Signatures of copy number alterations in human cancer.

Gains and losses of DNA are prevalent in cancer and emerge as a consequence of inter-related processes of replication stress, mitotic errors, spindle multipolarity and breakage-fusion-bridge cycles, among others, which may lead to chromosomal instability and aneuploidy1,2. These copy number alterations contribute to cancer initiation, progression and therapeutic resistance3-5. Here we present a conceptual framework to examine the patterns of copy number alterations in human cancer that is widely applicable to diverse data types, including whole-genome sequencing, whole-exome sequencing, reduced representation bisulfite sequencing, single-cell DNA sequencing and SNP6 microarray data. Deploying this framework to 9,873 cancers representing 33 human cancer types from The Cancer Genome Atlas6 revealed a set of 21 copy number signatures that explain the copy number patterns of 97% of samples. Seventeen copy number signatures were attributed to biological phenomena of whole-genome doubling, aneuploidy, loss of heterozygosity, homologous recombination deficiency, chromothripsis and haploidization. The aetiologies of four copy number signatures remain unexplained. Some cancer types harbour amplicon signatures associated with extrachromosomal DNA, disease-specific survival and proto-oncogene gains such as MDM2. In contrast to base-scale mutational signatures, no copy number signature was associated with many known exogenous cancer risk factors. Our results synthesize the global landscape of copy number alterations in human cancer by revealing a diversity of mutational processes that give rise to these alterations.

Mutations in ALK signaling pathways conferring resistance to ALK inhibitor treatment lead to collateral vulnerabilities in neuroblastoma cells.

BACKGROUND: Development of resistance to targeted therapies has tempered initial optimism that precision oncology would improve poor outcomes for cancer patients. Resistance mechanisms, however, can also confer new resistance-specific vulnerabilities, termed collateral sensitivities. Here we investigated anaplastic lymphoma kinase (ALK) inhibitor resistance in neuroblastoma, a childhood cancer frequently affected by activating ALK alterations. METHODS: Genome-wide forward genetic CRISPR-Cas9 based screens were performed to identify genes associated with ALK inhibitor resistance in neuroblastoma cell lines. Furthermore, the neuroblastoma cell line NBLW-R was rendered resistant by continuous exposure to ALK inhibitors. Genes identified to be associated with ALK inhibitor resistance were further investigated by generating suitable cell line models. In addition, tumor and liquid biopsy samples of four patients with ALK-mutated neuroblastomas before ALK inhibitor treatment and during tumor progression under treatment were genomically profiled. RESULTS: Both genome-wide CRISPR-Cas9-based screens and preclinical spontaneous ALKi resistance models identified NF1 loss and activating NRASQ61K mutations to confer resistance to chemically diverse ALKi. Moreover, human neuroblastomas recurrently developed de novo loss of NF1 and activating RAS mutations after ALKi treatment, leading to therapy resistance. Pathway-specific perturbations confirmed that NF1 loss and activating RAS mutations lead to RAS-MAPK signaling even in the presence of ALKi. Intriguingly, NF1 loss rendered neuroblastoma cells hypersensitive to MEK inhibition. CONCLUSIONS: Our results provide a clinically relevant mechanistic model of ALKi resistance in neuroblastoma and highlight new clinically actionable collateral sensitivities in resistant cells.

Ultra-Sensitive Mutation Detection and Genome-Wide DNA Copy Number Reconstruction by Error-Corrected Circulating Tumor DNA Sequencing.

BACKGROUND: Circulating free DNA sequencing (cfDNA-Seq) can portray cancer genome landscapes, but highly sensitive and specific technologies are necessary to accurately detect mutations with often low variant frequencies. METHODS: We developed a customizable hybrid-capture cfDNA-Seq technology using off-the-shelf molecular barcodes and a novel duplex DNA molecule identification tool for enhanced error correction. RESULTS: Modeling based on cfDNA yields from 58 patients showed that this technology, requiring 25 ng of cfDNA, could be applied to >95% of patients with metastatic colorectal cancer (mCRC). cfDNA-Seq of a 32-gene, 163.3-kbp target region detected 100% of single-nucleotide variants, with 0.15% variant frequency in spike-in experiments. Molecular barcode error correction reduced false-positive mutation calls by 97.5%. In 28 consecutively analyzed patients with mCRC, 80 out of 91 mutations previously detected by tumor tissue sequencing were called in the cfDNA. Call rates were similar for point mutations and indels. cfDNA-Seq identified typical mCRC driver mutations in patients in whom biopsy sequencing had failed or did not include key mCRC driver genes. Mutations only called in cfDNA but undetectable in matched biopsies included a subclonal resistance driver mutation to anti-EGFR antibodies in KRAS, parallel evolution of multiple PIK3CA mutations in 2 cases, and TP53 mutations originating from clonal hematopoiesis. Furthermore, cfDNA-Seq off-target read analysis allowed simultaneous genome-wide copy number profile reconstruction in 20 of 28 cases. Copy number profiles were validated by low-coverage whole-genome sequencing. CONCLUSIONS: This error-corrected, ultradeep cfDNA-Seq technology with a customizable target region and publicly available bioinformatics tools enables broad insights into cancer genomes and evolution. CLINICALTRIALSGOV IDENTIFIER: NCT02112357.

Coexistent hyperdiploidy does not abrogate poor prognosis in myeloma with adverse cytogenetics and may precede IGH translocations.

The acquisition of the cytogenetic abnormalities hyperdiploidy or translocations into the immunoglobulin gene loci are considered as initiating events in the pathogenesis of myeloma and were often assumed to be mutually exclusive. These lesions have clinical significance; hyperdiploidy or the presence of the t(11;14) translocation is associated with a favorable outcome, whereas t(4;14), t(14;16), and t(14;20) are unfavorable. Poor outcomes are magnified when lesions occur in association with other high-risk features, del17p and +1q. Some patients have coexistence of both good and poor prognostic lesions, and there has been no consensus on their risk status. To address this, we have investigated their clinical impact using cases in the Myeloma IX study (ISRCTN68454111) and shown that the coexistence of hyperdiploidy or t(11;14) does not abrogate the poor prognosis associated with adverse molecular lesions, including translocations. We have also used single-cell analysis to study cases with coexistent translocations and hyperdiploidy to determine how these lesions cosegregate within the clonal substructure, and we have demonstrated that hyperdiploidy may precede IGH translocation in a proportion of patients. These findings have important clinical and biological implications, as we conclude patients with coexistence of adverse lesions and hyperdiploidy should be considered high risk and treated accordingly.

A molecular diagnostic approach able to detect the recurrent genetic prognostic factors typical of presenting myeloma.

Risk stratification in myeloma requires an accurate assessment of the presence of a range of molecular abnormalities including the differing IGH translocations and the recurrent copy number abnormalities that can impact clinical behavior. Currently, interphase fluorescence in situ hybridization is used to detect these abnormalities. High failure rates, slow turnaround, cost, and labor intensiveness make it difficult and expensive to use in routine clinical practice. Multiplex ligation-dependent probe amplification (MLPA), a molecular approach based on a multiplex polymerase chain reaction method, offers an alternative for the assessment of copy number changes present in the myeloma genome. Here, we provide evidence showing that MLPA is a powerful tool for the efficient detection of copy number abnormalities and when combined with expression assays, MLPA can detect all of the prognostically relevant molecular events which characterize presenting myeloma. This approach opens the way for a molecular diagnostic strategy that is efficient, high throughput, and cost effective.

Tumor evolution metrics predict recurrence beyond 10 years in locally advanced prostate cancer.

Cancer evolution lays the groundwork for predictive oncology. Testing evolutionary metrics requires quantitative measurements in controlled clinical trials. We mapped genomic intratumor heterogeneity in locally advanced prostate cancer using 642 samples from 114 individuals enrolled in clinical trials with a 12-year median follow-up. We concomitantly assessed morphological heterogeneity using deep learning in 1,923 histological sections from 250 individuals. Genetic and morphological (Gleason) diversity were independent predictors of recurrence (hazard ratio (HR) = 3.12 and 95% confidence interval (95% CI) = 1.34-7.3; HR = 2.24 and 95% CI = 1.28-3.92). Combined, they identified a group with half the median time to recurrence. Spatial segregation of clones was also an independent marker of recurrence (HR = 2.3 and 95% CI = 1.11-4.8). We identified copy number changes associated with Gleason grade and found that chromosome 6p loss correlated with reduced immune infiltration. Matched profiling of relapse, decades after diagnosis, confirmed that genomic instability is a driving force in prostate cancer progression. This study shows that combining genomics with artificial intelligence-aided histopathology leads to the identification of clinical biomarkers of evolution.

Durable responses to ATR inhibition with ceralasertib in tumors with genomic defects and high inflammation.

BACKGROUNDPhase 1 study of ATRinhibition alone or with radiation therapy (PATRIOT) was a first-in-human phase I study of the oral ATR (ataxia telangiectasia and Rad3-related) inhibitor ceralasertib (AZD6738) in advanced solid tumors.METHODSThe primary objective was safety. Secondary objectives included assessment of antitumor responses and pharmacokinetic (PK) and pharmacodynamic (PD) studies. Sixty-seven patients received 20-240 mg ceralasertib BD continuously or intermittently (14 of a 28-day cycle).RESULTSIntermittent dosing was better tolerated than continuous, which was associated with dose-limiting hematological toxicity. The recommended phase 2 dose of ceralasertib was 160 mg twice daily for 2 weeks in a 4-weekly cycle. Modulation of target and increased DNA damage were identified in tumor and surrogate PD. There were 5 (8%) confirmed partial responses (PRs) (40-240 mg BD), 34 (52%) stable disease (SD), including 1 unconfirmed PR, and 27 (41%) progressive disease. Durable responses were seen in tumors with loss of AT-rich interactive domain-containing protein 1A (ARID1A) and DNA damage-response defects. Treatment-modulated tumor and systemic immune markers and responding tumors were more immune inflamed than nonresponding.CONCLUSIONCeralasertib monotherapy was tolerated at 160 mg BD intermittently and associated with antitumor activity.TRIAL REGISTRATIONClinicaltrials.gov: NCT02223923, EudraCT: 2013-003994-84.FUNDINGCancer Research UK, AstraZeneca, UK Department of Health (National Institute for Health Research), Rosetrees Trust, Experimental Cancer Medicine Centre.

Baseline Mutations and ctDNA Dynamics as Prognostic and Predictive Factors in ER-Positive/HER2-Negative Metastatic Breast Cancer Patients.

PURPOSE: Prognostic and predictive biomarkers to cyclin-dependent kinases 4 and 6 inhibitors are lacking. Circulating tumor DNA (ctDNA) can be used to profile these patients and dynamic changes in ctDNA could be an early predictor of treatment efficacy. Here, we conducted plasma ctDNA profiling in patients from the PEARL trial comparing palbociclib+fulvestrant versus capecitabine to investigate associations between baseline genomic landscape and on-treatment ctDNA dynamics with treatment efficacy. EXPERIMENTAL DESIGN: Correlative blood samples were collected at baseline [cycle 1-day 1 (C1D1)] and prior to treatment [cycle 1-day 15 (C1D15)]. Plasma ctDNA was sequenced with a custom error-corrected capture panel, with both univariate and multivariate Cox models used for treatment efficacy associations. A prespecified methodology measuring ctDNA changes in clonal mutations between C1D1 and C1D15 was used for the on-treatment ctDNA dynamic model. RESULTS: 201 patients were profiled at baseline, with ctDNA detection associated with worse progression-free survival (PFS)/overall survival (OS). Detectable TP53 mutation showed worse PFS and OS in both treatment arms, even after restricting population to baseline ctDNA detection. ESR1 mutations were associated with worse OS overall, which was lost when restricting population to baseline ctDNA detection. PIK3CA mutations confer worse OS only to patients on the palbociclib+fulvestrant treatment arm. ctDNA dynamics analysis (n = 120) showed higher ctDNA suppression in the capecitabine arm. Patients without ctDNA suppression showed worse PFS in both treatment arms. CONCLUSIONS: We show impaired survival irrespective of endocrine or chemotherapy-based treatments for patients with hormone receptor-positive/HER2-negative metastatic breast cancer harboring plasma TP53 mutations. Early ctDNA suppression may provide treatment efficacy predictions. Further validation to fully demonstrate clinical utility of ctDNA dynamics is warranted.

Genomic Patterns of Malignant Peripheral Nerve Sheath Tumor (MPNST) Evolution Correlate with Clinical Outcome and Are Detectable in Cell-Free DNA.

Malignant peripheral nerve sheath tumor (MPNST), an aggressive soft-tissue sarcoma, occurs in people with neurofibromatosis type 1 (NF1) and sporadically. Whole-genome and multiregional exome sequencing, transcriptomic, and methylation profiling of 95 tumor samples revealed the order of genomic events in tumor evolution. Following biallelic inactivation of NF1, loss of CDKN2A or TP53 with or without inactivation of polycomb repressive complex 2 (PRC2) leads to extensive somatic copy-number aberrations (SCNA). Distinct pathways of tumor evolution are associated with inactivation of PRC2 genes and H3K27 trimethylation (H3K27me3) status. Tumors with H3K27me3 loss evolve through extensive chromosomal losses followed by whole-genome doubling and chromosome 8 amplification, and show lower levels of immune cell infiltration. Retention of H3K27me3 leads to extensive genomic instability, but an immune cell-rich phenotype. Specific SCNAs detected in both tumor samples and cell-free DNA (cfDNA) act as a surrogate for H3K27me3 loss and immune infiltration, and predict prognosis. SIGNIFICANCE: MPNST is the most common cause of death and morbidity for individuals with NF1, a relatively common tumor predisposition syndrome. Our results suggest that somatic copy-number and methylation profiling of tumor or cfDNA could serve as a biomarker for early diagnosis and to stratify patients into prognostic and treatment-related subgroups. This article is highlighted in the In This Issue feature, p. 517.

Molecular Characterization of Circulating Tumor DNA in Pediatric Rhabdomyosarcoma: A Feasibility Study.

PURPOSE: Rhabdomyosarcomas (RMS) are rare neoplasms affecting children and young adults. Efforts to improve patient survival have been undermined by a lack of suitable disease markers. Plasma circulating tumor DNA (ctDNA) has shown promise as a potential minimally invasive biomarker and monitoring tool in other cancers; however, it remains underexplored in RMS. We aimed to determine the feasibility of identifying and quantifying ctDNA in plasma as a marker of disease burden and/or treatment response using blood samples from RMS mouse models and patients. METHODS: We established mouse models of RMS and applied quantitative polymerase chain reaction (PCR) and droplet digital PCR (ddPCR) to detect ctDNA within the mouse plasma. Potential driver mutations, copy-number alterations, and DNA breakpoints associated with PAX3/7-FOXO1 gene fusions were identified in the RMS samples collected at diagnosis. Patient-matched plasma samples collected from 28 patients with RMS before, during, and after treatment were analyzed for the presence of ctDNA via ddPCR, panel sequencing, and/or whole-exome sequencing. RESULTS: Human tumor-derived DNA was detectable in plasma samples from mouse models of RMS and correlated with tumor burden. In patients, ctDNA was detected in 14/18 pretreatment plasma samples with ddPCR and 7/7 cases assessed by sequencing. Levels of ctDNA at diagnosis were significantly higher in patients with unfavorable tumor sites, positive nodal status, and metastasis. In patients with serial plasma samples (n = 18), fluctuations in ctDNA levels corresponded to treatment response. CONCLUSION: Comprehensive ctDNA analysis combining high sensitivity and throughput can identify key molecular drivers in RMS models and patients, suggesting potential as a minimally invasive biomarker. Preclinical assessment of treatments using mouse models and further patient testing through prospective clinical trials are now warranted.

DIPG Harbors Alterations Targetable by MEK Inhibitors, with Acquired Resistance Mechanisms Overcome by Combinatorial Inhibition.

The survival of children with diffuse intrinsic pontine glioma (DIPG) remains dismal, with new treatments desperately needed. In a prospective biopsy-stratified clinical trial, we combined detailed molecular profiling and drug screening in newly established patient-derived models in vitro and in vivo. We identified in vitro sensitivity to MEK inhibitors in DIPGs harboring MAPK pathway alterations, but treatment of patient-derived xenograft models and a patient at relapse failed to elicit a significant response. We generated trametinib-resistant clones in a BRAFG469V model through continuous drug exposure and identified acquired mutations in MEK1/2 with sustained pathway upregulation. These cells showed hallmarks of mesenchymal transition and expression signatures overlapping with inherently trametinib-insensitive patient-derived cells, predicting sensitivity to dasatinib. Combined trametinib and dasatinib showed highly synergistic effects in vitro and on ex vivo brain slices. We highlight the MAPK pathway as a therapeutic target in DIPG and show the importance of parallel resistance modeling and combinatorial treatments for meaningful clinical translation. SIGNIFICANCE: We report alterations in the MAPK pathway in DIPGs to confer initial sensitivity to targeted MEK inhibition. We further identify for the first time the mechanism of resistance to single-agent targeted therapy in these tumors and suggest a novel combinatorial treatment strategy to overcome it in the clinic. This article is highlighted in the In This Issue feature, p. 587.

Circulating tumour DNA sequencing to determine therapeutic response and identify tumour heterogeneity in patients with paediatric solid tumours.

OBJECTIVE: Clinical diagnostic sequencing of circulating tumour DNA (ctDNA) is well advanced for adult patients, but application to paediatric cancer patients lags behind. METHODS: To address this, we have developed a clinically relevant (67 gene) NGS capture panel and accompanying workflow that enables sensitive and reliable detection of low-frequency genetic variants in cell-free DNA (cfDNA) from children with solid tumours. We combined gene panel sequencing with low pass whole-genome sequencing of the same library to inform on genome-wide copy number changes in the blood. RESULTS: Analytical validity was evaluated using control materials, and the method was found to be highly sensitive (0.96 for SNVs and 0.97 for INDEL), specific (0.82 for SNVs and 0.978 for INDEL), repeatable (>0.93 [95% CI: 0.89-0.95]) and reproducible (>0.87 [95% CI: 0.87-0.95]). Potential for clinical application was demonstrated in 39 childhood cancer patients with a spectrum of solid tumours in which the single nucleotide variants expected from tumour sequencing were detected in cfDNA in 94.4% (17/18) of cases with active extracranial disease. In 13 patients, where serial samples were available, we show a close correlation between events detected in cfDNA and treatment response, demonstrate that cfDNA analysis could be a useful tool to monitor disease progression, and show cfDNA sequencing has the potential to identify targetable variants that were not detected in tumour samples. CONCLUSIONS: This is the first pan-cancer DNA sequencing panel that we know to be optimised for cfDNA in children for blood-based molecular diagnostics in paediatric solid tumours.

Droplet digital PCR-based detection of circulating tumor DNA from pediatric high grade and diffuse midline glioma patients.

BACKGROUND: The use of liquid biopsy is of potential high importance for children with high grade (HGG) and diffuse midline gliomas (DMG), particularly where surgical procedures are limited, and invasive biopsy sampling not without risk. To date, however, the evidence that detection of cell-free DNA (cfDNA) or circulating tumor DNA (ctDNA) could provide useful information for these patients has been limited, or contradictory. METHODS: We optimized droplet digital PCR (ddPCR) assays for the detection of common somatic mutations observed in pediatric HGG/DMG, and applied them to liquid biopsies from plasma, serum, cerebrospinal fluid (CSF), and cystic fluid collected from 32 patients. RESULTS: Although detectable in all biomaterial types, ctDNA presented at significantly higher levels in CSF compared to plasma and/or serum. When applied to a cohort of 127 plasma specimens from 41 patients collected from 2011 to 2018 as part of a randomized clinical trial in pediatric non-brainstem HGG/DMG, ctDNA profiling by ddPCR was of limited use due to the small volumes (mean = 0.49 mL) available. In anecdotal cases where sufficient material was available, cfDNA concentration correlated with disease progression in two examples each of poor response in H3F3A_K27M-mutant DMG, and longer survival times in hemispheric BRAF_V600E-mutant cases. CONCLUSION: Tumor-specific DNA alterations are more readily detected in CSF than plasma. Although we demonstrate the potential of the approach to assessing tumor burden, our results highlight the necessity for adequate sample collection and approach to improve detection if plasma samples are to be used.

Triplet Therapy with Palbociclib, Taselisib, and Fulvestrant in PIK3CA-Mutant Breast Cancer and Doublet Palbociclib and Taselisib in Pathway-Mutant Solid Cancers.

Cyclin-dependent kinase 4/6 (CDK4/6) and PI3K inhibitors synergize in PIK3CA-mutant ER-positive HER2-negative breast cancer models. We conducted a phase Ib trial investigating the safety and efficacy of doublet CDK4/6 inhibitor palbociclib plus selective PI3K inhibitor taselisib in advanced solid tumors, and triplet palbociclib plus taselisib plus fulvestrant in 25 patients with PIK3CA-mutant, ER-positive HER2-negative advanced breast cancer. The triplet therapy response rate in PIK3CA-mutant, ER-positive HER2-negative cancer was 37.5% [95% confidence interval (CI), 18.8-59.4]. Durable disease control was observed in PIK3CA-mutant ER-negative breast cancer and other solid tumors with doublet therapy. Both combinations were well tolerated at pharmacodynamically active doses. In the triplet group, high baseline cyclin E1 expression associated with shorter progression-free survival (PFS; HR = 4.2; 95% CI, 1.3-13.1; P = 0.02). Early circulating tumor DNA (ctDNA) dynamics demonstrated high on-treatment ctDNA association with shorter PFS (HR = 5.2; 95% CI, 1.4-19.4; P = 0.04). Longitudinal plasma ctDNA sequencing provided genomic evolution evidence during triplet therapy. SIGNIFICANCE: The triplet of palbociclib, taselisib, and fulvestrant has promising efficacy in patients with heavily pretreated PIK3CA-mutant ER-positive HER2-negative advanced breast cancer. A subset of patients with PIK3CA-mutant triple-negative breast cancer derived clinical benefit from palbociclib and taselisib doublet, suggesting a potential nonchemotherapy targeted approach for this population.This article is highlighted in the In This Issue feature, p. 1.

Validation of the EuroClonality-NGS DNA capture panel as an integrated genomic tool for lymphoproliferative disorders.

Current diagnostic standards for lymphoproliferative disorders include multiple tests for detection of clonal immunoglobulin (IG) and/or T-cell receptor (TCR) rearrangements, translocations, copy-number alterations (CNAs), and somatic mutations. The EuroClonality-NGS DNA Capture (EuroClonality-NDC) assay was designed as an integrated tool to characterize these alterations by capturing IGH switch regions along with variable, diversity, and joining genes of all IG and TCR loci in addition to clinically relevant genes for CNA and mutation analysis. Diagnostic performance against standard-of-care clinical testing was assessed in a cohort of 280 B- and T-cell malignancies from 10 European laboratories, including 88 formalin-fixed paraffin-embedded samples and 21 reactive lesions. DNA samples were subjected to the EuroClonality-NDC protocol in 7 EuroClonality-NGS laboratories and analyzed using a bespoke bioinformatic pipeline. The EuroClonality-NDC assay detected B-cell clonality in 191 (97%) of 197 B-cell malignancies and T-cell clonality in 71 (97%) of 73 T-cell malignancies. Limit of detection (LOD) for IG/TCR rearrangements was established at 5% using cell line blends. Chromosomal translocations were detected in 145 (95%) of 152 cases known to be positive. CNAs were validated for immunogenetic and oncogenetic regions, highlighting their novel role in confirming clonality in somatically hypermutated cases. Single-nucleotide variant LOD was determined as 4% allele frequency, and an orthogonal validation using 32 samples resulted in 98% concordance. The EuroClonality-NDC assay is a robust tool providing a single end-to-end workflow for simultaneous detection of B- and T-cell clonality, translocations, CNAs, and sequence variants.

Genomic landscape of platinum resistant and sensitive testicular cancers.

While most testicular germ cell tumours (TGCTs) exhibit exquisite sensitivity to platinum chemotherapy, ~10% are platinum resistant. To gain insight into the underlying mechanisms, we undertake whole exome sequencing and copy number analysis in 40 tumours from 26 cases with platinum-resistant TGCT, and combine this with published genomic data on an additional 624 TGCTs. We integrate analyses for driver mutations, mutational burden, global, arm-level and focal copy number (CN) events, and SNV and CN signatures. Albeit preliminary and observational in nature, these analyses provide support for a possible mechanistic link between early driver mutations in RAS and KIT and the widespread copy number events by which TGCT is characterised.

Homologous recombination DNA repair deficiency and PARP inhibition activity in primary triple negative breast cancer.

Triple negative breast cancer (TNBC) encompasses molecularly different subgroups, with a subgroup harboring evidence of defective homologous recombination (HR) DNA repair. Here, within a phase 2 window clinical trial, RIO trial (EudraCT 2014-003319-12), we investigate the activity of PARP inhibitors in 43 patients with untreated TNBC. The primary end point, decreased Ki67, occured in 12% of TNBC. In secondary end point analyses, HR deficiency was identified in 69% of TNBC with the mutational-signature-based HRDetect assay. Cancers with HRDetect mutational signatures of HR deficiency had a functional defect in HR, assessed by impaired RAD51 foci formation on end of treatment biopsy. Following rucaparib treatment there was no association of Ki67 change with HR deficiency. In contrast, early circulating tumor DNA dynamics identified activity of rucaparib, with end of treatment ctDNA levels suppressed by rucaparib in mutation-signature HR-deficient cancers. In ad hoc analysis, rucaparib induced expression of interferon response genes in HR-deficient cancers. The majority of TNBCs have a defect in DNA repair, identifiable by mutational signature analysis, that may be targetable with PARP inhibitors.

Inactivating NF1 Mutations Are Enriched in Advanced Breast Cancer and Contribute to Endocrine Therapy Resistance.

PURPOSE: Advanced breast cancer (ABC) has not been subjected to the same degree of molecular scrutiny as early primary cancer. Breast cancer evolves with time and under the selective pressure of treatment, with the potential to acquire mutations with resistance to treatment and disease progression. To identify potentially targetable mutations in advanced breast cancer, we performed prospective molecular characterization of a cohort of patients with ABC. EXPERIMENTAL DESIGN: Biopsies from patients with advanced breast cancer were sequenced with a 41 genes targeted panel in the ABC Biopsy (ABC-Bio) study. Blood samples were collected at disease progression for circulating tumor DNA (ctDNA) analysis, along with matched primary tumor to assess for acquisition in ABC in a subset of patients. RESULTS: We sequenced 210 ABC samples, demonstrating enrichment compared with primary disease for potentially targetable mutations in HER2 (in 6.19% of samples), AKT1 (7.14%), and NF1 (8.10%). Of these enriched mutations, we show that NF1 mutations were frequently acquired in ABC, not present in the original primary disease. In ER-positive cancer cell line models, loss of NF1 resulted in endocrine therapy resistance, through both ER-dependent and -independent mechanisms. NF1 loss promoted ER-independent cyclin D1 expression, which could be therapeutically targeted with CDK4/6 inhibitors in vitro. Patients with NF1 mutations detected in baseline circulating tumor DNA had a good outcome on the CDK4/6 inhibitor palbociclib and fulvestrant. CONCLUSIONS: Our research identifies multiple therapeutic opportunities for advanced breast cancer and identifies the previously underappreciated acquisition of NF1 mutations.

Infant High-Grade Gliomas Comprise Multiple Subgroups Characterized by Novel Targetable Gene Fusions and Favorable Outcomes.

Infant high-grade gliomas appear clinically distinct from their counterparts in older children, indicating that histopathologic grading may not accurately reflect the biology of these tumors. We have collected 241 cases under 4 years of age, and carried out histologic review, methylation profiling, and custom panel, genome, or exome sequencing. After excluding tumors representing other established entities or subgroups, we identified 130 cases to be part of an "intrinsic" spectrum of disease specific to the infant population. These included those with targetable MAPK alterations, and a large proportion of remaining cases harboring gene fusions targeting ALK (n = 31), NTRK1/2/3 (n = 21), ROS1 (n = 9), and MET (n = 4) as their driving alterations, with evidence of efficacy of targeted agents in the clinic. These data strongly support the concept that infant gliomas require a change in diagnostic practice and management. SIGNIFICANCE: Infant high-grade gliomas in the cerebral hemispheres comprise novel subgroups, with a prevalence of ALK, NTRK1/2/3, ROS1, or MET gene fusions. Kinase fusion-positive tumors have better outcome and respond to targeted therapy clinically. Other subgroups have poor outcome, with fusion-negative cases possibly representing an epigenetically driven pluripotent stem cell phenotype.See related commentary by Szulzewsky and Cimino, p. 904.This article is highlighted in the In This Issue feature, p. 890.