Heterogeneity of small duct- and large duct-type intrahepatic cholangiocarcinoma.

AIMS: The histological subtype of intrahepatic cholangiocarcinoma (iCCA) is associated with different mutational characteristics that impact clinical management. So far, data are lacking on the presence of small duct iCCA (SD-iCCA) and large duct iCCA (LD-iCCA) in a single patient. The aim of the current study was to determine the presence and degree of intratumoural heterogeneity of SD- and LD-iCCA features in different tumour regions. METHODS AND RESULTS: All patients treated with surgically resected iCCA at Frankfurt University Hospital between December 2005 and March 2023 were retrospectively analysed. Histomorphological features of SD- and LD-iCCA were evaluated by an expert hepatobiliary pathologist. Tissue samples suspicious for subtype heterogeneity were further investigated. Immunohistochemistry for N-cadherin, S100P, MUC5AC, MUC6, TFF1 and AGR2 and mutational profiling with the Illumina TruSight Oncology 500 (TSO500) assay were performed separately for the SD- and LD-iCCA regions. Of 129 patients with surgically resected iCCA, features of either SD- or LD-iCCA were present in 67.4% (n = 87) and 24.8% of the patients (n = 32), respectively; 7.8% (n = 10) had histomorphological features of both SD- and LD-iCCA, seven patients (5.4%) of which had sufficient formalin-fixed, paraffin-embedded tissue for further analysis. Heterogeneity of both subtypes could be confirmed with immunohistochemistry. In five of seven (71.4%) patients, molecular profiling revealed intratumoural differences in genetic alterations between the SD- and LD-iCCA region. In one patient, a BRAF mutation (p.V600E) was found in the SD-iCCA but not in the LD-iCCA region of the tumour. CONCLUSIONS: A marked portion of patients with iCCA exhibits both SD- and LD-iCCA in different tumour regions. In case of the presence of histopathological heterogeneity, mutational profiling should be considered to avoid missing therapeutically relevant genetic alterations.

Increased MCL1 dependency leads to new applications of BH3-mimetics in drug-resistant neuroblastoma.

BACKGROUND: Neuroblastoma is a paediatric cancer that is characterised by poor prognosis for chemoresistant disease, highlighting the need for better treatment options. Here, we asked whether BH3-mimetics inhibiting BCL2 proteins may eliminate chemoresistant neuroblastoma cells. METHODS: We utilised cisplatin-adapted neuroblastoma cell lines as well as patient tissues before and after relapse to study alterations of BCL2 proteins upon chemoresistance. RESULTS: In a direct comparison of cisplatin-resistant cells we identified a prominent loss of sensitivity to BCL2/BCL-XL inhibitors that is associated with an increase in MCL1 dependency and high expression of MCL1 in patient tumour tissues. Screening of FDA-approved anti-cancer drugs in chemoresistant cells identified therapeutics that may be beneficial in combination with the clinically tested BH3-mimetic ABT263, but no synergistic drug interactions with the selective MCL1 inhibitor S63845. Further exploration of potential treatment options for chemoresistant neuroblastoma identified immunotherapy based on NK cells as highly promising, since NK cells are able to efficiently kill both parental and chemoresistant cells. CONCLUSIONS: These data highlight that the application of BH3-mimetics may differ between first line treatment and relapsed disease. Combination of NK cell-based immunotherapy with BH3-mimetics may further increase killing of chemoresistant neuroblastoma, outlining a new treatment strategy for relapsed neuroblastoma.

Deep learning based on hematoxylin-eosin staining outperforms immunohistochemistry in predicting molecular subtypes of gastric adenocarcinoma.

In gastric cancer (GC), there are four molecular subclasses that indicate whether patients respond to chemotherapy or immunotherapy, according to the TCGA. In clinical practice, however, not every patient undergoes molecular testing. Many laboratories have used well-implemented in situ techniques (IHC and EBER-ISH) to determine the subclasses in their cohorts. Although multiple stains are used, we show that a staining approach is unable to correctly discriminate all subclasses. As an alternative, we trained an ensemble convolutional neuronal network using bagging that can predict the molecular subclass directly from hematoxylin-eosin histology. We also identified patients with predicted intra-tumoral heterogeneity or with features from multiple subclasses, which challenges the postulated TCGA-based decision tree for GC subtyping. In the future, deep learning may enable targeted testing for molecular subtypes and targeted therapy for a broader group of GC patients. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Metachronous Osteosarcoma, A Differential Diagnosis to be Considered in Children With Osteosarcoma: A Review of Literature and a Case From Our Center.

Metachronous osteosarcomas (MOS) are currently defined as tumors that arise in a way and site unusual for typical metastasis. In this article, we reviewed the recent literature on the occurrence of metachronous osteosarcoma and presented a case from our center. Our patient, a 10-year-old girl, presented with metachronous osteoblastic osteosarcoma of the left distal femur ∼5 years after the successful treatment for osteosarcoma of the right distal femur. Even after several relapses, complete remission (CR) was achieved after the first osteosarcoma and after the metachronous osteosarcoma. The literature research revealed that metachronous osteosarcoma occurs in 3.4 to 5.4% of osteosarcoma patients. The time interval between the diagnosis of the initial osteosarcoma and the metachronous tumor ranged from 0.2 to 14.3 years (median 2.5 y). MOS appears to have differences in localization and metastatic spread, as well as a different survival pattern compared with primary osteosarcoma and osteosarcoma recurrence. Survival (median 4.3 y, range 0 to 24.6 y) appears to be associated with the time interval to diagnosis of MOS. In particular, early MOS (<24 mo after primary diagnosis) seem to have a poorer prognosis. Therefore, the occurrence of MOS at oncological unusual sites should be considered as a differential diagnosis in osteosarcoma survivors.

Increased HRD score in cisplatin resistant penile cancer cells.

BACKGROUND/INTRODUCTION: Penile cancer is a rare disease in demand for new therapeutic options. Frequently used combination chemotherapy with 5 fluorouracil (5-FU) and cisplatin (CDDP) in patients with metastatic penile cancer mostly results in the development of acquired drug resistance. Availability of cell culture models with acquired resistance against standard therapy could help to understand molecular mechanisms underlying chemotherapy resistance and to identify candidate treatments for an efficient second line therapy. METHODS: We generated a cell line from a humanpapilloma virus (HPV) negative penile squamous cell carcinoma (UKF-PEC-1). This cell line was subject to chronic exposure to chemotherapy with CDDP and / or 5-FU to induce acquired resistance in the newly established chemo-resistant sublines (PEC-1rCDDP2500, adapted to 2500 ng/ml CDDP; UKF-PEC-1r5-FU500, adapted to 500 ng/ml 5- FU; UKF-PEC1rCDDP2500/r5-FU500, adapted to 2500 ng/ml CDDP and 500 ng/ml 5 -FU). Afterwards cell line pellets were formalin-fixed, paraffin embedded and subject to sequencing as well as testing for homologous recombination deficiency (HRD). Additionally, exemplary immunohistochemical stainings for p53 and gammaH2AX were applied for verification purposes. Finally, UKF-PEC-1rCDDP2500, UKF-PEC-1r5-FU500, UKF-PEC1rCDDP2500/r5-FU500, and UKF-PEC-3 (an alternative penis cancer cell line) were tested for sensitivity to paclitaxel, docetaxel, olaparib, and rucaparib. RESULTS AND CONCLUSIONS: The chemo-resistant sublines differed in their mutational landscapes. UKF-PEC-1rCDDP2500 was characterized by an increased HRD score, which is supposed to be associated with increased PARP inhibitor and immune checkpoint inhibitor sensitivity in cancer. However, UKF-PEC-1rCDDP2500 did not display sensitivity to PARP inhibitors.

Molecular matched targeted therapies for primary brain tumors-a single center retrospective analysis.

PURPOSE: Molecular diagnostics including next generation gene sequencing are increasingly used to determine options for individualized therapies in brain tumor patients. We aimed to evaluate the decision-making process of molecular targeted therapies and analyze data on tolerability as well as signals for efficacy. METHODS: Via retrospective analysis, we identified primary brain tumor patients who were treated off-label with a targeted therapy at the University Hospital Frankfurt, Goethe University. We analyzed which types of molecular alterations were utilized to guide molecular off-label therapies and the diagnostic procedures for their assessment during the period from 2008 to 2021. Data on tolerability and outcomes were collected. RESULTS: 413 off-label therapies were identified with an increasing annual number for the interval after 2016. 37 interventions (9%) were targeted therapies based on molecular markers. Glioma and meningioma were the most frequent entities treated with molecular matched targeted therapies. Rare entities comprised e.g. medulloblastoma and papillary craniopharyngeoma. Molecular targeted approaches included checkpoint inhibitors, inhibitors of mTOR, FGFR, ALK, MET, ROS1, PIK3CA, CDK4/6, BRAF/MEK and PARP. Responses in the first follow-up MRI were partial response (13.5%), stable disease (29.7%) and progressive disease (46.0%). There were no new safety signals. Adverse events with fatal outcome (CTCAE grade 5) were not observed. Only, two patients discontinued treatment due to side effects. Median progression-free and overall survival were 9.1/18 months in patients with at least stable disease, and 1.8/3.6 months in those with progressive disease at the first follow-up MRI. CONCLUSION: A broad range of actionable alterations was targeted with available molecular therapeutics. However, efficacy was largely observed in entities with paradigmatic oncogenic drivers, in particular with BRAF mutations. Further research on biomarker-informed molecular matched therapies is urgently necessary.

The routine use of LCD-Array hybridisation technique for HPV subtyping in the diagnosis of penile carcinoma compared to other methods.

BACKGROUND: Routine human papillomavirus (HPV) testing is performed in cervival cancer and is required for classification of some head and neck cancers. In penile cancer a statement on HPV association of the carcinoma is required. In most cases p16 immunohistochemistry as a surrogate marker is applied in this setting. Since differing clinical outcomes for HPV positive and HPV negative tumors are described we await HPV testing to be requested more frequently by clinicians, also in the context of HPV vaccination, where other HPV subtypes are expected to emerge. METHOD: Therefore, a cohort of archived, formalin-fixed paraffin embedded (FFPE) penile neoplasias was stained for p16 and thereafter tested for HPV infection status via PCR based methods. Additionally to Sanger sequencing, we chose LCD-Array technique (HPV 3.5 LCD-Array Kit, Chipron; LCD-Array) for the detection of HPV in our probes expecting a less time consuming and sensitive HPV test for our probes. RESULTS: We found that LCD-Array is a sensitive and feasible method for HPV testing in routine diagnostics applicable to FFPE material in our cohort. Our cohort of penile carcinomas and carcinomas in situ was associated with HPV infection in 61% of cases. We detected no significant association between HPV infection status and histomorphological tumor characteristics as well as overall survival. CONCLUSIONS: We showed usability of molecular HPV testing on a cohort of archived penile carcinomas. To the best of our knowledge, this is the first study investigating LCD-Array technique on a cohort of penile neoplasias.

Complete Pathological Response After Neoadjuvant Short-Course Immunotherapy with Ipilimumab and Nivolumab in Locally Advanced MSI-H/dMMR Rectal Cancer.

BACKGROUND: Patients with colorectal carcinoma and high-grade microsatellite instability (MSI-H) or deficiency in mismatch repair (dMMR) exceptionally respond to immune checkpoint inhibitors (ICIs). ICIs are more active in treatment-naïve patients than in patients with refractory MSI-H/dMMR metastatic colorectal cancer and even more active in patients with locally advanced tumors. MATERIAL AND METHODS: A 33-year-old male patient with Lynch syndrome was diagnosed with a locally advanced rectal cancer and refused standard neoadjuvant chemoradiation because of the potential harm of sexual dysfunction. MMR and microsatellite instability status were analyzed by immunohistochemistry and fragment length polymerase chain reaction followed by capillary electrophoresis. RESULTS: After MSI-H/dMMR was confirmed, the patient was treated with ICIs (1 mg/kg ipilimumab at day 1 and 3 mg/kg nivolumab at day 1 and 15). A complete clinical response was documented at day 21 after start of treatment. The patient underwent a total mesorectal excision at day 30. In the extirpated tissue, a complete pathological response was confirmed. CONCLUSION: In MSI-H/dMMR locally advanced rectal cancer short-course ICI treatment is highly effective and may be discussed in patients with dMMR locally advanced rectal cancer. KEY POINTS: Immune checkpoint inhibitors are more active in treatment-naïve patients than in patients with refractory high-grade microsatellite instability (MSI-H)/deficiency in mismatch repair (dMMR) colorectal cancer. Standard neoadjuvant chemoradiation is less effective in MSI-H/dMMR rectal cancer patients than in patients with proficient mismatch repair. A young patient with Lynch syndrome and MSI-H/dMMR locally advanced rectal cancer refused chemoradiation in order to preserve his fertility. After neoadjuvant treatment with one dose of ipilimumab and two doses of nivolumab a complete clinical and pathological response was documented. Clinical trials are needed to first establish neoadjuvant treatment with immune checkpoint inhibitors in patients with locally advanced MSI-H/dMMR rectal cancer and thereafter to evaluate organ-preservation strategies.

[Metastatic castration-resistant prostate cancer]. / Das metastasierte kastrationsresistente Prostatakarzinom.

Metastatic castration-resistant prostate cancer (mCRPC) is considered as the final stage of the disease with limited therapeutic options to date. In this article, we review recent histopathologic and molecular pathologic findings that may expand our understanding of the disease and may lay, or have already laid the groundwork for the development of novel and individualized therapies. These include the detection of pathogenic mutations in the DNA repair genes BRCA1/2, androgen receptor splice variant 7 (AR-V7), deletion of the tumor suppressor gene PTEN, and evidence of neuroendocrine prostate cancer (t-NEPC) arising under antiandrogenic therapy. The theoretical and diagnostic basis behind the increasing relevance of pathology for therapeutic guidance in this stage of disease are presented.

Development, testing and validation of a targeted NGS-panel for the detection of actionable mutations in lung cancer (NSCLC) using anchored multiplex PCR technology in a multicentric setting.

Lung cancer is a paradigm for a genetically driven tumor. A variety of drugs were developed targeting specific biomarkers requiring testing for tumor genetic alterations in relevant biomarkers. Different next-generation sequencing technologies are available for library generation: 1) anchored multiplex-, 2) amplicon based- and 3) hybrid capture-based-PCR. Anchored multiplex PCR-based sequencing was investigated for routine molecular testing within the national Network Genomic Medicine Lung Cancer (nNGM). Four centers applied the anchored multiplex ArcherDX-Variantplex nNGMv2 panel to re-analyze samples pre-tested during routine diagnostics. Data analyses were performed by each center and compiled centrally according to study design. Pre-defined standards were utilized, and panel sensitivity was determined by dilution experiments. nNGMv2 panel sequencing was successful in 98.9% of the samples (N = 90). With default filter settings, all but two potential MET exon 14 skipping variants were identified at similar allele frequencies. Both MET variants were found with an adapted calling filter. Three additional variants (KEAP1, STK11, TP53) were called that were not identified in pre-testing analyses. Only total DNA amount but not a qPCR-based DNA quality score correlated with average coverage. Analysis was successful with a DNA input as low as 6.25 ng. Anchored multiplex PCR-based sequencing (nNGMv2) and a sophisticated user-friendly Archer-Analysis pipeline is a robust and specific technology to detect tumor genetic mutations for precision medicine of lung cancer patients.

Indirect comparison of capmatinib treatment from GEOMETRY mono-1 trial to SOC in German patients with locally advanced or metastatic NSCLC harboring METex14 skipping mutations.

BACKGROUND: This study provides comparative evidence of the selective MET inhibitor capmatinib versus standard of care (SOC) in first-line (1 L) and second-line (2 L) non-small cell lung cancer (NSCLC) patients with METex14 mutations in German routine care. METHODS: SOC data were collected from German routine care via retrospective chart review. Analyses were conducted as naive and propensity score adjusted (PSA) comparisons to capmatinib-treated patients within the GEOMETRY mono-1 trial. Effectiveness endpoints included overall survival (OS), progression-free survival (PFS), overall response rate (ORR), time to CNS progression (CNSprog), and exploratory safety endpoints. RESULTS: The SOC arm included 119 patients in 1 L and 46 in 2 L versus 60 patients in 1 L and 81 in 2 L treated with capmatinib, with balanced baseline characteristics after PSA. In 1 L, the naive comparison showed a significant benefit of capmatinib versus SOC for OS (median: 25.49 vs 14.59 months; HR 0.58; 95 % CI 0.39-0.87; P = 0.011), PFS (median: 12.45 vs 5.03 months; HR: 0.44; 95 % CI: 0.31-0.63; P < 0.001), and ORR (event rate: 68.3 vs 26.9 %; RR 2.54; 95 % CI 1.80-3.58; P < 0.001). In 2 L, OS, PFS, and ORR showed positive trends favoring capmatinib over SOC. Capmatinib treatment in the 1 L and 2 L led to significant benefit in CNSprog. PSA analyses showed consistent results to naive analysis. Exploratory safety endpoints indicated a manageable safety profile for capmatinib. CONCLUSIONS: The present study demonstrates the important role of capmatinib in providing robust clinically meaningful benefit to patients with NSCLC harboring METex14 mutations and its significant role in preventing the development of brain metastases.

Feasibility of Next-generation Sequencing of Liquid Biopsy (Circulating Tumor DNA) Samples and Tumor Tissue from Patients with Metastatic Prostate Cancer in a Real-world Clinical Setting in Germany.

BACKGROUND AND OBJECTIVE: With European Medicines Agency approval of PARP inhibitors in metastatic castration-resistant prostate cancer and ongoing trials in metastatic hormone-sensitive prostate cancer, detection of genetic alterations in BRCA1/2 and other homologous recombination repair genes has gained an important role. Our aim was to investigate the feasibility and comparability of comprehensive next-generation sequencing (NGS) of liquid biopsy (LB; circulating tumor DNA) and tumor tissue (TT) samples in a real-world clinical setting. METHODS: The study cohort consisted of 50 patients with metastatic prostate cancer (mPC) who had TT NGS performed for BRCA1/2 alterations and consent for additional LB NGS. The Oncomine Comprehensive Assay v3 (Thermo Fisher Scientific, Waltham, MA, USA) was used for TT NGS. The Guardant360 83-gene assay (Guardant Health, Palo Alto, CA, USA) was used for LB NGS, including all types of somatic alterations, microsatellite instability, and blood tumor mutational burden. We calculated BRCA1/2 alteration rates and the negative percentage agreement (NPA) and positive percentage agreement (PPA) between TT and LB results. KEY FINDINGS AND LIMITATIONS: TT NGS was successful in 44/50 patients (88%), with pathogenic BRCA1/2 alterations detected in four (9%). LB NGS was successful in all 50 patients (100%), with BRCA1/2 alterations detected in ten (20%). In a subgroup analysis for the 44 patients with successful TT NGS, NPA was 85% and PPA was 50%. The median time between TT sample collection and blood sampling for NGS was 132 wk (IQR 94-186). The limited sample size and differences in the time of NGS assessment are limitations. CONCLUSIONS AND CLINICAL IMPLICATIONS: LB NGS resulted in a higher detection rate for BRCA1/2 alterations in comparison to conventional TT NGS (20% vs 9%). Ideally, BRCA1/2 testing should be based on both approaches to identify all patients with mPC eligible for PARP inhibitor therapy. PATIENT SUMMARY: Our study shows that genetic tests for both tumor tissue and blood samples results in higher rates of detection of BRCA1/2 gene alterations in patients with metastatic prostate cancer.

High Concordance of Different Assays in the Determination of Homologous Recombination Deficiency-Associated Genomic Instability in Ovarian Cancer.

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

[Ovarian cancer: molecular pathology and molecularly targeted therapy]. / Ovarialkarzinom: Molekularpathologie und molekular gesteuerte Therapie.

Ovarian cancer is a common malignant genital neoplasm in women. Due to its frequent diagnosis only in advanced stages, prognosis is poor, although biomarker-driven targeted therapeutic approaches are evolving. These are currently based on molecular pathological analyses of homologous recombination deficiency (HRD) and the BRCA1/2 mutational status. The current CME article focuses on the histo- and molecular pathology of ovarian cancer, relevant molecular mechanisms, and test systems. These are discussed in the context of biomarker-driven targeted therapy.

A Comparison of Two Different FFPE Tissue Dissection Methods for Routine Diagnostics in Molecular Pathology: Manual Macrodissection versus Automated Microdissection Using the Roche "AVENIO Millisect" System.

Currently, in routine diagnostics, most molecular testing is performed on formalin-fixed, paraffin-embedded tissue after a histomorphological assessment. In order to find the best possible and targeted individual therapy, knowing the mutational status of the tumour is crucial. The "AVENIO Millisect" system Roche introduced an automation solution for the dissection of tissue on slides. This technology allows the precise and fully automated dissection of the tumour area without wasting limited and valuable patient material. In this study, the digitally guided microdissection was directly compared to the manual macrodissection regarding the precision and duration of the procedure, their DNA concentrations as well as DNA qualities, and the overall costs in 24 FFPE samples. In 21 of 24 cases (87.5%), the DNA yields of the manually dissected samples were higher in comparison to the automatically dissected samples. Shorter execution times and lower costs were also benefits of the manual scraping process. Nevertheless, the DNA quality achieved with both methods was comparable, which is essential for further molecular testing. Therefore, it could be used as an additional tool for precise tumour enrichment.

Impact of IDH1 mutation on clinical course of patients with intrahepatic cholangiocarcinoma: a retrospective analysis from a German tertiary center.

PURPOSE: IDH1 mutation is a known biomarker for targeted therapy of intrahepatic cholangiocarcinoma (iCCA), while its prognostic relevance for current palliative chemotherapy is still unclear. Aim of this study was to analyze clinicopathological characteristics of patients with IDH1 mutations and to outline a potential impact on the outcome after state-of-the-art palliative chemotherapy regimens. METHODS: All patients with iCCA receiving large panel molecular profiling and follow-up treatment at Frankfurt University Hospital until 04/2022 were retrospectively analyzed. Clinicopathological characteristics were assessed for IDH1 mutated (mut) and IDH1 wild type (wt) patients, and progression-free survival (PFS) and overall survival (OS) were determined. RESULTS: In total, 75 patients with iCCA received molecular profiling. Of the patients with available DNA data, pathogenic mutations in IDH1 were found in 14.5% (n = 10). IDH1 mut status was associated with lower serum CA-19/9 (p = 0.023), lower serum lactate dehydrogenase (p = 0.006), and a higher proportion of primary resectability (p = 0.028) as well as response to chemotherapy after recurrence (p = 0.009). Median PFS was 5.9 months (95% CI 4.4-7.3 months) for IDH1 wt in comparison to 9.8 months (95% CI 7.7-12 months) for patients with IDH1 mut (p = 0.031). IDH1 wt was a significant risk factor for shortened PFS in univariate (p = 0.043), but not in multivariate analysis (p = 0.061). There was no difference in OS between both groups. CONCLUSION: Patients with IDH1 mutated iCCA seem to have a favorable tumor biology including a longer PFS for palliative chemotherapy regimens compared to IDH1 wild type.

Machine-Learning-Aided Prediction of Brain Metastases Development in Non-Small-Cell Lung Cancers.

PURPOSE: Non-small-cell lung cancer (NSCLC) shows a high incidence of brain metastases (BM). Early detection is crucial to improve clinical prospects. We trained and validated classifier models to identify patients with a high risk of developing BM, as they could potentially benefit from surveillance brain MRI. METHODS: Consecutive patients with an initial diagnosis of NSCLC from January 2011 to April 2019 and an in-house chest-CT scan (staging) were retrospectively recruited at a German lung cancer center. Brain imaging was performed at initial diagnosis and in case of neurological symptoms (follow-up). Subjects lost to follow-up or still alive without BM at the data cut-off point (12/2020) were excluded. Covariates included clinical and/or 3D-radiomics-features of the primary tumor from staging chest-CT. Four machine learning models for prediction (80/20 training) were compared. Gini Importance and SHAP were used as measures of importance; sensitivity, specificity, area under the precision-recall curve, and Matthew's Correlation Coefficient as evaluation metrics. RESULTS: Three hundred and ninety-five patients compromised the clinical cohort. Predictive models based on clinical features offered the best performance (tuned to maximize recall: sensitivity∼70%, specificity∼60%). Radiomics features failed to provide sufficient information, likely due to the heterogeneity of imaging data. Adenocarcinoma histology, lymph node invasion, and histological tumor grade were positively correlated with the prediction of BM, age, and squamous cell carcinoma histology were negatively correlated. A subgroup discovery analysis identified 2 candidate patient subpopulations appearing to present a higher risk of BM (female patients + adenocarcinoma histology, adenocarcinoma patients + no other distant metastases). CONCLUSION: Analysis of the importance of input features suggests that the models are learning the relevant relationships between clinical features/development of BM. A higher number of samples is to be prioritized to improve performance. Employed prospectively at initial diagnosis, such models can help select high-risk subgroups for surveillance brain MRI.

NTRK fusion events and targeted treatment of advanced radioiodine refractory thyroid cancer.

PURPOSE: Pathogenic fusion events involving neurotrophic receptor tyrosine kinase (NTRK) have been described in ~ 2% of differentiated thyroid cancer (DTC). The selective tropomyosin receptor kinase (TRK) inhibitors entrectinib and larotrectinib have been approved in a tumor agnostic manner based on phase 1/2 clinical trials. In a real-world setting at five referral centers, we aimed to describe the prevalence of NTRK gene fusions and the efficacy and safety of TRK inhibitor treatment for non-medullary, advanced thyroid cancer (TC). METHODS: A total of 184 TC patients with testing for NTRK gene fusions were included. Progression-free survival (PFS) and overall survival (OS) probabilities were estimated using the Kaplan-Meier method in six patients with NTRK fusion-positive TC who underwent TRK inhibitor therapy. RESULTS: 8/184 (4%) patients harbored NTRK gene fusions. Six patients with radioiodine (RAI)-refractory TC harboring NTRK1 (n = 4) and NTRK3 (n = 2) gene fusions were treated with larotrectinib. Five patients (83%) had received ≥ 1 prior systemic therapy and one patient did not receive prior systemic therapy. All patients had morphologically progressive disease before treatment initiation. Objective response rate was 83%, including two complete remissions. Median PFS from start of TRK inhibitor treatment was 23 months (95% confidence interval [CI], 0-57.4) and median OS was not reached (NR) (95% CI, NR). Adverse events were of grade 1-3. CONCLUSION: The prevalence of NTRK gene fusions in our cohort of RAI-refractory TC is slightly higher than reported for all TC patients. Larotrectinib is an effective treatment option in the majority of NTRK gene fusion-positive advanced TC patients after prior systemic treatment and has a favorable safety profile.

Sequencing of BRCA1/2-alterations using NGS-based technology: annotation as a challenge.

In this study, the molecular profile of different BRCA-associated tumor types was assessed with regard to the classification and annotation of detected BRCA1/2 variants. The aim was to establish guidelines in order to facilitate the interpretation of BRCA1/2 alterations in routine diagnostics. Annotation of detected variants was evaluated compared to background mutations found in normal tissue samples and manually reviewed according to distinct online databases. This retrospective study included 48 samples (45 tumors, three non-tumors), which were sequenced with the GeneReader (QIAGEN). Thereof ten samples were additionally analyzed with the Ion S5™ (Thermo Fisher) and 20 samples with the MiSeq™ (Illumina®) to compare the different NGS devices, as well as the sequencing results and their quality. The analysis showed that the individual NGS platforms detected different numbers of BRCA1/2 alterations in the respective tumor sample. In addition, the GeneReader revealed variability in the detection and classification of pathogenic alterations within the platform itself as well as in comparison with the other platforms or online databases. The study concluded that the Ion S5™ in combination with the Oncomine™ Comprehensive Assay v3 is most recommendable for current and prospective requirements of molecular analysis in routine diagnostics. In addition to the two BRCA1/2 genes, a broad number of other genes (BRCAness genes and genes involved in the repair pathway) is covered by the panel, which may open up new treatment options for patients depending on the respective eligibility criteria.

Site of analysis matters - Ongoing complete response to Nivolumab in a patient with HIV/HPV related metastatic anal cancer and MLH1 mutation.

Anal cancer is a rare disease with increasing incidence. In patients with locally recurrent or metastatic disease which cannot be treated with chemoradiotherapy or salvage surgery systemic first-line chemotherapy with carboplatin and paclitaxel is standard of care. For patients who progress after first-line therapy and are still eligible for second-line therapy Programmed cell death protein 1 (PD-1) antibodies are potential therapeutic options. However, prediction of response to immunotherapy is still challenging including anal cancer. We report here to our knowledge the first anal cancer case with microsatellite instability (MSI) due to MLH1 mutation and a deep and ongoing response to Nivolumab treatment. Namely, thorough analysis of the primary tumor as well as metastatic sites by next generation sequencing (NGS) revealed that MSI was formally only found in the metastatic sites but not in the primary tumor. Concomitantly, tumor mutational burden (TMB) was higher in the metastatic site than in the primary tumor. Therefore, we conclude that all anal cancer patients should be tested for MSI and whenever possible molecular analysis should be performed rather from metastatic sites than from the primary tumor.