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.

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.

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.

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.

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.

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.

Alterations in BAP1 Are Associated with Cisplatin Resistance through Inhibition of Apoptosis in Malignant Pleural Mesothelioma.

PURPOSE: The clinical standard treatment for patients with malignant pleural mesothelioma (MPM) includes a cisplatin-based chemotherapy, leading to reduction of tumor size in only a minority of patients. Predicting response to chemotherapy in patients with MPM by using a genetic marker would, therefore, enable patient stratification. EXPERIMENTAL DESIGN: In this retrospective biomarker study, eligible patients had resectable MPM, measurable disease, and available primary MPM tissue. All patients underwent first-line treatment with cisplatin and pemetrexed, followed by surgery. Thorough molecular analysis was performed (whole-exome and targeted deep sequencing, and copy-number analyses), and also mechanistic in vitro data (viability assays, Western blots, and immunoprecipitation) using mesothelioma cell lines with and without siRNA-mediated BRCA1-associated protein 1 (BAP1) knockdown were provided. RESULTS: In a training cohort of patients with MPM (n = 28), mutations or deletions of BAP1 each predicted resistance to chemotherapy in patients with primary MPM. The negative predictive value of BAP1 loss in patients with MPM was confirmed by amplicon sequencing and copy-number array technology in an independent test cohort (n = 39). Preliminary mechanistic studies using siRNA-based knockdown of BAP1 in MPM cell culture models along with immunoprecipitation assays confirmed chemoresistance in vitro, possibly through inhibition of apoptosis and transcriptional regulation of the BAP1/HCF1/E2F1 axis. CONCLUSIONS: Alterations in BAP1 in MPM were a negative predictor for response to chemotherapy and could possibly be used as a companion biomarker for treatment decision.