Gastric Cancer and Conversion in Matched Lymph Node Metastasis.

OBJECTIVE: To establish the gene copy number status of receptor tyrosine kinase (RTK) and downstream signaling (DSS) genes genes in primary gastric cancer (primGC) and matched lymph node metastases (LNmet). BACKGROUND: Evidence suggests that coamplification between RTKs and DSSs and conversion between primGC and LNmet are associated with resistance to targeted therapy. METHODS: DNA from 237 Japanese primGC and 103 matched LNmet was analyzed using a newly developed multiplex ligation-dependent probe amplification (MLPA) probemix to investigate RTK (EGFR, HER2, FGFR2, and MET) and DSS (PIK3CA, KRAS, MYC, and CCNE1) gene copy number status. Results were compared between primGC and LNmet and related to clinicopathological data including survival. RESULTS: A total of 150 (63%) primGC had either RTK or DSS amplification. DSS coamplification was more frequent than RTK coamplification in primGC and LNmets. Moreover, 70 (30%) GC showed a disconcordant RTK and/or DSS gene copy number status between primGC and LNmet, most common was negative conversion for DSS genes (n=40 GC). The presence of RTK amplification in primGC was related to poorer survival in univariate analysis (P=0.04). CONCLUSIONS: This is the first and most comprehensive study in gastric cancer investigating the concordance between gene copy number status of targetable RTKs and downstream signaling oncogenes in primGC and LNmets. Future studies need to establish whether the relative high frequency of RTK and DSS coamplification and/or the relative high rate of negative conversion in LNmet can potentially explain recent failures of RTK targeted therapy in gastric cancer patients.

Biopsy proportion of tumour predicts pathological tumour response and benefit from chemotherapy in resectable oesophageal carcinoma: results from the UK MRC OE02 trial.

BACKGROUND: Neoadjuvant chemotherapy followed by surgery is the standard of care for UK patients with locally advanced resectable oesophageal carcinoma (OeC). However, not all patients benefit from multimodal treatment and there is a clinical need for biomarkers which can identify chemotherapy responders. This study investigated whether the proportion of tumour cells per tumour area (PoT) measured in the pre-treatment biopsy predicts chemotherapy benefit for OeC patients. PATIENTS AND METHODS: PoT was quantified using digitized haematoxylin/eosin stained pre-treatment biopsy slides from 281 OeC patients from the UK MRC OE02 trial (141 treated by surgery alone (S); 140 treated by 5-fluorouracil/cisplatin followed by surgery (CS)). The relationship between PoT and clinicopathological data including tumour regression grade (TRG), overall survival and treatment interaction was investigated. RESULTS: PoT was associated with chemotherapy benefit in a non-linear fashion (test for interaction, P=0.006). Only patients with a biopsy PoT between 40% and 70% received a significant survival benefit from neoadjuvant chemotherapy (N=129; HR (95%CI):1.94 (1.39-2.71), unlike those with lower or higher PoT (PoT<40%, N=39, HR:1.25 (0.66-2.35); PoT>70% (N=28, HR:0.65 (0.36-1.18)). High pre-treatment PoT was related to lack of primary tumour regression (TRG 4 or 5), P=0.0402. CONCLUSIONS: This is the first study to identify in a representative subgroup of OeC patients from a large randomized phase III trial that the proportion of tumour in the pre-chemotherapy biopsy predicts benefit from chemotherapy and may be a clinically useful biomarker for patient treatment stratification.Proportion of tumour is a novel biomarker which can be measured in the pre-treatment diagnostic biopsy and which may enable the identification of chemotherapy responders and non-responders among patients with oesophageal carcinoma. Proportion of tumour could easily become part of the routine reporting of oesophageal cancer biopsies and may aid in managing patients with borderline resectable cancer.

Reduced genomic tumor heterogeneity after neoadjuvant chemotherapy is related to favorable outcome in patients with esophageal adenocarcinoma.

Neoadjuvant chemo(radio)therapy followed by surgery is the standard of care for patients with locally advanced resectable esophageal adenocarcinoma (EAC). There is increasing evidence that drug resistance might be related to genomic heterogeneity. We investigated whether genomic tumor heterogeneity is different after cytotoxic chemotherapy and is associated with EAC patient survival. We used arrayCGH and a quantitative assessment of the whole genome DNA copy number aberration patterns ('DNA copy number entropy') to establish the level of genomic tumor heterogeneity in 80 EAC treated with neoadjuvant chemotherapy followed by surgery (CS group) or surgery alone (S group). The association between DNA copy number entropy, clinicopathological variables and survival was investigated.DNA copy number entropy was reduced after chemotherapy, even if there was no morphological evidence of response to therapy (p<0.001). Low DNA copy number entropy was associated with improved survival in the CS group (p=0.011) but not in the S group (p=0.396).Our results suggest that cytotoxic chemotherapy reduces DNA copy number entropy, which might be a more sensitive tumor response marker than changes in the morphological tumor phenotype. The use of DNA copy number entropy in clinical practice will require validation of our results in a prospective study.

Technical reproducibility of single-nucleotide and size-based DNA biomarker assessment using DNA extracted from formalin-fixed, paraffin-embedded tissues.

DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissues has been used in the past to analyze genetic polymorphisms. We evaluated the technical reproducibility of different types of assays for gene polymorphisms using DNA extracted from FFPE material. By using the MassARRAY iPLEX system, we investigated polymorphisms in DPYD (rs1801159 and rs3918290), UMPS (rs1801019), ERCC1 (rs11615), ERCC1 (rs3212986), and ERCC2 (rs13181) in 56 FFPE DNA samples. By using PCR, followed by size-based gel electrophoresis, we also examined TYMS 5' untranslated region 2R/3R repeats and GSTT1 deletions in 50 FFPE DNA samples and 34 DNAs extracted from fresh-frozen tissues and cell lines. Each polymorphism was analyzed by two independent runs. We found that iPLEX biomarker assays measuring single-nucleotide polymorphisms provided consistent concordant results. However, by using FFPE DNA, size-based PCR biomarkers (GSTT1 and TYMS 5' untranslated region) were discrepant in 32.7% (16/49, with exact 95% CI, 19.9%-47.5%; exact binomial confidence limit test) and 4.2% (2/48, with exact 95% CI, 0.5%-14.3%) of cases, respectively, whereas no discrepancies were observed using intact genomic DNA. Our findings suggest that DNA from FFPE material can be used to reliably test single-nucleotide polymorphisms. However, results based on size-based PCR biomarkers, and particularly GSTT1 deletions, using FFPE DNA need to be interpreted with caution. Independent repeated assays should be performed on all cases to assess potential discrepancies.