We integrated clinical, genomic, and transcriptomic data from 224 primaries and 95 metastases from 289 patients to characterize progression of pancreatic ductal adenocarcinoma (PDAC). Driver gene ...alterations and mutational and expression-based signatures were preserved, with truncations, inversions, and translocations most conserved. Cell cycle progression (CCP) increased with sequential inactivation of tumor suppressors, yet remained higher in metastases, perhaps driven by cell cycle regulatory gene variants. Half of the cases were hypoxic by expression markers, overlapping with molecular subtypes. Paired tumor heterogeneity showed cancer cell migration by Halstedian progression. Multiple PDACs arising synchronously and metachronously in the same pancreas were actually intra-parenchymal metastases, not independent primary tumors. Established clinical co-variates dominated survival analyses, although CCP and hypoxia may inform clinical practice.
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•Higher cell cycle progression in PDAC metastases; increases with driver gene loss•Half of PDACs are hypoxic and are associated with subtypes and treatment response•Paired tumors show molecular conservation and Halstedian progression•Multiple PDACs arising in the same pancreas are intra-parenchymal metastases
Connor et al. molecularly characterize primary and metastatic PDAC and show conserved alterations between primary and metastatic lesions. Clinical features outperform molecular alterations in survival analyses, but cell cycle progression and hypoxia signatures may inform clinical practice.
Abstract
Intermediate risk prostate cancer (CaP) with Gleason score (GS) of 7 show up to 100x variability in genetic instability. As CaP is multifocal and likely multiclonal, there is a need to ...characterize heterogeneity for patient stratification, which would increase the ability to act on genomic information by adding adjuvant therapies to offset systemic occult metastases that currently limit cure in ∼30% of patients. Individual genetic portraits could be used to improve cure on combined clinical-molecular staging criteria.
We undertook a pilot study to assess the genetic heterogeneity of potentially curable GS=7 CaP. We selected 10 men with GS=7 CaP; 5 treated with external beam radiotherapy (frozen pre-treatment biopsies) and 5 treated with radical prostatectomy (RadP, frozen tumour). Additionally, DNA from 18 distinct formalin-fixed, paraffin-embedded (FFPE) foci from the 5 RadP were analysed. Each of these 28 foci were subjected to whole-genome sequencing (WGS) and OncoScan SNP arrays to yield comprehensive genetic profiles. mRNA expression was evaluated on frozen RadP by microarray. Germline DNA from whole-blood was also analysed.
Following independent pathology reviews and manual macro-dissection of tumour areas of ≥70% cellularity, WGS (≥50x tumour, ≥30x germline) was performed on as little as 50 ng genomic DNA, and OncoScan arrays were performed using as little as 30ng DNA using either amplified or innate genomic DNA. Regions of CaP in FFPE RadP were recorded using a tissue map to identify independent malignant foci, and ERG immunostaining was performed to assist in the identification. In cases where ERG-positive and -negative foci were adjacent, ERG staining was repeated on an un-stained slide to confirm separate foci based on 3D multi-section analyses. ERG fusion status was also assessed in frozen samples by aCGH or IHC.
Validation of SNVs via SNP array and deep-resequencing showed ∼99% accuracy. Tumour cellularity was estimated using Qpure and was >60% for all samples. Phylogenetic techniques were used to demonstrate clear multi-clonality in two tumours. Across all tumours, ∼50% of SNVs were specific to an individual tumour-region. Phylogenies were confirmed with both SNVs and CNAs, but CNAs generally exhibited greater concordance amongst different regions of the same tumour. Some previously observed recurrent mutations were previously identified as recurrent in CaP (e.g. SPOP), and the overall mutation rate for intermediate-risk CaP was only somewhat below that reported for castrate-resistant disease (11,230 somatic SNVs per tumour).
Our studies support the concept that a complete characterization of inter- and intra-CaP heterogeneity is possible in fresh and archival tissues; the latter is important for correlations to clinical outcome. These approaches can then be streamlined for high-throughput analyses within personalized medicine laboratories leading to “point of care” molecular tests and individualization of therapy.
Citation Format: Michael E. Fraser, Richard de Borja, Dominique Trudel, Nicholas J. Harding, Pablo H. Hennings-Yeomans, Alice Meng, Emilie R. Lalonde, Andrew Brown, Natalie S. Fox, Taryne Chong, Amin Zia, Michelle Sam, Jianxin Wang, Michelle A. Chan-Seng-Yue, Jeremy Johns, Lee Timms, Nicholas Buchner, Ada Wong, Fouad Yousif, Rob Denroche, Gaetano Zafarana, Maud HW Starmans, Hanbert Chen, Shaylan Govind, Francis Nguyen, Melania Pintilie, Neil Fleshner, Stanislav Volik, Lakshmi Muthuswamy, Colin C. Collins, Thomas J. Hudson, Lincoln D. Stein, Timothy Beck, John D. McPherson, Theodorus van der Kwast, Paul C. Boutros, Rob G. Bristow. A molecular portrait of potentially curable prostate cancer. abstract. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2003. doi:10.1158/1538-7445.AM2013-2003
Lifelong blood production requires long-term hematopoietic stem cells (LT-HSCs), marked by stemness states involving quiescence and self-renewal, to transition into activated short-term HSCs ...(ST-HSCs) with reduced stemness. As few transcriptional changes underlie this transition, we used single-cell and bulk assay for transposase-accessible chromatin sequencing (ATAC-seq) on human HSCs and hematopoietic stem and progenitor cell (HSPC) subsets to uncover chromatin accessibility signatures, one including LT-HSCs (LT/HSPC signature) and another excluding LT-HSCs (activated HSPC Act/HSPC signature). These signatures inversely correlated during early hematopoietic commitment and differentiation. The Act/HSPC signature contains CCCTC-binding factor (CTCF) binding sites mediating 351 chromatin interactions engaged in ST-HSCs, but not LT-HSCs, enclosing multiple stemness pathway genes active in LT-HSCs and repressed in ST-HSCs. CTCF silencing derepressed stemness genes, restraining quiescent LT-HSCs from transitioning to activated ST-HSCs. Hence, 3D chromatin interactions centrally mediated by CTCF endow a gatekeeper function that governs the earliest fate transitions HSCs make by coordinating disparate stemness pathways linked to quiescence and self-renewal.
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•Divergent chromatin accessibility signatures discriminate activated HSPCs from LT-HSCs•CTCF silencing in vivo restrains LT-HSCs from transitioning to ST-HSCs•CTCF sites engaged during activation govern 3D interactions repressing stemness genes•Dynamic 3D chromatin interactions coordinately regulate multiple stemness pathways
Long-term hematopoietic stem cells (LT-HSCs) inactivate core stemness programs during activation and transition to hematopoietic stem and progenitor cells. Takayama et al. show that CTCF binding site acquisition is a gatekeeper of this transition, coordinately repressing multiple LT-HSC stemness genes, by mediating 3D rearrangements specific to activated HSPCs.
Polymorphous low-grade adenocarcinoma (PLGA) is the second most frequent type of malignant tumor of the minor salivary glands. We identified PRKD1 hotspot mutations encoding p.Glu710Asp in 72.9% of ...PLGAs but not in other salivary gland tumors. Functional studies demonstrated that this kinase-activating alteration likely constitutes a driver of PLGA.
The driver mutations causing Hematopoietic Stem Cell (HSC) expansion in Clonal Hematopoiesis (CH) are inherited by HSC progeny and in some settings alter the function of mutant immune cells. It is ...unclear how CH mutations impact immune cell function in response to immune checkpoint blockade (ICB), which is a critical cancer therapy for many solid tumours and hematological cancers. Understanding how CH influences ICB could provide a novel biomarker for patient stratification or personalized therapeutics and may offer insight into immunotherapy mechanisms, yielding novel drug targets to synergistically improve patient outcomes. Somatic mutations inactivating TET2, which is responsible for 5-methylcytosine hydroxylation and demethylation, are one of the most frequent drivers of CH. TET2 inactivation can enhance inflammation from monocytes/macrophages, CD4 + and CD8 + T cells, but its impact on ICB is unknown. Here we sought to define the impact of TET2-mutant CH (TET2-CH) on ICB response. First, we modelled TET2-CH by rescuing lethally irradiated C57BL6/j mice with Tet2-knockout (-KO), Tet2-heterozygous null (-het), or wild type (WT) bone marrow. Mice were subsequently implanted with the syngeneic colorectal adenocarcinoma line MC38 and treated with Programmed Cell Death Protein-1 (PD-1) targeted ICB or Isotype Control (control) antibodies. PD-1 ICB, but not control, treated tumors were significantly smaller in mice with Tet2-het or Tet2-KO versus WT hematopoiesis (Figure 1). There was a trend towards a dose-responsive effect, with more Tet2-het mice responding to PD-1 ICB than wt, and more Tet2-KO mice responding than Tet2-het or wt mice. Therefore, Tet2-mutant leukocytes promote response to PD-1 blockade. PD-1 ICB with targeted cell depletion revealed that the enhanced response in Tet2-het mice requires CD4 + T Cells, CD8 + T Cells and macrophages, but not NK cells. To understand the mechanism through which Tet2-inactivation promotes ICB responses we performed 10X single cell RNA-sequencing on 38,994 CD45 + tumor-infiltrating leukocytes (TILs) from Tet2-het and wt mice treated with PD-1 ICB or control. Tet2-het mutations dramatically changed the TIL landscape with PD-1 blockade by promoting the acquisition of cell fates that drive durable immunotherapy responses including CD8 + effector T cells, CD8 + memory T cells, CD4 + interferon-responsive cells, and macrophages expressing an inflammatory anti-tumour gene signature (Figure 2). In contrast, wt TILs from PD-1 treated tumours were significantly enriched for exhausted CD8 + T cells, CD4 + regulatory T cells, and immunosuppressive macrophages that can promote tumor growth (Figure 2). TIL DNA methylation analysis using Infinium Beadchip arrays revealed promoter hypermethylation in genes defining the immunosuppressive macrophage state. Collectively, this suggests that Tet2-CH reshapes TIL fate following ICB by promoting anti-tumor phenotypes and protecting cells from immunosuppressive states. To investigate the potential clinical relevance of our findings, we retrospectively tested for an association between TET2-CH and immunotherapy outcome in 569 ICB-treated solid tumor patients. CH mutations were detected in publicly-available exome-sequencing data at VAF >0.02 using Mutect2. While CH as a composite of all driver mutations did not correlate with outcome, in multivariate analysis TET2-CH was associated with 6-times greater odds of achieving 6 month clinical benefit in n=205 melanoma patients (95% CI 1.01-66.6). To explore whether TET2-CH might identify patients primed for ICB response, we screened for CH in the prospectively collected baseline PBMCs from 90 solid tumour patients treated in a Phase 2 trial of the PD-1 inhibitor Pembrolizumab (NCT02644369). While underpowered to draw conclusions, we noted that one patient, treated for the skin cancer Merkel Cell Carcinoma, was positive for TET2-CH and responded to ICB, and also survived longer than the cohort median and Merkel Cell Carcinoma median. In summary, our results show that TET2-CH promotes immunotherapy response by shifting TILs from immunosuppressive to anti-tumor fates. Mechanistically, this occurs through lineage-specific promoter hypermethylation and gene silencing. Further study of TET2-CH as a potential biomarker of ICB response is warranted and targeting TET2-regulated pathways may yield novel strategies to improve immunotherapy outcomes.
It is critical to understand how human quiescent long-term hematopoietic stem cells (LT-HSCs) sense demand from daily and stress-mediated cues and then transition into bioenergetically active progeny ...to differentiate and meet these cellular needs. However, the demand-adapted regulatory circuits of these early steps of hematopoiesis are largely unknown. Here we show that lysosomes, sophisticated nutrient-sensing and signaling centers, are regulated dichotomously by transcription factor EB (TFEB) and MYC to balance catabolic and anabolic processes required for activating LT-HSCs and guiding their lineage fate. TFEB-mediated induction of the endolysosomal pathway causes membrane receptor degradation, limiting LT-HSC metabolic and mitogenic activation, promoting quiescence and self-renewal, and governing erythroid-myeloid commitment. In contrast, MYC engages biosynthetic processes while repressing lysosomal catabolism, driving LT-HSC activation. Our study identifies TFEB-mediated control of lysosomal activity as a central regulatory hub for proper and coordinated stem cell fate determination.
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•TFEB nuclear localization and lysosomal activity decreases upon LT-HSC activation•MYC drives LT-HSC metabolic and mitogenic activation and inhibits lysosomal genes•TFEB induces lysosomal degradation of TfR1, balancing myeloid/erythroid fate choices•TFEB and lysosomal activity preserve quiescence and enhance self-renewal of LT-HSCs
García-Prat et al. show that lysosomes are regulated dichotomously by TFEB and c-MYC and are crucial for regulating human LT-HSC quiescence, self-renewal, and erythroid/myeloid lineage specification. Anabolic-catabolic lysosomal activity, including endolysosomal degradation of membrane receptors such as TfR1, is required for environmental sensing and activation of LT-HSCs.
Pancreatic cancer, a highly aggressive tumour type with uniformly poor prognosis, exemplifies the classically held view of stepwise cancer development. The current model of tumorigenesis, based on ...analyses of precursor lesions, termed pancreatic intraepithelial neoplasm (PanINs) lesions, makes two predictions: first, that pancreatic cancer develops through a particular sequence of genetic alterations (KRAS, followed by CDKN2A, then TP53 and SMAD4); and second, that the evolutionary trajectory of pancreatic cancer progression is gradual because each alteration is acquired independently. A shortcoming of this model is that clonally expanded precursor lesions do not always belong to the tumour lineage, indicating that the evolutionary trajectory of the tumour lineage and precursor lesions can be divergent. This prevailing model of tumorigenesis has contributed to the clinical notion that pancreatic cancer evolves slowly and presents at a late stage. However, the propensity for this disease to rapidly metastasize and the inability to improve patient outcomes, despite efforts aimed at early detection, suggest that pancreatic cancer progression is not gradual. Here, using newly developed informatics tools, we tracked changes in DNA copy number and their associated rearrangements in tumour-enriched genomes and found that pancreatic cancer tumorigenesis is neither gradual nor follows the accepted mutation order. Two-thirds of tumours harbour complex rearrangement patterns associated with mitotic errors, consistent with punctuated equilibrium as the principal evolutionary trajectory. In a subset of cases, the consequence of such errors is the simultaneous, rather than sequential, knockout of canonical preneoplastic genetic drivers that are likely to set-off invasive cancer growth. These findings challenge the current progression model of pancreatic cancer and provide insights into the mutational processes that give rise to these aggressive tumours.
Disease recurrence causes significant mortality in B-progenitor acute lymphoblastic leukemia (B-ALL). Genomic analysis of matched diagnosis and relapse samples shows relapse often arising from minor ...diagnosis subclones. However, why therapy eradicates some subclones while others survive and progress to relapse remains obscure. Elucidation of mechanisms underlying these differing fates requires functional analysis of isolated subclones. Here, large-scale limiting dilution xenografting of diagnosis and relapse samples, combined with targeted sequencing, identified and isolated minor diagnosis subclones that initiate an evolutionary trajectory toward relapse termed diagnosis Relapse Initiating clones (dRI). Compared with other diagnosis subclones, dRIs were drug-tolerant with distinct engraftment and metabolic properties. Transcriptionally, dRIs displayed enrichment for chromatin remodeling, mitochondrial metabolism, proteostasis programs, and an increase in stemness pathways. The isolation and characterization of dRI subclones reveals new avenues for eradicating dRI cells by targeting their distinct metabolic and transcriptional pathways before further evolution renders them fully therapy-resistant. SIGNIFICANCE: Isolation and characterization of subclones from diagnosis samples of patients with B-ALL who relapsed showed that relapse-fated subclones had increased drug tolerance and distinct metabolic and survival transcriptional programs compared with other diagnosis subclones. This study provides strategies to identify and target clinically relevant subclones before further evolution toward relapse.
IntroductionInotropes are used to prevent or treat postoperative low cardiac output syndrome. Common variants in adrenergic (ADR) signaling genes can influence inotrope response. We investigated ...association of both common and rare variants in ADR signaling genes with inotrope requirements in tetralogy of Fallot (TOF) patients undergoing surgical repair.MethodsOf 276 TOF eligible patients, 120 with complete 72 hour post-operative data were analyzed. All common and rare (<1% minor allele frequency) exonic variants in 167 ADR signaling genes were extracted from whole genome sequencing data. SNP-set (Sequence) Kernel Association Test-Optimised (SKAT-O) test was used to analyze association of mutation burden in the 167 genes with inotrope requirements after adjusting for gender, reported ethnicity, age at surgery, cardiopulmonary bypass (CPB), and aortic cross clamp (ACC) times.ResultsOf 120 patients, 55 had high inotrope requirement (i.e. inotropes for >48hrs, doubling of dose, or initiation of additional inotrope). Mean CPB time was higher in the high-inotrope vs low inotrope group (128±58 vs 100±34 mins, p =0.002), as was ACC time (88±35 vs 76±25 mins, p=0.038). After adjusting for CPB and ACC time, high inotrope group had lower 24 hour mean blood pressures (p<0.01), higher plasma lactate (p=0.016), lower 24 hr urine output (p=0.001), longer ventilator duration (p=0.001) and longer ICU stay (p<0.001). After quality filtering, 689 exonic variants were identified in 129 of the 167 candidate genes; 403 were novel/rare. On preliminary analysis, variants in 9 genes were associated with high inotrope requirements (p<0.05). Three (PPP1R13B, PPP1R3F, PPP1R3A) belonged to the protein phosphatase-1 (PP1) family. PP1 is a critical negative regulator of Ca2+ cycling and contractility in cardiomyocytes and mediates inotropic response to β-adrenergic stimulation.ConclusionsThere was considerable variability in inotrope requirements in children undergoing TOF repair. Variants in PP1 gene were associated with inotrope requirements. Genomic analysis on a larger cohort is underway to identify other pharmacogenetic variants that influence inotrope response. This may guide individualization of post-operative inotrope choices in the future.
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