Guangwu Guo

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.

Whole exome sequencing reveals frequent genetic alterations in BAP1, NF2, CDKN2A and CUL1 in malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is an aggressive neoplasm associated with asbestos exposure. Although previous studies based on candidate gene approaches have identified important common somatic mutations in MPM, these studies have focused on small sets of genes and have provided a limited view of the genetic alterations underlying this disease. Here, we performed whole-exome sequencing on DNA from 22 MPMs and matched blood samples, and identified 517 somatic mutations across 490 mutated genes. Integrative analysis of mutations and somatic copy-number alterations revealed frequent genetic alterations in BAP1, NF2, CDKN2A, and CUL1. Our study presents the first unbiased view of the genomic basis of MPM.

Genetic interrogation of circulating multiple myeloma cells at single-cell resolution

Circulating tumor cells provide a practical and noninvasive way to monitor multiple myeloma. Single-cell approach to multiple myeloma Multiple myeloma is a cancer of plasma cells in the bone marrow, and it remains very difficult to treat. Unfortunately, it is hard to tell when a patient is developing chemotherapy resistance because monitoring response to treatment in this cancer normally requires a bone marrow biopsy, an invasive and painful procedure that cannot be repeated frequently. Lohr et al. now demonstrate that circulating tumor cells in peripheral blood of multiple myeloma patients can provide the same genetic information as bone marrow samples, and sometimes more. This finding suggests that noninvasive monitoring of peripheral blood samples in multiple myeloma should be useful for tracking patients’ response to therapy and optimizing the treatment approach for each patient. Multiple myeloma (MM) remains an incurable disease, with a treatment-refractory state eventually developing in all patients. Constant clonal evolution and genetic heterogeneity of MM are a likely explanation for the emergence of drug-resistant disease. Monitoring of MM genomic evolution on therapy by serial bone marrow biopsy is unfortunately impractical because it involves an invasive and painful procedure. We describe how noninvasive and highly sensitive isolation and characterization of circulating tumor cells (CTCs) from peripheral blood at single-cell resolution recapitulate MM in the bone marrow. We demonstrate that CTCs provide the same genetic information as bone marrow MM cells and even reveal mutations with greater sensitivity than bone marrow biopsies in some cases. Single CTC RNA sequencing enables classification of MM and quantitative assessment of genes that are relevant for prognosis. We propose that the genomic characterization of CTCs should be included in clinical trials to follow the emergence of resistant subclones after MM therapy.

Insertions and Deletions Target Lineage-Defining Genes in Human Cancers

Certain cell types function as factories, secreting large quantities of one or more proteins that are central to the physiology of the respective organ. Examples include surfactant proteins in lung alveoli, albumin in liver parenchyma, and lipase in the stomach lining. Whole-genome sequencing analysis of lung adenocarcinomas revealed noncoding somatic mutational hotspots near VMP1/MIR21 and indel hotspots in surfactant protein genes (SFTPA1, SFTPB, and SFTPC). Extrapolation to other solid cancers demonstrated highly recurrent and tumor-type-specific indel hotspots targeting the noncoding regions of highly expressed genes defining certain secretory cellular lineages: albumin (ALB) in liver carcinoma, gastric lipase (LIPF) in stomach carcinoma, and thyroglobulin (TG) in thyroid carcinoma. The sequence contexts of indels targeting lineage-defining genes were significantly enriched in the AATAATD DNA motif and specific chromatin contexts, including H3K27ac and H3K36me3. Our findings illuminate a prevalent and hitherto unrecognized mutational process linking cellular lineage and cancer.

Genomic discovery and clonal tracking in multiple myeloma by cell-free DNA sequencing

Development of drug resistance [1, 2] and clonal evolution [3–5] is a major problem in multiple myeloma (MM) but its comprehensive longitudinal genetic characterization has been impractical thus far. We asked if copy number variations (CNV) and somatic mutations can be called robustly across the entire genome or exome, from cell-free DNA (cfDNA) in patients with active disease, and if clonal somatic mutations and CNVs found in the bone marrow (BM) are reliably reproduced by cfDNA. We hypothesized that (i) the subclonal composition of somatic mutations and CNVs differs, indicating that cfDNA and BM MM cells reveal distinct genetic information [6, 7] and (ii) cfDNA can be used to track disease load and clonal evolution of MM, to provide longitudinal genetic information about disease evolution that is not accessible by bone marrow biopsy [8, 9]. To first demonstrate whether low-pass whole-genome sequencing of cfDNA from MM patients is feasible, cfDNA was extracted from a total of 147 samples obtained from blood of 93 randomly selected MM patients, as well as from 12 healthy donors (Supplementary Table 1). We performed library construction, low-pass whole-genome sequencing (average depth of 0.22; range, 0.01–1×) and predicted segments of CNVs and estimates of tumor fraction (fraction of MM-derived cfDNA) using the ichorCNA [10] algorithm (Supplementary Figure 1). While CNVs were readily detected in cfDNA from MM patients, no CNVs were detected in cfDNA from healthy blood donors (predicted tumor fraction <0.05). Clinical data were available for 67 of 93 patients. Of those, we found that tumor fraction was ≥0.05 (detectable) in 16/26 (62%) cfDNA samples from patients with relapsed/refractory MM and 3/4 (75%) in cfDNA samples from patients with newly diagnosed MM, while tumor fraction in cfDNA from MM patients receiving treatment (35 patients with response and 2 patients with stable disease) was <0.05 (Fig. 1a). To evaluate the concordance of CNVs and mutations between cfDNA and matched BM myeloma, we performed whole-exome sequencing (WES) of cfDNA, BM-derived CD138CD45 sorted MM cells and CD138CD45 white blood cells as matched normal control from 10 patients to a median depth of 142× (range, 97–250×), 57× (11–137×) and 53× (34–80×), respectively (Supplementary Figure 1 and Supplementary Table 2). In an index patient (R13) These authors contributed equally: Guangwu Guo, Noopur S Raje.

Peptidomimetic Blockade of MYB in Acute Myeloid Leukemia

Aberrant gene expression is a hallmark of acute leukemias. However, therapeutic strategies for its blockade are generally lacking, largely due to the pharmacologic challenges of drugging transcription factors. MYB-driven gene trans-activation with CREB-binding protein (CBP)/P300 is required for the initiation and maintenance of a variety of acute lymphoblastic and myeloid leukemias, including refractory MLL-rearranged leukemias. Using structure-guided molecular design, we developed a prototypical peptidomimetic inhibitor MYBMIM that interferes with the assembly of the molecular MYB:CBP/P300 complex at micromolar concentrations and rapidly accumulates in the nuclei of AML cells. We found that treatment of AML cells with MYBMIM, led to the displacement of the MYB:CBP/P300 complex in cells, displacement of MYB from oncogenic enhancers and promoters enriched for MYB binding sites, and downregulation of MYB-dependent gene expression, including of MYC and BCL2 oncogenes. Both human MLL-rearranged and non-rearranged AML cells, underwent mitochondrial apoptosis in response to MYBMIM treatment, which could be partially rescued by ectopic expression of BCL2. We observed that MYBMIM treatment impeded leukemia growth and extended survival of immunodeficient mice engrafted with primary patient-derived MLL-rearranged leukemia cells. These findings emphasize the exquisite dependence of human AML on MYB:CBP/P300 transcriptional dysregulation, and establish a pharmacologic approach for its therapeutic blockade.

Abstract A1-01: Translational genomics of urologic cancer genes

Next generation sequencing (NGS) of urologic tumor genomes has led to the detection of frequently altered cancer genes. We have characterized bladder (BCa), prostate (PCa), and kidney (RCC) tumor genomes using exome, whole genome, and transcriptome NGS; copy number and SNP arrays, and bisulfite sequencing for methylation to detect genomic alterations associated with disease. We recently examined BCa tumors from 54 U.S. patients and compared these to 99 Chinese patients to identify novel alterations of cancer genes encoding proteins associated with DNA and chromatin, including telomerase ( TERT ), stromal antigen 2 ( STAG2 ), and BRCA1-associated protein-1 ( BAP1 ). We are the first to show the TERT promoter is altered in BCa by both somatic and germline nucleotide changes in 37/54 (69%) and 30/54 (56%) tumors, respectively; and that TERT somatic alterations do not correlate with other cancer gene mutations examined by exome NGS. Most TERT germline variants were novel (19/20), and three variants were confirmed as both somatic and germline in distinct tumors, including the most frequent variant, c. – 245 T>C (rs2853669). We show somatic promoter alterations are associated with increased TERT expression but, paradoxically, with significantly shorter telomeres in tumors as compared to matched normal tissue. STAG2 was altered by somatic deleterious sequence changes, genomic deletions, and promoter CpG hypermethylation. Mutations are associated with reduced patient survival and tumor cell aneuploidy based on an increased number of chromosomal arm copy number changes. Somatic BAP1 mutations are primarily deleterious sequence changes that occurred preferentially in U.S. Caucasian as compared to Chinese BCa patients. BAP1 mutations are associated with papillary histologic features in a subset of tumors and with mutations in the histone lysine–specific demethylase 6A ( KDM6A / UTX ) gene. BAP1 mutations contribute to a significant number of tumors with somatic and rare germline variants in BRCA pathway genes, including BRCA1 , BRCA2 , PALB2 , and ATM . We experimentally examined the function of KDM6A since it is the most frequently altered chromatin modifying gene in 24% of tumors. KDM6A loss in human BCa cells enhanced in vitro proliferation, in vivo tumor growth, and cell migration, confirming KDM6A loss drives the BCa phenotype. A comparison of BCa and other urologic tumor genomes allows us to identify several emergent common characteristics. Significant numbers of tumors are altered by rare, disease-associated germline cancer gene variants that likely have substantive effects on cancer risk and which likely influence the subsequent accrual of somatic alterations. These likely contribute to the missing heritability that is observed by cancer epidemiology studies. Many cancer genes are altered by characteristic frequencies of nonsynonymous sequence changes, promoter variants, copy number variation, and hypermethylation, that are routinely observed for a given gene-tissue-patient ethnicity combination. Approximately 4-5 tumor suppressors are altered for each oncogene, and interactions between altered cancer genes affect the resulting biological functions. In BCa, we show KDM6A loss increases cell proliferation which likely results in shorter telomeres even though TERT expression is increased. We identified altered cancer genes such as STAG2 that are associated with discrete clinical aspects of the disease phenotype, namely aneuploidy and reduced patient survival indicating a lethal subtype of disease. Cancer genes on the X chromosome such as STAG2 and KDM6A have a single copy in men and likely contribute to the gender bias observed in cancer. Finally, cancer gene alterations can identify patients likely to respond to a currently approved therapy. BAP1 and BRCA pathway alterations define BCa, PCa, and RCC tumors with a common DNA repair deficiency that might be exploited by targeted therapies such as poly (ADP ribose) polymerase inhibitors. Citation Format: Michael L. Nickerson, Kate M. Im, Sevilay Turan, Guangwu Guo, Lee E. Moore, Dan Theodorescu, Michael Dean. Translational genomics of urologic cancer genes. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-01.