Whole Genome Analysis of Ovarian Granulosa Cell Tumors Reveals Tumor Heterogeneity and a High-Grade TP53-Specific Subgroup

Adult granulosa cell tumors (AGCTs) harbor a somatic FOXL2 c.402C>G mutation in ~95% of cases and are mainly surgically removed due to limited systemic treatment effect. In this study, potentially targetable genomic alterations in AGCTs were investigated by whole genome sequencing on 46 tumor samples and matched normal DNA. Copy number variant (CNV) analysis confirmed gain of chromosome 12 and 14, and loss of 22. Pathogenic TP53 mutations were identified in three patients with highest tumor mutational burden and mitotic activity, defining a high-grade AGCT subgroup. Within-patient tumor comparisons showed 29–80% unique somatic mutations per sample, suggesting tumor heterogeneity. A higher mutational burden was found in recurrent tumors, as compared to primary AGCTs. FOXL2-wildtype AGCTs harbored DICER1, TERT(C228T) and TP53 mutations and similar CNV profiles as FOXL2-mutant tumors. Our study confirms that absence of the FOXL2 c.402C>G mutation does not exclude AGCT diagnosis. The lack of overlapping variants in targetable cancer genes indicates the need for personalized treatment for AGCT patients

In Vitro Systematic Drug Testing Reveals Carboplatin, Paclitaxel, and Alpelisib as a Potential Novel Combination Treatment for Adult Granulosa Cell Tumors

Simple Summary: Granulosa cell tumor treatment is challenging as there are few effective options besides surgery. In this study, we obtained tumor tissue from patients at surgery and cultured tumor cells in the laboratory. After sufficient expansion, we tested the effects of current treatments, such as chemotherapy and anti-hormonal treatment, and novel anti-cancer treatment options on cell survival. Results were generated within three weeks after tissue collection. We found that all drugs were ineffective when used as single treatments; however, some combinations were very effective. The PI3K protein inhibitor alpelisib was effective in combination with chemotherapy and achieved 50% cell death at assumed tolerable patient plasma concentrations. In conclusion, this study shows an approach to rapidly establish patient-derived cell lines for drug screens. The effectiveness of combined treatment with alpelisib and chemotherapy in granulosa cell tumors should be further investigated and may be a promising novel treatment option in patients with a granulosa cell tumor. Adult granulosa cell tumors (AGCTs) arise from the estrogen-producing granulosa cells. Treatment of recurrence remains a clinical challenge, as systemic anti-hormonal treatment or chemotherapy is only effective in selected patients. We established a method to rapidly screen for drug responses in vitro using direct patient-derived cell lines in order to optimize treatment selection. The response to 11 monotherapies and 12 combination therapies, including chemotherapeutic, anti-hormonal, and targeted agents, were tested in 12 AGCT-patient-derived cell lines and an AGCT cell line (KGN). Drug screens were performed within 3 weeks after tissue collection by measurement of cell viability 72 h after drug application. The potential synergy of drug combinations was assessed. The human maximum drug plasma concentration (Cmax) and steady state (Css) thresholds obtained from available phase I/II clinical trials were used to predict potential toxicity in patients. Patient-derived AGCT cell lines demonstrated resistance to all monotherapies. All cell lines showed synergistic growth inhibition by combination treatment with carboplatin, paclitaxel, and alpelisib at a concentration needed to obtain 50% cell death (IC50) that are below the maximum achievable concentration in patients (IC50 <Cmax). We show that AGCT cell lines can be rapidly established and used for patient-specific in vitro drug testing, which may guide treatment decisions. Combination treatment with carboplatin, paclitaxel, and alpelisib was consistently effective in AGCT cell lines and should be further studied as a potential effective combination for AGCT treatment in patients

Formalin-Fixed, Paraffin-Embedded–Targeted Locus Capture:A Next-Generation Sequencing Technology for Accurate DNA-Based Gene Fusion Detection in Bone and Soft Tissue Tumors

Chromosomal rearrangements are important drivers in cancer, and their robust detection is essential for diagnosis, prognosis, and treatment selection, particularly for bone and soft tissue tumors. Current diagnostic methods are hindered by limitations, including difficulties with multiplexing targets and poor quality of RNA. A novel targeted DNA-based next-generation sequencing method, formalin-fixed, paraffin-embedded–targeted locus capture (FFPE-TLC), has shown advantages over current diagnostic methods when applied on FFPE lymphomas, including the ability to detect novel rearrangements. We evaluated the utility of FFPE-TLC in bone and soft tissue tumor diagnostics. FFPE-TLC sequencing was successfully applied on noncalcified and decalcified FFPE samples (n = 44) and control samples (n = 19). In total, 58 rearrangements were identified in 40 FFPE tumor samples, including three previously negative samples, and none was identified in the FFPE control samples. In all five discordant cases, FFPE-TLC could identify gene fusions where other methods had failed due to either detection limits or poor sample quality. FFPE-TLC achieved a high specificity and sensitivity (no false positives and negatives). These results indicate that FFPE-TLC is applicable in cancer diagnostics to simultaneously analyze many genes for their involvement in gene fusions. Similar to the observation in lymphomas, FFPE-TLC is a good DNA-based alternative to the conventional methods for detection of rearrangements in bone and soft tissue tumors.</p

Prognostic Significance of POLE Proofreading Mutations in Endometrial Cancer

Background: Current risk stratification in endometrial cancer (EC) results in frequent over- and underuse of adjuvant therapy, and may be improved by novel biomarkers. We examined whether POLE proofreading mutations, recently reported in about 7% of ECs, predict prognosis. Methods: We performed targeted POLE sequencing in ECs from the PORTEC-1 and -2 trials (n = 788), and analyzed clinical outcome according to POLE status. We combined these results with those from three additional series (n = 628) by meta-analysis to generate multivariable-adjusted, pooled hazard ratios (HRs) for recurrence-free survival (RFS) and cancer-specific survival (CSS) of POLE-mutant ECs. All statistical tests were two-sided. Results: POLE mutations were detected in 48 of 788 (6.1%) ECs from PORTEC-1 and-2 and were associated with high tumor grade (P < .001). Women with POLE-mutant ECs had fewer recurrences (6.2% vs 14.1%) and EC deaths (2.3% vs 9.7%), though, in the total PORTEC cohort, differences in RFS and CSS were not statistically significant (multivariable-adjusted HR = 0.43, 95% CI = 0.13 to 1.37, P = .15; HR = 0.19, 95% CI = 0.03 to 1.44, P = .11 respectively). However, of 109 grade 3 tumors, 0 of 15 POLE-mutant ECs recurred, compared with 29 of 94 (30.9%) POLE wild-type cancers; reflected in statistically significantly greater RFS (multivariable-adjusted HR = 0.11, 95% CI = 0.001 to 0.84, P = .03). In the additional series, there were no EC-related events in any of 33 POLE-mutant ECs, resulting in a multivariable-adjusted, pooled HR of 0.33 for RFS (95% CI = 0.12 to 0.91, P = .03) and 0.26 for CSS (95% CI = 0.06 to 1.08, P = .06). Conclusion: POLE proofreading mutations predict favorable EC prognosis, independently of other clinicopathological variables, with the greatest effect seen in high-grade tumors. This novel biomarker may help to reduce overtreatment in E

Prognostic Integrated Image-Based Immune and Molecular Profiling in Early-Stage Endometrial Cancer

Optimum risk stratification in early-stage endometrial cancer (EC) combines clinicopathological factors and the molecular EC classification defined by The Cancer Genome Atlas (TCGA). It is unclear whether analysis of intratumoral immune infiltrate improves this. We developed a machine-learning image-based algorithm to quantify density of CD8+ and CD103+ immune cells in tumor epithelium and stroma in 695 stage I endometrioid ECs from the PORTEC-1&amp;-2 trials. The relationship between immune cell density and clinicopathological/molecular factors was analyzed by hierarchical clustering and multiple regression. The prognostic value of immune infiltrate by cell type and location was analyzed by univariable and multivariable Cox regression, incorporating the molecular EC classification. Tumor-infiltrating immune cell density varied substantially between cases, and more modestly by immune cell type and location. Clustering revealed three groups with high, intermediate and low densities, with highly significant variation in the proportion of molecular EC subgroups between them. Univariable analysis revealed intraepithelial CD8+ cell density as the strongest predictor of EC recurrence; multivariable analysis confirmed this was independent of pathological factors and molecular subgroup. Exploratory analysis suggested this association was not uniform across molecular subgroups, but greatest in tumors with mutant p53 and absent in DNA mismatch repair deficient cancers. Thus, this work identified that quantification of intraepithelial CD8+ cells improved upon the prognostic utility of the molecular EC classification in early-stage EC

Comparison of NTRK fusion detection methods in microsatellite-instability-high metastatic colorectal cancer

Tropomyosin receptor kinase (TRK) inhibitors have been approved for metastatic solid tumors harboring NTRK fusions, but the detection of NTRK fusions is challenging. International guidelines recommend pan-TRK immunohistochemistry (IHC) screening followed by next generation sequencing (NGS) in tumor types with low prevalence of NTRK fusions, including metastatic colorectal cancer (mCRC). RNA-based NGS is preferred, but is expensive, time-consuming, and extracting good-quality RNA from FFPE tissue is challenging. Alternatives in daily clinical practice are warranted. We assessed the diagnostic performance of RNA-NGS, FFPE-targeted locus capture (FFPE-TLC), fluorescence in situ hybridization (FISH), and the 5'/3' imbalance quantitative RT-PCR (qRT-PCR) after IHC screening in 268 patients with microsatellite-instability-high mCRC, the subgroup in which NTRK fusions are most prevalent (1-5%). A consensus result was determined after review of all assay results. In 16 IHC positive tumors, 10 NTRK fusions were detected. In 33 IHC negative samples, no additional transcribed NTRK fusions were found, underscoring the high sensitivity of IHC. Sensitivity of RNA-NGS, FFPE-TLC, FISH, and qRT-PCR was 90%, 90%, 78%, and 100%, respectively. Specificity was 100% for all assays. Robustness, defined as the percentage of samples that provided an interpretable result in the first run, was 100% for FFPE-TLC, yet more limited for RNA-NGS (85%), FISH (70%), and qRT-PCR (70%). Overall, we do not recommend FISH for the detection of NTRK fusions in mCRC due to its low sensitivity and limited robustness. We conclude that RNA-NGS, FFPE-TLC, and qRT-PCR are appropriate assays for NTRK fusion detection, after enrichment with pan-TRK IHC, in routine clinical practice

Reconstructing single-cell karyotype alterations in colorectal cancer identifies punctuated and gradual diversification patterns

Central to tumor evolution is the generation of genetic diversity. However, the extent and patterns by which de novo karyotype alterations emerge and propagate within human tumors are not well understood, especially at single-cell resolution. Here, we present 3D Live-Seq—a protocol that integrates live-cell imaging of tumor organoid outgrowth and whole-genome sequencing of each imaged cell to reconstruct evolving tumor cell karyotypes across consecutive cell generations. Using patient-derived colorectal cancer organoids and fresh tumor biopsies, we demonstrate that karyotype alterations of varying complexity are prevalent and can arise within a few cell generations. Sub-chromosomal acentric fragments were prone to replication and collective missegregation across consecutive cell divisions. In contrast, gross genome-wide karyotype alterations were generated in a single erroneous cell division, providing support that aneuploid tumor genomes can evolve via punctuated evolution. Mapping the temporal dynamics and patterns of karyotype diversification in cancer enables reconstructions of evolutionary paths to malignant fitness

Targeted locus amplification to develop robust patient-specific assays for liquid biopsies in pediatric solid tumors

Background: Liquid biopsies combine minimally invasive sample collection with sensitive detection of residual disease. Pediatric malignancies harbor tumor-driving copy number alterations or fusion genes, rather than recurrent point mutations. These regions contain tumor-specific DNA breakpoint sequences. We investigated the feasibility to use these breakpoints to design patient-specific markers to detect tumor-derived cell-free DNA (cfDNA) in plasma from patients with pediatric solid tumors. Materials and methods: Regions of interest (ROI) were identified through standard clinical diagnostic pipelines, using SNP array for CNAs, and FISH or RT-qPCR for fusion genes. Using targeted locus amplification (TLA) on tumor organoids grown from tumor material or targeted locus capture (TLC) on FFPE material, ROI-specific primers and probes were designed, which were used to design droplet digital PCR (ddPCR) assays. cfDNA from patient plasma at diagnosis and during therapy was analyzed. Results: TLA was performed on material from 2 rhabdomyosarcoma, 1 Ewing sarcoma and 3 neuroblastoma. FFPE-TLC was performed on 8 neuroblastoma tumors. For all patients, at least one patient-specific ddPCR was successfully designed and in all diagnostic plasma samples the patient-specific markers were detected. In the rhabdomyosarcoma and Ewing sarcoma patients, all samples after start of therapy were negative. In neuroblastoma patients, presence of patient-specific markers in cfDNA tracked tumor burden, decreasing during induction therapy, disappearing at complete remission and re-appearing at relapse. Conclusion: We demonstrate the feasibility to determine tumor-specific breakpoints using TLA/TLC in different pediatric solid tumors and use these for analysis of cfDNA from plasma. Considering the high prevalence of CNAs and fusion genes in pediatric solid tumors, this approach holds great promise and deserves further study in a larger cohort with standardized plasma sampling protocols

Patient-Derived Ovarian Cancer Organoids Mimic Clinical Response and Exhibit Heterogeneous Inter- and Intrapatient Drug Responses

There remains an unmet need for preclinical models to enable personalized therapy for ovarian cancer (OC) patients. Here we evaluate the capacity of patient-derived organoids (PDOs) to predict clinical drug response and functional consequences of tumor heterogeneity. We included 36 whole-genome-characterized PDOs from 23 OC patients with known clinical histories. OC PDOs maintain the genomic features of the original tumor lesion and recapitulate patient response to neoadjuvant carboplatin/paclitaxel combination treatment. PDOs display inter- and intrapatient drug response heterogeneity to chemotherapy and targeted drugs, which can be partially explained by genetic aberrations. PDO drug screening identifies high responsiveness to at least one drug for 88% of patients. PDOs are valuable preclinical models that can provide insights into drug response for individual patients with OC, complementary to genetic testing. Generating PDOs of multiple tumor locations can improve clinical decision making and increase our knowledge of genetic and drug response heterogeneity. De Witte et al. employ patient-derived organoids (PDOs) for ex vivo drug screening. Ovarian cancer (OC) PDOs often recapitulate patient drug response to first-line chemotherapy. In addition, OC PDOs display inter- and intrapatient drug response heterogeneity to chemotherapy and targeted drugs, which can be partly explained by genetic aberrations

Targeted locus amplification to develop robust patient-specific assays for liquid biopsies in pediatric solid tumors

BACKGROUND: Liquid biopsies combine minimally invasive sample collection with sensitive detection of residual disease. Pediatric malignancies harbor tumor-driving copy number alterations or fusion genes, rather than recurrent point mutations. These regions contain tumor-specific DNA breakpoint sequences. We investigated the feasibility to use these breakpoints to design patient-specific markers to detect tumor-derived cell-free DNA (cfDNA) in plasma from patients with pediatric solid tumors. MATERIALS AND METHODS: Regions of interest (ROI) were identified through standard clinical diagnostic pipelines, using SNP array for CNAs, and FISH or RT-qPCR for fusion genes. Using targeted locus amplification (TLA) on tumor organoids grown from tumor material or targeted locus capture (TLC) on FFPE material, ROI-specific primers and probes were designed, which were used to design droplet digital PCR (ddPCR) assays. cfDNA from patient plasma at diagnosis and during therapy was analyzed. RESULTS: TLA was performed on material from 2 rhabdomyosarcoma, 1 Ewing sarcoma and 3 neuroblastoma. FFPE-TLC was performed on 8 neuroblastoma tumors. For all patients, at least one patient-specific ddPCR was successfully designed and in all diagnostic plasma samples the patient-specific markers were detected. In the rhabdomyosarcoma and Ewing sarcoma patients, all samples after start of therapy were negative. In neuroblastoma patients, presence of patient-specific markers in cfDNA tracked tumor burden, decreasing during induction therapy, disappearing at complete remission and re-appearing at relapse. CONCLUSION: We demonstrate the feasibility to determine tumor-specific breakpoints using TLA/TLC in different pediatric solid tumors and use these for analysis of cfDNA from plasma. Considering the high prevalence of CNAs and fusion genes in pediatric solid tumors, this approach holds great promise and deserves further study in a larger cohort with standardized plasma sampling protocols