Whole-Genome Sequencing Analysis of Male Breast Cancer Unveils Novel Structural Events and Potential Therapeutic Targets.

The molecular characterization of male breast cancer (MaBC) has received limited attention in research, mostly because of its low incidence rate, accounting for only 0.5% to 1% of all reported cases of breast cancer each year. Managing MaBC presents significant challenges, with most treatment protocols being adapted from those developed for female breast cancer. Utilizing whole-genome sequencing (WGS) and state-of-the-art analyses, the genomic features of 10 MaBC cases (n = 10) were delineated and correlated with clinical and histopathologic characteristics. Using fluorescence in situ hybridization, an additional cohort of 18 patients was interrogated to supplement WGS findings. The genomic landscape of MaBC uncovered significant genetic alterations that could influence diagnosis and treatment. We found common somatic mutations in key driver genes, such as FAT1, GATA3, SMARCA4, and ARID2. Our study also mapped out structural variants that impact cancer-associated genes, such as ARID1A, ESR1, GATA3, NTRK1, and NF1. Using a WGS-based classifier, homologous recombination deficiency (HRD) was identified in 2 cases, both presenting with deleterious variants in BRCA2. Noteworthy was the observation of FGFR1 amplification in 21% of cases. Altogether, we identified at least 1 potential therapeutic target in 8 of the 10 cases, including high tumor mutational burden, FGFR1 amplification, and HRD. Our study is the first WGS characterization of MaBC, which uncovered potentially relevant variants, including structural events in cancer genes, HRD signatures, and germline pathogenic mutations. Our results demonstrate unique genetic markers and potential treatment targets in MaBC, thereby underlining the necessity of tailoring treatment strategies for this understudied patient population. These WGS-based findings add to the growing knowledge of MaBC genomics and highlight the need to expand research on this type of cancer.

NIPBL::NACC1 Fusion Hepatic Carcinoma.

Several reports describing a rare primary liver tumor with histologic features reminiscent of follicular thyroid neoplasms have been published under a variety of descriptive terms including thyroid-like, solid tubulocystic, and cholangioblastic cholangiocarcinoma. Although these tumors are considered to represent histologic variants, they lack classic features of cholangiocarcinoma and have unique characteristics, namely immunoreactivity for inhibin and NIPBL::NACC1 fusions. The purpose of this study is to present clinicopathologic and molecular data for a large series of these tumors to better understand their pathogenesis. We identified 11 hepatic tumors with these features. Immunohistochemical and NACC1 and NIPBL fluorescence in situ hybridization assays were performed on all cases. Four cases had available material for whole-genome sequencing (WGS) analysis. Most patients were adult women (mean age: 42 y) who presented with abdominal pain and large hepatic masses (mean size: 14 cm). Ten patients had no known liver disease. Of the patients with follow-up information, 3/9 (33%) pursued aggressive behavior. All tumors were composed of bland cuboidal cells with follicular and solid/trabecular growth patterns in various combinations, were immunoreactive for inhibin, showed albumin mRNA by in situ hybridization, and harbored the NIPBL::NACC1 fusion by fluorescence in situ hybridization. WGS corroborated the presence of the fusion in all 4 tested cases, high tumor mutational burden in 2 cases, and over 30 structural variants per case in 3 sequenced tumors. The cases lacked mutations typical of conventional intrahepatic cholangiocarcinoma. In this report, we describe the largest series of primary inhibin-positive hepatic neoplasms harboring a NIPBL::NACC1 fusion and the first WGS analysis of these tumors. We propose to name this neoplasm NIPBL:NACC1 fusion hepatic carcinoma.

Subclonal Somatic Copy-Number Alterations Emerge and Dominate in Recurrent Osteosarcoma.

Multiple large-scale genomic profiling efforts have been undertaken in osteosarcoma to define the genomic drivers of tumorigenesis, therapeutic response, and disease recurrence. The spatial and temporal intratumor heterogeneity could also play a role in promoting tumor growth and treatment resistance. We conducted longitudinal whole-genome sequencing of 37 tumor samples from 8 patients with relapsed or refractory osteosarcoma. Each patient had at least one sample from a primary site and a metastatic or relapse site. Subclonal copy-number alterations were identified in all patients except one. In 5 patients, subclones from the primary tumor emerged and dominated at subsequent relapses. MYC gain/amplification was enriched in the treatment-resistant clones in 6 of 7 patients with multiple clones. Amplifications in other potential driver genes, such as CCNE1, RAD21, VEGFA, and IGF1R, were also observed in the resistant copy-number clones. A chromosomal duplication timing analysis revealed that complex genomic rearrangements typically occurred prior to diagnosis, supporting a macroevolutionary model of evolution, where a large number of genomic aberrations are acquired over a short period of time followed by clonal selection, as opposed to ongoing evolution. A mutational signature analysis of recurrent tumors revealed that homologous repair deficiency (HRD)-related SBS3 increases at each time point in patients with recurrent disease, suggesting that HRD continues to be an active mutagenic process after diagnosis. Overall, by examining the clonal relationships between temporally and spatially separated samples from patients with relapsed/refractory osteosarcoma, this study sheds light on the intratumor heterogeneity and potential drivers of treatment resistance in this disease. SIGNIFICANCE: The chemoresistant population in recurrent osteosarcoma is subclonal at diagnosis, emerges at the time of primary resection due to selective pressure from neoadjuvant chemotherapy, and is characterized by unique oncogenic amplifications.

Clonal evolution during metastatic spread in high-risk neuroblastoma.

Patients with high-risk neuroblastoma generally present with widely metastatic disease and often relapse despite intensive therapy. As most studies to date focused on diagnosis-relapse pairs, our understanding of the genetic and clonal dynamics of metastatic spread and disease progression remain limited. Here, using genomic profiling of 470 sequential and spatially separated samples from 283 patients, we characterize subtype-specific genetic evolutionary trajectories from diagnosis through progression and end-stage metastatic disease. Clonal tracing timed disease initiation to embryogenesis. Continuous acquisition of structural variants at disease-defining loci (MYCN, TERT, MDM2-CDK4) followed by convergent evolution of mutations targeting shared pathways emerged as the predominant feature of progression. At diagnosis metastatic clones were already established at distant sites where they could stay dormant, only to cause relapses years later and spread via metastasis-to-metastasis and polyclonal seeding after therapy.

Diagnostic utility of whole genome sequencing in adults with B-other acute lymphoblastic leukemia.

Genomic profiling during the diagnosis of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) in adults is used to guide disease classification, risk stratification, and treatment decisions. Patients for whom diagnostic screening fails to identify disease-defining or risk-stratifying lesions are classified as having B-other ALL. We screened a cohort of 652 BCP-ALL cases enrolled in UKALL14 to identify and perform whole genome sequencing (WGS) of paired tumor-normal samples. For 52 patients with B-other, we compared the WGS findings with data from clinical and research cytogenetics. WGS identified a cancer-associated event in 51 of 52 patients, including an established subtype defining genetic alterations that were previously missed with standard-of-care (SoC) genetics in 5 of them. Of the 47 true B-other ALL, we identified a recurrent driver in 87% (41). A complex karyotype via cytogenetics emerges as a heterogeneous group, including distinct genetic alterations associated with either favorable (DUX4-r) or poor outcomes (MEF2D-r and IGK::BCL2). For a subset of 31 cases, we integrated the findings from RNA sequencing (RNA-seq) analysis to include fusion gene detection and classification based on gene expression. Compared with RNA-seq, WGS was sufficient to detect and resolve recurrent genetic subtypes; however, RNA-seq can provide orthogonal validation of findings. In conclusion, we demonstrated that WGS can identify clinically relevant genetic abnormalities missed with SoC testing as well as identify leukemia driver events in virtually all cases of B-other ALL.

Unified classification and risk-stratification in Acute Myeloid Leukemia.

Clinical recommendations for Acute Myeloid Leukemia (AML) classification and risk-stratification remain heavily reliant on cytogenetic findings at diagnosis, which are present in <50% of patients. Using comprehensive molecular profiling data from 3,653 patients we characterize and validate 16 molecular classes describing 100% of AML patients. Each class represents diverse biological AML subgroups, and is associated with distinct clinical presentation, likelihood of response to induction chemotherapy, risk of relapse and death over time. Secondary AML-2, emerges as the second largest class (24%), associates with high-risk disease, poor prognosis irrespective of flow Minimal Residual Disease (MRD) negativity, and derives significant benefit from transplantation. Guided by class membership we derive a 3-tier risk-stratification score that re-stratifies 26% of patients as compared to standard of care. This results in a unified framework for disease classification and risk-stratification in AML that relies on information from cytogenetics and 32 genes. Last, we develop an open-access patient-tailored clinical decision support tool.

A randomized phase 3 trial of interferon-α vs hydroxyurea in polycythemia vera and essential thrombocythemia.

The goal of therapy for patients with essential thrombocythemia (ET) and polycythemia vera (PV) is to reduce thrombotic events by normalizing blood counts. Hydroxyurea (HU) and interferon-α (IFN-α) are the most frequently used cytoreductive options for patients with ET and PV at high risk for vascular complications. Myeloproliferative Disorders Research Consortium 112 was an investigator-initiated, phase 3 trial comparing HU to pegylated IFN-α (PEG) in treatment-naïve, high-risk patients with ET/PV. The primary endpoint was complete response (CR) rate at 12 months. A total of 168 patients were treated for a median of 81.0 weeks. CR for HU was 37% and 35% for PEG (P = .80) at 12 months. At 24 to 36 months, CR was 20% to 17% for HU and 29% to 33% for PEG. PEG led to a greater reduction in JAK2V617F at 24 months, but histopathologic responses were more frequent with HU. Thrombotic events and disease progression were infrequent in both arms, whereas grade 3/4 adverse events were more frequent with PEG (46% vs 28%). At 12 months of treatment, there was no significant difference in CR rates between HU and PEG. This study indicates that PEG and HU are both effective treatments for PV and ET. With longer treatment, PEG was more effective in normalizing blood counts and reducing driver mutation burden, whereas HU produced more histopathologic responses. Despite these differences, both agents did not differ in limiting thrombotic events and disease progression in high-risk patients with ET/PV. This trial was registered at www.clinicaltrials.gov as #NCT01259856.

Bone Marrow Surveillance of Pediatric Cancer Survivors Identifies Clones that Predict Therapy-Related Leukemia.

PURPOSE: Therapy-related myelodysplastic syndrome and acute leukemias (t-MDS/AL) are a major cause of nonrelapse mortality among pediatric cancer survivors. Although the presence of clonal hematopoiesis (CH) in adult patients at cancer diagnosis has been implicated in t-MDS/AL, there is limited published literature describing t-MDS/AL development in children. EXPERIMENTAL DESIGN: We performed molecular characterization of 199 serial bone marrow samples from 52 patients treated for high-risk neuroblastoma, including 17 with t-MDS/AL (transformation), 14 with transient cytogenetic abnormalities (transient), and 21 without t-MDS/AL or cytogenetic alterations (neuroblastoma-treated control). We also evaluated for CH in a cohort of 657 pediatric patients with solid tumor. RESULTS: We detected at least one disease-defining alteration in all cases at t-MDS/AL diagnosis, most commonly TP53 mutations and KMT2A rearrangements, including involving two novel partner genes (PRDM10 and DDX6). Backtracking studies identified at least one t-MDS/AL-associated mutation in 13 of 17 patients at a median of 15 months before t-MDS/AL diagnosis (range, 1.3-32.4). In comparison, acquired mutations were infrequent in the transient and control groups (4/14 and 1/21, respectively). The relative risk for development of t-MDS/AL in the presence of an oncogenic mutation was 8.8 for transformation patients compared with transient. Unlike CH in adult oncology patients, TP53 mutations were only detectable after initiation of cancer therapy. Last, only 1% of pediatric patients with solid tumor evaluated had CH involving myeloid genes. CONCLUSIONS: These findings demonstrate the clinical relevance of identifying molecular abnormalities in predicting development of t-MDS/AL and should guide the formation of intervention protocols to prevent this complication in high-risk pediatric patients.

Comprehensive Molecular Profiling of Desmoplastic Small Round Cell Tumor.

Desmoplastic small round cell tumor (DSRCT) is characterized by the EWSR1-WT1 t(11;22) (p13:q12) translocation. Few additional putative drivers have been identified, and research has suffered from a lack of model systems. Next-generation sequencing (NGS) data from 68 matched tumor-normal samples, whole-genome sequencing data from 10 samples, transcriptomic and affymetrix array data, and a bank of DSRCT patient-derived xenograft (PDX) are presented. EWSR1-WT1 fusions were noted to be simple, balanced events. Recurrent mutations were uncommon, but were noted in TERT (3%), ARID1A (6%), HRAS (5%), and TP53 (3%), and recurrent loss of heterozygosity (LOH) at 11p, 11q, and 16q was identified in 18%, 22%, and 34% of samples, respectively. Comparison of tumor-normal matched versus unmatched analysis suggests overcalling of somatic mutations in prior publications of DSRCT NGS data. Alterations in fibroblast growth factor receptor 4 (FGFR4) were identified in 5 of 68 (7%) of tumor samples, whereas differential overexpression of FGFR4 was confirmed orthogonally using 2 platforms. PDX models harbored the pathognomic EWSR1-WT1 fusion and were highly representative of corresponding tumors. Our analyses confirm DSRCT as a genomically quiet cancer defined by the balanced translocation, t(11;22)(p13:q12), characterized by a paucity of secondary mutations but a significant number of copy number alterations. Against this genomically quiet background, recurrent activating alterations of FGFR4 stood out, and suggest that this receptor tyrosine kinase, also noted to be highly expressed in DSRCT, should be further investigated. Future studies of DSRCT biology and preclinical therapeutic strategies should benefit from the PDX models characterized in this study. IMPLICATIONS: These data describe the general quiescence of the desmoplastic small round cell tumor (DSRCT) genome, present the first available bank of DSRCT model systems, and nominate FGFR4 as a key receptor tyrosine kinase in DSRCT, based on high expression, recurrent amplification, and recurrent activating mutations.

Isabl Platform, a digital biobank for processing multimodal patient data.

BACKGROUND: The widespread adoption of high throughput technologies has democratized data generation. However, data processing in accordance with best practices remains challenging and the data capital often becomes siloed. This presents an opportunity to consolidate data assets into digital biobanks-ecosystems of readily accessible, structured, and annotated datasets that can be dynamically queried and analysed. RESULTS: We present Isabl, a customizable plug-and-play platform for the processing of multimodal patient-centric data. Isabl's architecture consists of a relational database (Isabl DB), a command line client (Isabl CLI), a RESTful API (Isabl API) and a frontend web application (Isabl Web). Isabl supports automated deployment of user-validated pipelines across the entire data capital. A full audit trail is maintained to secure data provenance, governance and ensuring reproducibility of findings. CONCLUSIONS: As a digital biobank, Isabl supports continuous data utilization and automated meta analyses at scale, and serves as a catalyst for research innovation, new discoveries, and clinical translation.

Phase 2 study of ruxolitinib and decitabine in patients with myeloproliferative neoplasm in accelerated and blast phase.

Myeloproliferative neoplasms (MPN) that have evolved into accelerated or blast phase disease (MPN-AP/BP) have poor outcomes with limited treatment options and therefore represent an urgent unmet need. We have previously demonstrated in a multicenter, phase 1 trial conducted through the Myeloproliferative Neoplasms Research Consortium that the combination of ruxolitinib and decitabine is safe and tolerable and is associated with a favorable overall survival (OS). In this phase 2 trial, 25 patients with MPN-AP/BP were treated at the recommended phase 2 dose of ruxolitinib 25 mg twice daily for the induction cycle followed by 10 mg twice daily for subsequent cycles in combination with decitabine 20 mg/m2 for 5 consecutive days in a 28-day cycle. Nineteen patients died during the study follow-up. The median OS for all patients on study was 9.5 months (95% confidence interval, 4.3-12.0). Overall response rate (complete remission + incomplete platelet recovery + partial remission) was 11/25 (44%) and response was not associated with improved survival. We conclude that the combination of decitabine and ruxolitinib was well tolerated, demonstrated favorable OS, and represents a therapeutic option for this high-risk patient population. This trial was registered at www.clinicaltrials.gov as #NCT02076191.

Implications of TP53 allelic state for genome stability, clinical presentation and outcomes in myelodysplastic syndromes.

Tumor protein p53 (TP53) is the most frequently mutated gene in cancer1,2. In patients with myelodysplastic syndromes (MDS), TP53 mutations are associated with high-risk disease3,4, rapid transformation to acute myeloid leukemia (AML)5, resistance to conventional therapies6-8 and dismal outcomes9. Consistent with the tumor-suppressive role of TP53, patients harbor both mono- and biallelic mutations10. However, the biological and clinical implications of TP53 allelic state have not been fully investigated in MDS or any other cancer type. We analyzed 3,324 patients with MDS for TP53 mutations and allelic imbalances and delineated two subsets of patients with distinct phenotypes and outcomes. One-third of TP53-mutated patients had monoallelic mutations whereas two-thirds had multiple hits (multi-hit) consistent with biallelic targeting. Established associations with complex karyotype, few co-occurring mutations, high-risk presentation and poor outcomes were specific to multi-hit patients only. TP53 multi-hit state predicted risk of death and leukemic transformation independently of the Revised International Prognostic Scoring System (IPSS-R)11. Surprisingly, monoallelic patients did not differ from TP53 wild-type patients in outcomes and response to therapy. This study shows that consideration of TP53 allelic state is critical for diagnostic and prognostic precision in MDS as well as in future correlative studies of treatment response.

Accelerated single cell seeding in relapsed multiple myeloma.

Multiple myeloma (MM) progression is characterized by the seeding of cancer cells in different anatomic sites. To characterize this evolutionary process, we interrogated, by whole genome sequencing, 25 samples collected at autopsy from 4 patients with relapsed MM and an additional set of 125 whole exomes collected from 51 patients. Mutational signatures analysis showed how cytotoxic agents introduce hundreds of unique mutations in each surviving cancer cell, detectable by bulk sequencing only in cases of clonal expansion of a single cancer cell bearing the mutational signature. Thus, a unique, single-cell genomic barcode can link chemotherapy exposure to a discrete time window in a patient's life. We leveraged this concept to show that MM systemic seeding is accelerated at relapse and appears to be driven by the survival and subsequent expansion of a single myeloma cell following treatment with high-dose melphalan therapy and autologous stem cell transplant.

In vivo interactions between myosin XI, vesicles and filamentous actin are fast and transient in Physcomitrella patens.

The actin cytoskeleton and active membrane trafficking machinery are essential for polarized cell growth. To understand the interactions between myosin XI, vesicles and actin filaments in vivo, we performed fluorescence recovery after photobleaching and showed that the dynamics of myosin XIa at the tip of the spreading earthmoss Physcomitrella patens caulonemal cells are actin-dependent and that 50% of myosin XI is bound to vesicles. To obtain single-particle information, we used variable-angle epifluorescence microscopy in protoplasts to demonstrate that protein myosin XIa and VAMP72-labeled vesicles localize in time and space over periods lasting only a few seconds. By tracking data with Hidden Markov modeling, we showed that myosin XIa and VAMP72-labeled vesicles exhibit short runs of actin-dependent directed transport. We also found that the interaction of myosin XI with vesicles is short-lived. Together, this vesicle-bound fraction, fast off-rate and short average distance traveled seem be crucial for the dynamic oscillations observed at the tip, and might be vital for regulation and recycling of the exocytosis machinery, while simultaneously promoting vesicle focusing and vesicle secretion at the tip, necessary for cell wall expansion.

Patient-Driven Discovery, Therapeutic Targeting, and Post-Clinical Validation of a Novel AKT1 Fusion-Driven Cancer.

Despite the important role of the PI3K/AKT/mTOR axis in the pathogenesis of cancer, to date there have been few functional oncogenic fusions identified involving the AKT genes. A 12-year-old female with a histopathologically indeterminate epithelioid neoplasm was found to harbor a novel fusion between the LAMTOR1 and AKT1 genes. Through expanded use access, she became the first pediatric patient to be treated with the oral ATP-competitive pan-AKT inhibitor ipatasertib. Treatment resulted in dramatic tumor regression, demonstrating through patient-driven discovery that the fusion resulted in activation of AKT1, was an oncogenic driver, and could be therapeutically targeted with clinical benefit. Post-clinical validation using patient-derived model systems corroborated these findings, confirmed a membrane-bound and constitutively active fusion protein, and identified potential mechanisms of resistance to single-agent treatment with ipatasertib. SIGNIFICANCE: This study describes the patient-driven discovery of the first AKT1 fusion-driven cancer and its treatment with the AKT inhibitor ipatasertib. Patient-derived in vitro and in vivo model systems are used to confirm the LAMTOR1-AKT1 fusion as a tumorigenic driver and identify potential mechanisms of resistance to AKT inhibition.This article is highlighted in the In This Issue feature, p. 565.