Whole-genome analysis identifies novel drivers and high-risk double-hit events in relapsed/refractory myeloma.

Large-scale analyses of genomic data from patients with newly diagnosed multiple myeloma (ndMM) have been undertaken, however, large-scale analysis of relapsed/refractory MM (rrMM) has not been performed. We hypothesize that somatic variants chronicle the therapeutic exposures and clonal structure of myeloma from ndMM to rrMM stages. We generated whole-genome sequencing (WGS) data from 418 tumors (386 patients) derived from 6 rrMM clinical trials and compared them with WGS from 198 unrelated patients with ndMM in a population-based case-control fashion. We identified significantly enriched events at the rrMM stage, including drivers (DUOX2, EZH2, TP53), biallelic inactivation (TP53), noncoding mutations in bona fide drivers (TP53BP1, BLM), copy number aberrations (CNAs; 1qGain, 17pLOH), and double-hit events (Amp1q-ISS3, 1qGain-17p loss-of-heterozygosity). Mutational signature analysis identified a subclonal defective mismatch repair signature enriched in rrMM and highly active in high mutation burden tumors, a likely feature of therapy-associated expanding subclones. Further analysis focused on the association of genomic aberrations enriched at different stages of resistance to immunomodulatory agent (IMiD)-based therapy. This analysis revealed that TP53, DUOX2, 1qGain, and 17p loss-of-heterozygosity increased in prevalence from ndMM to lenalidomide resistant (LENR) to pomalidomide resistant (POMR) stages, whereas enrichment of MAML3 along with immunoglobulin lambda (IGL) and MYC translocations distinguished POM from the LEN subgroup. Genomic drivers associated with rrMM are those that confer clonal selective advantage under therapeutic pressure. Their role in therapy evasion should be further evaluated in longitudinal patient samples, to confirm these associations with the evolution of clinical resistance and to identify molecular subsets of rrMM for the development of targeted therapies.

Loss of COP9 signalosome genes at 2q37 is associated with IMiD resistance in multiple myeloma.

The acquisition of a multidrug refractory state is a major cause of mortality in myeloma. Myeloma drugs that target the cereblon (CRBN) protein include widely used immunomodulatory drugs (IMiDs), and newer CRBN E3 ligase modulator drugs (CELMoDs), in clinical trials. CRBN genetic disruption causes resistance and poor outcomes with IMiDs. Here, we investigate alternative genomic associations of IMiD resistance, using large whole-genome sequencing patient datasets (n = 522 cases) at newly diagnosed, lenalidomide (LEN)-refractory and lenalidomide-then-pomalidomide (LEN-then-POM)-refractory timepoints. Selecting gene targets reproducibly identified by published CRISPR/shRNA IMiD resistance screens, we found little evidence of genetic disruption by mutation associated with IMiD resistance. However, we identified a chromosome region, 2q37, containing COP9 signalosome members COPS7B and COPS8, copy loss of which significantly enriches between newly diagnosed (incidence 5.5%), LEN-refractory (10.0%), and LEN-then-POM-refractory states (16.4%), and may adversely affect outcomes when clonal fraction is high. In a separate dataset (50 patients) with sequential samples taken throughout treatment, we identified acquisition of 2q37 loss in 16% cases with IMiD exposure, but none in cases without IMiD exposure. The COP9 signalosome is essential for maintenance of the CUL4-DDB1-CRBN E3 ubiquitin ligase. This region may represent a novel marker of IMiD resistance with clinical utility.

Multiple cereblon genetic changes are associated with acquired resistance to lenalidomide or pomalidomide in multiple myeloma.

Emergence of drug resistance to all available therapies is the major challenge to improving survival in myeloma. Cereblon (CRBN) is the essential binding protein of the widely used immunomodulatory drugs (IMiDs) and novel CRBN E3 ligase modulator drugs (CELMoDs) in myeloma, as well as certain proteolysis targeting chimeras (PROTACs), in development for a range of diseases. Using whole-genome sequencing (WGS) data from 455 patients and RNA sequencing (RNASeq) data from 655 patients, including newly diagnosed (WGS, n = 198; RNASeq, n = 437), lenalidomide (LEN)-refractory (WGS, n = 203; RNASeq, n = 176), and pomalidomide (POM)-refractory cohorts (WGS, n = 54; RNASeq, n = 42), we found incremental increases in the frequency of 3 CRBN aberrations, namely point mutations, copy losses/structural variations, and a specific variant transcript (exon 10 spliced), with progressive IMiD exposure, until almost one-third of patients had CBRN alterations by the time they were POM refractory. We found all 3 CRBN aberrations were associated with inferior outcomes to POM in those already refractory to LEN, including those with gene copy losses and structural variations, a finding not previously described. This represents the first comprehensive analysis and largest data set of CBRN alterations in myeloma patients as they progress through therapy. It will help inform patient selection for sequential therapies with CRBN-targeting drugs.

Genetic legacy of state centralization in the Kuba Kingdom of the Democratic Republic of the Congo.

Few phenomena have had as profound or long-lasting consequences in human history as the emergence of large-scale centralized states in the place of smaller scale and more local societies. This study examines a fundamental, and yet unexplored, consequence of state formation: its genetic legacy. We studied the genetic impact of state centralization during the formation of the eminent precolonial Kuba Kingdom of the Democratic Republic of the Congo (DRC) in the 17th century. We analyzed genome-wide data from over 690 individuals sampled from 27 different ethnic groups from the Kasai Central Province of the DRC. By comparing genetic patterns in the present-day Kuba, whose ancestors were part of the Kuba Kingdom, with those in neighboring non-Kuba groups, we show that the Kuba today are more genetically diverse and more similar to other groups in the region than expected, consistent with the historical unification of distinct subgroups during state centralization. We also found evidence of genetic mixing dating to the time of the Kingdom at its most prominent. Using this unique dataset, we characterize the genetic history of the Kasai Central Province and describe the historic late wave of migrations into the region that contributed to a Bantu-like ancestry component found across large parts of Africa today. Taken together, we show the power of genetics to evidence events of sociopolitical importance and highlight how DNA can be used to better understand the behaviors of both people and institutions in the past.

Group-based pharmacogenetic prediction: is it feasible and do current NHS England ethnic classifications provide appropriate data?

Inter-individual variation of drug metabolising enzymes (DMEs) leads to variable efficacy of many drugs and even adverse drug responses. Consequently, it would be desirable to test variants of many DMEs before drug treatment. Inter-ethnic differences in frequency mean that the choice of SNPs to test may vary across population groups. Here we examine the utility of testing representative groups as a way of assessing what variants might be tested. We show that publicly available population information is potentially useful for determining loci for pre-treatment genetic testing, and for determining the most prevalent risk haplotypes in defined groups. However, we also show that the NHS England classifications have limitations for grouping for these purposes, in particular for people of African descent. We conclude: (1) genotyping of hospital patients and people from the hospital catchment area confers no advantage over using samples from appropriate existing ethnic group collections or publicly available data, (2) given the current NHS England Black African grouping, a decision as to whether to test, would have to apply to all patients of recent Black African ancestry to cover reported risk alleles and (3) the current scarcity of available genome and drug effect data from Africans is a problem for both testing and treatment decisions.

Association of MSY haplotype background with nonobstructive azoospermia is AZF-dependent: A case-control study.

Identifying causal genes of spermatogenic failure on the male-specific region of Y chromosome (MSY) has been a challenging process. Due to the nonrecombining nature of MSY, haplotype-based approaches have recently been shown to be promising in identifying associated MSY haplogroups. We conducted an MSY analysis of nonobstructive azoospermia (NOA) patients in a case-control setting (N = 278 and 105 respectively) to identify modal haplogroups strongly associated with NOA. Patients with AZF deletions (AZF+) and no AZF deletions (AZF-) were compared with the control group. Given the larger sample set of AZF- NOA patients, we further investigated the association based on histopathological severity, namely Sertoli cell-only syndrome and maturation arrest subtypes. We observed no significant enrichment of MSY haplogroups in AZF- azoospermic patients (or its subtypes). However, we observed a strongly significant association between haplogroup J2a* and AZF+ patients (FDR-corrected p = .0056; OR = 7.02, 95%CI 1.89 to 39.20), a haplogroup which also showed significant enrichment for AZFa/b deletions (p = 4x10-4 ). We conclude that unlike AZF+ patients, AZF- NOA are less likely to have an MSY causative factor with large effect size, thus indicating that the aetiology of AZF- NOA, and to some extent AZFc NOA, is more likely to be based on non-MSY factors.

The Genetic Legacy of Zoroastrianism in Iran and India: Insights into Population Structure, Gene Flow, and Selection.

Zoroastrianism is one of the oldest extant religions in the world, originating in Persia (present-day Iran) during the second millennium BCE. Historical records indicate that migrants from Persia brought Zoroastrianism to India, but there is debate over the timing of these migrations. Here we present genome-wide autosomal, Y chromosome, and mitochondrial DNA data from Iranian and Indian Zoroastrians and neighboring modern-day Indian and Iranian populations and conduct a comprehensive genome-wide genetic analysis in these groups. Using powerful haplotype-based techniques, we find that Zoroastrians in Iran and India have increased genetic homogeneity relative to other sampled groups in their respective countries, consistent with their current practices of endogamy. Despite this, we infer that Indian Zoroastrians (Parsis) intermixed with local groups sometime after their arrival in India, dating this mixture to 690-1390 CE and providing strong evidence that Iranian Zoroastrian ancestry was maintained primarily through the male line. By making use of the rich information in DNA from ancient human remains, we also highlight admixture in the ancestors of Iranian Zoroastrians dated to 570 BCE-746 CE, older than admixture seen in any other sampled Iranian group, consistent with a long-standing isolation of Zoroastrians from outside groups. Finally, we report results, and challenges, from a genome-wide scan to identify genomic regions showing signatures of positive selection in present-day Zoroastrians that might correlate to the prevalence of particular diseases among these communities.

Identification of a missense variant in CLDN2 in obstructive azoospermia.

Obstructive azoospermia (OA), defined as an obstruction in any region of the male genital tract, accounts for 40% of all azoospermia cases. Of all OA cases, ~30% are thought to have a genetic origin, however, hitherto, the underlying genetic etiology of the majority of these cases remain unknown. To address this, we took a family-based whole-exome sequencing approach to identify causal variants of OA in a multiplex family with epidydimal obstruction. A novel gain-of-function missense variant in CLDN2 (c.481G>C; p.Gly161Arg) was found to co-segregate with the phenotype, consistent with the X-linked inheritance pattern observed in the pedigree. To assess the pathogenicity of this variant, the wild and mutant protein structures were modeled and their potential for strand formation in multimeric form was assessed and compared. The results showed that dimeric and tetrameric arrangements of Claudin-2 were not only reduced, but were also significantly altered by this single residue change. We, therefore, envisage that this amino acid change likely forms a polymeric discontinuous strand, which may lead to the disruption of tight junctions among epithelial cells. This missense variant is thus likely to be responsible for the disruption of the blood-epididymis barrier, causing dislodged epithelial cells to clog the genital tract, hence causing OA. This study not only sheds light on the underlying pathobiology of OA, but also provides a basis for more efficient diagnosis in the clinical setting.

Mosaic PPM1D mutations are associated with predisposition to breast and ovarian cancer.

Improved sequencing technologies offer unprecedented opportunities for investigating the role of rare genetic variation in common disease. However, there are considerable challenges with respect to study design, data analysis and replication. Using pooled next-generation sequencing of 507 genes implicated in the repair of DNA in 1,150 samples, an analytical strategy focused on protein-truncating variants (PTVs) and a large-scale sequencing case-control replication experiment in 13,642 individuals, here we show that rare PTVs in the p53-inducible protein phosphatase PPM1D are associated with predisposition to breast cancer and ovarian cancer. PPM1D PTV mutations were present in 25 out of 7,781 cases versus 1 out of 5,861 controls (P = 1.12 × 10(-5)), including 18 mutations in 6,912 individuals with breast cancer (P = 2.42 × 10(-4)) and 12 mutations in 1,121 individuals with ovarian cancer (P = 3.10 × 10(-9)). Notably, all of the identified PPM1D PTVs were mosaic in lymphocyte DNA and clustered within a 370-base-pair region in the final exon of the gene, carboxy-terminal to the phosphatase catalytic domain. Functional studies demonstrate that the mutations result in enhanced suppression of p53 in response to ionizing radiation exposure, suggesting that the mutant alleles encode hyperactive PPM1D isoforms. Thus, although the mutations cause premature protein truncation, they do not result in the simple loss-of-function effect typically associated with this class of variant, but instead probably have a gain-of-function effect. Our results have implications for the detection and management of breast and ovarian cancer risk. More generally, these data provide new insights into the role of rare and of mosaic genetic variants in common conditions, and the use of sequencing in their identification.

Testis-Specific Y-Centric Protein-Protein Interaction Network Provides Clues to the Etiology of Severe Spermatogenic Failure.

Pinpointing causal genes for spermatogenic failure (SpF) on the Y chromosome has been an ever daunting challenge with setbacks during the past decade. Since complex diseases result from the interaction of multiple genes and also display considerable missing heritability, network analysis is more likely to explicate an etiological molecular basis. We therefore took a network medicine approach by integrating interactome (protein-protein interaction (PPI)) and transcriptome data to reconstruct a Y-centric SpF network. Two sets of seed genes (Y genes and SpF-implicated genes (SIGs)) were used for network reconstruction. Since no PPI was observed among Y genes, we identified their common immediate interactors. Interestingly, 81% (N = 175) of these interactors not only interacted directly with SIGs, but also they were enriched for differentially expressed genes (89.6%; N = 43). The SpF network, formed mainly by the dys-regulated interactors and the two seed gene sets, comprised three modules enriched for ribosomal proteins and nuclear receptors for sex hormones. Ribosomal proteins generally showed significant dys-regulation with RPL39L, thought to be expressed at the onset of spermatogenesis, strongly down-regulated. This network is the first global PPI network pertaining to severe SpF and if experimentally validated on independent data sets can lead to more accurate diagnosis and potential fertility recovery of patients.

Contrasting exome constancy and regulatory region variation in the gene encoding CYP3A4: an examination of the extent and potential implications.

OBJECTIVE: CYP3A4 expression varies up to 100-fold among individuals, and, to date, genetic causes remain elusive. As a major drug-metabolizing enzyme, elucidation of such genetic causes would increase the potential for introducing personalized dose adjustment of therapies involving CYP3A4 drug substrates. The foetal CYP3A isoform, CYP3A7, is reported to be expressed in ∼10% of European adults and may thus contribute towards the metabolism of endogenous substances and CYP3A drug substrates. However, little is known about the distribution of the variant expressed in the adult. METHODS: We resequenced the exons, flanking introns, regulatory elements and 3'UTR of CYP3A4 in five Ethiopian populations and incorporated data from the 1000 Genomes Project. Using bioinformatic analysis, we assessed likely consequences of observed CYP3A4 genomic variation. We also conducted the first extensive geographic survey of alleles associated with adult expression of CYP3A7 - that is, CYP3A7*1B and CYP3A7*1C. RESULTS AND CONCLUSION: Ethiopia contained 60 CYP3A4 variants (26 novel) and more variants (>1%) than all non-African populations combined. No nonsynonymous mutation was found in the homozygous form or at more than 2.8% in any population. Seventy-nine per cent of haplotypes contained 3'UTR and/or regulatory region variation with striking pairwise population differentiation, highlighting the potential for interethnic variation in CYP3A4 expression. Conversely, coding region variation showed that significant interethnic variation is unlikely at the protein level. CYP3A7*1C was found at up to 17.5% in North African populations and in significant linkage disequilibrium with CYP3A5*3, indicating that adult expression of the foetal isoform is likely to be accompanied by reduced or null expression of CYP3A5.

Palenque de San Basilio in Colombia: genetic data support an oral history of a paternal ancestry in Congo.

The Palenque, a black community in rural Colombia, have an oral history of fugitive African slaves founding a free village near Cartagena in the seventeenth century. Recently, linguists have identified some 200 words in regular use that originate in a Kikongo language, with Yombe, mainly spoken in the Congo region, being the most likely source. The non-recombining portion of the Y chromosome (NRY) and mitochondrial DNA were analysed to establish whether there was greater similarity between present-day members of the Palenque and Yombe than between the Palenque and 42 other African groups (for all individuals,n= 2799) from which forced slaves might have been taken. NRY data are consistent with the linguistic evidence that Yombe is the most likely group from which the original male settlers of Palenque came. Mitochondrial DNA data suggested substantial maternal sub-Saharan African ancestry and a strong founder effect but did not associate Palenque with any particular African group. In addition, based on cultural data including inhabitants' claims of linguistic differences, it has been hypothesized that the two districts of the village (Abajo and Arriba) have different origins, with Arriba founded by men originating in Congo and Abajo by those born in Colombia. Although significant genetic structuring distinguished the two from each other, no supporting evidence for this hypothesis was found.

Isoform-Level Gene Expression Profiles of Human Y Chromosome Azoospermia Factor Genes and Their X Chromosome Paralogs in the Testicular Tissue of Non-Obstructive Azoospermia Patients.

The human Y chromosome has an inevitable role in male fertility because it contains many genes critical for spermatogenesis and the development of the male gonads. Any genetic variation or epigenetic modification affecting the expression pattern of Y chromosome genes may thus lead to male infertility. In this study, we performed isoform-level gene expression profiling of Y chromosome genes within the azoospermia factor (AZF) regions, their X chromosome counterparts, and few autosomal paralogues in testicular biopsies of 12 men with preserved spermatogenesis and 68 men with nonobstructive azoospermia (NOA) (40 Sertoli-cell-only syndrome (SCOS) and 28 premiotic maturation arrest (MA)). This was undertaken using quantitative real-time PCR (qPCR) at the transcript level and Western blotting (WB) and immunohistochemistry (IHC) at the protein level. We profiled the expression of 41 alternative transcripts encoded by 14 AZFa, AZFb, and AZFc region genes (USP9Y, DDX3Y, XKRY, HSFY1, CYORF15A, CYORF15B, KDM5D, EIF1AY, RPS4Y2, RBMY1A1, PRY, BPY2, DAZ1, and CDY1) as well as their X chromosome homologue transcripts and a few autosomal homologues. Of the 41 transcripts, 18 were significantly down-regulated in men with NOA when compared with those of men with complete spermatogenesis. In contrast, the expression of five transcripts increased significantly in NOA patients. Furthermore, to confirm the qPCR results at the protein level, we performed immunoblotting and IHC experiments (based on 24 commercial and homemade antibodies) that detected 10 AZF-encoded proteins. In addition, their localization in testis cell types and organelles was determined. Interestingly, the two missing proteins, XKRY and CYORF15A, were detected for the first time. Finally, we focused on the expression patterns of the significantly altered genes in 12 MA patients with successful sperm retrieval compared to those of 12 MA patients with failed sperm retrieval to predict the success of sperm retrieval in azoospermic men. We showed that HSFY1-1, HSFY1-3, BPY2-1, KDM5C2, RBMX2, and DAZL1 transcripts could be used as potential molecular markers to predict the presence of spermatozoa in MA patients. In this study, we have identified isoform level signature that can be used to discriminate effectively between MA, SCOS, and normal testicular tissues and suggests the possibility of diagnosing the presence of mature sperm cell in azoospermic men to prevent additional testicular sperm extraction (TESE) surgery.

In silico engineering a CD80 variant with increased affinity to CTLA-4 and decreased affinity to CD28 for optimized cancer immunotherapy.

CD80 or cluster of differentiation 80, also known as B7-1, is a member of the immunoglobulin super family, which binds to CTLA-4 and CD28 T cell receptors and induces inhibitory and inductive signals respectively. Although CTLA-4 and CD28 receptors belong to the same protein family, slight differences in their structures leads to CD80 having a higher binding affinity to CTLA-4 (-14.55 kcal/mol) compared with CD28(-12.51 kcal/mol). In this study, we constructed a variant of CD80 protein with increased binding affinity to CTLA-4 and decreased binding affinity to CD28. This variant has no signaling capability, and can act as a cap for these receptors to protect them from natural CD80 proteins existing in the body. The first step was the evolutionary and alanine scanning analysis of CD80 protein to determine conserved regions in this protein. Next, complex alanine scanning technique was employed to determine CD80 protein hotspots in CD80-CTLA-4 and CD80-CD28 protein complexes. This information was fed into a computational model developed in R for in silico mutagenesis and CD80 variant library construction. The 3D structures of variants were modeled using the Swiss model webserver. After modeling the 3D structures, HADDOCK server was employed to build all protein-protein complexes, which contain CTLA-4-CD80 variant complexes, Wild type CD80-CD28 complexes and CD28-CD80 variant complexes. Protein-protein binding free energy was determined using FoldX and the variant number 316 with mutations at 29, 31, 33 positions showed increased binding affinity to CTLA-4 (-21.43 kcal/mol) and decreased binding affinity to CD28 (- 9.54 kcal/mol). Finally, molecular dynamics (MD) simulations confirmed the stability of variant 316. In conclusion, we designed a new CD80 protein variant with potential immunotherapeutic applications.

Landscape of Genetic Alterations Underlying Hallmark Signature Changes in Cancer Reveals TP53 Aneuploidy-driven Metabolic Reprogramming.

The hallmark signatures based on gene expression capture core cancer processes. Through a pan-cancer analysis, we describe the overview of hallmark signatures across tumor types/subtypes and reveal significant relationships between these signatures and genetic alterations. TP53 mutation exerts diverse changes, including increased proliferation and glycolysis, which are closely mimicked by widespread copy-number alterations. Hallmark signature and copy-number clustering identify a cluster of squamous tumors and basal-like breast and bladder cancers with elevated proliferation signatures, frequent TP53 mutation, and high aneuploidy. In these basal-like/squamous TP53-mutated tumors, a specific and consistent spectrum of copy-number alterations is preferentially selected prior to whole-genome duplication. Within Trp53-null breast cancer mouse models, these copy-number alterations spontaneously occur and recapitulate the hallmark signature changes observed in the human condition. Together, our analysis reveals intertumor and intratumor heterogeneity of the hallmark signatures, uncovering an oncogenic program induced by TP53 mutation and select aneuploidy events to drive a worsened prognosis. Significance: Our data demonstrate that TP53 mutation and a resultant selected pattern of aneuploidies cause an aggressive transcriptional program including upregulation of glycolysis signature with prognostic implications. Importantly, basal-like breast cancer demonstrates genetic and/or phenotypic changes closely related to squamous tumors including 5q deletion that reveal alterations that could offer therapeutic options across tumor types regardless of tissue of origin.

Cancer origin tracing and timing in two high-risk prostate cancers using multisample whole genome analysis: prospects for personalized medicine.

BACKGROUND: Prostate cancer (PrCa) genomic heterogeneity causes resistance to therapies such as androgen deprivation. Such heterogeneity can be deciphered in the context of evolutionary principles, but current clinical trials do not include evolution as an essential feature. Whether or not analysis of genomic data in an evolutionary context in primary prostate cancer can provide unique added value in the research and clinical domains remains an open question. METHODS: We used novel processing techniques to obtain whole genome data together with 3D anatomic and histomorphologic analysis in two men (GP5 and GP12) with high-risk PrCa undergoing radical prostatectomy. A total of 22 whole genome-sequenced sites (16 primary cancer foci and 6 lymph node metastatic) were analyzed using evolutionary reconstruction tools and spatio-evolutionary models. Probability models were used to trace spatial and chronological origins of the primary tumor and metastases, chart their genetic drivers, and distinguish metastatic and non-metastatic subclones. RESULTS: In patient GP5, CDK12 inactivation was among the first mutations, leading to a PrCa tandem duplicator phenotype and initiating the cancer around age 50, followed by rapid cancer evolution after age 57, and metastasis around age 59, 5 years prior to prostatectomy. In patient GP12, accelerated cancer progression was detected after age 54, and metastasis occurred around age 56, 3 years prior to prostatectomy. Multiple metastasis-originating events were identified in each patient and tracked anatomically. Metastasis from prostate to lymph nodes occurred strictly ipsilaterally in all 12 detected events. In this pilot, metastatic subclone content analysis appears to substantially enhance the identification of key drivers. Evolutionary analysis' potential impact on therapy selection appears positive in these pilot cases. CONCLUSIONS: PrCa evolutionary analysis allows tracking of anatomic site of origin, timing of cancer origin and spread, and distinction of metastatic-capable from non-metastatic subclones. This enables better identification of actionable targets for therapy. If extended to larger cohorts, it appears likely that similar analyses could add substantial biological insight and clinically relevant value.

Myeloma Genome Project Panel is a Comprehensive Targeted Genomics Panel for Molecular Profiling of Patients with Multiple Myeloma.

PURPOSE: We designed a comprehensive multiple myeloma targeted sequencing panel to identify common genomic abnormalities in a single assay and validated it against known standards. EXPERIMENTAL DESIGN: The panel comprised 228 genes/exons for mutations, 6 regions for translocations, and 56 regions for copy number abnormalities (CNA). Toward panel validation, targeted sequencing was conducted on 233 patient samples and further validated using clinical FISH (translocations), multiplex ligation probe analysis (MLPA; CNAs), whole-genome sequencing (WGS; CNAs, mutations, translocations), or droplet digital PCR (ddPCR) of known standards (mutations). RESULTS: Canonical immunoglobulin heavy chain translocations were detected in 43.2% of patients by sequencing, and aligned with FISH except for 1 patient. CNAs determined by sequencing and MLPA for 22 regions were comparable in 103 samples and concordance between platforms was R2 = 0.969. Variant allele frequency (VAF) for 74 mutations were compared between sequencing and ddPCR with concordance of R2 = 0.9849. CONCLUSIONS: In summary, we have developed a targeted sequencing panel that is as robust or superior to FISH and WGS. This molecular panel is cost-effective, comprehensive, clinically actionable, and can be routinely deployed to assist risk stratification at diagnosis or posttreatment to guide sequencing of therapies.

Clonal diversification and histogenesis of malignant germ cell tumours.

Germ cell tumours (GCTs) are a collection of benign and malignant neoplasms derived from primordial germ cells. They are uniquely able to recapitulate embryonic and extraembryonic tissues, which carries prognostic and therapeutic significance. The developmental pathways underpinning GCT initiation and histogenesis are incompletely understood. Here, we study the relationship of histogenesis and clonal diversification in GCTs by analysing the genomes and transcriptomes of 547 microdissected histological units. We find no correlation between genomic and histological heterogeneity. However, we identify unifying features including the retention of fetal developmental transcripts across tissues, expression changes on chromosome 12p, and a conserved somatic evolutionary sequence of whole genome duplication followed by clonal diversification. While this pattern is preserved across all GCTs, the developmental timing of the duplication varies between prepubertal and postpubertal cases. In addition, tumours of younger children exhibit distinct substitution signatures which may lend themselves as potential biomarkers for risk stratification. Our findings portray the extensive diversification of GCT tissues and genetic subclones as randomly distributed, while identifying overarching transcriptional and genomic features.

Whole-genome analysis of Nigerian patients with breast cancer reveals ethnic-driven somatic evolution and distinct genomic subtypes.

Black women across the African diaspora experience more aggressive breast cancer with higher mortality rates than white women of European ancestry. Although inter-ethnic germline variation is known, differential somatic evolution has not been investigated in detail. Analysis of deep whole genomes of 97 breast cancers, with RNA-seq in a subset, from women in Nigeria in comparison with The Cancer Genome Atlas (n = 76) reveal a higher rate of genomic instability and increased intra-tumoral heterogeneity as well as a unique genomic subtype defined by early clonal GATA3 mutations with a 10.5-year younger age at diagnosis. We also find non-coding mutations in bona fide drivers (ZNF217 and SYPL1) and a previously unreported INDEL signature strongly associated with African ancestry proportion, underscoring the need to expand inclusion of diverse populations in biomedical research. Finally, we demonstrate that characterizing tumors for homologous recombination deficiency has significant clinical relevance in stratifying patients for potentially life-saving therapies.

Genomic and evolutionary classification of lung cancer in never smokers.

Lung cancer in never smokers (LCINS) is a common cause of cancer mortality but its genomic landscape is poorly characterized. Here high-coverage whole-genome sequencing of 232 LCINS showed 3 subtypes defined by copy number aberrations. The dominant subtype (piano), which is rare in lung cancer in smokers, features somatic UBA1 mutations, germline AR variants and stem cell-like properties, including low mutational burden, high intratumor heterogeneity, long telomeres, frequent KRAS mutations and slow growth, as suggested by the occurrence of cancer drivers' progenitor cells many years before tumor diagnosis. The other subtypes are characterized by specific amplifications and EGFR mutations (mezzo-forte) and whole-genome doubling (forte). No strong tobacco smoking signatures were detected, even in cases with exposure to secondhand tobacco smoke. Genes within the receptor tyrosine kinase-Ras pathway had distinct impacts on survival; five genomic alterations independently doubled mortality. These findings create avenues for personalized treatment in LCINS.