Peer-to-peer validation of Ki-67 scoring in a pathology quality circle as a tool to assess interobserver variability: are we better than we thought?

Ki-67, a nuclear protein expressed in all stages of cellular proliferation, is a valuable tool to assess tumor proliferation and has been linked to more aggressive tumor behavior. However, interlaboratory staining heterogeneity and inter-observer variability challenge its reproducibility. Round Robin tests are a suitable tool to standardize and harmonize immunohistochemical and molecular analyses in histopathology. The study investigates the interrater and interlaboratory reproducibility of Ki-67-scoring using both manual and automated approaches. Unstained TMA slides comprising diverse tumor types (breast cancer, neuroendocrine tumors, lymphomas, and head and neck squamous cell carcinoma) were distributed to six pathology laboratories, each employing their routine staining protocols. Manual and automated scoring methods were applied, and interrater and interlaboratory agreement assessed using intraclass correlation coefficients (ICC). The results highlight good-to-excellent reliability overall, with automated scoring demonstrating higher consistency (ICC 0.955) than manual scoring (ICC 0.871). Results were more variable when looking at the individual entities. Reliability remained good for lymphomas (ICC 0.878) and breast cancer (ICC 0.784) and was poor in well-differentiated neuroendocrine tumors (ICC 0.354). This study clearly advocates standardized practices and training to ensure consistency in Ki-67-assessment, and it demonstrates that this can be achieved in a peer-to-peer approach in local quality-circles.

Mass Spectrometry-Based Profiling of Histone Post-Translational Modifications in Uveal Melanoma Tissues, Human Melanocytes, and Uveal Melanoma Cell Lines - A Pilot Study.

Purpose: Epigenetic alterations in uveal melanoma (UM) are still neither well characterized, nor understood. In this pilot study, we sought to provide a deeper insight into the possible role of epigenetic alterations in the pathogenesis of UM and their potential prognostic relevance. To this aim, we comprehensively profiled histone post-translational modifications (PTMs), which represent epigenetic features regulating chromatin accessibility and gene transcription, in UM formalin-fixed paraffin-embedded (FFPE) tissues, control tissues, UM cell lines, and healthy melanocytes. Methods: FFPE tissues of UM (n = 24), normal choroid (n = 4), human UM cell lines (n = 7), skin melanocytes (n = 6), and uveal melanocytes (n = 2) were analyzed through a quantitative liquid chromatography-mass spectrometry (LC-MS) approach. Results: Hierarchical clustering showed a clear separation with several histone PTMs that changed significantly in a tumor compared to normal samples, in both tissues and cell lines. In addition, several acetylations and H4K20me1 showed lower levels in BAP1 mutant tumors. Some of these changes were also observed when we compared GNA11 mutant tumors with GNAQ tumors. The epigenetic profiling of cell lines revealed that the UM cell lines MP65 and UPMM1 have a histone PTM pattern closer to the primary tissues than the other cell lines analyzed. Conclusions: Our results suggest the existence of different histone PTM patterns that may be important for diagnosis and prognosis in UM. However, further analyses are needed to confirm these findings in a larger cohort. The epigenetic characterization of a panel of UM cell lines suggested which cellular models are more suitable for epigenetic investigations.

Value of Diagnostic Tools in the Diagnosis of Osteomyelitis: Pilot Study to Establish an Osteomyelitis Score.

Osteomyelitis (OM) remains one of the most feared complications in bone surgery and trauma. Its diagnosis remains a major challenge due to lack of guidelines. The aim of this study was to prospectively analyze the value of the most common and available diagnostic tools and to establish an OM score to derive treatment recommendations. All patients with suspected OM were included in a prospective pilot study. All patients underwent blood sampling for C-reactive protein and white blood cell count analysis. Magnetic resonance imaging (MRI), and microbiologic and histopathologic samples, were taken from representative sites of initial debridement. All patients were treated according to their OM test results and followed for at least one year. Subsequently, the value of individual or combined diagnostic tools was analyzed in patients with confirmed OM and in patients in whom OM was ruled out. Based on these findings, an OM score was developed that included MRI, microbiology, and histopathology. The score identified all control patients and all but one OM patient, resulting in a correct diagnosis of 93.3%, which was validated in a second independent larger cohort. This was the first study to analyze the value of the most commonly used tools to diagnose OM. The proposed OM score provides a simple scoring system to safely interpret test results with high accuracy.

[Molecular cytology: Opportunities and challenges]. / Molekulare Zytologie: Chancen und Herausforderungen.

Predictive marker (re-)analysis of tumor material can be a real obstacle in several tumor entities, like non-small cell lung cancer (NSCLC), due to difficult anatomic conditions and small biopsy samples. As reported in the literature, cytological samples comprise excellent starting material for predictive marker analysis like fluorescence in situ hybridization and next generation sequencing. As for formalin-fixed paraffin-embedded tissue samples, rigorous quality control and standardized laboratory operating procedures are mandatory. Further advantages of cytological specimens are the rapid and straightforward inspection of representativeness, for example by rapid on-site evaluation (ROSE). Another striking advantage is that the fresh cellular material from smears and serous cavity fluids can be used for the generation of two- and three-dimensional cell culture models. Hereby, in addition to the conventional biomarker testing, complex complementary functional genomic assays can also be applied, for example, to assess the effects of multiple variants in one sample and unknown variants of tumor driver genes and tumor suppressor genes. This information may provide additional vulnerabilities of the tumor to be considered for the therapy decision, for example in the molecular tumor board.

Multicenter Evaluation of the Idylla GeneFusion in Non-Small-Cell Lung Cancer.

Targeted therapy in lung cancer requires the assessment of multiple oncogenic driver alterations, including fusion genes. This retrospective study evaluated the Idylla GeneFusion prototype, an automated and ease-of-use (<2 minutes) test, with a short turnaround time (3 hours) to detect fusions involving ALK, ROS1, RET, and NTRK1/2/3 genes and MET exon 14 skipping. This multicenter study (18 centers) included 313 tissue samples from lung cancer patients with 97 ALK, 44 ROS1, 20 RET, and 5 NTRKs fusions, 32 MET exon 14 skipping, and 115 wild-type samples, previously identified with reference methods (RNA-based next-generation sequencing/fluorescence in situ hybridization/quantitative PCR). Valid results were obtained for 306 cases (98%), overall concordance between Idylla and the reference methods was 89% (273/306); overall sensitivity and specificity were 85% (165/193) and 96% (108/113), respectively. Discordances were observed in 28 samples, where Idylla did not detect the alteration identified by the reference methods; and 5 samples where Idylla identified an alteration not detected by the reference methods. All of the ALK-, ROS1-, and RET-specific fusions and MET exon 14 skipping identified by Idylla GeneFusion were confirmed by reference method. To conclude, Idylla GeneFusion is a clinically valuable test that does not require a specific infrastructure, allowing a rapid result. The absence of alteration or the detection of expression imbalance only requires additional testing by orthogonal methods.

TargetPlex FFPE-Direct DNA Library Preparation Kit for SiRe NGS panel: an international performance evaluation study.

AIM: Next generation sequencing (NGS) represents a key diagnostic tool to identify clinically relevant gene alterations for treatment-decision making in cancer care. However, the complex manual workflow required for NGS has limited its implementation in routine clinical practice. In this worldwide study, we validated the clinical performance of the TargetPlex FFPE-Direct DNA Library Preparation Kit for NGS analysis. Impressively, this new assay obviates the need for separate, labour intensive and time-consuming pre-analytical steps of DNA extraction, purification and isolation from formalin-fixed paraffin embedded (FFPE) specimens in the NGS workflow. METHODS: The TargetPlex FFPE-Direct DNA Library Preparation Kit, which enables NGS analysis directly from FFPE, was specifically developed for this study by TargetPlex Genomics Pleasanton, California. Eleven institutions agreed to take part in the study coordinated by the Molecular Cytopathology Meeting Group (University of Naples Federico II, Naples, Italy). All participating institutions received a specific Library Preparation Kit to test eight FFPE samples previously assessed with standard protocols. The analytical parameters and mutations detected in each sample were then compared with those previously obtained with standard protocols. RESULTS: Overall, 92.8% of the samples were successfully analysed with the TargetPlex FFPE-Direct DNA Library Preparation Kit on Thermo Fisher Scientific and Illumina platforms. Altogether, in comparison with the standard workflow, the TargetPlex FFPE-Direct DNA Library Preparation Kit was able to detect 90.5% of the variants. CONCLUSION: The TargetPlex FFPE-Direct DNA Library Preparation Kit combined with the SiRe panel constitutes a convenient, practical and robust cost-saving solution for FFPE NGS analysis in routine practice.

[Incidental finding of an intestinally differentiated adenocarcinoma in a tailgut cyst after robotic-assisted resection]. / Zufallsbefund eines intestinal differenzierten Adenokarzinoms in einer Tailgut Zyste nach robotisch-assistierter Resektion.

Due to pelvic symptoms, a diagnostic sectional imaging was initiated in a 52-year-old female patient. This revealed a cystic, retrorectal mass, suspected to be a tailgut cyst. Due to the symptoms and the unclear dignity after several frustrating endosonographic punctures, a robotic-assisted resection of the cystic Tumor was performed after careful interdisciplinary consultation.The histological examination confirmed the diagnosis of a tailgut cyst but also revealed parts of an intestinally differentiated adenocarcinoma.Due to the unclear metastatic behaviour, robotic-assisted low anterior resection with total mesorectal excision was performed as oncological resection, similar to rectal carcinomas. No residuals or lymph node metastases were detectable in the histological examination, so that follow- up monitoring was recommended.Retrorectal tumours are an extremely rare entity, worldwide only 28 cases of an intestinally differentiated carcinoma in a tailgut cyst have been described so far. Since there are no clear recommendations in the literature regarding the diagnostic or therapeutic procedure, we would like to discuss a possible algorithm in case of a proven retrorectal mass in our case study.

Prognostic Impact of KRAS G12C Mutation in Patients With NSCLC: Results From the European Thoracic Oncology Platform Lungscape Project.

INTRODUCTION: KRAS mutations, the most frequent gain-of-function alterations in NSCLC, are currently emerging as potential predictive therapeutic targets. The role of KRAS-G12C (Kr_G12C) is of special interest after the recent discovery and preclinical analyses of two different Kr_G12C covalent inhibitors (AMG-510, MRTX849). METHODS: KRAS mutations were evaluated in formalin-fixed, paraffin-embedded tissue sections by a microfluidic-based multiplex polymerase chain reaction platform as a component of the previously published European Thoracic Oncology Platform Lungscape 003 Multiplex Mutation study, of clinically annotated, resected, stage I to III NSCLC. In this study, -Kr_G12C mutation prevalence and its association with clinicopathologic characteristics, molecular profiles, and postoperative patient outcome (overall survival, relapse-free survival, time-to-relapse) were explored. RESULTS: KRAS gene was tested in 2055 Lungscape cases (adenocarcinomas: 1014 [49%]) with I or II or III stage respective distribution of 53% or 24% or 22% and median follow-up of 57 months. KRAS mutation prevalence in the adenocarcinoma cohort was 38.0% (95% confidence interval (CI): 35.0% to 41.0%), with Kr_G12C mutation representing 17.0% (95% CI: 14.7% to 19.4%). In the "histologic-subtype" cohort, Kr_G12C prevalence was 10.5% (95% CI: 9.2% to 11.9%). When adjusting for clinicopathologic characteristics, a significant negative prognostic effect of Kr_G12C presence versus other KRAS mutations or nonexistence of KRAS mutation was identified in the adenocarcinoma cohort alone and in the "histologic-subtype" cohort. For overall survival in adenocarcinomas, hazard ratio (HR)G12C versus other KRAS is equal to 1.39 (95% CI: 1.03 to 1.89, p = 0.031) and HRG12C versus no KRAS is equal to 1.32 (95% CI: 1.03 to 1.69, p = 0.028) (both also significant in the "histologic-subtype" cohort). For time-to-relapse, HRG12C versus other KRAS is equal to 1.41 (95% CI: 1.03 to 1.92, p = 0.030). In addition, among all patients, for relapse-free survival, HRG12C versus no KRAS is equal to 1.27 (95% CI: 1.04 to 1.54, p = 0.017). CONCLUSIONS: In this large, clinically annotated stage I to III NSCLC cohort, the specific Kr_G12C mutation is significantly associated with poorer prognosis (adjusting for clinicopathologic characteristics) among adenocarcinomas and in unselected NSCLCs.

A requirement for p120-catenin in the metastasis of invasive ductal breast cancer.

We report here the effects of targeted p120-catenin (encoded by CTNND1; hereafter denoted p120) knockout (KO) in a PyMT mouse model of invasive ductal (mammary) cancer (IDC). Mosaic p120 ablation had little effect on primary tumor growth but caused significant pro-metastatic alterations in the tumor microenvironment, ultimately leading to a marked increase in the number and size of pulmonary metastases. Surprisingly, although early effects of p120-ablation included decreased cell-cell adhesion and increased invasiveness, cells lacking p120 were almost entirely unable to colonized distant metastatic sites in vivo The relevance of this observation to human IDC was established by analysis of a large clinical dataset of 1126 IDCs. As reported by others, p120 downregulation in primary IDC predicted worse overall survival. However, as in the mice, distant metastases were almost invariably p120 positive, even in matched cases where the primary tumors were p120 negative. Collectively, our results demonstrate a strong positive role for p120 (and presumably E-cadherin) during metastatic colonization of distant sites. On the other hand, downregulation of p120 in the primary tumor enhanced metastatic dissemination indirectly via pro-metastatic conditioning of the tumor microenvironment.

Conceptual framework for precision cancer medicine in Germany: Consensus statement of the Deutsche Krebshilfe working group 'Molecular Diagnostics and Therapy'.

Precision cancer medicine (PCM) holds great promises to offer more effective therapies to patients based on molecular profiling of their individual tumours. Although the PCM approach seems intuitive, multiple conceptional and structural challenges interfere with the broad implementation of PCM into clinical practice. Accordingly, concerted national and international efforts are needed to guide the further development and broad adoption of PCM in Germany. With support of the 'German Cancer Aid' (Deutsche Krebshilfe [DKH]) a task force 'Molecular Diagnostics and Therapy' was implemented. In two workshops supported by the DKH, delegates from the fourteen comprehensive cancer centresidentified key topics essential to implement quality-guided, harmonized and adaptable PCM. Based on an online questionnaire and using a modified Delphi approach, nine statements were drafted and evaluated within the group. These statements could serve as a basis to define a collaborative strategy for PCM in the future with the aim to sustain and further improve its quality.

Current Topics and Practical Considerations of Cytology Practice in Lung Cancer: Reflexions from the Lung Symposium at the 42nd European Congress of Cytology, Malmö, 2019.

In June 2019, a lung symposium was held at the 42nd European Congress of Cytology in Malmö, Sweden. Due to the current importance of cytological samples in the diagnoses and molecular analysis to set up the utmost management of lung cancer patients, cytologists from different countries shared the experience of their institutions. The place of the cytological samples gains more and more importance on the potential long-term survival gain through personalized medicine and this harbors the improvement of the guidelines both in pathology and cytology field. In this symposium, the new 6-tiered reporting system for pulmonary cytology proposed by the Papanicolaou Society of Cytopathology and detailed cytomorphological approach to lung carcinoma including lookalike lesions and DNA- and RNA-based analysis of cytology material have been discussed. The cytopathologist plays a pivotal role in ensuring success of a correct triage for the cytology material to be sure of the adequacy and quality of the yield from the rapid on-site evaluation till the report which should encompass molecular profile in rational patient management.

Correction to: Epigenetic regulation of L1CAM in endometrial carcinoma: comparison to cancer-testis (CT-X) antigens.

Following publication of the original article [1], we have been alerted to errors in Figs. 2 and 8. In Fig. 2B, the GAPDH loading control for Hec1A cells is shown twice in error (in Fig. 2B and Fig. 2C). In Fig. 8, in testis case 1 (first column) the MAGE-A4 staining panel was repeated and also appears as the NY-ESO-1 staining panel in error. The corrected versions of Fig. 2 and Fig. 8 are shown below. We apologize for this inconvenience.

Gemcitabine Synergizes with Immune Checkpoint Inhibitors and Overcomes Resistance in a Preclinical Model and Mesothelioma Patients.

PURPOSE: Combination of immune checkpoint inhibitors with chemotherapy is under investigation for cancer treatment. EXPERIMENTAL DESIGN: We studied the rationale of such a combination for treating mesothelioma, a disease with limited treatment options. RESULTS: The combination of gemcitabine and immune checkpoint inhibitors outperformed immunotherapy alone with regard to tumor control and survival in a preclinical mesothelioma model; however, the addition of dexamethasone to gemcitabine and immune checkpoint inhibitors nullified the synergistic clinical response. Furthermore, treatment with gemcitabine plus anti-PD-1 resulted in an objective clinical response in two patients with mesothelioma, who were resistant to gemcitabine or anti-PD-1 as monotherapy. CONCLUSIONS: Thus, treatment of mesothelioma with a combination of gemcitabine with immune checkpoint inhibitors is feasible and results in synergistic clinical response compared with single treatment in the absence of steroids.

Targeted next-generation-sequencing for reliable detection of targetable rearrangements in lung adenocarcinoma-a single center retrospective study.

Oncogenic rearrangements leading to targetable gene fusions are well-established cancer driver events in lung adenocarcinoma. Accurate and reliable detection of these gene fusions is crucial to select the appropriate targeted therapy for each patient. We compared the targeted next-generation-sequencing Oncomine Focus Assay (OFA; Thermo Fisher Scientific) with conventional ALK FISH and anti-Alk immunohistochemistry in a cohort of 52 lung adenocarcinomas (10 ALK rearranged, 18 non-ALK rearranged, and 24 untested cases). We found a sensitivity and specificity of 100% for detection of ALK rearrangements using the OFA panel. In addition, targeted next generation sequencing allowed us to analyze a set of 23 driver genes in a single assay. Besides EML4-ALK (11/52 cases), we detected EZR-ROS1 (1/52 cases), KIF5B-RET (1/52 cases) and MET-MET (4/52 cases) fusions. All EML4-ALK, EZR-ROS1 and KIF5B-RET fusions were confirmed by multiplexed targeted next generation sequencing assay (Oncomine Solid Tumor Fusion Transcript Kit, Thermo Fisher Scientific). All cases with EML4-ALK rearrangement were confirmed by Alk immunohistochemistry and all but one by ALK FISH. In our experience, targeted next-generation sequencing is a reliable and timesaving tool for multiplexed detection of targetable rearrangements. Therefore, targeted next-generation sequencing represents an efficient alternative to time-consuming single target assays currently used in molecular pathology.

Integrative genomic profiling of large-cell neuroendocrine carcinomas reveals distinct subtypes of high-grade neuroendocrine lung tumors.

Pulmonary large-cell neuroendocrine carcinomas (LCNECs) have similarities with other lung cancers, but their precise relationship has remained unclear. Here we perform a comprehensive genomic (n = 60) and transcriptomic (n = 69) analysis of 75 LCNECs and identify two molecular subgroups: "type I LCNECs" with bi-allelic TP53 and STK11/KEAP1 alterations (37%), and "type II LCNECs" enriched for bi-allelic inactivation of TP53 and RB1 (42%). Despite sharing genomic alterations with adenocarcinomas and squamous cell carcinomas, no transcriptional relationship was found; instead LCNECs form distinct transcriptional subgroups with closest similarity to SCLC. While type I LCNECs and SCLCs exhibit a neuroendocrine profile with ASCL1high/DLL3high/NOTCHlow, type II LCNECs bear TP53 and RB1 alterations and differ from most SCLC tumors with reduced neuroendocrine markers, a pattern of ASCL1low/DLL3low/NOTCHhigh, and an upregulation of immune-related pathways. In conclusion, LCNECs comprise two molecularly defined subgroups, and distinguishing them from SCLC may allow stratified targeted treatment of high-grade neuroendocrine lung tumors.

Systematic Kinase Inhibitor Profiling Identifies CDK9 as a Synthetic Lethal Target in NUT Midline Carcinoma.

Kinase inhibitors represent the backbone of targeted cancer therapy, yet only a limited number of oncogenic drivers are directly druggable. By interrogating the activity of 1,505 kinase inhibitors, we found that BRD4-NUT-rearranged NUT midline carcinoma (NMC) cells are specifically killed by CDK9 inhibition (CDK9i) and depend on CDK9 and Cyclin-T1 expression. We show that CDK9i leads to robust induction of apoptosis and of markers of DNA damage response in NMC cells. While both CDK9i and bromodomain inhibition over time result in reduced Myc protein expression, only bromodomain inhibition induces cell differentiation and a p21-induced cell-cycle arrest in these cells. Finally, RNA-seq and ChIP-based analyses reveal a BRD4-NUT-specific CDK9i-induced perturbation of transcriptional elongation. Thus, our data provide a mechanistic basis for the genotype-dependent vulnerability of NMC cells to CDK9i that may be of relevance for the development of targeted therapies for NMC patients.

Prevalence and clinical association of MET gene overexpression and amplification in patients with NSCLC: Results from the European Thoracic Oncology Platform (ETOP) Lungscape project.

INTRODUCTION: In a well-defined NSCLC cohort of the ETOP Lungscape program, we explored the epidemiology of IHC MET overexpression and amplification, their inter-correlation, and their association to outcome. METHODS: Resected NSCLC were assessed for MET gene copy number (GCN) and expression using silver in-situ hybridization (SISH) and immunohistochemistry (IHC) on TMAs in a multicenter setting. MET amplification was defined as MET/centromere ratio≥2 (with average MET GCN≥4), high MET GCN as CGN≥5 and MET IHC+ as ≥2+ intensity in ≥50% of tumor cells. A total of 182 MET IHC+ and EGFR/KRAS WT tumors were analyzed for METex14 skipping mutation. RESULTS: MET IHC+ was found in 23.8% of 2432 patients, significantly associated with female gender, small tumor size, and adenocarcinoma histology. We observed a high inter-laboratory variability in IHC and SISH analysis. MET amplification prevailed in 4.6% and MET GCN≥5 in 4.1% of 1572 patients. MET amplification and MET GCN≥5 were not significantly associated with any tumor characteristics or stage. Both were significantly associated with IHC MET positivity (p<0.001). METex14 skipping mutation prevailed in 5 of 182 (2.7%) MET IHC+ WT EGFR/KRAS NSCLC, 4 of which within the 88 adenocarcinomas (4.5%). No association of IHC MET overexpression, SISH MET amplification or high MET GCN was found with OS, RFS or TTR. CONCLUSION: MET overexpression is found in 23.8% of surgically resected NSCLC. MET amplification prevails in 4.6% and is associated with MET overexpression. Both have no influence on prognosis. The large inter-laboratory variability in IHC highlights the challenge of MET IHC analysis in routine practice.