Mara Rosenberg

Widespread Genetic Heterogeneity in Multiple Myeloma: Implications for Targeted Therapy

We performed massively parallel sequencing of paired tumor/normal samples from 203 multiple myeloma (MM) patients and identified significantly mutated genes and copy number alterations and discovered putative tumor suppressor genes by determining homozygous deletions and loss of heterozygosity. We observed frequent mutations in KRAS (particularly in previously treated patients), NRAS, BRAF, FAM46C, TP53, and DIS3 (particularly in nonhyperdiploid MM). Mutations were often present in subclonal populations, and multiple mutations within the same pathway (e.g., KRAS, NRAS, and BRAF) were observed in the same patient. In vitro modeling predicts only partial treatment efficacy of targeting subclonal mutations, and even growth promotion of nonmutated subclones in some cases. These results emphasize the importance of heterogeneity analysis for treatment decisions.

Landscape of genomic alterations in cervical carcinomas

Cervical cancer is responsible for 10–15% of cancer-related deaths in women worldwide. The aetiological role of infection with high-risk human papilloma viruses (HPVs) in cervical carcinomas is well established. Previous studies have also implicated somatic mutations in PIK3CA, PTEN, TP53, STK11 and KRAS as well as several copy-number alterations in the pathogenesis of cervical carcinomas. Here we report whole-exome sequencing analysis of 115 cervical carcinoma–normal paired samples, transcriptome sequencing of 79 cases and whole-genome sequencing of 14 tumour–normal pairs. Previously unknown somatic mutations in 79 primary squamous cell carcinomas include recurrent E322K substitutions in the MAPK1 gene (8%), inactivating mutations in the HLA-B gene (9%), and mutations in EP300 (16%), FBXW7 (15%), NFE2L2 (4%), TP53 (5%) and ERBB2 (6%). We also observe somatic ELF3 (13%) and CBFB (8%) mutations in 24 adenocarcinomas. Squamous cell carcinomas have higher frequencies of somatic nucleotide substitutions occurring at cytosines preceded by thymines (Tp*C sites) than adenocarcinomas. Gene expression levels at HPV integration sites were statistically significantly higher in tumours with HPV integration compared with expression of the same genes in tumours without viral integration at the same site. These data demonstrate several recurrent genomic alterations in cervical carcinomas that suggest new strategies to combat this disease.

Whole exome sequencing of circulating tumor cells provides a window into metastatic prostate cancer

Comprehensive analyses of cancer genomes promise to inform prognoses and precise cancer treatments. A major barrier, however, is inaccessibility of metastatic tissue. A potential solution is to characterize circulating tumor cells (CTCs), but this requires overcoming the challenges of isolating rare cells and sequencing low-input material. Here we report an integrated process to isolate, qualify and sequence whole exomes of CTCs with high fidelity using a census-based sequencing strategy. Power calculations suggest that mapping of >99.995% of the standard exome is possible in CTCs. We validated our process in two patients with prostate cancer, including one for whom we sequenced CTCs, a lymph node metastasis and nine cores of the primary tumor. Fifty-one of 73 CTC mutations (70%) were present in matched tissue. Moreover, we identified 10 early trunk and 56 metastatic trunk mutations in the non-CTC tumor samples and found 90% and 73% of these mutations, respectively, in CTC exomes. This study establishes a foundation for CTC genomics in the clinic.

Mutations driving CLL and their evolution in progression and relapse

Which genetic alterations drive tumorigenesis and how they evolve over the course of disease and therapy are central questions in cancer biology. Here we identify 44 recurrently mutated genes and 11 recurrent somatic copy number variations through whole-exome sequencing of 538 chronic lymphocytic leukaemia (CLL) and matched germline DNA samples, 278 of which were collected in a prospective clinical trial. These include previously unrecognized putative cancer drivers (RPS15, IKZF3), and collectively identify RNA processing and export, MYC activity, and MAPK signalling as central pathways involved in CLL. Clonality analysis of this large data set further enabled reconstruction of temporal relationships between driver events. Direct comparison between matched pre-treatment and relapse samples from 59 patients demonstrated highly frequent clonal evolution. Thus, large sequencing data sets of clinically informative samples enable the discovery of novel genes associated with cancer, the network of relationships between the driver events, and their impact on disease relapse and clinical outcome.

MAP Kinase Pathway Alterations in BRAF-Mutant Melanoma Patients with Acquired Resistance to Combined RAF/MEK Inhibition

Treatment of BRAF-mutant melanoma with combined dabrafenib and trametinib, which target RAF and the downstream MAP-ERK kinase (MEK)1 and MEK2 kinases, respectively, improves progression-free survival and response rates compared with dabrafenib monotherapy. Mechanisms of clinical resistance to combined RAF/MEK inhibition are unknown. We performed whole-exome sequencing (WES) and whole-transcriptome sequencing (RNA-seq) on pretreatment and drug-resistant tumors from five patients with acquired resistance to dabrafenib/trametinib. In three of these patients, we identified additional mitogen-activated protein kinase (MAPK) pathway alterations in the resistant tumor that were not detected in the pretreatment tumor, including a novel activating mutation in MEK2 (MEK2(Q60P)). MEK2(Q60P) conferred resistance to combined RAF/MEK inhibition in vitro, but remained sensitive to inhibition of the downstream kinase extracellular signal-regulated kinase (ERK). The continued MAPK signaling-based resistance identified in these patients suggests that alternative dosing of current agents, more potent RAF/MEK inhibitors, and/or inhibition of the downstream kinase ERK may be needed for durable control of BRAF-mutant melanoma.

Whole-exome sequencing identifies a recurrent NAB2-STAT6 fusion in solitary fibrous tumors.

Solitary fibrous tumors (SFTs) are rare mesenchymal tumors. Here, we describe the identification of a NAB2-STAT6 fusion from whole-exome sequencing of 17 SFTs. Analysis in 53 tumors confirmed the presence of 7 variants of this fusion transcript in 29 tumors (55%), representing a lower bound for fusion frequency at this locus and suggesting that the NAB2-STAT6 fusion is a distinct molecular feature of SFTs.

Genomic Characterization of Brain Metastases Reveals Branched Evolution and Potential Therapeutic Targets

UNLABELLED Brain metastases are associated with a dismal prognosis. Whether brain metastases harbor distinct genetic alterations beyond those observed in primary tumors is unknown. We performed whole-exome sequencing of 86 matched brain metastases, primary tumors, and normal tissue. In all clonally related cancer samples, we observed branched evolution, where all metastatic and primary sites shared a common ancestor yet continued to evolve independently. In 53% of cases, we found potentially clinically informative alterations in the brain metastases not detected in the matched primary-tumor sample. In contrast, spatially and temporally separated brain metastasis sites were genetically homogenous. Distal extracranial and regional lymph node metastases were highly divergent from brain metastases. We detected alterations associated with sensitivity to PI3K/AKT/mTOR, CDK, and HER2/EGFR inhibitors in the brain metastases. Genomic analysis of brain metastases provides an opportunity to identify potentially clinically informative alterations not detected in clinically sampled primary tumors, regional lymph nodes, or extracranial metastases. SIGNIFICANCE Decisions for individualized therapies in patients with brain metastasis are often made from primary-tumor biopsies. We demonstrate that clinically actionable alterations present in brain metastases are frequently not detected in primary biopsies, suggesting that sequencing of primary biopsies alone may miss a substantial number of opportunities for targeted therapy.

Comprehensive Genomic Analysis of Rhabdomyosarcoma Reveals a Landscape of Alterations Affecting a Common Genetic Axis in Fusion-Positive and Fusion-Negative Tumors

UNLABELLED Despite gains in survival, outcomes for patients with metastatic or recurrent rhabdomyosarcoma remain dismal. In a collaboration between the National Cancer Institute, Childrens Oncology Group, and Broad Institute, we performed whole-genome, whole-exome, and transcriptome sequencing to characterize the landscape of somatic alterations in 147 tumor/normal pairs. Two genotypes are evident in rhabdomyosarcoma tumors: those characterized by the PAX3 or PAX7 fusion and those that lack these fusions but harbor mutations in key signaling pathways. The overall burden of somatic mutations in rhabdomyosarcoma is relatively low, especially in tumors that harbor a PAX3/7 gene fusion. In addition to previously reported mutations in NRAS, KRAS, HRAS, FGFR4, PIK3CA, and CTNNB1, we found novel recurrent mutations in FBXW7 and BCOR, providing potential new avenues for therapeutic intervention. Furthermore, alteration of the receptor tyrosine kinase/RAS/PIK3CA axis affects 93% of cases, providing a framework for genomics-directed therapies that might improve outcomes for patients with rhabdomyosarcoma. SIGNIFICANCE This is the most comprehensive genomic analysis of rhabdomyosarcoma to date. Despite a relatively low mutation rate, multiple genes were recurrently altered, including NRAS, KRAS, HRAS, FGFR4, PIK3CA, CTNNB1, FBXW7, and BCOR. In addition, a majority of rhabdomyosarcoma tumors alter the receptor tyrosine kinase/RAS/PIK3CA axis, providing an opportunity for genomics-guided intervention.

Distinct patterns of somatic genome alterations in lung adenocarcinomas and squamous cell carcinomas

To compare lung adenocarcinoma (ADC) and lung squamous cell carcinoma (SqCC) and to identify new drivers of lung carcinogenesis, we examined the exome sequences and copy number profiles of 660 lung ADC and 484 lung SqCC tumor–normal pairs. Recurrent alterations in lung SqCCs were more similar to those of other squamous carcinomas than to alterations in lung ADCs. New significantly mutated genes included PPP3CA, DOT1L, and FTSJD1 in lung ADC, RASA1 in lung SqCC, and KLF5, EP300, and CREBBP in both tumor types. New amplification peaks encompassed MIR21 in lung ADC, MIR205 in lung SqCC, and MAPK1 in both. Lung ADCs lacking receptor tyrosine kinase–Ras–Raf pathway alterations had mutations in SOS1, VAV1, RASA1, and ARHGAP35. Regarding neoantigens, 47% of the lung ADC and 53% of the lung SqCC tumors had at least five predicted neoepitopes. Although targeted therapies for lung ADC and SqCC are largely distinct, immunotherapies may aid in treatment for both subtypes.

Response and Acquired Resistance to Everolimus in Anaplastic Thyroid Cancer

Everolimus, an inhibitor of the mammalian target of rapamycin (mTOR), is effective in treating tumors harboring alterations in the mTOR pathway. Mechanisms of resistance to everolimus remain undefined. Resistance developed in a patient with metastatic anaplastic thyroid carcinoma after an extraordinary 18-month response. Whole-exome sequencing of pretreatment and drug-resistant tumors revealed a nonsense mutation in TSC2, a negative regulator of mTOR, suggesting a mechanism for exquisite sensitivity to everolimus. The resistant tumor also harbored a mutation in MTOR that confers resistance to allosteric mTOR inhibition. The mutation remains sensitive to mTOR kinase inhibitors.