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Purpose A wide therapeutic repertoire has become available to oncologists including radio- and chemotherapy, small molecules and monoclonal antibodies. However, drug efficacy can be limited by genetic heterogeneity. Here, we designed a webtool that facilitates the data analysis of the in vitro drug sensitivity data on 265 approved compounds from the GDSC database in association with a plethora of genetic changes documented for 1001 cell lines in the CCLE data. Methods The webtool computes odds ratios of drug resistance for a queried set of genetic alterations. It provides results on the efficacy of single compounds or groups of compounds assigned to cellular signaling pathways. Webtool availability: https://tools.hornlab.org/GDSC/. Results We first replicated established associations of genetic driver mutations in BRAF, RAS genes and EGFR with drug response. We then tested the ‘BRCAness’ hypothesis and did not find increased sensitivity to the assayed PARP inhibitors. Analyzing specific PIK3CA mutations related to cancer and mendelian overgrowth, we found support for the described sensitivity of H1047 mutants to GSK690693 targeting the AKT pathway. Testing a co-mutated gene pair, GATA3 activation abolished PTEN-related sensitivity to PI3K/mTOR inhibition. Finally, the pharmacogenomic modifier ABCB1 was associated with olaparib resistance. Conclusions This tool could identify potential drug candidates in the presence of custom sets of genetic changes and moreover, improve the understanding of signaling pathways. The underlying computer code can be adapted to larger drug response datasets to help structure and accommodate the increasingly large biomedical knowledge base.

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 immune-related 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.

Objectives In patients with NSCLC, current ESTS and ESMO guidelines recommend invasive lymph node (LN) staging with EBUS-TBNA even if FDG-PET/CT is negative for mediastinal LNs if at least one of three risk factors is present (cN1, non-peripheral primary or primary >3 cm). Modified workflows to avoid unnecessary invasive procedures were evaluated. Materials and methods Monocentric retrospective analysis of pretherapeutic FDG-PET/CT in 247 patients with NSCLC (62 % male; age, 68 [43–88] years) using an analog or digital PET/CT scanner. PET windowing was standardized. LNs were positive if ‘LN uptake > mediastinal blood pool’ or short axis >10 mm. Surgery or EBUS-TBNA served as reference for diagnostic accuracy per LN station. In all patients with negative mediastinal LNs by PET/CT, LN histology from surgery was available. Results Among 700 L N stations analyzed, 180 were malignant. Sensitivity and specificity of PET/CT per LN station were 93 % and 71 %. Following current guidelines, 76 patients with mediastinal negative PET/CT required confirmatory invasive staging. Only 5/76 patients had unexpected pN2 (all had adenocarcinoma). In a modified approach, confirmatory invasive staging was confined to patients with mediastinal negative PET/CT who showed all three risk factors. Using this modification, EBUS-TBNA could have been omitted in 62 (82 %) of the 76 patients who required EBUS-TBNA based on current recommendation. Among these 62 patients, only one patient had unsuspected pN2 (single-level) while the remaining 61 of 62 omitted EBUS-TBNA were deemed unnecessary because mediastinal LNs were confirmed to be negative. No multi-level pN2 would have been missed. Conclusion In the current analysis, 82 % of EBUS-TBNA procedures in patients with mediastinal negative PET/CT could have been omitted by modifying the current guideline workflow as proposed (i.e., restricting EBUS-TBNA in patients with cN0/1 to those with all three risk factors). This was consistent with different PET/CT scanners. Prospective confirmation is required.

Thyroid transcription factor 1 (TTF-1) is a prognostic biomarker in lung adenocarcinoma; however, TTF-1-positive patients also display more favorable factors like actionable target mutations. In contrast, TTF-1-negative cancer is a poorly described entity. We performed a retrospective study to characterize a TTF-1-negative phenotype and to evaluate outcome depending on the chemotherapy regimen applied in the EGFR/ALK-negative population.Phenotypic traits were analyzed in 741 patients with evaluable TTF-1 expression status, among them 529 patients with platinum-based first-line chemotherapy, with disease diagnosed between 2009 and 2016 at a tertiary referral university hospital. The influence of TTF-1 and several cofactors on progression-free survival and overall survival (OS) were analyzed using a 1:1 propensity score matching model, depending on the platinum doublet chemotherapy's incorporating pemetrexed or not, with subsequent Cox regression.TTF-1 negativity implied a distinct cancer phenotype with the predominance of male sex, worse Eastern Cooperative Oncology Group performance status, greater metastatic burden at primary diagnosis, and more adrenal gland metastases. These patients had improved progression-free survival (hazard ratio, 0.42; P = .001) and OS (hazard ratio, 0.40; P .001) when gemcitabine-, taxane-, or vinorelbine-based regimens were provided instead of pemetrexed. None of the regimens was superior in TTF-1-positive patients with regard to OS. Overall, TTF-1 expression was strongly prognostic with a substantial increase in progression-free survival (hazard ratio, 0.54; P .001) and OS (hazard ratio, 0.53; P .001).TTF-1 negativity is associated with a distinct cancer phenotype. Incorporation of this biomarker may be helpful when choosing an appropriate therapy regimen.

Objectives We aim to provide a better understanding of the molecular landscape of primary lung adenocarcinomas with intestinal differentiation. Material and Methods Five invasive mucinous adenocarcinomas (IMA) and seven pulmonary enteric adenocarcinomas (PEAD) were included in this study. Furthermore, we analyzed six pulmonary colloid adenocarcinomas (CAD), including one primary tumor, one metastasis, and two sample pairs consisting of the primary colloid lung tumor and a matching metastasis and an acinar component, respectively. All samples were characterized using immunohistochemistry (TTF-1, CK7, CK20, CDX2, Ki-67, ALK and PD-L1) and a next generation sequencing panel covering 404 cancer-related genes (FoundationOne® gene panel). Results and Conclusion While Ki-67 expression was comparably low in IMA (range: 8-15%) and in primary CAD (range: 5-8%), we observed considerably higher proliferation rates in the non-colloid tumor compartment (16%) and metastases (72%) from CAD, as well as in the PEAD-group (36-71%). The overall tumor mutational burden was lowest in IMA (2.5 mutations per megabase), intermediate in CAD (5.8 mutations per megabase) and highest in PEAD (16.8 mutations per megabase). KRAS mutations were frequent in all three tumor subtypes, but TP53 mutations were mostly limited to PEAD. While chromosomal alterations were rare in IMA, we discovered MYC amplifications in three of four CAD. Comparing primary and metastatic CAD, we observed the acquisition of multiple mutations and chromosomal alterations. PEAD had a variety of chromosomal alterations, including two cases with RICTOR amplification. PD-L1 expression (20%, 50% and 80% of tumor cells) was limited to three PEAD samples, only. In conclusion, we provide a detailed insight into the molecular alterations across and within the different subtypes of pulmonary adenocarcinomas with intestinal differentiation. From a clinical perspective, we provide data on potential treatment strategies for patients with PEAD, including immunotherapy.

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.

Hintergrund und zielsetzung Patienten mit malignen Melanomen im Fruhstadium haben ein unterschiedliches Gesamtuberleben. Derzeit existieren keine etablierten prognostischen Marker. Unser Ziel war es, zu einem besseren Verstandnis der potenziell prognostischen immunhistochemischen Marker fur die Risikostratifizierung beizutragen. Patienten und methoden 161 operativ entfernte maligne Melanome (Stadien pT1 und pT2) wurden immunhistochemisch auf die Expression von 20 verschiedenen Proteinen untersucht. Die Ergebnisse wurden mit dem Gesamtuberleben korreliert. Die Kohorte wurde randomisiert in eine Entdeckungs- und eine Validierungskohorte aufgeteilt. Ergebnisse Eine hohe Bcl-2-Expression, eine hohe nukleare S100A4-Expression und ein Ki67-Proliferationsindex von ≥ 20 % waren mit einem kurzeren Gesamtuberleben assoziiert. Eine starke MITF-Immunreaktivitat erwies sich als pradiktiv fur eine gute Prognose. Die Kombination dieser vier Marker ergab einen Multimarker-Score mit signifikantem prognostischem Wert in der multivariaten Uberlebensanalyse (HR: 3,704; 95 %-KI 1,484-9,246; p = 0,005). Zusatzlich gelang es mit dem Score, drei prognostische Gruppen zu identifizieren: eine Niedrigrisikogruppe mit sehr guten Gesamtuberlebensraten (Funf-Jahres-Uberlebensrate 100 %), eine Gruppe mit mittlerem Risiko (Funf-Jahres-Uberlebensrate 81,8 %) und eine Hochrisikogruppe (Funf-Jahres-Uberlebensrate 52,6 %). Der prognostische Aussagewert bestatigte sich in der Validierungskohorte. Schlussfolgerungen Die kombinierte immunhistochemische Analyse von Bcl-2, nuklearem S100A4, Ki67 und MITF konnte zu einer besseren Risikostratifizierung bei Patienten mit malignen Melanomen im Fruhstadium beitragen.

Pulmonary enteric adenocarcinoma is a rare non-small cell lung cancer subtype. It is poorly characterized and cannot be distinguished from metastatic colorectal or upper gastrointestinal adenocarcinomas by means of routine pathological methods. As DNA methylation patterns are known to be highly tissue specific, we aimed to develop a methylation-based algorithm to differentiate these entities. To this end, genome-wide methylation profiles of 600 primary pulmonary, colorectal, and upper gastrointestinal adenocarcinomas obtained from The Cancer Genome Atlas and the Gene Expression Omnibus database were used as a reference cohort to train a machine learning algorithm. The resulting classifier correctly classified all samples from a validation cohort consisting of 680 primary pulmonary, colorectal and upper gastrointestinal adenocarcinomas, demonstrating the ability of the algorithm to reliably distinguish these three entities. We then analyzed methylation data of 15 pulmonary enteric adenocarcinomas as well as four pulmonary metastases and four primary colorectal adenocarcinomas with the algorithm. All 15 pulmonary enteric adenocarcinomas were reliably classified as primary pulmonary tumors and all four metastases as well as all four primary colorectal cancer samples were identified as colorectal adenocarcinomas. In a t-distributed stochastic neighbor embedding analysis, the pulmonary enteric adenocarcinoma samples did not form a separate methylation subclass but rather diffusely intermixed with other pulmonary cancers. Additional characterization of the pulmonary enteric adenocarcinoma series using fluorescence in situ hybridization, next-generation sequencing and copy number analysis revealed KRAS mutations in nine of 15 samples (60%) and a high number of structural chromosomal changes. Except for an unusually high rate of chromosome 20 gain (67%), the molecular data was mostly reminiscent of standard pulmonary adenocarcinomas. In conclusion, we provide sound evidence of the pulmonary origin of pulmonary enteric adenocarcinomas and in addition provide a publicly available machine learning-based algorithm to reliably distinguish these tumors from metastatic colorectal cancer.

BACKGROUND: Pembrolizumab is a standard of care as first line palliative therapy in PD-L1 overexpressing (≥50%) non-small cell lung cancer (NSCLC). This study aimed at the identification of KRAS and TP53-defined mutational subgroups in the PD-L1 high population to distinguish long-term responders from those with limited benefit. METHODS: In this retrospective, observational study, patients from 4 certified lung cancer centers in Berlin, Germany, having received pembrolizumab monotherapy as first line palliative treatment for lung adenocarcinoma (LuAD) from 2017 to 2018, with PD-L1 expression status and targeted NGS data available, were evaluated. RESULTS: A total of 119 patients were included. Rates for KRAS, TP53 and combined mutations were 52.1%, 47.1% and 21.9%, respectively, with no association given between KRAS and TP53 mutations (P=0.24). By trend, PD-L1 expression was higher in KRAS-positive patients (75% vs. 65%, P=0.13). Objective response rate (ORR), median progression-free survival (PFS) and overall survival (OS) in the KRAS(G12C) group (n=32, 51.6%) were 63.3%, 19.8 months (mo.) and not estimable (NE), respectively. Results in KRAS(other) and wild type patients were similar and by far lower (42.7%, P=0.06; 6.2 mo., P

With this external quality assessment (EQA) scheme, we aim to investigate the diagnostic performance of the currently available methods for the detection of ALK alterations in non-small cell lung cancer on a national scale, namely, in situ hybridization (ISH), immunohistochemistry (IHC), and RNA/DNA sequencing (NGS). The EQA scheme cohort consisted of ten specimens, including four ALK positive and six ALK negative samples, which were thoroughly pretested using IHC, ISH, and RNA/DNA NGS. Unstained tumor sections were provided to the 57 participants, and the results were retrieved via an online questionnaire. ISH was used by 29, IHC by 38, and RNA/DNA sequencing by 19 participants. Twenty-eight institutions (97%) passed the ring trial using ISH, 33 (87%) by using IHC, and 18 (95%) by using NGS. The highest sensitivity and interrater agreement (Fleiss ‘ kappa) was observed for RNA/DNA sequencing (99%, 0.975), followed by ISH (94%, 0.898) and IHC (92%, 0.888). However, the proportion of samples that were not evaluable due to bad tissue quality was also higher for RNA/DNA sequencing (4%) compared with ISH (0.7%) and IHC (0.5%). While all three methods produced reliable results between the different institutions, the highest sensitivity and concordance were observed for RNA/DNA sequencing. These findings encourage the broad implementation of this method in routine diagnostic, although the application might be limited by technical capacity, economical restrictions, and tissue quality of formalin-fixed samples. Supplementary Information The online version contains supplementary material available at 10.1007/s00428-021-03106-5.

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. (c) 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Head and neck squamous cell carcinoma (HNSC) patients are at risk of suffering from both pulmonary metastases or a second squamous cell carcinoma of the lung (LUSC). Differentiating pulmonary metastases from primary lung cancers is of high clinical importance, but not possible in most cases with current diagnostics. To address this, we performed DNA methylation profiling of primary tumors and trained three different machine learning methods to distinguish metastatic HNSC from primary LUSC. We developed an artificial neural network that correctly classified 96.4% of the cases in a validation cohort of 279 patients with HNSC and LUSC as well as normal lung controls, outperforming support vector machines (95.7%) and random forests (87.8%). Prediction accuracies of more than 99% were achieved for 92.1% (neural network), 90% (support vector machine), and 43% (random forest) of these cases by applying thresholds to the resulting probability scores and excluding samples with low confidence. As independent clinical validation of the approach, we analyzed a series of 51 patients with a history of HNSC and a second lung tumor, demonstrating the correct classifications based on clinicopathological properties. In summary, our approach may facilitate the reliable diagnostic differentiation of pulmonary metastases of HNSC from primary LUSC to guide therapeutic decisions.

In patients with non-small-cell lung cancer (NSCLC), asphericity (ASP) of the primary tumor's metabolic tumor volume (MTV) has shown prognostic significance. This study aimed at validation in an independent and sufficiently large cohort.A retrospective study was performed of 311 NSCLC patients undergoingEvents (progression and relapse) occurred in 167 of 311 patients; 137 died (median survivor follow-up, 37 months). In multivariable Cox regression for OS, ASP33.3% (hazard ratio, 1.58 [1.04-2.39]), male sex (1.84), age (1.04 per year), Eastern Cooperative Oncology Group performance status ≥ 2 versus 0/1 (2.68), stage II versus I (1.96), and Rx/1 versus R0 resection (2.1) were significant. Among separate UICC stages, ASP only predicted OS in stage II (optimal,19.5%; median OS, 33 vs. 59 months). Regarding PFS, ASP21.2%, male sex, Eastern Cooperative Oncology Group performance status ≥ 2, stage II versus I disease, and Rx/1 resection were prognostic. ASP remained prognostic for stage II disease (optimal,19.5%; PFS, 12 vs. 47 months). Log-rank test for ASP was significant at any cutoff ≥ 18% (OS) or from 9% to 59% (PFS).ASP was validated as prognostic factor for PFS and OS in patients with NSCLC and curative treatment intent, especially stage II. High ASP in stage II could imply intensified treatment or intensified follow-up.

Aims Whereas current cancer diagnosis largely relies on the well-established organ and tissue typing of tumours, partially complemented by molecular properties, the comprehensive molecular profiling efforts of recent years have stimulated proposals for molecular reclassifications of tumours independently of anatomical origin. Proposals based only on mutational profiles show the least concordance with histotypes, whereas greater concordance is achieved when various genomic and proteomic data are included. Methods and results The most comprehensive molecular reclassification of tumours, by Hoadley et al (Cell, 158, 2014; 929) and Hoadley et al (Cell, 173, 2018; 291), integrated multi-omics data, and proposes novel molecular tumour classes. To investigate the relationship between the proposed molecular classes and the original histological tumour types, we re-examined the histomorphology of molecularly reclassified cases. Our results show that the claimed molecular reclassification is associated with and explainable by specific histological subtypes in 70% of the reclassified cases. Conclusion Therefore, in contrast to the proclaimed reclassification and independence of molecular and histological tumour types, our analysis demonstrates that comprehensive molecular profiling, which includes gene expression and methylation as well as proteomic profiling in addition to mutational analyses, is largely consistent with histomorphological tumour properties.

Ziel/Aim: In patients with non-small cell lung cancer (NSCLC) undergoing treatment with curative intent, the asphericity (ASP) of the primary tumor’s metabolic tumor volume (MTV) has been demonstrated as a prognostic factor. This study aimed at validation in an independent cohort with sufficient sample size. Methodik/Methods: Retrospective study in 313 NSCLC patients (203 men; median age, 67 [41-87] a) undergoing FDG-PET/CT with the same scanner prior to treatment in curative intent (always including resection of the primary tumor). 137 patients had UICC stage I, 79 patients stage II and 97 patients stage III disease (adenocarcinoma [ADC], 153; squamous cell carcinoma [SCC], 143, other, 17). Delineation of primary tumor MTV with semiautomated background-adapted threshold relative to its SUVmax. Univariable Cox regression for progression-free (PFS) and overall survival (OS) for PET parameters (MTV, ASP, SUVmax, SUVmean), clinical (UICC stage I vs. II vs. III), histological (SCC vs. ADC) and treatment variables (Rx/1 vs. R0 resection, chemotherapy yes/no, radiotherapy yes/no). Multivariable Cox of parameters significant in univariate Cox. Ergebnisse/Results: Events (progression, relapse, death) occurred in 169/313 patients, 139 patients died (median follow-up in survivors, 37 months). In multivariable Cox for OS, ASP >33.3% (hazard ratio [HR], 1.53 [95%-confidence interval, 1.02-2.3]), Rx/1 vs. R0 resection (HR, 2.47 [1.5-4.2]) and SCC vs. ADC (HR, 1.53 [1.1-2.2]) were significant. Log-rank test for ASP was significant at any cut-off from 18% upwards. Among separate UICC stages, ASP was only prognostic for OS in stage II (optimal, >19.5%; median OS, 33 vs. 59 months; p21.2% (HR, 1.75 [1.2-2.5]) and Rx/1 vs. R0 (HR, 2.48 [1.5-4.1]) were significant. Log-rank test for ASP was significant at any cut-off from 10-60%. Schlussfolgerungen/Conclusions: ASP was validated as an independent predictor of PFS and OS in NSCLC patients with curative treatment intent. Subdividing UICC stages, ASP remained prognostic in stage II.

e23540 Background: Early presentation of soft-tissue sarcoma (STS) patients to a specialized sarcoma center including discussion in the interdisciplinary sarcoma board (ISB) prior to surgery is essential to the treatment of sarcomas. This approach significantly improves patient survival and guideline coherence. However, there exists only limited information on the adherence to the recommendations of the ISB. Accordingly, we decided to analyze a STS cohort at a large German sarcoma center focusing on outcome parameters and adherence to quality parameters defined by the German Cancer Society (Deutsche Krebsgesellschaft). Methods: In a retrospective data analysis, we identified n = 230 adult patients presented at the ISB of the Charité–Universitätsmedizin Berlin in Germany from January 2015 until December 2019. Inclusion criteria were as follows: newly diagnosed STS, presentation during first-line therapy and recommendation of at least one treatment modality such as surgery, chemotherapy, radiotherapy, regional hyperthermia or follow-up by the ISB. Clinical and follow up data was collected by using the hospital information system and the outpatient network. Results: Our patient cohort included 53% male and 47% female patients with a median age of 58 years (range 19-96). The majority (86%) showed a localized tumor stage, while 14% already had metastases. In 24% of the cases the sarcomas were classified as low-grade, in 76% as high-grade. Surgery was recommended for 66% of the cases, for 80% of them combined with chemotherapy, radiotherapy or hyperthermia. 14% of the patients received a recommendation for all 4 modalities. For 9% of the patients, chemotherapy alone was recommended. Both overall survival and progression-free survival was significantly higher in the group with complete adherence to the recommendations of the ISB (p < 0.001). The worst prognosis was found in patients unable to adhere to the recommendations due to rapid progression or complications of the therapy (HR for death 15.06, 95%CI 7.94-32.22). If one recommended modality was not carried out, there also was a higher risk of death (HR 4.38, 95%CI 1.75-10.96). Most common reasons were patient refusal or individual decision by the treating physician. A metastasized tumor stage was associated with an increased risk of death (HR 2.62, 95%CI 1.45-4.75). In contrast, neither the histological grading (low vs. high) nor age did influence the mortality significantly. Conclusions: In our cohort of STS patients, survival depends significantly on adherence to the recommendations of the ISB. Our analysis at a German sarcoma center is in line with previous international reports demonstrating the importance of interdisciplinary decisions and therapeutic adherence. We hereby underline the essential role of interdisciplinary approaches in care of STS patients and the effort to implement defined quality parameters.

Fluorescence in-situ hybridization (FISH) for the detection of ALK-rearrangements in non-small cell lung cancer (NSCLC) is based on at first sight clear cut-off criteria (≥15% of tumor cells) for split signals (SS) and single red signals (SRS). However, NSCLC with SS-counts around the cut-off may cause interpretation problems.Tissue microarrays containing 753 surgically resected NSCLCs were independently tested for ALK-alterations by FISH and immunohistochemistry (IHC). Our analysis focused on samples with SS/SRS in the range between 10% and 20% (ALK-FISH borderline group). To better understand the role of these samples in routine diagnostics, we performed statistical analyses to systematically estimate the probability of ALK-FISH-misclassification (false negative or positive) for different numbers of evaluated tumor cell nuclei (30, 50, 100, and 200).94.3% (710/753) of the cases were classified as unequivocally (10% or ≥20%) ALK-FISH-negative (93%; 700/753) or positive (1.3%; 10/753) and showed concordant IHC results. 5.7% (43/753) of the samples showed SS/SRS between 10% and 20% of the tumor cells. Out of these, 7% (3/43; ALK-FISH: 14%, 18% and 20%) were positive by ALK-IHC, while 93% (40/43) had no detectable expression of the ALK-protein. Statistical analysis showed that ALK-FISH misclassifications occur frequently for samples with rearrangements between 10% and 20% if ALK-characterization is based on a sharp cut-off point (15%). If results in this interval are defined as equivocal (borderline), statistical sampling-related ALK-FISH misclassifications will occur in less than 1% of the cases if 100 tumor cells are evaluated.While ALK status can be determined robustly for the majority of NSCLC by FISH our analysis showed that ∼6% of the cases belong to a borderline group for which ALK-FISH evaluation has only limited reliability due to statistical sampling effects. These cases should be considered equivocal and therapy decisions should include additional tests and clinical considerations.

Key Clinical Message We report a case of several autoimmune disorders eventually presenting as severe multi organ dysfunction syndrome caused by adult hemophagocytic lymphohistiocytosis (HLH). Clinical and laboratory tests might lead to fatal misinterpretation without awareness of its diagnostic evaluation, as HLH shares common features with sepsis and immune‐mediated systemic inflammatory response syndromes.

Background In patients with non–small-cell lung cancer (NSCLC), asphericity (ASP) of the primary tumor’s metabolic tumor volume (MTV) has shown prognostic significance. This study aimed at validation in an independent and sufficiently large cohort. Patients and Methods A retrospective study was performed of 311 NSCLC patients undergoing 18F-fluorodeoxyglucose positron emission tomography / computed tomography (18F-FDG PET/CT) before curatively intended treatment (always including surgery). A total of 140 patients had International Union Against Cancer (UICC) stage I disease, 78 had stage II disease, and 93 had stage III disease (adenocarcinoma, n = 153; squamous-cell carcinoma, n = 141). Primary tumor MTV was delineated with semiautomated background-adapted threshold relative to the standardized maximum uptake value (SUVmax). Cox regression (progression-free survival [PFS] and overall survival [OS]) analysis for positron emission tomography (MTV, ASP, SUVmax) as well as for clinical (T/N descriptor, UICC stages), histologic, and treatment variables (Rx/1 vs. R0 resection, chemotherapy/radiotherapy yes/no) were performed. Results Events (progression and relapse) occurred in 167 of 311 patients; 137 died (median survivor follow-up, 37 months). In multivariable Cox regression for OS, ASP > 33.3% (hazard ratio, 1.58 [1.04-2.39]), male sex (1.84), age (1.04 per year), Eastern Cooperative Oncology Group performance status ≥ 2 versus 0/1 (2.68), stage II versus I (1.96), and Rx/1 versus R0 resection (2.1) were significant. Among separate UICC stages, ASP only predicted OS in stage II (optimal, > 19.5%; median OS, 33 vs. 59 months). Regarding PFS, ASP > 21.2%, male sex, Eastern Cooperative Oncology Group performance status ≥ 2, stage II versus I disease, and Rx/1 resection were prognostic. ASP remained prognostic for stage II disease (optimal, > 19.5%; PFS, 12 vs. 47 months). Log-rank test for ASP was significant at any cutoff ≥ 18% (OS) or from 9% to 59% (PFS). Conclusion ASP was validated as prognostic factor for PFS and OS in patients with NSCLC and curative treatment intent, especially stage II. High ASP in stage II could imply intensified treatment or intensified follow-up.

To date, most studies investigating the immune tumor microenvironment of non-small cell lung cancers (NSCLC) only consider a small number of immune cell subsets or do not reflect the distribution of these cells between different tumor compartments as they were performed on tissue microarrays (TMA). To address this, we analyzed the immune infiltrate in surgically resected NSCLCs, focusing on potential spatial heterogeneity. We evaluated 45 NSCLCs based on whole-slide sections using immunohistochemistry with eleven different antibodies (CD3, CD4, CD8, CD20, CD68, Gata3, FOXP3, T-bet, kappa, lambda, PD-L1). While most markers were relatively evenly distributed among different tumor compartments as well as within the same tumor compartment, some immune cell subsets showed a considerable variance. Notably, the immune infiltrate at the tumor invasion front was dominated by B cells. Concerning markers for T cell differentiation, FoxP3 (Th2) was predominantly expressed in stromal lymphocytes, while T-bet (Th1) was most commonly expressed in intraepithelial immune cells. Although most immune cell subtypes showed a heterogenous distribution within in the intraepithelial compartment, the results from a simulated TMA and core biopsy were mostly in line with the results from whole slide evaluation. Regarding disease specific survival, there were no clear correlations. Interestingly, patients with intraepithelial T-bet positive lymphocytes had a significantly better outcome (p = 0.039), however, this difference was not preserved in multivariate analysis. In conclusion, our study shows that the immune tumor microenvironment of NSCLCs is complex and partially heterogenous, especially concerning markers for T cell differentiation.

Background Rearrangements of the anaplastic lymphoma kinase (ALK) belong to the promising targets in the therapy of advanced non-small cell lung cancer (NSCLC) and are predominantly detected by immunohistochemistry (IHC) and/or fluorescence in-situ hybridization (FISH). However, both methods occasionally produce discordant results, especially in so-called borderline (BL) cases, showing ALK FISH-positive signals in 10–20% of the tumor nuclei around the cutoff (15%). This leads to a diagnostic and thus to a therapeutic dilemma. Methods We selected 18 unequivocal (12 ALK IHC/FISH-negative; 6 ALK IHC/FISH-positive) and 15 equivocal samples with discordant results between FISH (Abbott, Vysis LSI ALK Dual Color) and IHC (Ventana, D5F3), including cases with FISH-BL results, for further RNA based-analysis. To detect ALK rearrangement at the transcriptional level, RNA was analyzed using a targeted multiplex-PCR panel followed by IonTorrent sequencing and by direct transcript counting using a digital probe-based assay (NanoString). Sensitivity of both methods was defined using RNA obtained from an ALK-positive cell line dilution series. Results Cases with unequivocal IHC/FISH results showed concordant data with both RNA-based methods, whereas the three IHC-negative/FISH-positive samples were negative. The four IHC-negative/FISH-BL-negative cases, as well as the five IHC-negative/FISH-BL-positive samples showed negative results by massive parallel sequencing (MPS) and digital probe-based assay. The two IHC-positive/FISH-BL-positive cases were both positive on the RNA-level, whereas a tumor with questionable IHC and FISH-BL-positive status displayed no ALK fusion transcript. Conclusions The comparison of methods for the confirmation of ALK rearrangements revealed that the detection of ALK protein by IHC and ALK fusion transcripts on transcriptional level by MPS and the probe-based assay leads to concordant results. Only a small proportion of clearly ALK FISH-positive cases are unable to express the ALK protein and ALK fusion transcript which might explain a non-responding to ALK inhibitors. Therefore, our findings led us to conclude that ALK testing should initially be based on IHC and/or RNA-based methods.

Background and objectives Overall survival (OS) in patients with early-stage malignant melanoma differs. To date, there are no established prognostic markers. We aimed to contribute to a better understanding of potential prognostic immunohistochemical markers for risk stratification. Patients and methods 161 surgically resected early-stage malignant melanomas (stage pT1 and pT2) were analyzed for expression of 20 different proteins using immunohistochemistry. The results were correlated with OS. The cohort was randomly split into a discovery and a validation cohort. Results High Bcl-2 expression, high nuclear S100A4 expression as well as a Ki67 proliferation index of ≥ 20 % were associated with shorter OS. Strong MITF immunoreactivity was a predictor for favorable prognosis. A combination of these four markers resulted in a multi-marker score with significant prognostic value in multivariate survival analysis (HR: 3.704; 95 % CI 1.484 to 9.246; p = 0.005). Furthermore, the score was able to differentiate a low-risk group with excellent OS rates (five-year survival rate: 100 %), an intermediate-risk group (five-year survival rate: 81.8 %) and a high-risk group (five-year survival rate: 52.6 %). The prognostic value was confirmed within the validation cohort. Conclusions Combined immunohistochemical analysis of Bcl-2, nuclear S100A4, Ki67 and MITF could contribute to better risk stratification of early-stage malignant melanoma patients.

International audience; Introduction Lung cancer is the most common cancer worldwide. Latest guidelines from the College of American Pathologist and the European society of medical oncologists indicate anaplastic lymphoma kinase (ALK) rearrangement testing is standard practice. Historically, diagnostics for ALK used fluorescence in situ hybridisation (FISH); however, immunohistochemical (IHC) assays are becoming common practice. Unfortunately, recent assessment of current practice indicated that not all patients who should be tested for ALK translocation are undergoing ALK testing. Methods From a series of European and Israeli labs, we collected patients with discordant IHC and FISH testing, which were subsequently treated with ALK-targeted therapy, for discussion of the question, to treat or not to treat? Results Our study may support ALK IHC testing as a better predictor of response to targeted therapy provided that the labs implement controlled preanalytical procedures, use correct clone, run protocols on automated staining platforms and validate using external quality assessments. © Author (s) (or their employer(s)) 2018.

Introduction: Detection of anaplastic lymphoma kinase (ALK)-gene rearrangements in non-small-cell lung cancer (NSCLC) is mainly performed by fluorescence in-situ hybridization (FISH). The question was raised if FISH might be replaced by immunohistochemistry (IHC) in a reliable and reproducible manner across different laboratories. Methods: After calibration of the staining instruments and training of the observers to binary interpretation (positive versus negative), 15 NSCLC were independently tested for ALK protein expression by IHC only in a multicenter setting (16 institutes). Each laboratory utilized the VENTANA ALK-D5F3 IHC assay. As demonstrated by FISH the samples displayed unequivocal ALK break-positivity (6x) and negativity (7x), as well as ALK positive-borderline character (2x), which is challenging for FISH diagnosis and thus was RT-PCR-confirmed. Results: All seven ALK FISH-negative cases were homogenously scored as ALK-IHC negative. All 16 participants scored the two ALK positive-borderline samples as unequivocally positive according to their protein expression. Concordant IHC interpretation was also noticed in four of six unequivocal ALK break positive cases. In two of six some observers described a weak/heterogeneous ALK-IHC staining. This would have resulted in a subsequent ALK-testing (FISH/PCR) in a routine diagnostic setting. Conclusions: This so-called ALK-Harmonization-Study shows for the first time that predictive semiquantitative IHC reveals reliable and reproducible results across several labs when methodology and interpretation are strictly defined and the pathologists are uniquely trained. The application of validated ALK IHC assays and its comparison to ALK-FISH is highly needed in future clinical trials. This might answer the question if ALK-IHC cannot only serve as a prescreening tool, but as a stand-alone test at least in cases displaying an unequivocally staining pattern as well as an alternative predictive test in samples with reduced FISH interpretability.

Background The reliable identification of non-small cell lung cancers (NSCLC) with chromosomal breaks in the gene of the anaplastic lymphoma kinase (ALK) is crucial for the induction of therapy with ALK-inhibitors. In order to ensure a reliable detection of ALK-breaks by means of fluorescence in situ hybridization (FISH) testing, round robin tests are essential. In preparation of a nation (German)-wide round robin test we initiated a pre-testing phase involving 8 experts in FISH-diagnostics to identify NSCLC cases (n = 10) with a pre-tested ALK-status. In addition, ALK immunohistochemistry (IHC) was performed to assess ALK protein expression. Material and methods Sections derived from a tissue microarray, each consisting of 3 cores from 10 NSCLC cases, were independently tested for ALK protein expression by IHC and genomic ALK-breaks by FISH involving 8 institutes of pathology. Based on a pre-screening, 5 cases were identified to be clearly ALK-break negative, whereas the remaining 5 cases were ALK-break positive including one case with low percentage (20%) of positive cells. The latter had been additionally tested by RT-PCR. Results The 5 unequivocal ALK-break negative NSCLC were almost consistently scored negative by means of FISH and IHC by all 8 experts. Interestingly, 4 of the 5 cases with pre-defined ALK-breaks revealed homogenous FISH results whereas IHC for the detection of ALK protein expression showed heterogeneous results. The remaining case (low number of ALK-break positive cells) was scored negative by 3 experts and positive by the other 5. RT-PCR revealed the expression of an EML4-ALK fusion gene variant 1. Conclusion ALK-break negative NSCLC cases revealed concordant homogeneous results by means of FISH and IHC (score 0–1) by all 8 experts. Discordant FISH results were raised in one ALK-break positive case with a low number of affected tumor cells. The remaining 4 ALK-break positive cases revealed concordant FISH data whereas the ALK-IHC revealed very diverse results. The cases with concordant FISH results provide an excellent basis for round robin ALK-FISH testing. As long as standardized ALK-IHC protocols are missing, ALK protein expression cannot by regarded as the method of choice for identification of patients eligible for treatment with ALK inhibitors.

The non-classical MHC I paralogue HLA-G is expressed by cytotrophoblast cells and implicated with fetomaternal tolerance by downregulating the maternal adaptive and innate immune response against the fetus. HLA-G expression correlates with favorable graft outcome in humans and recently promising immunosuppressive effects of therapeutic HLA-G in experimental transplantation (skin allograft acceptance) were shown. Consequently, we examined this novel therapeutic approach in solid organ transplantation. In this study, therapeutic recombinant HLA-G5 was evaluated for the first time in a solid organ model of acute rejection (ACR) after orthotopic intestinal transplantation (ITX). Allogenic ITX was performed in rats (Brown Norway to Lewis) with and without HLA-G treatment. It was found that HLA-G treatment significantly reduced histologically proven ACR at both an early and late postoperative timepoint (POD 4/7), concomitant to a functionally preserved graft contractility at POD 7. Interestingly, graft infiltration by myeloperoxidase+ cells was significantly reduced at POD7 by HLA-G treatment. Moreover, HLA-G treatment showed an effect on the allogenic T-cell immune response as assessed by flow cytometry: The influx of recipient-derived CD8+ T-cells into the graft mesenteric lymphnodes at POD7 was significantly reduced while CD4+ populations were not affected. As a potential mechanism of action, an induction of T-reg populations in the mesenteric lymphnodes was postulated, but flow cytometric analysis of classical CD4+/CD25+/FoxP3+Treg-cells showed no significant alteration by HLA-G treatment. The novel therapeutic approach using recombinant HLA-G5 reported herein demonstrates a significant immunosuppressive effect in this model of allogenic experimental intestinal transplantation. This effect may be mediated via inhibition of recipient-derived CD8+ T-cell populations either directly or by induction of non-classical Treg populations.

The effect of imatinib on T and B cells in patients with chronic myelogenous leukaemia (CML) is not well understood. An upregulation of the transcription factor Broad-complex-Tramtrack-Bric-a-Brac and Cap‘n’collar 1 bZip transcription factor 2 (BACH2), which is involved in the development and differentiation of B cells, was demonstrated in a CML cell line treated with imatinib. The present study retrospectively analysed the expression and distribution of cluster of differentiation (CD)3, CD20 and BACH2 (per 1,000 cells), as well as the co-expression of CD20 and BACH2, using immunohistochemistry in serial bone marrow trephines obtained from 14 CML patients treated with imatinib in comparison to 17 patients with newly diagnosed CML and 6 control trephines. Bone marrow trephines of CML patients in remission under imatinib therapy exhibited significantly higher numbers of CD3 and CD20 infiltrates (partly ordered in aggregates) compared with patients with newly diagnosed CML and control individuals. Similarly, nuclear expression of BACH2 in granulopoietic cells was increased in CML patients treated with imatinib, which may represent the histological correlate of the positive treatment effect. Furthermore, since BACH2 is involved in B cell development, its altered expression patterns by imatinib may be one explanation for high B cell numbers, as revealed by CD20/BACH2 (nuclear)-positive cells. As the present data are preliminary, future prospective studies are required to assess the prognostic and predictive role of BACH2 in patients with CML under targeted therapy.

Background There is currently no Europe-wide consensus on the appropriate preanalytical measures and workflow to optimise procedures for tissue-based molecular testing of non-small-cell lung cancer (NSCLC). To address this, a group of lung cancer experts (see list of authors) convened to discuss and propose standard operating procedures (SOPs) for NSCLC. Methods Based on earlier meetings and scientific expertise on lung cancer, a multidisciplinary group meeting was aligned. The aim was to include all relevant aspects concerning NSCLC diagnosis. After careful consideration, the following topics were selected and each was reviewed by the experts: surgical resection and sampling; biopsy procedures for analysis; preanalytical and other variables affecting quality of tissue; tissue conservation; testing procedures for epidermal growth factor receptor, anaplastic lymphoma kinase and ROS proto-oncogene 1, receptor tyrosine kinase (ROS1) in lung tissue and cytological specimens; as well as standardised reporting and quality control (QC). Finally, an optimal workflow was described. Results Suggested optimal procedures and workflows are discussed in detail. The broad consensus was that the complex workflow presented can only be executed effectively by an interdisciplinary approach using a well-trained team. Conclusions To optimise diagnosis and treatment of patients with NSCLC, it is essential to establish SOPs that are adaptable to the local situation. In addition, a continuous QC system and a local multidisciplinary tumour-type-oriented board are essential.

Background: The term ‘pilonidal sinus’ (PS) reflects an acute exacerbating, purulent, fistulating chronic inflammation located in the coccyx region. Systematic histological investigations are scarce, and the etiology has remained controversial. Methods: Histological and immunohistochemical characterization of totally excised material of 27 patients (68 specimens) with PS (no antecedent and no current clinical signs of hidradenitis suppurativa, HS) and its correlation with data on HS which we published earlier. Results: Follicular hyperplasia/hyperkeratosis and interfollicular epidermal hyperplasia are main features of PS. Early pathology seems to take place at terminal hair follicles, whereas sinus tract formations are a secondary event. Focused regions show an inflammatory mixed infiltrate consisting of CD3+, CD4+, CD8+, CD68+ and CD79+ cells. Conclusions: PS and HS have various common characteristics at the histological and immunohistochemical level. Considering PS as a unilocalized type of HS, risk factors known in the latter should henceforth be evaluated in PS as well.

Hidradenitis suppurativa (acne inversa) is a chronic suppurative and scarring inflammatory disease with predilection in the apocrine gland-bearing areas. Histological investigations in the 1990s showed keratotic occlusion of the terminal follicle structure to be the initial cause. Our investigations describe and reproduce the morphology and try to figure out very early lesions of HS. A total of 262 operative specimens from 60 patients were investigated by routine histology and 11 operative specimens by immunohistochemistry: HS is dominated by a heterogeneous histological image. 82% of the surgical specimens showed mild or pronounced follicular hyperkeratosis, whereas an isotopic hyperplasia of follicular epithelium was evident in 77%. Pronounced perifolliculitis was seen in 68% and rupture of the follicle structure in 28%. Features which had not so far been described in detail were: epidermal psoriasiform hyperplasia (43%) and subepidermal interfollicular inflammatory infiltrate (78%). In all 11 specimens, immunohistochemical investigations showed a perifollicular and subepidermal inflammation of CD-3-, CD-4-, CD-68-, CD-79- and CD-8-cells, the latter with a striking selective epitheliotropism. To conclude, we could show follicular hyperkeratosis and lymphocytic perifollicular inflammation as early patterns in pathogenesis, whereas rupture of the follicle structure takes place later. Finally, it seems that there are two hot spots of inflammatory events (perifollicular and subepidermal) composed of a comparable inflammatory cell mixture. The CD-8 cell epitheliotropism (follicular and epidermal) described here and its influence in follicular hyperkeratosis, in hyperplasia of follicular epithelium and in epidermal psoriasiform hyperplasia will be of further interest, for instance, concerning early pharmacological intervention.

In April 2013 our group published a review on predictive molecular pathology in this journal. Although only 2 years have passed many new facts and stimulating developments have happened in diagnostic molecular pathology rendering it worthwhile to present an up-date on this topic. A major technical improvement is certainly given by the introduction of next-generation sequencing (NGS; amplicon, whole exome, whole genome) and its application to formalin-fixed paraffin-embedded (FFPE) tissue in routine diagnostics. Based on this 'revolution' the analyses of numerous genetic alterations in parallel has become a routine approach opening the chance to characterize patients' malignant tumors much more deeply without increasing turn-around time and costs. In the near future this will open new strategies to apply 'off-label' targeted therapies, e.g. for rare tumors, otherwise resistant tumors etc. The clinically relevant genetic aberrations described in this review include mutation analyses of RAS (KRAS and NRAS), BRAF and PI3K in colorectal cancer, KIT or PDGFR alpha as well as BRAF, NRAS and KIT in malignant melanoma. Moreover, we present several recent advances in the molecular characterization of malignant lymphoma. Beside the well-known mutations in NSCLC (EGFR, ALK) a number of chromosomal aberrations (KRAS, ROS1, MET) have become relevant. Only very recently has the clinical need for analysis of BRCA1/2 come up and proven as a true challenge for routine diagnostics because of the genes' special structure and hot-spot-free mutational distribution. The genetic alterations are discussed in connection with their increasingly important role in companion diagnostics to apply targeted drugs as efficient as possible. As another aspect of the increasing number of druggable mutations, we discuss the challenges personalized therapies pose for the design of clinical studies to prove optimal efficacy particularly with respect to combination therapies of multiple targeted drugs and conventional chemotherapy. Such combinations would lead to an extremely high complexity that would hardly be manageable by applying conventional study designs for approval, e.g. by the FDA or EMA. Up-coming challenges such as the application of methylation assays and proteomic analyses on FFPE tissue will also be discussed briefly to open the door towards the ultimate goal of reading a patients' tissue as 'deeply' as possible. Although it is yet to be shown, which levels of biological information are most informative for predictive pathology, an integrated molecular characterization of tumors will likely offer the most comprehensive view for individualized therapy approaches. To optimize cancer treatment we need to understand tumor biology in much more detail on morphological, genetic, proteomic as well as epigenetic grounds. Finally, the complex challenges on the level of drug design, molecular diagnostics, and clinical trials make necessary a close collaboration among academic institutions, regulatory authorities and pharmaceutical companies.

a b s t r a c t Objectives: ALK, MET and ROS1 are prognostic and predictive markers in NSCLC, which need to be imple- mented in daily routine. To evaluate different detection approaches and scoring systems for optimal stratification of patients eligible for mutation testing in the future, we screened a large and unselected cohort of NSCLCs for all three alterations. Material and methods: Using tissue microarrays, 473 surgically resected NSCLCs were tested for ALK and MET expression by IHC and genomic alterations in the ALK, MET and ROS1 gene by FISH. For MET IHC, two different criteria (MetMAb and H-score), for MET FISH, three different scoring systems (UCCC, Cappuzzo, PathVysion) were investigated. Results: ALK and ROS1 positivity was seen in 2.6% and 1.3% of all ADCs, respectively, but not in pure SCCs. One ROS1 translocated tumor showed additional ROS1 amplification. MET IHC+/FISH+ cases were found in both histological subtypes (8.6% in all NSCLCs; 10.6% in ADCs; 5.0% in SCCs) and were associated with pleural invasion, lymphatic vessel invasion and lymph node metastasis. MET altered ADCs more frequently showed a papillary growth pattern. Whereas ALK testing revealed homogenous results in IHC and FISH, we saw discordant results for MET in about 10% of cases. Both METIHC scoring systems revealed almost identical results. We did not encounter any combined FISH positivity for ALK, MET or ROS1. However, three ALK positive cases harbored MET overexpression. Conclusion: In daily routine, IHC could support FISH in the identification of ALK altered NSCLCs. Further research is needed to assess the role of discordant MET results by means of IHC and FISH as well as the relevance of tumors with an increased ROS1 gene copy number. © 2014 Elsevier Ireland Ltd. All rights reserved.

Background The anaplastic lymphoma kinase (ALK) inhibitor crizotinib has recently received approval for the treatment of patients with locally advanced or metastatic ALK-positive non–small-cell lung cancer (NSCLC). As the therapeutic prescription postulates the detection of ALK rearrangements, reliable diagnostic approaches are of utmost importance. With this study, we present the data of the first German ALK-round robin test based on genomic DNA in situ hybridization (ISH). The application of immunohistochemistry (IHC) for ALK protein detection was optional and not required for certification. Methods Two tissue microarrays, each consisting of the same 10 NSCLC but in different arrangement of the cases, were generated (five unequivocally ALK-positive and five unequivocally ALK-negative cases). ISH-based results and optional IHC data had to be submitted within 10 working days. A successful participation (certification) was reached if at least 19 of the 20 possible points were scored (2 points for a correct case classification). Results Fifty-three of 59 participants (89.8%) provided their data for ISH-ALK detection within the submission period. Thirty-two of 53 participants (60.3%) received at least 19 points required for certification. Remarkably, the range of cells with aberrant ALK signal configuration was broad in ALK-negative (0–13%) and in ALK-positive cases (15–95%). Thirty-five participants supported the round robin test with optional ALK IHC results, which displayed a great heterogeneity in the ALK ISH-positive cases. Conclusion In essence, our ALK ISH round robin test clearly demonstrates that there is accumulating need for improvement of ALK testing. Although ISH may be regarded as a well-established procedure, its broad application in a diagnostic setting is challenging and requires standardized methods and harmonized interpretation to achieve sound results for therapeutic decisions. The same is true for ALK IHC which, however if standardized, might improve the diagnostic approach.

Hidradenitis suppurativa (acne inversa) is a chronic suppurative and scarring inflammatory disease with predilection in the apocrine gland-bearing areas. Histological investigations in the 1990s showed keratotic occlusion of the terminal follicle structure to be the initial cause. Our investigations describe and reproduce the morphology and try to figure out very early lesions of HS. A total of 262 operative specimens from 60 patients were investigated by routine histology and 11 operative specimens by immunohistochemistry: HS is dominated by a heterogeneous histological image. 82% of the surgical specimens showed mild or pronounced follicular hyperkeratosis, whereas an isotopic hyperplasia of follicular epithelium was evident in 77%. Pronounced perifolliculitis was seen in 68% and rupture of the follicle structure in 28%. Features which had not so far been described in detail were: epidermal psoriasiform hyperplasia (43%) and subepidermal interfollicular inflammatory infiltrate (78%). In all 11 specimens, immunohistochemical investigations showed a perifollicular and subepidermal inflammation of CD-3-, CD-4-, CD-68-, CD-79- and CD-8-cells, the latter with a striking selective epitheliotropism. To conclude, we could show follicular hyperkeratosis and lymphocytic perifollicular inflammation as early patterns in pathogenesis, whereas rupture of the follicle structure takes place later. Finally, it seems that there are two hot spots of inflammatory events (perifollicular and subepidermal) composed of a comparable inflammatory cell mixture. The CD-8 cell epitheliotropism (follicular and epidermal) described here and its influence in follicular hyperkeratosis, in hyperplasia of follicular epithelium and in epidermal psoriasiform hyperplasia will be of further interest, for instance, concerning early pharmacological intervention.