Search

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.

Purpose: Cisplatin resistance is a major obstacle in the treatment of ovarian carcinoma. ABCC2 is commonly localized in apical cell membranes and could confer cisplatin resistance. Here, we show that ABCC2 can be localized in the cytoplasmic membrane as well as in the nuclear membrane of various human tissues including ovarian carcinoma cells.Experimental Design: For the subcellular detection of ABCC2, immunohistochemistry was done using 41 Federation Internationale des Gynaecologistes et Obstetristes stage III ovarian carcinoma specimens prepared before treatment with cisplatin-based schemes and 35 specimens from the same group after chemotherapy. Furthermore, 11 ovarian carcinoma cell lines as well as tissue microarrays consisting of various human tissues were analyzed.Results: Nuclear membranous localization of ABCC2 was associated with response to first-line chemotherapy at primary (P = 0.0013) and secondary surgery (P = 0.0060). Cases with relapse showed higher nuclear membrane expression at primary (P = 0.0003) and secondary surgery (P = 0.0024). Kaplan-Meier analyses showed that weak nuclear membrane ABCC2 expression before treatment was associated with significantly longer overall (P = 0.04) and progression-free survival (P = 0.001); following chemotherapy, it correlated with significantly longer progression-free survival (P = 0.038). Tissue microarrays confirmed nuclear membranous localization of ABCC2, in particular, in poorly differentiated cells. In ovarian carcinoma cells, it correlated with resistance against cisplatin, whereas localization in the cytoplasmic membrane did not.Conclusions: ABCC2 confers resistance to cisplatin of ovarian carcinoma in cell culture systems and in clinics when expressed in the nuclear membrane. Thus, ABCC2 localization can predict platinum therapy outcome. Furthermore, expression of ABCC2 in nuclear membranes in human tissues is specific for poorly differentiated cells including stem cells.

Supplementary Figures S1-S2 from ABCC2 (MRP2, cMOAT) Can Be Localized in the Nuclear Membrane of Ovarian Carcinomas and Correlates with Resistance to Cisplatin and Clinical Outcome

Purpose: This phase I clinical trial evaluated safety, feasibility, and efficiency of nonviral intratumoral jet-injection gene transfer in patients with skin metastases from melanoma and breast cancer.Experimental Design: Seventeen patients were enrolled. The patients received five jet injections with a total dose of 0.05 mg β-galactosidase (LacZ)-expressing plasmid DNA (pCMVβ) into a single cutaneous lesion. Clinical and laboratory safety monitoring were done. Systemic plasmid clearance was monitored by quantitative real-time PCR of blood samples throughout the study. All lesions were resected after 2 to 6 days. Intratumoral plasmid DNA load, DNA distribution, and LacZ expression was analyzed by quantitative real-time PCR, quantitative reverse transcription-PCR, Western blot, immunohistochemistry, and 5-bromo-4-chloro-3-indolyl-β-d-galactoside staining.Results: Jet injection of plasmid DNA was safely done in all patients. No serious side effects were observed. Thirty minutes after jet injection, peak plasmid DNA levels were detected in the blood followed by rapid decline and clearance. Plasmid DNA and LacZ mRNA and protein expression were detected in all treated lesions. Quantitative analysis revealed a correlation of plasmid DNA load and LacZ-mRNA expression confirmed by Western blot. Immunohistochemistry and 5-bromo-4-chloro-3-indolyl-β-d-galactoside staining showed LacZ-protein throughout the tumor. Transfected tumor areas were found close and distant to the jet-injection site with varying levels of DNA load and transgene expression.Conclusion: Intratumoral jet injection of plasmid DNA led to efficient LacZ reporter gene expression in all patients. No side effects were experienced, supporting safety and applicability of this novel nonviral approach. A next step with a therapeutic gene product should determine antitumor efficacy of jet-injection gene transfer.

EPI-14-1149 . EGFR mutation type by tissue source tested

Supplemental Methods, Figures 1-2, Table S1. Supplemental Methods providing details on the study cohort and the image analysis algorithm. Supp. Figure 1: A) Ki67 stained breast cancer tissue. B) Haematoxylin Signal derived from color deconvolution (1) and C) histogram showing frequencies (y axis) of specific heamatoxylin intensities (x axis) of the image background (red) and foreground (green). Arrows show representative locations of different intensity classes: no tissue (red), tissue but no cell nuclei (blue), Ki67 negative cell (green) and Ki67 positive cell nuclei (orange). Figure 2: Construction of the borderline between plausible and non-plausible thresholds by combining the threshold and the resulting ratio of positive cells. Supplementary table S1: Clinico-pathological data of the study cohort

Supplemental Table S3. Tissue source and testing method used for EGFR mutation analysis

Introduction: EGFR mutations confer sensitivity to EGFR tyrosine kinase inhibitors (TKI) in advanced non–small cell lung cancer (NSCLC). We investigated the clinicopathologic characteristics associated with EGFR mutations and their impact on real-world treatment decisions and outcomes in Caucasian patients with advanced NSCLC.Methods: REASON (NCT00997230) was a noninterventional multicenter study in patients (≥18 years) with stage IIIb/IV NSCLC, who were candidates for EGFR mutation testing and first-line systemic treatment, but not eligible for surgery or radiotherapy. Patients were followed up according to normal clinical practice and assessed for primary (correlation of mutation status with baseline characteristics) and secondary endpoints (first-line treatment decision).Results: Baseline data were obtained for 4,200 patients; 4,196 fulfilled the inclusion criteria; EGFR mutations were detected in 431 patients; no EGFR mutations were detected in 3,590 patients; mutation status was not evaluable in 175 patients. In multivariate analysis, the odds of EGFR mutations were significantly higher (P < 0.0001) in females versus males (odds ratio: 1.85; 95% confidence interval, 1.48–2.32), never-smokers versus smokers (3.64; 2.91–4.56), and patients with adenocarcinoma versus other histologic subtypes (2.94; 2.17–4.08). The most commonly prescribed first-line systemic treatments were: EGFR-TKIs in EGFR mutation–positive NSCLC (56.6%) and combination chemotherapy in EGFR mutation–negative NSCLC (78.5%).Conclusions: This represents the largest dataset for EGFR mutations in Caucasian patients and shows EGFR mutations to be most prevalent in females with adenocarcinoma who had never smoked.Impact: These findings add to our understanding of the prognostic and predictive factors of NSCLC, supporting future improved treatment selection. Cancer Epidemiol Biomarkers Prev; 24(8); 1254–61. ©2015 AACR.

Supplementary Figure S1 from Novel Jet-Injection Technology for Nonviral Intratumoral Gene Transfer in Patients with Melanoma and Breast Cancer

Purpose: Recently, several studies reported a strong functional link between histone deacetylases (HDAC) and the development of tumors of the large intestine. However, despite the importance of these molecules, comparably little is known on expression patterns and functions of specific HDAC isoforms in colorectal cancer.Experimental Design: We characterized class I HDAC isoform expression patterns in a cohort of 140 colorectal carcinomas by immunohistochemistry. In addition, effects of HDAC inhibition by valproic acid and suberoylanilide hydroxamic acid, and specific HDAC isoform knockdown by short interfering RNA, were investigated in a cell culture model.Results: We found class I HDACs highly expressed in a subset of colorectal carcinomas with positivity for HDAC1 in 36.4%, HDAC2 in 57.9%, and HDAC3 in 72.9% of cases. Expression was significantly enhanced in strongly proliferating (P = 0.002), dedifferentiated (P = 0.022) tumors. High HDAC expression levels implicated significantly reduced patient survival (P = 0.001), with HDAC2 expression being an independent survival prognosticator (hazard ratio, 2.6; P = 0.03). Short interfering RNA–based inhibition of HDAC1 and HDAC2 but not HDAC3 suppressed growth of colon cancer cells in vitro, although to a lesser extent than chemical HDAC inhibitors did.Conclusions: The strong prognostic impact of HDAC isoforms in colorectal cancer, the interactions of HDACs with tumor cell proliferation and differentiation in vivo, and our finding that HDACs are differentially expressed in colorectal tumors suggest that the evaluation of HDAC expression in clinical trials for HDAC inhibitors might help to identify a patient subgroup who will exceptionally profit from such a treatment.

Purpose: Scoring proliferation through Ki67 immunohistochemistry is an important component in predicting therapy response to chemotherapy in patients with breast cancer. However, recent studies have cast doubt on the reliability of “visual” Ki67 scoring in the multicenter setting, particularly in the lower, yet clinically important, proliferation range. Therefore, an accurate and standardized Ki67 scoring is pivotal both in routine diagnostics and larger multicenter studies.Experimental Design: We validated a novel fully automated Ki67 scoring approach that relies on only minimal a priori knowledge on cell properties and requires no training data for calibration. We applied our approach to 1,082 breast cancer samples from the neoadjuvant GeparTrio trial and compared the performance of automated and manual Ki67 scoring.Results: The three groups of autoKi67 as defined by low (≤15%), medium (15.1%–35%), and high (>35%) automated scores showed pCR rates of 5.8%, 16.9%, and 29.5%, respectively. AutoKi67 was significantly linked to prognosis with overall and progression-free survival P values POS < 0.0001 and PPFS < 0.0002, compared with POS < 0.0005 and PPFS < 0.0001 for manual Ki67 scoring. Moreover, automated Ki67 scoring was an independent prognosticator in the multivariate analysis with POS = 0.002, PPFS = 0.009 (autoKi67) versus POS = 0.007, PPFS = 0.004 (manual Ki67).Conclusions: The computer-assisted Ki67 scoring approach presented here offers a standardized means of tumor cell proliferation assessment in breast cancer that correlated with clinical endpoints and is deployable in routine diagnostics. It may thus help to solve recently reported reliability concerns in Ki67 diagnostics. Clin Cancer Res; 21(16); 3651–7. ©2014 AACR.

Supplementary Table S1-S2 from ABCC2 (MRP2, cMOAT) Can Be Localized in the Nuclear Membrane of Ovarian Carcinomas and Correlates with Resistance to Cisplatin and Clinical Outcome

Supplementary Materials and Methods, Tables 1-4, Figures 1-5 from Hyperactivation of the Insulin-like Growth Factor Receptor I Signaling Pathway Is an Essential Event for Cisplatin Resistance of Ovarian Cancer Cells

Supplementary Figure 1 from Mass Spectrometry–Based Metabolic Profiling Reveals Different Metabolite Patterns in Invasive Ovarian Carcinomas and Ovarian Borderline Tumors

Supplementary Figure 2 from Mass Spectrometry–Based Metabolic Profiling Reveals Different Metabolite Patterns in Invasive Ovarian Carcinomas and Ovarian Borderline Tumors

Supplementary Tables 1-3 from Mass Spectrometry–Based Metabolic Profiling Reveals Different Metabolite Patterns in Invasive Ovarian Carcinomas and Ovarian Borderline Tumors

R scripts to reproduce the statistical tests for the TCGA survival analyses.

Metabolites are the end products of cellular regulatory processes, and their levels can be regarded as the ultimate response of biological systems to genetic or environmental changes. We have used a metabolite profiling approach to test the hypothesis that quantitative signatures of primary metabolites can be used to characterize molecular changes in ovarian tumor tissues. Sixty-six invasive ovarian carcinomas and nine borderline tumors of the ovary were analyzed by gas chromatography/time-of-flight mass spectrometry (GC-TOF MS) using a novel contamination-free injector system. After automated mass spectral deconvolution, 291 metabolites were detected, of which 114 (39.1%) were annotated as known compounds. By t test statistics with P < 0.01, 51 metabolites were significantly different between borderline tumors and carcinomas, with a false discovery rate of 7.8%, estimated with repeated permutation analysis. Principal component analysis (PCA) revealed four principal components that were significantly different between both groups, with the highest significance found for the second component (P = 0.00000009). PCA as well as additional supervised predictive models allowed a separation of 88% of the borderline tumors from the carcinomas. Our study shows for the first time that large-scale metabolic profiling using GC-TOF MS is suitable for analysis of fresh frozen human tumor samples, and that there is a consistent and significant change in primary metabolism of ovarian tumors, which can be detected using multivariate statistical approaches. We conclude that metabolomics is a promising high-throughput, automated approach in addition to functional genomics and proteomics for analyses of molecular changes in malignant tumors. (Cancer Res 2006; 66(22): 10795-804)

Ovarian cancer is a heterogeneous disease of low prevalence, but poor survival. Early diagnosis is critical for survival, but it is often challenging because the symptoms of ovarian cancer are subtle and become apparent only during advanced stages of the disease. Therefore, the identification of robust biomarkers of early disease is a clinical priority. Metabolomic profiling is an emerging diagnostic tool enabling the detection of biomarkers reflecting alterations in tumor metabolism, a hallmark of cancer. In this study, we performed metabolomic profiling of serum and tumor tissue from 158 patients with high-grade serous ovarian cancer (HGSOC) and 100 control patients with benign or non-neoplastic lesions. We report metabolites of hydroxybutyric acid (HBA) as novel diagnostic and prognostic biomarkers associated with tumor burden and patient survival. The accumulation of HBA metabolites caused by HGSOC was also associated with reduced expression of succinic semialdehyde dehydrogenase (encoded by ALDH5A1), and with the presence of an epithelial-to-mesenchymal transition gene signature, implying a role for these metabolic alterations in cancer cell migration and invasion. In conclusion, our findings represent the first comprehensive metabolomics analysis in HGSOC and propose a new set of metabolites as biomarkers of disease with diagnostic and prognostic capabilities. Cancer Res; 76(4); 796–804. ©2015 AACR.

Results of statistical tests for all metabolites in serum samples.

Supplementary Tables related to clinicopathological data (S1), metabolomics results (S3, S5, S6), gene expression and copy number survival analyses (S7-S8) and gene set enrichment analyses (S9-10) as well as Supplementary Figures (S1-S6) related to metabolomics results.

Supplementary Figure Legends 1-3 from Mass Spectrometry–Based Metabolic Profiling Reveals Different Metabolite Patterns in Invasive Ovarian Carcinomas and Ovarian Borderline Tumors

Supplementary Figure Legends

Supplementary Figure 3 from Mass Spectrometry–Based Metabolic Profiling Reveals Different Metabolite Patterns in Invasive Ovarian Carcinomas and Ovarian Borderline Tumors

Platinum plays a central role in the therapy of ovarian cancer, and the emergence of platinum resistance is a major obstacle for clinical management of the disease. We treated A2780 ovarian cancer cells by weekly cycles of cisplatin over a period of 6 months and unveiled that enhanced insulin-like growth factor I receptor (IGF-IR) expression and autocrine IGF-I are associated with hyperactivation of the IGF-IR and phosphatidylinositol-3-OH kinase (PI3K) pathways in cisplatin-resistant cells. IGF-IR expression levels increased during treatment cycles and correlated with cisplatin resistance. Purified IGF-I induced cisplatin resistance in diverse ovarian cancer cell lines, and small molecule inhibitors proved that IGF-IR and PI3K are essential for cisplatin resistance. Similar results were obtained with BG-1 ovarian cancer cells. Cytogenetic and array comparative genomic hybridization analyses revealed selection and de novo formation of chromosomal alterations during resistance development. An analysis of gene expression profiles of primary ovarian carcinomas identified the regulatory subunit PIK3R2 of PI3-kinase as a significant negative prognosis factor for ovarian cancer. We conclude that targeting the IGF-IR and the PI3K pathways is a promising new strategy to treat cisplatin-resistant ovarian carcinomas. [Cancer Res 2009;69(7):2996–3003]

Based on new trial data regarding immune checkpoint inhibitors (ICIs), the detection of high-grade microsatellite instability (MSI-H) or underlying deficient mismatch repair protein (dMMR) is now becoming increasingly important for predicting treatment response. For the first time, a PD‑1 ICI (pembrolizumab) has been approved by the European Medicines Agency (EMA) for first-line treatment of advanced (stage IV) dMMR/MSI‑H colorectal cancer (CRC). Further indications, such as dMMR/MSI‑H endometrial carcinoma (EC), have already succeeded (Dostarlimab, 2nd line treatment) and others are expected to follow before the end of 2021. The question of optimal testing in routine diagnostics should therefore be re-evaluated. Based on a consideration of the strengths and weaknesses of the widely available methods (immunohistochemistry and PCR), a test algorithm is proposed that allows quality assured, reliable, and cost-effective dMMR/MSI‑H testing. For CRC and EC, testing is therefore already possible at the primary diagnosis stage, in line with international recommendations (NICE, NCCN). The clinician is therefore enabled from the outset to consider not only the predictive but also the prognostic and predispositional implications of such a test when counseling patients and formulating treatment recommendations. As a basis for quality assurance, participation in interlaboratory comparisons and continuous documentation of results (e.g., QuIP Monitor) are strongly recommended.

Based on new trial data regarding immune checkpoint inhibitors (ICIs), the detection of high-grade microsatellite instability (MSI-H) or underlying deficient mismatch repair protein (dMMR) is now becoming increasingly important for predicting treatment response. For the first time, a PD‑1 ICI (pembrolizumab) has been approved by the European Medicines Agency (EMA) for first-line treatment of advanced (stage IV) dMMR/MSI‑H colorectal cancer (CRC). Further indications, such as dMMR/MSI‑H endometrial carcinoma (EC), have already succeeded (Dostarlimab, 2nd line treatment) and others are expected to follow before the end of 2021. The question of optimal testing in routine diagnostics should therefore be re-evaluated. Based on a consideration of the strengths and weaknesses of the widely available methods (immunohistochemistry and PCR), a test algorithm is proposed that allows quality assured, reliable, and cost-effective dMMR/MSI‑H testing. For CRC and EC, testing is therefore already possible at the primary diagnosis stage, in line with international recommendations (NICE, NCCN). The clinician is therefore enabled from the outset to consider not only the predictive but also the prognostic and predispositional implications of such a test when counseling patients and formulating treatment recommendations. As a basis for quality assurance, participation in interlaboratory comparisons and continuous documentation of results (e.g., QuIP Monitor) are strongly recommended.

Die „Qualitatssicherung-Initiative Pathologie“ (QuIP) bietet den Pathologen die Moglichkeit, die methodischen Ablaufe der immunhistologischen und molekularen Diagnostik ergebnisorientiert uberprufen und zertifizieren zu lassen. Fur die In-situ-Hybridisierungs- (ISH-)Diagnostik wurden 2019 5 Ringversuche angeboten, 2 wiederkehrende (HER2-ISH-Magenkarzinome und HER2-ISH-Mammakarzinome) sowie 3 prototypische (ROS1-NSCLC, ALK1-NSCLC, NTRK). Dabei richten sich die angebotenen RV nach der Entwicklung in der Diagnostik und der Bedeutung der therapeutischen Relevanz, die mit den abzufragenden Molekulen und der damit einhergehenden Methoden verbunden ist. Die Ergebnisse der RV zeigten 2019 eine Sensitivitat von mindestens 94,4 % und eine Spezifitat von mindestens 96,6 % sowie einer Erfolgsquote von 85–99 %. Dies spricht fur einen hohen Qualitatsstandard der RV und der teilnehmenden Institute

We here report on the first neuropathological round robin trials initiated by the Quality Assurance Initiative Pathology (QuIP) in Germany in the years 2018 and 2019. Testing services as external laboratory controls were offered for IDH1-R132H immunohistochemistry in 2018 followed by a molecular trial for IDH1 and IDH2 mutations in 2019 including the rare mutational variants. Also in 2019, a trial on MGMT promoter methylation testing was offered. On a national scale, trial offers were well received with around 40 participating institutions. The international announcement of the molecular IDH1/IDH2 mutational trial achieved only moderate European outspread. Success rates in all three trials were excellent (IDH1-R132H immunohistochemistry 2018: 94%, 18 out of 20 possible points required; IDH1/IDH2 mutational status 2019: 100%, 19 out of 20 possible points required; MGMT promoter methylation 2019: 94%, 19 out of 20 possible points required) indicating that quality standards are high in the broad majority of the institutions. Trial participation also involved filling in a questionnaire asking for background information on local testing procedures. We here present a first assessment of the information collected providing unique insights in the landscape of molecular testing in neuropathology. Derived from this information we identify future challenges and provide an outlook on the development of quality assurance in the field of neuropathology.

Gene fusions involving the three neurotrophic tyrosine receptor kinase genes NTRK1, NTRK2, or NTRK3 were identified as oncogenic drivers in many cancer types. Two small molecule inhibitors have been tested in clinical trials recently and require the detection of a NTRK fusion gene prior to therapeutic application. Fluorescence in situ hybridization (FISH) and targeted next-generation sequencing (tNGS) assays are commonly used for diagnostic profiling of gene fusions. In the presented study we applied an external quality assessment (EQA) scheme in order to investigate the suitability of FISH and RNA-/DNA-based tNGS for detection of NTRK fusions in a multinational and multicentric ring trial. In total 27 participants registered for this study. Nine institutions took part in the FISH-based and 18 in the NGS-based round robin test, the latter additionally subdivided into low-input and high-input NGS methods (regarding nucleic acid input). Regardless of the testing method applied, all participants received tumor sections of 10 formalin-fixed and paraffin-embedded (FFPE) tissue blocks for in situ hybridization or RNA/DNA extraction, and the results were submitted via an online questionnaire. For FISH testing, eight of nine (88.8%) participants, and for NGS-based testing 15 of 18 (83.3%) participants accomplished the round robin test successfully. The overall high success rate demonstrates that FISH- and tNGS-based NTRK testing can be well established in a routine diagnostic setting. Complementing this dataset, we provide an updated in silico analysis on the coverage of more than 150 NTRK fusion variants by several commercially available RNA-based tNGS panels.

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.

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.

Tumor programmed death ligand 1 (PD-L1) expression is associated with improved clinical benefit from immunotherapies targeting the PD-1 pathway. We conducted a global, multicenter, retrospective observational study to determine real-world prevalence of tumor PD-L1 expression in patients with NSCLC.Patients ≥18 years with histologically confirmed stage IIIB/IV NSCLC and a tumor tissue block (≤5 years old) obtained before treatment were identified in 45 centers across 18 countries. Tumor samples from eligible patients were selected consecutively, when possible. PD-L1 expression was evaluated at each center using the PD-L1 IHC 22C3 pharmDx kit (Agilent, Santa Clara, CA, USA).Of 2617 patients who met inclusion criteria, 2368 (90%) had PD-L1 data; 530 (22%) patients had PD-L1 TPS ≥ 50%, 1232 (52%) had PD-L1 TPS ≥ 1%, and 1136 (48%) had PD-L1 TPS 1%. The most common reason for not having PD-L1 data (n = 249) was insufficient tumor cells (100) on the slide (n = 170 [6%]). Percentages of patients with PD-L1 TPS ≥ 50% and TPS ≥ 1%, respectively were: 22%/52% in Europe; 22%/53% in Asia Pacific; 21%/47% in the Americas, and 24%/55% in other countries. Prevalence of EGFR mutations (19%) and ALK alterations (3%) was consistent with prior reports from metastatic NSCLC studies. Among 1064 patients negative for both EGFR mutation and ALK alteration, the percentage with PD-L1 TPS ≥ 50% and TPS ≥ 1%, respectively, were 27% and 53%.This is the largest real-world study in advanced NSCLC to date evaluating PD-L1 tumor expression using the 22C3 pharmDx kit. Testing failure rate was low with local evaluation of PD-L1 TPS across a large number of centers. Prevalence of PD-L1 TPS ≥ 50% and TPS ≥ 1% among patients with stage IIIB/IV NSCLC was similar across geographic regions and broadly consistent with central testing results from clinical trial screening populations.