Non-genetic determinants of malignant clonal fitness at single-cell resolution.

All cancers emerge after a period of clonal selection and subsequent clonal expansion. Although the evolutionary principles imparted by genetic intratumour heterogeneity are becoming increasingly clear1, little is known about the non-genetic mechanisms that contribute to intratumour heterogeneity and malignant clonal fitness2. Here, using single-cell profiling and lineage tracing (SPLINTR)-an expressed barcoding strategy-we trace isogenic clones in three clinically relevant mouse models of acute myeloid leukaemia. We find that malignant clonal dominance is a cell-intrinsic and heritable property that is facilitated by the repression of antigen presentation and increased expression of the secretory leukocyte peptidase inhibitor gene (Slpi), which we genetically validate as a regulator of acute myeloid leukaemia. Increased transcriptional heterogeneity is a feature that enables clonal fitness in diverse tissues and immune microenvironments and in the context of clonal competition between genetically distinct clones. Similar to haematopoietic stem cells3, leukaemia stem cells (LSCs) display heritable clone-intrinsic properties of high, and low clonal output that contribute to the overall tumour mass. We demonstrate that LSC clonal output dictates sensitivity to chemotherapy and, although high- and low-output clones adapt differently to therapeutic pressure, they coordinately emerge from minimal residual disease with increased expression of the LSC program. Together, these data provide fundamental insights into the non-genetic transcriptional processes that underpin malignant clonal fitness and may inform future therapeutic strategies.

Genomic analysis of low-grade serous ovarian carcinoma to identify key drivers and therapeutic vulnerabilities.

Low-grade serous ovarian carcinoma (LGSOC) is associated with a poor response to existing chemotherapy, highlighting the need to perform comprehensive genomic analysis and identify new therapeutic vulnerabilities. The data presented here represent the largest genetic study of LGSOCs to date (n = 71), analysing 127 candidate genes derived from whole exome sequencing cohorts to generate mutation and copy-number variation data. Additionally, immunohistochemistry was performed on our LGSOC cohort assessing oestrogen receptor, progesterone receptor, TP53, and CDKN2A status. Targeted sequencing identified 47% of cases with mutations in key RAS/RAF pathway genes (KRAS, BRAF, and NRAS), as well as mutations in putative novel driver genes including USP9X (27%), MACF1 (11%), ARID1A (9%), NF2 (4%), DOT1L (6%), and ASH1L (4%). Immunohistochemistry evaluation revealed frequent oestrogen/progesterone receptor positivity (85%), along with CDKN2A protein loss (10%) and CDKN2A protein overexpression (6%), which were linked to shorter disease outcomes. Indeed, 90% of LGSOC samples harboured at least one potentially actionable alteration, which in 19/71 (27%) cases were predictive of clinical benefit from a standard treatment, either in another cancer's indication or in LGSOC specifically. In addition, we validated ubiquitin-specific protease 9X (USP9X), which is a chromosome X-linked substrate-specific deubiquitinase and tumour suppressor, as a relevant therapeutic target for LGSOC. Our comprehensive genomic study highlighted that there is an addiction to a limited number of unique 'driver' aberrations that could be translated into improved therapeutic paths. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Whole-genome characterization of chemoresistant ovarian cancer.

Patients with high-grade serous ovarian cancer (HGSC) have experienced little improvement in overall survival, and standard treatment has not advanced beyond platinum-based combination chemotherapy, during the past 30 years. To understand the drivers of clinical phenotypes better, here we use whole-genome sequencing of tumour and germline DNA samples from 92 patients with primary refractory, resistant, sensitive and matched acquired resistant disease. We show that gene breakage commonly inactivates the tumour suppressors RB1, NF1, RAD51B and PTEN in HGSC, and contributes to acquired chemotherapy resistance. CCNE1 amplification was common in primary resistant and refractory disease. We observed several molecular events associated with acquired resistance, including multiple independent reversions of germline BRCA1 or BRCA2 mutations in individual patients, loss of BRCA1 promoter methylation, an alteration in molecular subtype, and recurrent promoter fusion associated with overexpression of the drug efflux pump MDR1.

Therapeutic options for mucinous ovarian carcinoma.

OBJECTIVE: Mucinous ovarian carcinoma (MOC) is an uncommon ovarian cancer histotype that responds poorly to conventional chemotherapy regimens. Although long overall survival outcomes can occur with early detection and optimal surgical resection, recurrent and advanced disease are associated with extremely poor survival. There are no current guidelines specifically for the systemic management of recurrent MOC. We analyzed data from a large cohort of women with MOC to evaluate the potential for clinical utility from a range of systemic agents. METHODS: We analyzed gene copy number (n = 191) and DNA sequencing data (n = 184) from primary MOC to evaluate signatures of mismatch repair deficiency and homologous recombination deficiency, and other genetic events. Immunohistochemistry data were collated for ER, CK7, CK20, CDX2, HER2, PAX8 and p16 (n = 117-166). RESULTS: Molecular aberrations noted in MOC that suggest a match with current targeted therapies include amplification of ERBB2 (26.7%) and BRAF mutation (9%). Observed genetic events that suggest potential efficacy for agents currently in clinical trials include: KRAS/NRAS mutations (66%), TP53 missense mutation (49%), RNF43 mutation (11%), ARID1A mutation (10%), and PIK3CA/PTEN mutation (9%). Therapies exploiting homologous recombination deficiency (HRD) may not be effective in MOC, as only 1/191 had a high HRD score. Mismatch repair deficiency was similarly rare (1/184). CONCLUSIONS: Although genetically diverse, MOC has several potential therapeutic targets. Importantly, the lack of response to platinum-based therapy observed clinically corresponds to the lack of a genomic signature associated with HRD, and MOC are thus also unlikely to respond to PARP inhibition.

Atypical ductal hyperplasia is a multipotent precursor of breast carcinoma.

The current model for breast cancer progression proposes independent 'low grade (LG)-like' and 'high grade (HG)-like' pathways but lacks a known precursor to HG cancer. We applied low-coverage whole-genome sequencing to atypical ductal hyperplasia (ADH) with and without carcinoma to shed light on breast cancer progression. Fourteen out of twenty isolated ADH cases harboured at least one copy number alteration (CNA), but had fewer aberrations than LG or HG ductal carcinoma in situ (DCIS). ADH carried more HG-like CNA than LG DCIS (e.g. 8q gain). Correspondingly, 64% (7/11) of ADH cases with synchronous HG carcinoma were clonally related, similar to LG carcinoma (67%, 6/9). This study represents a significant shift in our understanding of breast cancer progression, with ADH as a common precursor lesion to the independent 'low grade-like' and 'high grade-like' pathways. These data suggest that ADH can be a precursor of HG breast cancer and that LG and HG carcinomas can evolve from a similar ancestor lesion. We propose that although LG DCIS may be committed to a LG molecular pathway, ADH may remain multipotent, progressing to either LG or HG carcinoma. This multipotent nature suggests that some ADH cases could be more clinically significant than LG DCIS, requiring biomarkers for personalising management. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Massively-parallel sequencing assists the diagnosis and guided treatment of cancers of unknown primary.

The clinical management of patients with cancer of unknown primary (CUP) is hampered by the absence of a definitive site of origin. We explored the utility of massively-parallel (next-generation) sequencing for the diagnosis of a primary site of origin and for the identification of novel treatment options. DNA enrichment by hybridization capture of 701 genes of clinical and/or biological importance, followed by massively-parallel sequencing, was performed on 16 CUP patients who had defied attempts to identify a likely site of origin. We obtained high quality data from both fresh-frozen and formalin-fixed, paraffin-embedded samples, demonstrating accessibility to routine diagnostic material. DNA copy-number obtained by massively-parallel sequencing was comparable to that obtained using oligonucleotide microarrays or quantitatively hybridized fluorescently tagged oligonucleotides. Sequencing to an average depth of 458-fold enabled detection of somatically acquired single nucleotide mutations, insertions, deletions and copy-number changes, and measurement of allelic frequency. Common cancer-causing mutations were found in all cancers. Mutation profiling revealed therapeutic gene targets and pathways in 12/16 cases, providing novel treatment options. The presence of driver mutations that are enriched in certain known tumour types, together with mutational signatures indicative of exposure to sunlight or smoking, added to clinical, pathological, and molecular indicators of likely tissue of origin. Massively-parallel DNA sequencing can therefore provide comprehensive mutation, DNA copy-number, and mutational signature data that are of significant clinical value for a majority of CUP patients, providing both cumulative evidence for the diagnosis of primary site and options for future treatment.

MAC-Seq: Coupling Low-Cost, High-Throughput RNA-Seq with Image-Based Phenotypic Screening in 2D and 3D Cell Models.

Transcriptomic profiling has fundamentally influenced our understanding of cancer pathophysiology and response to therapeutic intervention and has become a relatively routine approach. However, standard protocols are usually low-throughput, single-plex assays and costs are still quite prohibitive. With the evolving complexity of in vitro cell model systems, there is a need for resource-efficient high-throughput approaches that can support detailed time-course analytics, accommodate limited sample availability, and provide the capacity to correlate phenotype to genotype at scale. MAC-seq (multiplexed analysis of cells) is a low-cost, ultrahigh-throughput RNA-seq workflow in plate format to measure cell perturbations and is compatible with high-throughput imaging. Here we describe the steps to perform MAC-seq in 384-well format and apply it to 2D and 3D cell cultures. On average, our experimental conditions identified over ten thousand expressed genes per well when sequenced to a depth of one million reads. We discuss technical aspects, make suggestions on experimental design, and document critical operational procedures. Our protocol highlights the potential to couple MAC-seq with high-throughput screening applications including cell phenotyping using high-content cell imaging.

The Th17-defining transcription factor RORγt promotes glomerulonephritis.

Although Th17 responses may contribute to the pathogenesis of glomerulonephritis, whether the key transcription factor in Th17 cell development, RORγt, also promotes glomerulonephritis is unknown. Here, we induced crescentic glomerulonephritis in wild-type and RORγt-deficient (RORγt(-/-)) mice. RORγt(-/-) mice were protected from disease, with reduced histologic and functional injury and decreased leukocyte infiltration. Because RORγt(-/-) mice lack lymph nodes, which may influence the development of nephritis, we performed cell-transfer studies. We reconstituted Rag1(-/-) mice, which lack adaptive immunity but otherwise have normal architecture of the lymphatic system, with splenocytes from naïve wild-type or RORγt(-/-) mice. Mice receiving wild-type splenocytes exhibited high mortality from renal failure after induction of nephritis whereas mice receiving RORγt(-/-) cells were protected. To determine the effect of RORγt deficiency specifically in T helper cells, we isolated naïve CD4(+) T cells from wild-type and RORγt(-/-) mice and transferred them into Rag1(-/-) animals. Recipients of wild-type CD4(+) T cells developed severe glomerulonephritis whereas recipients of RORγt(-/-) cells developed less severe disease. To exclude effects of altered regulatory T cell (Treg) development caused by RORγt deficiency, we transferred naïve CD4(+) T cells depleted of Tregs into Rag1(-/-) mice. Recipients of wild-type, Treg-depleted, CD4(+) T cells developed severe glomerulonephritis whereas recipients of RORγt(-/-), Treg-depleted CD4(+) T cells did not. Taken together, this study demonstrates that RORγt promotes the development of crescentic glomerulonephritis by directing nephritogenic Th17 responses.

TRACEBACK: Testing of Historical Tubo-Ovarian Cancer Patients for Hereditary Risk Genes as a Cancer Prevention Strategy in Family Members.

PURPOSE: Tubo-ovarian cancer (TOC) is a sentinel cancer for BRCA1 and BRCA2 pathogenic variants (PVs). Identification of a PV in the first member of a family at increased genetic risk (the proband) provides opportunities for cancer prevention in other at-risk family members. Although Australian testing rates are now high, PVs in patients with TOC whose diagnosis predated revised testing guidelines might have been missed. We assessed the feasibility of detecting PVs in this population to enable genetic risk reduction in relatives. PATIENTS AND METHODS: In this pilot study, deceased probands were ascertained from research cohort studies, identification by a relative, and gynecologic oncology clinics. DNA was extracted from archival tissue or stored blood for panel sequencing of 10 risk-associated genes. Testing of deceased probands ascertained through clinic records was performed with a consent waiver. RESULTS: We identified 85 PVs in 84 of 787 (11%) probands. Familial contacts of 39 of 60 (65%) deceased probands with an identified recipient (60 of 84; 71%) have received a written notification of results, with follow-up verbal contact made in 85% (33 of 39). A minority of families (n = 4) were already aware of the PV. For many (29 of 33; 88%), the genetic result provided new information and referral to a genetic service was accepted in most cases (66%; 19 of 29). Those who declined referral (4 of 29) were all male next of kin whose family member had died more than 10 years before. CONCLUSION: We overcame ethical and logistic challenges to demonstrate that retrospective genetic testing to identify PVs in previously untested deceased probands with TOC is feasible. Understanding reasons for a family member's decision to accept or decline a referral will be important for guiding future TRACEBACK projects.

Epigenetic Activation of Plasmacytoid DCs Drives IFNAR-Dependent Therapeutic Differentiation of AML.

Pharmacologic inhibition of epigenetic enzymes can have therapeutic benefit against hematologic malignancies. In addition to affecting tumor cell growth and proliferation, these epigenetic agents may induce antitumor immunity. Here, we discovered a novel immunoregulatory mechanism through inhibition of histone deacetylases (HDAC). In models of acute myeloid leukemia (AML), leukemia cell differentiation and therapeutic benefit mediated by the HDAC inhibitor (HDACi) panobinostat required activation of the type I interferon (IFN) pathway. Plasmacytoid dendritic cells (pDC) produced type I IFN after panobinostat treatment, through transcriptional activation of IFN genes concomitant with increased H3K27 acetylation at these loci. Depletion of pDCs abrogated panobinostat-mediated induction of type I IFN signaling in leukemia cells and impaired therapeutic efficacy, whereas combined treatment with panobinostat and IFNα improved outcomes in preclinical models. These discoveries offer a new therapeutic approach for AML and demonstrate that epigenetic rewiring of pDCs enhances antitumor immunity, opening the possibility of exploiting this approach for immunotherapies. SIGNIFICANCE: We demonstrate that HDACis induce terminal differentiation of AML through epigenetic remodeling of pDCs, resulting in production of type I IFN that is important for the therapeutic effects of HDACis. The study demonstrates the important functional interplay between the immune system and leukemias in response to HDAC inhibition. This article is highlighted in the In This Issue feature, p. 1397.

Single-Cell Gene Expression, Clonality, and Feature Barcoding of Melanoma Tumor-Infiltrating Lymphocytes.

We describe here a protocol to measure gene expression, T cell receptor (TCR) sequence, and protein expression by single T cells extracted from melanoma, using 10× Chromium technology. This method includes freezing and thawing of the melanoma infiltrating lymphocytes, staining of cells with fluorescent and barcode-conjugated antibodies, sorting of T cells, and loading the cells on the 10× Chromium Controller. After sequencing, analysis includes quality control, genetic demultiplexing to resolve genetically different samples, and T cell clonality and clustering analysis. Single cell RNA sequencing paints the complete portrait of individual T cells, including their clonality and phenotype, and it reconstructs a complete picture of the T cell infiltrate in a tumor that is represented as cell clustering similar to a pointillism painting.

SUGAR-seq enables simultaneous detection of glycans, epitopes, and the transcriptome in single cells.

Multimodal single-cell RNA sequencing enables the precise mapping of transcriptional and phenotypic features of cellular differentiation states but does not allow for simultaneous integration of critical posttranslational modification data. Here, we describe SUrface-protein Glycan And RNA-seq (SUGAR-seq), a method that enables detection and analysis of N-linked glycosylation, extracellular epitopes, and the transcriptome at the single-cell level. Integrated SUGAR-seq and glycoproteome analysis identified tumor-infiltrating T cells with unique surface glycan properties that report their epigenetic and functional state.

Th1 and Th17 cells induce proliferative glomerulonephritis.

Th1 effector CD4+ cells contribute to the pathogenesis of proliferative and crescentic glomerulonephritis, but whether effector Th17 cells also contribute is unknown. We compared the involvement of Th1 and Th17 cells in a mouse model of antigen-specific glomerulonephritis in which effector CD4+ cells are the only components of adaptive immunity that induce injury. We planted the antigen ovalbumin on the glomerular basement membrane of Rag1(-/-) mice using an ovalbumin-conjugated non-nephritogenic IgG1 monoclonal antibody against alpha3(IV) collagen. Subsequent injection of either Th1- or Th17-polarized ovalbumin-specific CD4+ effector cells induced proliferative glomerulonephritis. Mice injected with Th1 cells developed progressive albuminuria over 21 d, histologic injury including 5.5 +/- 0.9% crescent formation/segmental necrosis, elevated urinary nitrate, and increased renal NOS2, CCL2, and CCL5 mRNA. Mice injected with Th17 cells developed albuminuria by 3 d; compared with Th1-injected mice, their glomeruli contained more neutrophils and greater expression of renal CXCL1 mRNA. In conclusion, Th1 and Th17 effector cells can induce glomerular injury. Understanding how these two subsets mediate proliferative forms of glomerulonephritis may lead to targeted therapies.

Improved next-generation sequencing pre-capture library yields and sequencing parameters using on-bead PCR.

Tumor DNA sequencing results can have important clinical implications. However, its use is often limited by low DNA input, owing to small tumor biopsy size. To help overcome this limitation we have developed a simple improvement to a commonly used next-generation sequencing (NGS) capture-based library preparation method using formalin-fixed paraffin-embedded-derived tumor DNA. By using on-bead PCR for pre-capture library generation we show that library yields are dramatically increased, resulting in decreased sample failure rates. Improved yields allowed for a reduction in PCR cycles, which translated into improved sequencing parameters without affecting variant calling. This methodology should be applicable to any NGS system in which input DNA is a limiting factor.

Characterization of the immune profile of oral tongue squamous cell carcinomas with advancing disease.

We investigated whether a unique immune response was instigated with the development of oral tongue squamous cell carcinomas (OTSCC), with/without nodal involvement, with/without recurrent metastatic disease, or within tumor involved nodes. One hundred and ten formalin-fixed paraffin-embedded samples were collected from a retrospective cohort of 67 OTSCC patients and 10 non-cancerous tongue samples. Targets including CD4, CD8, FOXP3, PD-L1, and PD-1 were analyzed by immunohistochemistry. The Nanostring PanCancer Immune Profiling Panel was used for gene expression profiling. Data were externally validated in the The Cancer Genome Atlas (TCGA) head and neck (HNSCC), melanoma and lung squamous cell carcinoma (LSCC) cohorts. A 24-immune gene signature was identified that discriminated more aggressive OTSCC cases, and although not prognostic in HNSCC was associated with survival in other TCGA cohorts (improved survival for melanoma, P < .001 and worse survival for LSCC, P = .038). OTSCC exhibited concordant gene and immunohistochemical (IHC) features characterized by a TH-2 biased, proinflammatory profile with upregulated B cell and neutrophil gene activity and increased CD4, FOXP3, and PD-L1 expression (P < .001 for all by IHC). Compared to less advanced disease, nodal involvement and recurrent OTSCC did not induce a different immune response although recurrent disease was characterized by significantly higher PD-L1 expression (P = .004 by SP263, P = .013 by 22C3, P = .004 for gene expression). Identification of a gene signature associated with different prognostic effects in other cancers highlights common pathways of immune dysregulation that are impacted by the tumor origin. The significant immunosuppressive signaling in OTSCC indicates primary failure of immune system to control carcinogenesis emphasizing the need for early, combination therapeutic approaches.

The genetic architecture of breast papillary lesions as a predictor of progression to carcinoma.

Intraductal papillomas (IDP) are challenging breast findings because of their variable risk of progression to malignancy. The molecular events driving IDP development and genomic features of malignant progression are poorly understood. In this study, genome-wide CNA and/or targeted mutation analysis was performed on 44 cases of IDP, of which 20 cases had coexisting ductal carcinoma in situ (DCIS), papillary DCIS or invasive ductal carcinoma (IDC). CNA were rare in pure IDP, but 69% carried an activating PIK3CA mutation. Among the synchronous IDP cases, 55% (11/20) were clonally related to the synchronous DCIS and/or IDC, only one of which had papillary histology. In contrast to pure IDP, PIK3CA mutations were absent from clonal cases. CNAs in any of chromosomes 1, 16 or 11 were significantly enriched in clonal IDP lesions compared to pure and non-clonal IDP. The observation that 55% of IDP are clonal to DCIS/IDC indicates that IDP can be a direct precursor for breast carcinoma, not limited to the papillary type. The absence of PIK3CA mutations and presence of CNAs in IDP could be used clinically to identify patients at high risk of progression to carcinoma.

Endogenous CD100 promotes glomerular injury and macrophage recruitment in experimental crescentic glomerulonephritis.

CD100 participates in adaptive immune responses and is important in neural cell migration. To determine the role of endogenous CD100 in severe glomerular inflammation, we induced experimental crescentic glomerulonephritis by planting a foreign antigen in glomeruli of sensitized normal and CD100-deficient (CD100(-/-)) mice. Fewer CD100(-/-) glomeruli exhibited crescent formation or severe histological changes. Antigen-specific immune responses were reduced in CD100(-/-) mice. There was less interferon (IFN)-gamma and interleukin (IL)-4 production by splenocytes and fewer activated T and B cells were present in lymph nodes of immunized CD100(-/-) mice. Serum antigen-specific immunoglobulin (IgG) levels were also decreased. Glomerular macrophage and CD4(+) cell infiltration, and IgG and C3 deposition were attenuated. Normal kidneys expressed mRNA for CD100 and plexin-B1 (the tissue receptor of CD100). Direct immunofluorescence showed that renal-CD100 protein was predominantly in tubules, while plexin-B1 was present in both glomeruli and tubules. To determine whether glomerular plexin-B1 mediates leucocyte recruitment via leucocyte CD100, recruitment was studied after passive transfer of heterologous antibody (attracting neutrophils) or isologous antibody (attracting macrophages). Glomerular macrophages were reduced in CD100(-/-) mice, but neutrophil recruitment was equivalent, consistent with CD100 expression on macrophages, but not neutrophils. CD100 promotes severe nephritogenic immune responses and leucocyte CD100-glomerular plexin-B1 interactions enhance macrophage recruitment to glomeruli.

IL-1RI deficiency ameliorates early experimental renal interstitial fibrosis.

BACKGROUND: IL-1beta has the potential to promote progressive renal disease by effects on macrophage recruitment and activation or by effects mediated through tubular cell transforming growth factor (TGF)-beta production, previously demonstrated in vitro. METHODS: The in vivo roles of endogenous IL-1beta and its type I receptor (IL-1RI) in renal fibrosis were studied using wild-type C57BL/6 mice, IL-1beta(-/-) and IL-1RI(-/-) mice with unilateral ureteric obstruction. RESULTS: After 7 days, IL-1RI(-/-) mice (IL-1alpha and IL-1beta deficient) were protected from injury and collagen accumulation. IL-1beta(-/-) mice demonstrated some histological protection, but no reduction in alpha1(1) procollagen mRNA or biochemically measured collagen accumulation. Compared with obstructed kidneys from wild-type mice, TGF-beta1 mRNA was reduced in IL-1RI(-/-) mice (with trends to reduced TGF-beta2 and TGF-beta3). Expression of a downstream TGF-beta effector, connective tissue growth factor, was decreased in IL-1RI(-/-) mice. IL-1RI(-/-) mice exhibited less tubulointerstitial apoptosis compared with wild-type mice. Macrophage infiltration and adhesion molecule mRNA expression was unchanged in IL-1beta(-/-) or IL-1RI(-/-) mice. While TNF expression was similar to wild-type mice, IFN-gamma expression was reduced in both IL-1beta(-/-) and IL-1RI(-/-) mice. IL-1RI(-/-) mice at 14 days showed a catch-up in fibrosis compared with wild-type mice. CONCLUSION: IL-1/IL-1RI interactions are profibrotic in renal fibrosis. IL-1RI(-/-) mice were more protected at an early stage, associated with changes in TGF-beta and downstream mediators of fibrosis, but independent of the presence of infiltrating macrophages.