Large-scale genomic characterization of tumors from prospective cohort studies may yield new insights into cancer pathogenesis. We performed whole-exome sequencing of 619 incident colorectal cancers ...(CRCs) and integrated the results with tumor immunity, pathology, and survival data. We identified recurrently mutated genes in CRC, such as BCL9L, RBM10, CTCF, and KLF5, that were not previously appreciated in this disease. Furthermore, we investigated the genomic correlates of immune-cell infiltration and found that higher neoantigen load was positively associated with overall lymphocytic infiltration, tumor-infiltrating lymphocytes (TILs), memory T cells, and CRC-specific survival. The association with TILs was evident even within microsatellite-stable tumors. We also found positive selection of mutations in HLA genes and other components of the antigen-processing machinery in TIL-rich tumors. These results may inform immunotherapeutic approaches in CRC. More generally, this study demonstrates a framework for future integrative molecular epidemiology research in colorectal and other malignancies.
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•Whole-exome sequencing of 619 colorectal cancers with clinicopathologic annotations•Discovery of significantly mutated genes in colorectal cancer•Neoantigen load correlation with infiltrating lymphocytes and memory T cells•Positive selection for HLA mutations in immune-cell-infiltrated tumors
Through whole-exome sequencing of annotated colorectal tumors, Giannakis et al. identify additional colorectal cancer driver genes and correlate high neoantigen load with increased lymphocytic infiltration and improved survival. They also find positive selection for HLA mutations in immune-cell-infiltrated tumors. These results may inform immunotherapeutic approaches in colorectal cancer.
BRAF mutations occur in approximately 10% of colorectal cancers. Although RAF inhibitor monotherapy is highly effective in BRAF-mutant melanoma, response rates in BRAF-mutant colorectal cancer are ...poor. Recent clinical trials of combined RAF/EGFR or RAF/MEK inhibition have produced improved efficacy, but patients ultimately develop resistance. To identify molecular alterations driving clinical acquired resistance, we performed whole-exome sequencing on paired pretreatment and postprogression tumor biopsies from patients with BRAF-mutant colorectal cancer treated with RAF inhibitor combinations. We identified alterations in MAPK pathway genes in resistant tumors not present in matched pretreatment tumors, including KRAS amplification, BRAF amplification, and a MEK1 mutation. These alterations conferred resistance to RAF/EGFR or RAF/MEK combinations through sustained MAPK pathway activity, but an ERK inhibitor could suppress MAPK activity and overcome resistance. Identification of MAPK pathway reactivating alterations upon clinical acquired resistance underscores the MAPK pathway as a critical target in BRAF-mutant colorectal cancer and suggests therapeutic options to overcome resistance.
RAF inhibitor combinations represent promising approaches in clinical development for BRAF-mutant colorectal cancer. Initial characterization of clinical acquired resistance mechanisms to these regimens identified several MAPK pathway alterations driving resistance by reactivating MAPK signaling, highlighting the critical dependence of BRAF-mutant colorectal cancers on MAPK signaling and offering potential strategies to overcome resistance.
African-American men have the highest incidence of and mortality from prostate cancer. Whether a biological basis exists for this disparity remains unclear. Exome sequencing (
= 102) and targeted ...validation (
= 90) of localized primary hormone-naïve prostate cancer in African-American men identified several gene mutations not previously observed in this context, including recurrent loss-of-function mutations in
, an ETS transcriptional repressor, in 5% of cases. Analysis of existing prostate cancer cohorts revealed
deletions in 3% of primary prostate cancers and mutations or deletions in
in 3% to 5% of lethal castration-resistant prostate cancers. Knockdown of
confers increased anchorage-independent growth and generates a gene expression signature associated with oncogenic ETS activation and androgen signaling. Together, these results suggest that
is a prostate cancer tumor-suppressor gene. More generally, our findings support the application of systematic cancer genomic characterization in settings of broader ancestral diversity to enhance discovery and, eventually, therapeutic applications.
Systematic genomic sequencing of prostate cancer in African-American men revealed new insights into prostate cancer, including the identification of
as a prostate cancer gene; somatic copy-number alteration differences; and uncommon
and
alterations. This study highlights the importance of inclusion of underrepresented minorities in cancer sequencing studies.
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Background: The melanocortin-4 receptor (MC4R) pathway is critical for the regulation of energy balance. Variants within genes comprising this pathway, including POMC, PCSK1, LEPR, SH2B1, and NCOA1 ...(also known as SRC1), have a well-established association with severe obesity. However, the frequency of variants in these genes has not been assessed systematically in a clinically relevant US population. Methods: We sequenced POMC, PCSK1, LEPR, SH2B1, and NCOA1 exons and intron-exon boundaries in 45,866 US individuals with severe obesity (<18 years old, >97th percentile BMI for age; >18 years old, BMI >40kg/m2). This cohort is comprised of individuals sequenced across multiple initiatives, including the Uncovering Rare Obesity® diagnostic genetic testing program. In the current analysis, we included rare variants classified as pathogenic/likely pathogenic (P/LP) or as a variant of uncertain significance (VUS) according to ACMG criteria. Results: 8.2% of individuals with severe obesity carried >1 rare variants in >1 of the 5 studied genes, including 0.3% who carried a P/LP variant and 7.9% who carried a VUS variant. Within the context of a community-focused clinical diagnostic tool, Uncovering Rare Obesity® results demonstrated a higher frequency of P/LP of 0.6% and a 10.0% frequency of VUS genotypes. Conclusions: Overall, in our large US-based cohort of individuals with severe, early-onset obesity, ~8% of individuals carry >1 potentially clinically relevant rare variants in the 5 MC4R pathway genes POMC, PCSK1, LEPR, SH2B1, and NCOA1. Understanding the role of these variants in the pathophysiology of obesity may improve the clinical care of individuals living with these rare genetic diseases of obesity.
Background: The melanocortin 4 receptor (MC4R) pathway is critical for the regulation of energy balance. Variants within genes comprising this pathway, including POMC, PCSK1, LEPR, SH2B1, and SRC1, ...have a well-established association with severe obesity. However, the frequency of variants in these genes have not been assessed systematically in a clinically relevant US population. Methods: We sequenced POMC, PCSK1, LEPR, SH2B1, and SRC1 exons and intron-exon boundaries in 35,276 US individuals with severe obesity (<18 years old, >97th percentile BMI for age; >18 years old, BMI >40kg/m2). This cohort is comprised of individuals sequenced across multiple initiatives, including the Uncovering Rare Obesity (URO) diagnostic genetic testing program. In the current analysis, we included rare variants classified as pathogenic/likely pathogenic (P/LP) or as a variant of uncertain significance (VUS) according to ACMG criteria. We additionally included one non-rare variant, PCSK1 p.N221D, which for which published functional and population studies suggests a potential contribution to obesity. Results: 10.2% of individuals with severe obesity carried >1 rare variants in >1 of the 5 studied genes, including 0.7% who carry a P/ LP variant and 9.5% who carry a VUS variant. An additional 5.4% carried the PCSK1 p.N221D variant. Within the context of a community focused clinical diagnostic tool, URO demonstrated a slightly higher frequency of P/LP and PCSK1 N221D genotypes, 1.2% and 6.9%, respectively, and a 9.8% frequency of VUS genotypes. Conclusions: Overall, in our large US-based cohort of individuals with severe early-onset obesity, 15.6% of individuals carry a potentially clinically relevant variant in the MC4R pathway genes POMC, PCSK1, LEPR, SH2B1, and SRC1. Understanding the role of these variants in the pathophysiology of obesity may improve the clinical care of individuals living with these rare genetic diseases of obesity.
Cisplatin-based chemotherapy is the standard of care for patients with muscle-invasive urothelial carcinoma. Pathologic downstaging to pT0/pTis after neoadjuvant cisplatin-based chemotherapy is ...associated with improved survival, although molecular determinants of cisplatin response are incompletely understood. We performed whole-exome sequencing on pretreatment tumor and germline DNA from 50 patients with muscle-invasive urothelial carcinoma who received neoadjuvant cisplatin-based chemotherapy followed by cystectomy (25 pT0/pTis "responders," 25 pT2+ "nonresponders") to identify somatic mutations that occurred preferentially in responders. ERCC2, a nucleotide excision repair gene, was the only significantly mutated gene enriched in the cisplatin responders compared with nonresponders (q < 0.01). Expression of representative ERCC2 mutants in an ERCC2-deficient cell line failed to rescue cisplatin and UV sensitivity compared with wild-type ERCC2. The lack of normal ERCC2 function may contribute to cisplatin sensitivity in urothelial cancer, and somatic ERCC2 mutation status may inform cisplatin-containing regimen usage in muscle-invasive urothelial carcinoma.
Somatic ERCC2 mutations correlate with complete response to cisplatin-based chemosensitivity in muscle-invasive urothelial carcinoma, and clinically identified mutations lead to cisplatin sensitivity in vitro. Nucleotide excision repair pathway defects may drive exceptional response to conventional chemotherapy.
Pam and its homologs (the PHR protein family) are large E3 ubiquitin ligases that function to regulate synapse formation and growth in mammals, zebrafish, Drosophila, and Caenorhabditis elegans. ...Phr1-deficient mouse models (Phr1Δ8,9 and Phr1Magellan, with deletions in the N-terminal putative guanine exchange factor region and the C-terminal ubiquitin ligase region, respectively) exhibit axon guidance/outgrowth defects and striking defects of major axon tracts in the CNS. Our earlier studies identified Pam to be associated with tuberous sclerosis complex (TSC) proteins, ubiquitinating TSC2 and regulating mammalian/mechanistic target of rapamycin (mTOR) signaling. Here, we examine the potential involvement of the TSC/mTOR complex 1(mTORC1) signaling pathway in Phr1-deficient mouse models. We observed attenuation of mTORC1 signaling in the brains of both Phr1Δ8,9 and Phr1Magellan mouse models. Our results establish that Pam regulates TSC/mTOR signaling in vitro and in vivo through two distinct domains. To further address whether Pam regulates mTORC1 through two functionally independent domains, we undertook heterozygous mutant crossing between Phr1Δ8,9 and Phr1Magellan mice to generate a compound heterozygous model to determine whether these two domains can complement each other. mTORC1 signaling was not attenuated in the brains of double mutants (Phr1Δ8,9/Mag), confirming that Pam displays dual regulation of the mTORC1 pathway through two functional domains. Our results also suggest that although dysregulation of mTORC1 signaling may be responsible for the corpus callosum defects, other neurodevelopmental defects observed with Phr1 deficiency are independent of mTORC1 signaling. The ubiquitin ligase complex containing Pam-Fbxo45 likely targets additional synaptic and axonal proteins, which may explain the overlapping neurodevelopmental defects observed in Phr1 and Fbxo45 deficiency.
Background: Pam and its homologs act as key regulators of axon guidance and outgrowth and synapse development.
Results: Pam regulates mTORC1 signaling in vivo, and attenuated mTORC1 signaling may partly explain the axonal defects in Phr1-deficient mice.
Conclusion: mTORC1-dependent and -independent mechanisms contribute to neurodevelopmental defects in Phr1 deficiency.
Significance: Understanding how Pam regulates synapse development will have direct relevance for diverse aspects of neural development.
Several recent next generation sequencing studies have uncovered mutations in key genes that contribute to CLL pathogenesis. Most CLLs with deletions of chromosome 13q (del(13q), incidence 50-60%) or ...normal cytogenetics (incidence 15-20%) do not carry these recurrent driver mutations. Clues to the genetic basis of disease in this more indolent group likely lie elsewhere in the genome.
We present preliminary data from whole-genome sequencing (WGS) of a cohort of CLL patients with del(13q) (n=13) and normal cytogenetics (n=10), sequenced at 30X and 60X coverage for tumor and normal (saliva) respectively. Of the 23 patients, 43% were male and the median age at diagnosis was 53 years. The median time from diagnosis to sampling was 1.9 years, and 22 of 23 patients were previously untreated. With a median follow-up of 4.8 years in the overall cohort, 8 patients (35%) had been treated at a median 2.5 years from diagnosis. Although indolent by cytogenetics and of low Rai stage (median 1), the cohort includes balanced numbers of ZAP70± and IGHV mutated/unmutated cases.
A total of 68,928 point mutations with an average of 2,995 mutations per genome were detected in our cohort using the MuTect algorithm, which is approximately 60% fewer mutations than in multiple myeloma. The pattern of mutations in protein coding regions was similar to previous exome sequencing studies, with an average of 18.7 ±7.4 non-synonymous mutations per case (0.57±0.2 per Mb). The rate of mutations was significantly lower in patients diagnosed at a younger age (p=0.016, 2 sided t test). As expected based on the cytogenetic makeup of our cohort, the incidence of mutations in the 20 known CLL driver genes did not reach genome wide significance. However, the incidence of mutations in these driver genes was significantly higher in patients with normal cytogenetics as compared to del(13q) (60% vs 18%, p=0.039).
Rearrangement events were identified using the dRanger algorithm followed by mapping of the precise breakpoint to single base pair resolution using the Breakpointer algorithm. A total of 78 rearrangements were identified, corresponding to a median of 3 (range 0-15) per CLL genome, significantly fewer than in most other cancers. Similar to other hematologic malignancies, the incidence of deletions (70%) was significantly higher than that of tandem duplications (9%) (Wilcoxon signed rank test, p=0.0001). In addition, inter-chromosomal translocations exhibited a strong positive correlation with long range deletions (Pearson’s r=0.72, p=0.0001) and a moderate positive correlation with inversions (Pearson’s r=0.64, p=0.0009), suggesting a common mechanism. The only clinical feature associated with the presence of structural rearrangements was ZAP70 positivity (p=0.046).
Of the 13 patients with del(13q), 4 were due to inter-chromosomal rearrangement that led to loss of the common minimal region of deletion. The other most recurrent rearrangement partners were chr.2 (n=12 cases, 52%), chr.14 (n=9 cases, 39%) and chr.1 (n=5 cases, 21%), while no rearrangements were seen in chromosomes 8,11,17,18 and 19. The remaining chromosomes were affected in <20% cases. Chr.2 events accounted for 32% of rearrangements (25/78) with breakpoints scattered throughout the chromosome and consisting of 32% inversions, 28% long-range deletions, 20% inter-chromosomal translocations, 16% deletions and 4% tandem-duplications. Chr. 2p16 gain, a recurrent chromosomal abnormality in CLL, was not detected in our cohort, and therefore not associated with these rearrangements.
Chr.14 accounted for 17% of rearrangements (13/78), with breakpoints concentrated in the 5’IgH region (chr.14q32.33), in the vicinity of the KIAA0125 gene. Rearrangements involving chr.14q32.33 (n=11 total, 7 deletions) were associated with a shorter time to first therapy (TTFT) (HR=2.88, p=0.013), despite no association with IGHV or ZAP70 status.
In summary, this cohort of CLLs with del(13q) and normal cytogenetics represents the largest WGS cohort reported to date and underscores the stability of the CLL genome. Nonetheless, structural rearrangements are common, and the number of non-13q rearrangements was associated with higher risk ZAP70 status. Furthermore 5’IgH rearrangements enriched in deletions were associated with shorter TTFT within this lower risk subgroup. Additional WGS data on novel somatic mutations and regulatory regions will be presented at the meeting.
No relevant conflicts of interest to declare.
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