Global Ring Study to Investigate the Comparability of Total Assay Performance of Commercial Claudin 18 Antibodies for Evaluation in Gastric Cancer.

Claudin 18.2 (CLDN18.2), the dominant isoform of CLDN18 in gastric tissues, is a highly specific tight junction protein of the gastric mucosa with variably retained expressions in gastric and gastroesophageal junction cancers. Additionally, CLDN18.2-targeted treatment with zolbetuximab, in combination with chemotherapy, has recently been assessed in 2 phase-III studies of patients with HER2-negative, locally advanced, unresectable, or metastatic gastric or gastroesophageal junction adenocarcinoma. These trials used the investigational VENTANA CLDN18 (43-14A) RxDx immunohistochemistry (IHC) assay on the Ventana BenchMark platform to identify patients eligible for CLDN18.2-targeted treatment. We report the findings of a global ring study evaluating the analytical comparability of concordance of the results of 3 CLDN18 antibodies (Ventana, LSBio, and Novus) stained on 3 IHC-staining platforms (Ventana, Dako, and Leica). A tissue microarray (TMA), comprising 15 gastric cancer cases, was stained by 27 laboratories across 11 countries. Each laboratory stained the TMAs using at least 2 of the 3 evaluated CLDN18 antibodies. Stained TMAs were assessed and scored using an agreed IHC-scoring algorithm, and the results were collated for statistical analysis. The data confirmed a high level of concordance for the VENTANA CLDN18 (43-14A; Ventana platform only) and LSBio antibodies on both the Dako and Leica platforms, with accuracy, precision, sensitivity, and specificity rates all reaching a minimum acceptable ≥85% threshold and good-to-excellent levels of concordance as measured by Cohen's kappa coefficient. The Novus antibody showed the highest level of variability against the reference central laboratory results for the same antibody/platform combinations. It also failed to meet the threshold for accuracy and sensitivity when used on either the Dako or Leica platform. These results demonstrated the reliability of IHC testing for CLDN18 expression in gastric tumor samples when using commercially available platforms with an appropriate methodology and primary antibody selection.

High Interobserver Variability Among Pathologists Using Combined Positive Score to Evaluate PD-L1 Expression in Gastric, Gastroesophageal Junction, and Esophageal Adenocarcinoma.

Reliable, reproducible methods to interpret programmed death ligand-1 (PD-L1) expression on tumor cells (TC) and immune cells (IC) are needed for pathologists to inform decisions associated with checkpoint inhibitor therapies. Our international study compared interpathologist agreement of PD-L1 expression using the combined positive score (CPS) under standardized conditions on samples from patients with gastric/gastroesophageal junction/esophageal adenocarcinoma. Tissue sections from 100 adenocarcinoma pretreatment biopsies were stained in a single laboratory using the PD-L1 immunohistochemistry 28-8 and 22C3 (Agilent) pharmDx immunohistochemical assays. PD-L1 CPS was evaluated by 12 pathologists on scanned whole slide images of these biopsies before and after a 2-hour CPS training session by Agilent. Additionally, pathologists determined PD-L1-positive TC, IC, and total viable TC on a single tissue fragment from 35 of 100 biopsy samples. Scoring agreement among pathologists was assessed using the intraclass correlation coefficient (ICC). Interobserver variability for CPS for 100 biopsies was high, with only fair agreement among pathologists both pre- (range, 0.45-0.55) and posttraining (range, 0.56-0.57) for both assays. For the 35 single biopsy samples, poor/fair agreement was also observed for the total number of viable TC (ICC, 0.09), number of PD-L1-positive IC (ICC, 0.19), number of PD-L1-positive TC (ICC, 0.54), and calculated CPS (ICC, 0.14), whereas calculated TC score (positive TC/total TC) showed excellent agreement (ICC, 0.82). Retrospective histologic review of samples with the poorest interpathologist agreement revealed the following as possible confounding factors: (1) ambiguous identification of positively staining stromal cells, (2) faint or variable intensity of staining, (3) difficulty in distinguishing membranous from cytoplasmic tumor staining, and (4) cautery and crush artifacts. These results emphasize the need for objective techniques to standardize the interpretation of PD-L1 expression when using the CPS methodology on gastric/gastroesophageal junction cancer biopsies to accurately identify patients most likely to benefit from immune checkpoint inhibitor therapy.

High interobserver and intraobserver reproducibility among pathologists assessing PD-L1 CPS across multiple indications.

AIMS: A common concern among pathologists scoring PD-L1 immunohistochemical staining is interobserver and intraobserver variability. We assessed the interobserver and intraobserver reproducibility of PD-L1 scoring among trained pathologists using a combined positive score (CPS; tumour cell and tumour-associated immune cell staining). METHODS AND RESULTS: Data were collected for 2 years (2017-2019) from 456 pathologists worldwide. Digital training encompassed unique, tumour-specific training, and test sets. Samples were stained using PD-L1 IHC 22C3 pharmDx and evaluated at specific CPS cutoffs for gastric cancer (GC), cervical cancer (CC), urothelial cancer (UC), oesophageal cancer (OC), and head and neck squamous cell carcinoma (HNSCC). Pathologists underwent expert-to-peer training and scored 20 blinded samples on day 1 and 25 blinded samples on day 2 (including 15 of the day 1 samples). Interobserver and intraobserver reproducibility were assessed. For GC (120 observers) and CC (32 observers) samples assessed at CPS ≥1, average interobserver agreement was 91.5% and 91.0%, respectively, and average intraobserver agreement was 90.2% and 96.6%, respectively. For UC (139 observers) and OC (52 observers) samples measured at CPS ≥10, average interobserver agreement was 93.4% and 93.7%, respectively, and average intraobserver agreement was 92.0% and 92.5%, respectively. For HNSCC samples (113 observers), average interobserver agreement was 94.1% at CPS ≥1 and 86.5% at CPS ≥20; intraobserver agreement was 94.7% at CPS ≥1 and 90.5% at CPS ≥20. CONCLUSION: The consistently high interobserver and intraobserver concordance rates support the effectiveness of face-to-face training of many global pathologists for scoring PD-L1 CPS across multiple indications at several specific cutoffs.

ISH-based HER2 diagnostics.

A prerequisite for all HER2 directed therapies is the demonstration of HER2 receptor protein overexpression and/or gene amplification by in situ hybridization (ISH). ASCO and CAP have published several HER2 test guidelines over the past 15 years for both breast and gastric cancer. The latest version for breast cancer (2018) focuses on special issues of ISH related to the definitions of special diagnostic groups (1-5). The guidelines for gastroesophageal adenocarcinoma (2017), essentially based on ToGA trial data, are now also being used for other tumors such as pancreas, gallbladder, and non-small-cell lung cancer. For colorectal cancer, a modified testing procedure has been proposed. Recently, besides overexpression and amplification, a third type of HER gene alteration, namely mutation, has gained much interest. Next-generation sequencing (NGS) allows detection of both amplification and mutation of the HER2 gene providing new options of therapy especially in the case of activating mutations.

[ISH-based HER2 diagnostics]. / ISH-basierte HER2-Diagnostik.

A prerequisite for all HER2 directed therapies is the demonstration of HER2 receptor protein overexpression and/or gene amplification by in situ hybridization (ISH). ASCO and CAP have published several HER2 test guidelines over the past 15 years for both breast and gastric cancer. The latest version for breast cancer (2018) focuses on special issues of ISH related to the definitions of special diagnostic groups (1-5). The guidelines for gastroesophageal adenocarcinoma (2017), essentially based on ToGA trial data, are now also being used for other tumors such as pancreas, gallbladder, and non-small-cell lung cancer. For colorectal cancer, a modified testing procedure has been proposed. Recently, besides overexpression and amplification, a third type of HER gene alteration, namely mutation, has gained much interest. Next-generation sequencing (NGS) allows detection of both amplification and mutation of the HER2 gene providing new options of therapy especially in the case of activating mutations.

Optimization and validation of PD-L1 immunohistochemistry staining protocols using the antibody clone 28-8 on different staining platforms.

Several immunohistochemistry (IHC) assays have been developed to assess tumor programmed death-ligand 1 (PD-L1) expression levels in patients who are candidates for programmed death-1 (PD-1)/PD-L1 inhibitor therapy. The PD-L1 IHC 28-8 pharmDx kit is FDA-approved as a complementary diagnostic and CE-marked as an in vitro diagnostic device for nivolumab therapy in melanoma and specific lung cancer subtypes (and for squamous cell carcinoma of the head and neck/urothelial carcinoma in Europe only). Kit availability is limited outside the United States, and its use requires the Dako Autostainer Link 48 platform, which is unavailable in many laboratories. Validated laboratory-developed tests based on 28-8 concentrated antibody outside the kit are needed. This study compared the results from PD-L1 expression level analysis across four immunohistochemistry platforms (Dako Autostainer Link 48, Dako Omnis, Leica Bond-III, and Ventana BenchMark ULTRA) with the 28-8 pharmDx kit in lung cancer (multiple histologies), melanoma, and head and neck cancer (multiple histologies). Samples were prepared per protocol for each platform and stained using PD-L1 IHC 28-8 pharmDx kit on Dako Autostainer Link 48, and per protocol for each platform. The control samples (tonsil and placenta tissue; cell lines with prespecified PD-L1 expression levels) were tested to evaluate the specificity and the sensitivity of test assays. An agreement level of 0.90 with the pharmDx kit was set for each platform. Inter- and intra-assay reliability were assessed. Evaluable samples were lung cancer = 29; melanoma = 31; head and neck cancer = 30. Mean agreement was calculated for PD-L1 expression levels of ≥1%, ≥5%, ≥10%, and ≥50%. Mean overall agreement for all indications was 0.87-0.99. Inter- and intra-assay of scoring/classification repeatability was 100%. Analysis of PD-L1 expression levels using laboratory-developed immunohistochemistry assays with 28-8 antibody may be permissible if the platform is validated using reference samples with defined expression levels.

Interlaboratory concordance of PD-L1 immunohistochemistry for non-small-cell lung cancer.

AIMS: Programmed death ligand 1 (PD-L1) immunohistochemistry has become a mandatory diagnostic test in the treatment of lung cancer. Several research initiatives have started to harmonise the five PD-L1 immunohistochemistry assays that have been used in clinical trials. Here, we report data on interlaboratory and interassay concordance for commercial assays ('assays') and laboratory-developed tests (LDTs) at 10 German testing sites. METHODS AND RESULTS: To assess interlaboratory concordance, a tissue microarray containing 21 pulmonary carcinoma specimens was centrally prepared. Pre-cut sections were stained at 10 sites by the use of assays 28-8, 22C3, SP263, and SP142, as well as 11 LDTs. Assay performance was evaluated with a second tissue microarray containing 11 cell lines with defined PD-L1 expression. Quality control was centrally performed by manual and digital analyses. The assays yielded reproducible IHC staining patterns at all sites. In agreement with previous studies, 22C3, 28-8 and SP263 showed similar staining patterns, whereas SP142 was distinct. Among the LDTs, six of 11 protocols showed staining patterns similar to those of assays 22C3 and 28-8. Interlaboratory concordance of tumour cell scoring by use of a six-step system was moderate (Light's κ = 0.43-0.69), whereas the clinically approved cut-offs of ≥1% and ≥50% showed substantial concordance (κ = 0.73-0.89). Immune cell scoring by the use of SP142 yielded moderate concordance (κ = 0.42). CONCLUSIONS: The data confirm the previously described staining patterns of the assays, and show that they can be reproducibly employed at different sites. LDTs with staining results similar to those of the assays are implementable, but have to be carefully validated.

Impact of updated HER2 testing guidelines in breast cancer--re-evaluation of HERA trial fluorescence in situ hybridization data.

Recently the American Society of Clinical Oncology and the College of American Pathologists have updated their clinical practice guidelines for HER2 testing in breast cancer. In order to evaluate these new recommendations, we have re-assessed the HER2 status of 6018 breast cancer cases of the screening population for the HERceptin adjuvant (HERA) trial that were originally centrally tested by fluorescence in situ hybridization based on the FDA-released test guidelines. According to the most recent 2013 ASCO/CAP recommendations, 3380 (56.2%) cases were classified as HER2 positive compared with 3359 (55.8%) applying the HERA/FDA scheme and 3339 (55.5%) applying the 2007 ASCO/CAP guidelines. Twenty-one cases switched from negative (HERA/FDA scheme) to positive (2013 ASCO/CAP guidelines). This group is characterized by a mean HER2 gene copy number of ≥6.0, polysomy or co-amplification of CEP17 with an average CEP17 count of 5, and with HER2 receptor overexpression in 75% of cases. On the basis of the HER2 gene copy number alone, we observe 494 cases (8.2%) that are in the equivocal range. Most of these cases (>80%) were also nondecisive by immunohistochemistry (score 2+) irrespective of whether ratio was <2.0>. The number of equivocal cases that would require HER2 reflex testing decreases to 113 (1.9%) if in addition to the HER2 gene copy number also the ratio of HER2 and CEP17 copy numbers is considered via dual-color in situ hybridization. The combination of applying the HER2 mean gene copy number as well as the HER2/CEP17 ratio to define equivocal test decisions by fluorescence in situ hybridization as proposed by the current ASCO/CAP guidelines appears to be a more optimum approach to adopt in order to avoid or minimize reporting of false negative results. Using the mean HER2 gene copy number alone for decision making results in a significant increase of equivocal cases.

Epidermal growth factor receptor immunohistochemistry: new opportunities in metastatic colorectal cancer.

The treatment of cancer is becoming more precise, targeting specific oncogenic drivers with targeted molecular therapies. The epidermal growth factor receptor has been found to be over-expressed in a multitude of solid tumours. Immunohistochemistry is widely used in the fields of diagnostic and personalised medicine to localise and visualise disease specific proteins. To date the clinical utility of epidermal growth factor receptor immunohistochemistry in determining monoclonal antibody efficacy has remained somewhat inconclusive. The lack of an agreed reproducible scoring criteria for epidermal growth factor receptor immunohistochemistry has, in various clinical trials yielded conflicting results as to the use of epidermal growth factor receptor immunohistochemistry assay as a companion diagnostic. This has resulted in this test being removed from the licence for the drug panitumumab and not performed in clinical practice for cetuximab. In this review we explore the reasons behind this with a particular emphasis on colorectal cancer, and to suggest a way of resolving the situation through improving the precision of epidermal growth factor receptor immunohistochemistry with quantitative image analysis of digitised images complemented with companion molecular morphological techniques such as in situ hybridisation and section based gene mutation analysis.

Scoring PD-L1 Expression in Urothelial Carcinoma: An International Multi-Institutional Study on Comparison of Manual and Artificial Intelligence Measurement Model (AIM-PD-L1) Pathology Assessments.

Assessing programmed death ligand 1 (PD-L1) expression on tumor cells (TCs) using Food and Drug Administration-approved, validated immunoassays can guide the use of immune checkpoint inhibitor (ICI) therapy in cancer treatment. However, substantial interobserver variability has been reported using these immunoassays. Artificial intelligence (AI) has the potential to accurately measure biomarker expression in tissue samples, but its reliability and comparability to standard manual scoring remain to be evaluated. This multinational study sought to compare the %TC scoring of PD-L1 expression in advanced urothelial carcinoma, assessed by either an AI Measurement Model (AIM-PD-L1) or expert pathologists. The concordance among pathologists and between pathologists and AIM-PD-L1 was determined. The positivity rate of ≥ 1%TC PD-L1 was between 20-30% for 8/10 pathologists, and the degree of agreement and scoring distribution for among pathologists and between pathologists and AIM-PD-L1 was similar both scored as a continuous variable or using the pre-defined cutoff. Numerically higher score variation was observed with the 22C3 assay than with the 28-8 assay. A 2-h training module on the 28-8 assay did not significantly impact manual assessment. Cases exhibiting significantly higher variability in the assessment of PD-L1 expression (mean absolute deviation > 10) were found to have patterns of PD-L1 staining that were more challenging to interpret. An improved understanding of sources of manual scoring variability can be applied to PD-L1 expression analysis in the clinical setting. In the future, the application of AI algorithms could serve as a valuable reference guide for pathologists while scoring PD-L1.

Critical research gaps and translational priorities for the successful prevention and treatment of breast cancer.

INTRODUCTION: Breast cancer remains a significant scientific, clinical and societal challenge. This gap analysis has reviewed and critically assessed enduring issues and new challenges emerging from recent research, and proposes strategies for translating solutions into practice. METHODS: More than 100 internationally recognised specialist breast cancer scientists, clinicians and healthcare professionals collaborated to address nine thematic areas: genetics, epigenetics and epidemiology; molecular pathology and cell biology; hormonal influences and endocrine therapy; imaging, detection and screening; current/novel therapies and biomarkers; drug resistance; metastasis, angiogenesis, circulating tumour cells, cancer 'stem' cells; risk and prevention; living with and managing breast cancer and its treatment. The groups developed summary papers through an iterative process which, following further appraisal from experts and patients, were melded into this summary account. RESULTS: The 10 major gaps identified were: (1) understanding the functions and contextual interactions of genetic and epigenetic changes in normal breast development and during malignant transformation; (2) how to implement sustainable lifestyle changes (diet, exercise and weight) and chemopreventive strategies; (3) the need for tailored screening approaches including clinically actionable tests; (4) enhancing knowledge of molecular drivers behind breast cancer subtypes, progression and metastasis; (5) understanding the molecular mechanisms of tumour heterogeneity, dormancy, de novo or acquired resistance and how to target key nodes in these dynamic processes; (6) developing validated markers for chemosensitivity and radiosensitivity; (7) understanding the optimal duration, sequencing and rational combinations of treatment for improved personalised therapy; (8) validating multimodality imaging biomarkers for minimally invasive diagnosis and monitoring of responses in primary and metastatic disease; (9) developing interventions and support to improve the survivorship experience; (10) a continuing need for clinical material for translational research derived from normal breast, blood, primary, relapsed, metastatic and drug-resistant cancers with expert bioinformatics support to maximise its utility. The proposed infrastructural enablers include enhanced resources to support clinically relevant in vitro and in vivo tumour models; improved access to appropriate, fully annotated clinical samples; extended biomarker discovery, validation and standardisation; and facilitated cross-discipline working. CONCLUSIONS: With resources to conduct further high-quality targeted research focusing on the gaps identified, increased knowledge translating into improved clinical care should be achievable within five years.

FOCUS4 biomarker laboratories: from the benefits to the practical and logistical issues faced during 6 years of centralised testing.

AIMS: FOCUS4 was a phase II/III umbrella trial, recruiting patients with advanced or metastatic colorectal cancer, between 2014 and 2020. Molecular profiling of patients' formalin-fixed, paraffin-embedded tumour blocks was undertaken at two centralised biomarker laboratories (Leeds and Cardiff), and the results fed directly to the Medical Research Council Clinical Trials Unit, and used for subsequent randomisation. Here the laboratories discuss their experiences. METHODS: Following successful tumour content assessment, blocks were sectioned for DNA extraction and immunohistochemistry (IHC). Pyrosequencing was initially used to determine tumour mutation status (KRAS, NRAS, BRAF and PIK3CA), then from 2018 onwards, next-generation sequencing was employed to allow the inclusion of TP53. Protein expression of MLH1, MSH2, MSH6, PMS2 and pTEN was determined by IHC. An interlaboratory comparison programme was initiated, allowing sample exchanges, to ensure continued assay robustness. RESULTS: 1291 tumour samples were successfully analysed. Assay failure rates were very low; 1.9%-3.3% for DNA sequencing and 0.9%-1.3% for IHC. Concordance rates of >98% were seen for the interlaboratory comparisons, where a result was obtained by both laboratories. CONCLUSIONS: Practical and logistical problems were identified, including poor sample quality and difficulties with sample anonymisation. The often last-minute receipt of a sample for testing and a lack of integration with National Health Service mutation analysis services were challenging. The laboratories benefitted from both pretrial validations and interlaboratory comparisons, resulting in robust assay development and provided confidence during the implementation of new sequencing technologies. We conclude that our centralised approach to biomarker testing in FOCUS4 was effective and successful.

Tumor cell cytoplasmic metallothionein expression associates with differential tumor immunogenicity and prognostic outcome in high-grade serous ovarian carcinoma.

Background: The underlying mechanism of high T-cell presence as a favorable prognostic factor in high-grade serous ovarian carcinoma (HGSOC) is not yet understood. In addition to immune cells, various cofactors are essential for immune processes. One of those are metallothioneins (MTs), metal-binding proteins comprising various isoforms. MTs play a role in tumor development and drug resistance. Moreover, MTs influence inflammatory processes by regulating zinc homeostasis. In particular, T-cell function and polarization are particularly susceptible to changes in zinc status. The aim of the present study was to investigate a possible role of MT-mediated immune response and its association with prognostic outcome in ovarian cancer. Methods: A retrospective study was conducted on a clinically well-characterized cohort of 24 patients with HGSOC treated at the University Hospital of Essen. Gene expression patterns for anti-cancer immunogenicity-related targets were performed using the NanoString nCounter platform for digital gene expression analysis with the appurtenant PanCancer Immune Profiling panel, consisting of 770 targets and 30 reference genes. Tumor-associated immunohistochemical MT protein expression was evaluated using a semi-quantitative four-tier Immunohistochemistry (IHC) scoring. Results: MT immunoexpression was detected in 43% (10/23) of all HGSOC samples. MT immunoexpression levels showed a significant association to survival, leading to prolonged progression-free and overall survival in positively stained tumors. Furthermore, T-cell receptor signaling gene signature showed a strong activation in MT-positive tumors. Activated downstream signaling cascades resulting in elevated interferon-gamma expression with a shift in the balance between T helper cells (TH1 and TH2) could be observed in the MT-positive subgroup. In addition, a higher expression pattern of perforin and several granzymes could be detected, overall suggestive of acute, targeted anti-cancer immune response in MT-positive samples. Conclusion: This is the first study combining broad, digital mRNA screening of anti-tumor immune response-associated genes and their relation to MT-I/II in ovarian cancer. MT overexpression is associated with molecular characteristics of an anti-cancer immune response and is a strong prognostic marker in ovarian HGSOC. The observed immune cell activation associated with tumor MT expression comprises but is not limited to T cells and natural killer cells.

Addition of cetuximab to oxaliplatin-based first-line combination chemotherapy for treatment of advanced colorectal cancer: results of the randomised phase 3 MRC COIN trial.

BACKGROUND: In the Medical Research Council (MRC) COIN trial, the epidermal growth factor receptor (EGFR)-targeted antibody cetuximab was added to standard chemotherapy in first-line treatment of advanced colorectal cancer with the aim of assessing effect on overall survival. METHODS: In this randomised controlled trial, patients who were fit for but had not received previous chemotherapy for advanced colorectal cancer were randomly assigned to oxaliplatin and fluoropyrimidine chemotherapy (arm A), the same combination plus cetuximab (arm B), or intermittent chemotherapy (arm C). The choice of fluoropyrimidine therapy (capecitabine or infused fluouroracil plus leucovorin) was decided before randomisation. Randomisation was done centrally (via telephone) by the MRC Clinical Trials Unit using minimisation. Treatment allocation was not masked. The comparison of arms A and C is described in a companion paper. Here, we present the comparison of arm A and B, for which the primary outcome was overall survival in patients with KRAS wild-type tumours. Analysis was by intention to treat. Further analyses with respect to NRAS, BRAF, and EGFR status were done. The trial is registered, ISRCTN27286448. FINDINGS: 1630 patients were randomly assigned to treatment groups (815 to standard therapy and 815 to addition of cetuximab). Tumour samples from 1316 (81%) patients were used for somatic molecular analyses; 565 (43%) had KRAS mutations. In patients with KRAS wild-type tumours (arm A, n=367; arm B, n=362), overall survival did not differ between treatment groups (median survival 17·9 months [IQR 10·3-29·2] in the control group vs 17·0 months [9·4-30·1] in the cetuximab group; HR 1·04, 95% CI 0·87-1·23, p=0·67). Similarly, there was no effect on progression-free survival (8·6 months [IQR 5·0-12·5] in the control group vs 8·6 months [5·1-13·8] in the cetuximab group; HR 0·96, 0·82-1·12, p=0·60). Overall response rate increased from 57% (n=209) with chemotherapy alone to 64% (n=232) with addition of cetuximab (p=0·049). Grade 3 and higher skin and gastrointestinal toxic effects were increased with cetuximab (14 vs 114 and 67 vs 97 patients in the control group vs the cetuximab group with KRAS wild-type tumours, respectively). Overall survival differs by somatic mutation status irrespective of treatment received: BRAF mutant, 8·8 months (IQR 4·5-27·4); KRAS mutant, 14·4 months (8·5-24·0); all wild-type, 20·1 months (11·5-31·7). INTERPRETATION: This trial has not confirmed a benefit of addition of cetuximab to oxaliplatin-based chemotherapy in first-line treatment of patients with advanced colorectal cancer. Cetuximab increases response rate, with no evidence of benefit in progression-free or overall survival in KRAS wild-type patients or even in patients selected by additional mutational analysis of their tumours. The use of cetuximab in combination with oxaliplatin and capecitabine in first-line chemotherapy in patients with widespread metastases cannot be recommended. FUNDING: Cancer Research UK, Cancer Research Wales, UK Medical Research Council, Merck KGgA.

The PD-1/PD-L1 Pathway: A Perspective on Comparative Immuno-Oncology.

The programmed cell death protein 1/programmed death-ligand 1 (PD-1/PD-L1) pathway mainly attracted attention in immuno-oncology, leading to the development of immune checkpoint therapy. It has, however, much broader importance for tissue physiology and pathology. It mediates basic processes of immune tolerance and tissue homeostasis. In addition, it is involved in the pathogenesis of chronic infectious diseases, autoimmunity, and cancer. It is also an important paradigm for comparative pathology as well as the "one health one medicine" concept. The aim of this review is to provide an overview of novel research into the diverse facets of the PD-1/PD-L1 pathway and to give insights into its fine-tuning homeostatic role in a tissue-specific context. This review details early translational research from the discovery phase based on mice as animal models for understanding pathophysiological aspects in human tissues to more recent research extending the investigations to several animal species. The latter has the twofold goal of comparing this pathway between humans and different animal species and translating diagnostic tools and treatment options established for the use in human beings to animals and vice versa.

Comparison of HercepTest™ mAb pharmDx (Dako Omnis, GE001) with Ventana PATHWAY anti-HER-2/neu (4B5) in breast cancer: correlation with HER2 amplification and HER2 low status.

Performance of the new CE-IVD-marked HercepTest™ mAb pharmDx (Dako Omnis) assay (HercepTest (mAb)) was compared against the PATHWAY® anti-HER-2/neu (4B5) (PATHWAY 4B5) assay using 119 pre-selected breast cancer samples covering the entire range of HER2 immunohistochemistry (IHC) expression scores (0, 1 + , 2 + , 3 +). The sensitivity and specificity of both assays were assessed based on consensus IHC scores and amplification status, as determined by fluorescence in situ hybridization (FISH) according to 2018 ASCO/CAP testing guidelines. There was a high concordance between results from the HercepTest (mAb) and PATHWAY 4B5 assays for HER2-negative (IHC 0, 1 + , 2 + and FISH negative) and HER2-positive (IHC 3 + , 2 + and FISH positive) breast carcinomas (98.2%). Regarding individual IHC scores, complete agreement was achieved in 69.7% (83/119) of cases, and all but one of the discordant cases were due to higher HER2-status scoring using the HercepTest (mAb). Thus, more tumors were overscored as IHC 2 + by HercepTest (mAb) (27 versus 15) as evidenced by their lower FISH positivity rate (48.1% versus 80%). However, two amplified tumors identified as IHC 2 + by HercepTest (mAb) were missed by PATHWAY 4B5 (IHC 1 +). Four additional cases identified as IHC 2 + by HercepTest (mAb), with FISH ratio < 2 but elevated gene counts (≥ 4 to < 6), were recorded negative by PATHWAY 4B5. The HercepTest (mAb) detects HER2 expression with higher sensitivity in tumors with gene amplification (ISH group 1) and increased gene counts (ISH group 4) as well as in HER2-low tumors (HER2 IHC2 + /FISH negative or IHC 1 +). Future studies will demonstrate whether this translates into improved patient selection especially for new HER2-directed therapies.

Glutathione S-transferases as molecular markers of tumour progression and prognosis in renal cell carcinoma.

AIMS: Renal cell carcinoma (RCC) often recurs as distant metastasis; there is thus a need for new indicators to identify high-risk patients. Glutathione S-transferases (GST)-α and -π are involved in the renal bioactivation of toxic metabolites. The aim was to investigate whether their expression is of diagnostic and prognostic value. METHODS AND RESULTS: Western blotting of microdissected normal kidney and immunostaining of histological RCC microarrays shows expression of GST-α in proximal tubular cells, while GST-π was found in the distal nephron. Of the primary 174 RCC cases examined, GST-α immunoreactivity was restricted to conventional RCC (n=76, 68% positive) and was not seen in any other RCC subtypes. The cross-tabulation of the GST-α scores with other prognostic indices demonstrated that GST-α immunostaining was significantly more frequent in low-grade tumours (χ(2): P<0.004), and that conventional GST-α-positive RCC patients had a mean disease-free survival of 6.0 years (95% confidence interval 5.33-6.63), compared with 4.7 years (3.54-5.90) in GST-α-negative tumours (Kaplan-Meier survival analysis, P=0.011, log-rank test). CONCLUSIONS: GST-α is a highly specific diagnostic marker for primary conventional RCC, where it is a prognostic marker if grade is omitted from the multivariate analysis.

Overexpression of the drug resistance-associated protein metallothionein does not correlate with response of sarcomas to isolated limb perfusion treatment.

BACKGROUND AND OBJECTIVES: Hyperthermic isolated limb perfusion with TNF-alpha and melphalan (HILP-TM) achieves high response rates in sarcomas. Melphalan resistance was previously reported to be associated with overexpression of metallothioneins (MTs). Objective of this study was to investigate the influence of MT expression on tumor responses in HILP-TM-treated soft tissue (STSs) and bone sarcomas (BS). METHODS: In primary biopsies of 41 HILP-TM-treated sarcomas (37 STSs and 4 BS), MT expression was assessed by an immunoreactive score. The pathologic response to HILP-TM was quantified in the corresponding tumor resection specimens. We studied the association of MT-IRS between histological regression (responder >90%, or non-responder

Does Simian Virus 40 (SV40) Have a Role in UK Malignant Pleural Mesothelioma? No Role is Identified in a Sensitive RNA In Situ Hybridization Study on Potentially Affected Birth Cohorts.

BACKGROUND: Simian virus 40 (SV40)-contaminated polio vaccine was accidentally administered to about one-third of the UK population receiving polio vaccines between 1956 and 1962. SV40 was subsequently demonstrated to be a carcinogenic virus in experimental and animal models. Since then, the SV40 oncogenic protein large T antigen (SV40 Tag) has been shown to cause malignant transformation of asbestos-treated human pleural mesothelial cells and malignant pleural mesotheliomas in asbestos-exposed SV40 Tag transgenic mice. The present study was designed to investigate the possible association of SV40 Tag with human malignant pleural mesothelioma samples from birth cohorts of the UK population exposed to combined peak levels of asbestos and SV40-contaminated polio vaccines. MATERIALS AND METHODS: Tumor and background lung tissue microarrays prepared from archival surgical specimens of 139 pleural mesothelioma cases, collected over a period of 8 years (1998 to 2005), were analyzed. These represented birth cohorts overlapping with the period 1950 to 1960, exposed to a high level of both asbestos and SV40-contaminated live polio vaccines. SV40 Tag mRNA expression was investigated using a highly sensitive and specific SV40 Tag RNA in situ hybridization detection method on the basis of the novel RNAscope technology. RESULTS: SV40 Tag RNA was not detected in any of the 127 evaluable tumor cases, despite appropriate results obtained for the external positive and negative controls included. CONCLUSION: The complete absence of SV40 Tag mRNA in this large series of cases contradicts experimental evidence suggestive of SV40 link with asbestos-exposed malignant pleural mesotheliomas in the UK population. Alternative explanations of the negative findings are discussed to exclude possible confounding factors.