Lung cancer, the leading cause of cancer-related mortality, presents major challenges for both standard therapies and chimeric antigen receptor (CAR) T cell therapy due to tumour heterogeneity and resistance. Preclinical models that capture patient-specific factors are essential for personalizing treatment decisions. Here we show that matched lung tumouroids and healthy lung organoids derived from patients provide a robust platform for studying therapy responses. The tumouroids faithfully retained the molecular and histological identity of the original tumours, as confirmed by genomic, epigenomic and proteomic analyses, and accurately replicated individual patient responses to standard-of-care therapies. Importantly, the platform also revealed patient-specific CAR T cell responses, uncovering a complex interplay between target antigen density and broader, tumour-intrinsic resistance programmes. By capturing these individualized factors, our model supports rational patient selection for CAR T cell therapy in lung cancer and provides a framework for designing CAR T cells tailored to overcome resistance mechanisms in solid tumours. A platform using matched patient-derived lung tumouroids and healthy lung organoids enables accurate examination of patient responses to CAR T therapy and offers a faithful framework for improved CAR T design.

Background: ESR1 mutations are biomarkers in breast cancer patients who develop metastatic disease after endocrine therapy (ET). Recently, the Food and Drug Administration (FDA) and European Medicines Agency (EMA) have approved Elacestrant, a selective estrogen receptor degrader for patients harboring ESR1 mutations. This has necessitated the establishment of reliable and sensitive NGS- or PCR-based assays to detect these ESR1 resistance mutations in liquid biopsy samples. Methods: We evaluated NGS results of a pan-cancer cohort of almost 6000 patients from two major German institutes of pathology, to show that the occurrence of ESR1 mutations is extremely rare (<1%) in ET-naïve patients. This suggests that ESR1 mutations arise almost exclusively under the pressure of ET. Therefore, we designed a breast cancer-specific hybrid capture-based NGS liquid biopsy assay covering 12 breast cancer-related genes, including ESR1, PIK3CA, AKT1, ERBB2, BRCA1/2, and TP53. We validated the HS2-Mamma-LIQ assay extensively using reference material to detect mutations to 0.1% variant allele frequency (VAF) and compared the performance to a commercially available ESR1 ddPCR assay. Results: We show the results of routine diagnostic analysis of the first consecutive 354 patients with activating ESR1 mutations rate of 43%, with 20% of patients harboring co-mutations in PIK3CA and other genes underlining the relevance of tumor heterogeneity. Our study highlights liquid biopsy as a preferred approach for monitoring ESR1 mutations in breast cancer patients by showing cases where NGS analysis suggests complex tumor heterogeneity with multiple ESR1 as well as PIK3CA mutations at different VAFs. Conclusions: Our findings not only corroborate prior research concerning the rarity of these mutations in unselected patients but also emphasize the importance of robust and broad molecular assays rather than single gene assays in their detection and characterization in the diagnostic setting. Advantages of different approaches are discussed to address the current clinical need.

In precision oncology, reliable testing of predictive molecular biomarkers is the prerequisite for optimal patient treatment. Interlaboratory comparisons are a crucial tool to verify diagnostic performance and reproducibility of one's approach. Here, we describe the design and results of the first recurrent, internationally performed PIK3CA Breast Cancer Tissue external quality assessment (EQA), which was organized by German Quality in Pathology (QuIP) GmbH and started in 2021. After the internal pretesting phase performed by the (lead) panel institutes, in both 2021 and 2022, each EQA test set comprised n=10 tissue samples of hormone receptor-positive, human epidermal growth factor receptor 2-negative invasive breast cancer (IBC) that had to be analyzed and reported by the participants. In 2021, the results were evaluated separately for German-speaking countries (part 1) and international laboratories (part 2). In 2022, the EQA was performed across the European Union. The EQA success rates were 84.6% (n=11/13), 88.6% (n=39/44), and 87.9% (n=29/33) for EQA 2021 part 1, EQA 2021 part 2, and EQA 2022, respectively. The most commonly used methodologies were next-generation sequencing and mutation-/allele-specific qualitative polymerase-chain-reaction-based assays. In summary, this recurrent PIK3CA EQA proved to be a suitable approach for quality assessment in predictive molecular biomarker testing, to obtain an international overview of methods used for PIK3CA mutation analysis and to identify the strengths and weaknesses of individual methods.

PURPOSE Poly(ADP-ribose) polymerase inhibitors (PARPi) have shown promising clinical results in the treatment of ovarian cancer. Analysis of biomarker subgroups consistently revealed higher benefits for patients with homologous recombination deficiency (HRD). The test that is most often used for the detection of HRD in clinical studies is the Myriad myChoice assay. However, other assays can also be used to assess biomarkers, which are indicative of HRD, genomic instability (GI), and BRCA1/ 2 mutation status. Many of these assays have high potential to be broadly applied in clinical routine diagnostics in a time-effective decentralized manner. Here, we compare the performance of a multitude of alternative assays in comparison with Myriad myChoice in high-grade serous ovarian cancer (HGSOC). METHODS DNA from HGSOC samples was extracted from formalin-fixed paraffin-embedded tissue blocks of cases previously run with the Myriad myChoice assay, and GI was measured by multiple molecular assays (CytoSNP, AmoyDx, Illumina TSO500 HRD, OncoScan, NOGGO GISv1, QIAseq HRD Panel and whole genome sequencing), applying different bioinformatics algorithms. RESULTS Application of different assays to assess GI, including Myriad myChoice, revealed high concordance of the generated scores ranging from very substantial to nearly perfect fit, depending on the assay and bioinformatics pipelines applied. Interlaboratory comparison of assays also showed high concordance of GI scores. CONCLUSION Assays for GI assessment not only show a high concordance with each other but also in correlation with Myriad myChoice. Thus, almost all of the assays included here can be used effectively to assess HRD-associated GI in the clinical setting. This is important as PARPi treatment on the basis of these tests is compliant with European Medicines Agency approvals, which are methodologically not test-bound.

In precision oncology, reliable testing of predictive molecular biomarkers is a prerequisite for optimal patient treatment. Interlaboratory comparisons are a crucial tool to verify diagnostic performance and reproducibility of one's approach. Herein is described the design and results of the fi rst recurrent, internationally performed PIK3CA (phosphatidylinositol-4,5-bisphosphate 3 kinase catalytic subunit a ) breast cancer tissue external quality assessment (EQA), organized by German Quality in Pathology GmbH and started in 2021. After the internal pretesting phase performed by the (lead) panel institutes, in both 2021 and 2022, each EQA test set comprised n = 10 tissue samples of hormone receptor- positive, human epidermal growth factor receptor 2- negative invasive breast cancer that had to be analyzed and reported by the participants. In 2021, the results were evaluated separately for German-speaking countries (part 1) and international laboratories (part 2). In 2022, the EQA was performed across the European Union. The EQA success rates were 84.6% (n n = 11/13), 88.6% (n n = 39/ 44), and 87.9% (n n = 29/33) for EQA 2021 part 1, part 2, and EQA 2022, respectively. The most commonly used methods were next-generation sequencing and mutation-/allele-specific fi c qualitative PCR-based assays. In summary, this recurrent PIK3CA EQA proved to be a suitable approach to obtain an international overview of methods used for PIK3CA mutation analysis, to evaluate them qualitatively, and identify the strengths and weaknesses of individual methods. (J Mol Diagn 2024, 26: 624-637;- 637; https://doi.org/10.1016/j.jmoldx.2024.04.003)

The worldwide approval of the combination maintenance therapy of olaparib and bevacizumab in advanced high-grade serous ovarian cancer requires complex molecular diagnostic assays that are sufficiently robust for the routine detection of driver mutations in homologous recombination repair (HRR) genes and genomic instability (GI), employing formalin-fixed (FFPE) paraffin-embedded tumor samples without matched normal tissue. We therefore established a DNA-based hybrid capture NGS assay and an associated bioinformatic pipeline that fulfils our institution's specific needs. The assay´s target regions cover the full exonic territory of relevant cancer-related genes and HRR genes and more than 20,000 evenly distributed single nucleotide polymorphism (SNP) loci to allow for the detection of genome-wide allele specific copy number alterations (CNA). To determine GI status, we implemented an %CNA score that is robust across a broad range of tumor cell content (25-85%) often found in routine FFPE samples. The assay was established using high-grade serous ovarian cancer samples for which BRCA1 and BRCA2 mutation status as well as Myriad MyChoice homologous repair deficiency (HRD) status was known. The NOGGO (Northeastern German Society for Gynecologic Oncology) GIS (GI-Score) v1 assay was clinically validated on more than 400 samples of the ENGOT PAOLA-1 clinical trial as part of the European Network for Gynaecological Oncological Trial groups (ENGOT) HRD European Initiative. The "NOGGO GIS v1 assay" performed using highly robust hazard ratios for progression-free survival (PFS) and overall survival (OS), as well a significantly lower dropout rate than the Myriad MyChoice clinical trial assay supporting the clinical utility of the assay. We also provide proof of a modular and scalable routine diagnostic method, that can be flexibly adapted and adjusted to meet future clinical needs, emerging biomarkers, and further tumor entities.

Claudins regulate paracellular permeability, contribute to epithelial polarization and are dysregulated during inflammation and carcinogenesis. Variants of the claudin-binding domain of Clostridium perfringens enterotoxin (cCPE) are highly sensitive protein ligands for generic detection of a broad spectrum of claudins. Here, we investigated the preferential binding of YFP- or GST-cCPE fusion proteins to non-junctional claudin molecules. Plate reader assays, flow cytometry and microscopy were used to assess the binding of YFP- or GST-cCPE to non-junctional claudins in multiple in vitro and ex vivo models of human and rat gastrointestinal epithelia and to monitor formation of a tight junction barrier. Furthermore, YFP-cCPE was used to probe expression, polar localization and dysregulation of claudins in patient-derived organoids generated from gastric dysplasia and gastric cancer. Live-cell imaging and immunocytochemistry revealed cell polarity and presence of tight junctions in glandular organoids (originating from intestinal-type gastric cancer and gastric dysplasia) and, in contrast, a disrupted diffusion barrier for granular organoids (originating from discohesive tumor areas). In sum, we report the use of cCPE fusion proteins as molecular probes to specifically and efficiently detect claudin expression, localization and tight junction dysregulation in cell lines, tissue explants and patient-derived organoids of the gastrointestinal tract.

Poly(ADP-ribose)-polymerase 1 inhibitors (PARPi) are currently in use in certain clinical settings for patients with ovarian cancer. Patients with homologous recombination repair deficiency (HRD) within their tumor which was defined as present if BRCA was mutated (BRCAm) and/or HRD-associated genomic instability (GIS) scores were high, revealed higher benefits, demonstrating the value of biomarker testing in this context. Currently the Myriad MyChoice assay is the most commonly used assay for HRD detection in clinical studies but other molecular assays can also be applied. Here, we compare the performance of multiple molecular assays in determining GIS in high grade ovarian cancer. DNA from high-grade epithelial ovarian cancer was extracted from formalin-fixed paraffin embedded tissue of cases with known Myriad MyChoice GIS scores. GIS was measured by CytoSNP, AmoyDX, Illumina TSO 500+, Thermo Fisher OCA+, Oncoscan, Agilent NOGGO GIS v1, Qiagen QIASeq HRD Panel, low pass whole genome sequencing and determined using different bioinformatics algorithms. Most assays were run twice, independently in two labs to allow for interlaboratory comparisons. GIS datasets were compared and correlated to BRCA mutation status. Up to 70 cases were analyzed per assay (range 13-70, final goal: assessment of 100 samples per assay). The application of different assays to assess GIS revealed high concordance/correlations of the GIS scores generated by the different assays, the concordance/correlation of GIS scores generated by the alternative assays to myChoice generated GIS also ranged from very substantial to nearly perfect fit, depending on the assay and bioinformatics pipelines applied. Interlaboratory comparison of assays also showed high concordance of the GIS scores generated. As expected, high GIS scores were significantly associated with the presence of BRCA mutations for all assays. Assays for GIS assessment show a concordance both between each other but also in correlation to the current standard assay, Myriad MyChoice. Interlaboratory variation was low. Our data suggest, that a variety of assays in principle could be used to asses GIS in the clinical setting.

Precision oncology based on specific molecular alterations requires precise and reliable detection of therapeutic targets in order to initiate the optimal treatment. In many European countries-including Germany-assays employed for this purpose are highly diverse and not prescribed by authorities, making inter-laboratory comparison difficult. To ensure reproducible molecular diagnostic results across many laboratories and different assays, ring trials are essential and a well-established tool. Here, we describe the design and results of the ring trial for the detection of therapeutically relevant PIK3CA hotspot mutations in HR+/HER2-breast cancer tissue and liquid biopsy (LB). For PIK3CA mutation detection in tissue samples, 43 of the 54 participants (80%) provided results compliant with the reference values. Participants using NGS-based assays showed higher success rate (82%) than those employing Sanger sequencing (57%). LB testing was performed with two reference materials differing in the length of the mutated DNA fragments. Most participants used NGS-based or commercial real-time PCR assays (70%). The 167 bp fragments led to a successful PIK3CA mutation detection by only 31% of participants whereas longer fragments of 490 bp were detectable even by non-optimal assays (83%). In conclusion, the first ring trial for PIK3CA mutation detection in Germany showed that PIK3CA mutation analysis is broadly established for tissue samples and that NGS-based tests seem to be more suitable than Sanger sequencing. PIK3CA mutation detection in LB should be carried out with assays specifically designed for this purpose in order to avoid false-negative results.

Introduction/Background Several clinical trials have demonstrated that the maintenance with a PARP inhibitor with or without bevacizumab following platinum-based therapy improved PFS in advanced ovarian cancer patients. The benefit was significant greater in homologous recombination deficient (HRD) patients according to Myriad myChoice test. The PAOLA-1 olaparib+bevacizumab maintenance regimen was approved in USA/Europe/Japan for HRD and BRCA positive patients. Using sample from the PAOLA-1/ENGOT-ov25 we evaluated the novel ‘NOGGO-GIS ASSAY’ as part of the ENGOT HRD-European-Initiative. Methodology A hybrid capture NGS assay based on the Agilent XTH2 chemistry and SNP backbone, was developed for the detection of somatic driver mutations in key cancer genes, BRCA1/2 mutations, HRR gene mutations and HRD in combination with a bioinformatic analysis pipeline based on publicly available tools. The assay was clinically validated using 468 ovarian cancer samples from the PAOLA-1/ENGOT-ov25 trial. Results Here we report the first results of the ‘NOGGO-GIS ASSAY’ validation compared to the Myriad myChoice clinical trial. The assay is based on widely available hybrid capture chemistry, to cover 57 genes and approx. 20.000 SNP loci, automated for parallel processing of 48 samples per run and requires 50 ng of genomic DNA extracted from tissue section with at least 30% of tumor content with a low failure rate of around 5%. The performance characteristics of the NOGGO GIS Assay are comparable to the PAOLA-1/ENGOT-ov25 clinical trial assay. The NOGGO GIS Assay showed a similar impact of olaparib+bevacizumab on PFS with a comparable Hazard Ratio for HRD positive patients. Conclusion The ‘NOGGO-GIS ASSAY’ based on widely available components was validated on clinical trial samples showing performance characteristics similar to the clinical trial assay. The low failure rate, low input material required, HRD and BRCA1/2 and mutation status in 57 clinically relevant genes makes this a highly attractive option for analysis of FFPE samples.

Objectives:The use of liquid biopsies (LB) in patients with solid malignancies enables comprehensive genomic profiling (CGP) of circulating tumor DNA (ctDNA) and has the potential to guide therapy stratification and support disease monitoring. To examine clinical uptake of LB in a real-world setting, LB implementation was analyzed at two German cancer centers (LMU Munich and Charité - Universitätsmedizin Berlin) between 2017 and 2021, with focus on colorectal cancer (CRC) patients. Methods:In this retrospective analysis, all patients who received a LB between January 2017 and December 2021 as part of routine clinical management were included. To provide adequate context, we collected disease characteristics and technical specifications of the LB methods applied. Additionally, we examined the concordance of RAS status in tumor tissue and LB. Finally, we discuss the potential of LB as a diagnostic tool to drive personalized treatment in CRC patients and how to implement LB in clinical routine. Results:In total, our cohort included 86 CRC patients and 161 LB conducted in these patients between 2017 and 2021. In 59 patients, comparison between tissue-based and liquid-based molecular diagnostics, revealed a divergence in 23 (39%) of the evaluable samples. Conclusion:Our real-world data analysis indicates that the possibilities of LB are not yet exploited in everyday clinical practice. Currently, the variety of methods and lack of standardization, as well as restricted reimbursement for liquid based CGP hinder the use of LB in clinical routine. To overcome these issues, prospective clinical trials are needed to provide evidence driving the implementation of LB into the management of CRC patients and to support their implementation into clinical guidelines.

The diagnosis of sinonasal tumors is challenging due to a heterogeneous spectrum of various differential diagnoses as well as poorly defined, disputed entities such as sinonasal undifferentiated carcinomas (SNUCs). In this study, we apply a machine learning algorithm based on DNA methylation patterns to classify sinonasal tumors with clinical-grade reliability. We further show that sinonasal tumors with SNUC morphology are not as undifferentiated as their current terminology suggests but rather reassigned to four distinct molecular classes defined by epigenetic, mutational and proteomic profiles. This includes two classes with neuroendocrine differentiation, characterized by IDH2 or SMARCA4/ARID1A mutations with an overall favorable clinical course, one class composed of highly aggressive SMARCB1-deficient carcinomas and another class with tumors that represent potentially previously misclassified adenoid cystic carcinomas. Our findings can aid in improving the diagnostic classification of sinonasal tumors and could help to change the current perception of SNUCs.

Approximately every second patient from everyday clinical practice receives pembrolizumab as first-line palliative treatment without clear evidence from clinical trials. Analyzing 153 consecutive patients, a comorbidity-defined PS >= 2, symptomatic brain metastases, or baseline steroids were associated with substantially reduced survival. All-in-all, real-world and clinical trial efficacy correspond well as long as baseline characteristics of real world and study patients match. Introduction: Pembrolizumab is a highly effective standard of care in PD-L1 overexpressing (>= 50%) non-small-cell lung cancer. However, a substantial share of patients from everyday clinical practice is treated without clear evidence from clinical trials. Patients and Methods: We performed a retrospective multicentric study including all consecutive patients from 6 certified lung cancer centers in Berlin, Germany, having received pembrolizumab as first-line palliative therapy from January 1 until December 31, 2017. Aims were to validate published clinical trials with a special focus on efficacy and outcome in patients with reduced performance status (PS), brain metastases, and steroids. Results: A total of 153 patients were included (median age 69 years, 58% men, 69% adenocarcinoma). Rates for PS >= 2, brain metastases, and steroids were 24.8%, 20.9%, and 24.2%, respectively. Median objective response rate, progression-free and overall survival were 48.5%, 8.2 and 22.0 months for all patients and 52.4%, 8.8 and 29.2 months in patients fulfilling the inclusion criteria for the KEYNOTE-024 trial. Patients with a comorbidity-defined PS >= 2, symptomatic brain metastases requiring upfront radiotherapy, or baseline steroids had significantly reduced survival. In contrast, durable responses occurred with a tumor-related PS >= 2 or asymptomatic brain metastases. Grade 3/4 and 5 immunerelated adverse events affected 13.7% and 2.0% of patients. Conclusion: Real-world and clinical trial efficacy with upfront pembrolizumab correspond well. Pembrolizumab may sufficiently control asymptomatic brain metastases and may improve a cancer-related reduced PS. However, the frail share of patients with a comorbidity-defined PS >= 2, symptomatic brain metastases, or baseline steroids derives no relevant benefit. (C) 2021 Elsevier Inc. All rights reserved.

Tumor mutation burden (TMB) is evaluated as a biomarker of response to immunotherapy. We present the efforts of the Onconetwork Immuno-Oncology Consortium to validate a commercial targeted sequencing test for TMB calculation. A three-phase study was designed to validate the Oncomine Tumor Mutational Load (OTML) assay at nine European laboratories. Phase 1 evaluated reproducibility and accuracy on seven control samples. In phase 2, six formalin-fixed, paraffin-embedded samples tested with FoundationOne were reanalyzed with the OTML panel to evaluate concordance and reproducibility. Phase 3 involved analysis of 90 colorectal cancer samples with known microsatellite instability (MSI) status to evaluate TMB and MSI association. High reproducibility of TMB was demonstrated among the sites in the first and second phases. Strong correlation was also detected between mean and expected TMB in phase 1 (r(2) = 0.998) and phase 2 (r(2) = 0.96). Detection of actionable mutations was also confirmed. In colorectal cancer samples, the expected pattern of MSI-high/high-TMB and microsatellite stability/low-TMB was present, and gene signatures produced by the panel suggested the presence of a POLE mutation in two samples. The OTML panel demonstrated robustness and reproducibility for TMB evaluation. Results also suggest the possibility of using the panel for mutational signatures and variant detection. Collaborative efforts between academia and companies are crucial to accelerate the translation of new biomarkers into clinical research.

Cutaneous, ocular, and mucosal melanomas are histologically indistinguishable tumors that are driven by a different spectrum of genetic alterations. With current methods, identification of the site of origin of a melanoma metastasis is challenging. DNA methylation profiling has shown promise for the identification of the site of tumor origin in various settings. Here we explore the DNA methylation landscape of melanomas from different sites and analyze if different melanoma origins can be distinguished by their epigenetic profile. We performed DNA methylation analysis, next generation DNA panel sequencing, and copy number analysis of 82 non‐cutaneous and 25 cutaneous melanoma samples. We further analyzed eight normal melanocyte cell culture preparations. DNA methylation analysis separated uveal melanomas from melanomas of other primary sites. Mucosal, conjunctival, and cutaneous melanomas shared a common global DNA methylation profile. Still, we observed location‐dependent DNA methylation differences in cancer‐related genes, such as low frequencies of RARB (7/63) and CDKN2A promoter methylation (6/63) in mucosal melanomas, or a high frequency of APC promoter methylation in conjunctival melanomas (6/9). Furthermore, all investigated melanomas of the paranasal sinus showed loss of PTEN expression (9/9), mainly caused by promoter methylation. This was less frequently seen in melanomas of other sites (24/98). Copy number analysis revealed recurrent amplifications in mucosal melanomas, including chromosomes 4q, 5p, 11q and 12q. Most melanomas of the oral cavity showed gains of chromosome 5p with TERT amplification (8/10), while 11q amplifications were enriched in melanomas of the nasal cavity (7/16). In summary, mucosal, conjunctival, and cutaneous melanomas show a surprisingly similar global DNA methylation profile and identification of the site of origin by DNA methylation testing is likely not feasible. Still, our study demonstrates tumor location‐dependent differences of promoter methylation frequencies in specific cancer‐related genes together with tumor site‐specific enrichment for specific chromosomal changes and genetic mutations. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

In the last years, Poly(ADP-ribose)-polymerase 1 inhibitors (PARPi) became a standard treatment option for patients with recurrent platinum-sensitive ovarian cancer and recently have been approved for maintenance use in the first-line setting. Biomarker subgroup analysis consistently revealed higher benefit for patients with homologous recombination deficiency (HRD; BRCA1/2m and/or genomic instability (GIS) positive), demonstrating the value of biomarker testing in this setting. The Myriad MyChoice assay has been the most commonly used HRD assay applied in the majority of clinical studies.

Disruption of the homologous recombination repair (HRR) pathway was reported in at least half of high-grade serous ovarian cancer (HGSOC) patients. HRR deficiency (HRD) is associated with superior response to PARP inhibition maintenance therapy in both primary and relapsed HGSOC. Moreover, HRD is a prerequisite for combination therapy with Bevacizumab and Olaparib following first-line chemotherapy. Aim of this study was to evaluate if a machine learning framework trained on matrix-assisted laser desorption/ionization imaging mass spectroscopy (MALDI-IMS) data can accurately predict the HRD status in HGSOC tumors. We collected during surgery paired formalin-fixed paraffin-embedded (FFPE) tumor samples from primary and at relapse in 50 HGSOC patients. HRD status was previously evaluated using the Myriad myChoice® CDx test, that was conclusive in 88 samples. HRD status served as the reference label for machine learning of MALDI-IMS data generated from the same samples. Models achieved a 96.8% mean accuracy and 0.995 mean area under the curve (AUC) on three randomized subsets of the data. Utilizing a majority vote scheme, all patients could be assigned the correct HRD status. Using an accessible, cheaper and easy to reproduce method such as MALDI-IMS technology combined with a machine learning framework we reached a similar performance to the Myriad myChoice® CDx test in predicting HRD status in HGSOC.

Mucosal melanomas arise from the mucosal lining of various organs. Their etiology is currently unknown and there are no tissue-based methods to differentiate it from cutaneous melanomas. Furthermore, prognostic and predictive markers (e.g. for immune checkpoint inhibition) are lacking. In this study, we aimed to assess the protein expression levels of cell cycle-associated proteins and immune checkpoint markers in a cohort of mucosal melanomas in comparison to cutaneous melanomas and evaluated the effect of potential regulatory mechanisms. We performed immunohistochemistry, DNA methylation analysis and copy number profiling of 47 mucosal and 28 cutaneous melanoma samples. Protein expression of CD117, Ki67 and p16 was higher in mucosal melanomas, while BCL2, Cyclin D1, PD-1 and PD-L1 were overexpressed in cutaneous melanomas. CDKN2A deletions were the most prevalent numeric chromosomal alterations in both mucosal and cutaneous melanoma and were associated with decreased p16 expression. KIT was frequently amplified in mucosal melanomas, but not associated with CD117 expression. On the other hand, amplification of CCND1 lead to Cyclin D1 overexpression. In mucosal melanoma patients high PD-1 expression and high PD-L1 promoter methylation levels were associated with improved survival. PD-L1 expression correlated with response to immune checkpoint inhibitor therapy in the combined group of melanoma patients. Mucosal and cutaneous melanomas show different expression levels of cell cycle-associated and immunomodulatory proteins that are partially regulated by DNA methylation and copy number alterations. PD-1 expression and PD-L1 promoter methylation levels might be a prognostic marker for mucosal melanomas.

Upfront KRAS and NRAS gene testing (‘RAS’) is the standard of care for metastatic colorectal cancer (mCRC), to guide first-line treatment. The presence of RAS mutation (MT) is a negative predictor for the efficacy of anti-EGFR antibodies and the use of cetuximab and panitumumab is restricted to RAS wild-type (WT) mCRC. Conversion from RAS WT to RAS MT mCRC after treatment with anti-EGFR antibodies is a known and well-described acquired resistance mechanism. The by far less frequent ‘NeoRAS wild-type’ phenomenon (reversion from RAS MT to RAS WT) has recently drawn attention. The proposed effect of chemotherapy on RAS status in mCRC patients is not fully understood. Because of the intriguing biological consequence of a RAS MT to RAS WT reversion, subsequent treatment of NeoRAS WT patients with anti-EGFR antibodies is increasingly being discussed. Here, we report three clinical cases of NeoRAS WT mCRC patients, which received standard-of-care regimens for RAS MT mCRC. Anti-EGFR antibodies were used in two out of three patients after progression of the disease. One of the patients had a long-term response. In line with our observations, NeoRAS WT phenomenon occurs in clinical practice. Retesting of RAS status during treatment should be discussed in patients with unusual long-term clinical courses of RAS MT mCRC to optimise treatment strategy and to evaluate the use of anti-EGFR antibodies.