Single-cell genomics is essential to chart tumor ecosystems. Although single-cell RNA-Seq (scRNA-Seq) profiles RNA from cells dissociated from fresh tumors, single-nucleus RNA-Seq (snRNA-Seq) is ...needed to profile frozen or hard-to-dissociate tumors. Each requires customization to different tissue and tumor types, posing a barrier to adoption. Here, we have developed a systematic toolbox for profiling fresh and frozen clinical tumor samples using scRNA-Seq and snRNA-Seq, respectively. We analyzed 216,490 cells and nuclei from 40 samples across 23 specimens spanning eight tumor types of varying tissue and sample characteristics. We evaluated protocols by cell and nucleus quality, recovery rate and cellular composition. scRNA-Seq and snRNA-Seq from matched samples recovered the same cell types, but at different proportions. Our work provides guidance for studies in a broad range of tumors, including criteria for testing and selecting methods from the toolbox for other tumors, thus paving the way for charting tumor atlases.
Despite rare cancers accounting for 25% of adult tumors
, they are difficult to study due to the low disease incidence and geographically dispersed patient populations, which has resulted in ...significant unmet clinical needs for patients with rare cancers. We assessed whether a patient-partnered research approach using online engagement can overcome these challenges, focusing on angiosarcoma, a sarcoma with an annual incidence of 300 cases in the United States. Here we describe the development of the Angiosarcoma Project (ASCproject), an initiative enabling US and Canadian patients to remotely share their clinical information and biospecimens for research. The project generates and publicly releases clinically annotated genomic data on tumor and germline specimens on an ongoing basis. Over 18 months, 338 patients registered for the ASCproject, which comprises a large proportion of all patients with angiosarcoma. Whole-exome sequencing (WES) of 47 tumors revealed recurrently mutated genes that included KDR, TP53, and PIK3CA. PIK3CA-activating mutations were observed predominantly in primary breast angiosarcoma, which suggested a therapeutic rationale. Angiosarcoma of the head, neck, face and scalp (HNFS) was associated with a high tumor mutation burden (TMB) and a dominant ultraviolet damage mutational signature, which suggested that for the subset of patients with angiosarcoma of HNFS, ultraviolet damage may be a causative factor and that immune checkpoint inhibition may be beneficial. Medical record review revealed that two patients with HNFS angiosarcoma had received off-label therapeutic use of antibody to the programmed death-1 protein (anti-PD-1) and had experienced exceptional responses, which highlights immune checkpoint inhibition as a therapeutic avenue for HNFS angiosarcoma. This patient-partnered approach has catalyzed an opportunity to discover the etiology and potential therapies for patients with angiosarcoma. Collectively, this proof-of-concept study demonstrates that empowering patients to directly participate in research can overcome barriers in rare diseases and can enable discoveries.
Synovial sarcoma (SyS) is an aggressive neoplasm driven by the SS18-SSX fusion, and is characterized by low T cell infiltration. Here, we studied the cancer-immune interplay in SyS using an ...integrative approach that combines single-cell RNA sequencing (scRNA-seq), spatial profiling and genetic and pharmacological perturbations. scRNA-seq of 16,872 cells from 12 human SyS tumors uncovered a malignant subpopulation that marks immune-deprived niches in situ and is predictive of poor clinical outcomes in two independent cohorts. Functional analyses revealed that this malignant cell state is controlled by the SS18-SSX fusion, is repressed by cytokines secreted by macrophages and T cells, and can be synergistically targeted with a combination of HDAC and CDK4/CDK6 inhibitors. This drug combination enhanced malignant-cell immunogenicity in SyS models, leading to induced T cell reactivity and T cell-mediated killing. Our study provides a blueprint for investigating heterogeneity in fusion-driven malignancies and demonstrates an interplay between immune evasion and oncogenic processes that can be co-targeted in SyS and potentially in other malignancies.
After a prolonged response, an anaplastic thyroid cancer developed resistance to mammalian target of rapamycin (mTOR) inhibition by somatic mutation of mTOR at the everolimus binding site. The mutant ...enzyme retained in vitro responsiveness to an mTOR kinase inhibitor.
A better understanding of the mechanisms of sensitivity and resistance to anticancer therapies may improve patient selection and allow the development of rational treatment designs. One approach involves studying paired biopsy samples of pretreatment and drug-resistant tumors obtained from patients with exquisite sensitivity or unusually durable responses to therapy.
Everolimus is a Food and Drug Administration–approved oral allosteric inhibitor of mTOR. Tumors that exhibit a dependency on the mTOR pathway might have enhanced sensitivity to mTOR inhibition. Inactivating mutations in the tumor-suppressor genes
TSC1, TSC2,
and
STK11
result in mTOR-pathway activation and are targetable by TOR inhibitors in hamartoma syndromes . . .
CDK4/6 inhibition is now part of the standard armamentarium for patients with estrogen receptor-positive (ER+) breast cancer, so that defining mechanisms of resistance is a pressing issue. Here, we ...identify increased CDK6 expression as a key determinant of acquired resistance after palbociclib treatment in ER+ breast cancer cells. CDK6 expression is critical for cellular survival during palbociclib exposure. The increased CDK6 expression observed in resistant cells is dependent on TGF-β pathway suppression via miR-432-5p expression. Exosomal miR-432-5p expression mediates the transfer of the resistance phenotype between neighboring cell populations. Levels of miR-432-5p are higher in primary breast cancers demonstrating CDK4/6 resistance compared to those that are sensitive. These data are further confirmed in pre-treatment and post-progression biopsies from a parotid cancer patient who had responded to ribociclib, demonstrating the clinical relevance of this mechanism. Finally, the CDK4/6 inhibitor resistance phenotype is reversible in vitro and in vivo by a prolonged drug holiday.
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•CDK4/6 inhibitor resistance is mediated by increased CDK6 expression•Resistance can be conferred between cell populations via extracellular signaling•Increased exosomal miR-432-5p suppresses the TGF-β pathway, increasing CDK6 levels•Resistance is reversible by prolonged drug removal in vivo
Cornell et al. demonstrate a mechanism of acquired CDK4/6 inhibitor resistance that is independent of inherent genetic mutations, is conferred through extracellular signaling, and is reversible in vitro and in vivo. Resistance was mediated by exosomal miRNA, causing increased expression of CDK6 to overcome G1 arrest and promote cell survival.
Crucial transitions in cancer—including tumor initiation, local expansion, metastasis, and therapeutic resistance—involve complex interactions between cells within the dynamic tumor ecosystem. ...Transformative single-cell genomics technologies and spatial multiplex in situ methods now provide an opportunity to interrogate this complexity at unprecedented resolution. The Human Tumor Atlas Network (HTAN), part of the National Cancer Institute (NCI) Cancer Moonshot Initiative, will establish a clinical, experimental, computational, and organizational framework to generate informative and accessible three-dimensional atlases of cancer transitions for a diverse set of tumor types. This effort complements both ongoing efforts to map healthy organs and previous large-scale cancer genomics approaches focused on bulk sequencing at a single point in time. Generating single-cell, multiparametric, longitudinal atlases and integrating them with clinical outcomes should help identify novel predictive biomarkers and features as well as therapeutically relevant cell types, cell states, and cellular interactions across transitions. The resulting tumor atlases should have a profound impact on our understanding of cancer biology and have the potential to improve cancer detection, prevention, and therapeutic discovery for better precision-medicine treatments of cancer patients and those at risk for cancer.
The Human Tumor Atlas Network outlines their ambitious plan to generate 3D, single-cell, multiparametric, and longitudinal maps of diverse tumor types.
Seventy percent of breast cancers express the estrogen receptor (ER), and agents that target the ER are the mainstay of treatment. However, virtually all people with ER
breast cancer develop ...resistance to ER-directed agents in the metastatic setting. Beyond mutations in the ER itself, which occur in 25-30% of people treated with aromatase inhibitors
, knowledge about clinical resistance mechanisms remains incomplete. We identified activating HER2 mutations in metastatic biopsies from eight patients with ER
metastatic breast cancer who had developed resistance to aromatase inhibitors, tamoxifen or fulvestrant. Examination of treatment-naive primary tumors in five patients showed no evidence of pre-existing mutations in four of five patients, suggesting that these mutations were acquired under the selective pressure of ER-directed therapy. The HER2 mutations and ER mutations were mutually exclusive, suggesting a distinct mechanism of acquired resistance to ER-directed therapies. In vitro analysis confirmed that the HER2 mutations conferred estrogen independence as well as-in contrast to ER mutations-resistance to tamoxifen, fulvestrant and the CDK4 and CDK6 inhibitor palbociclib. Resistance was overcome by combining ER-directed therapy with the irreversible HER2 kinase inhibitor neratinib.
Whole-exome sequencing of cell-free DNA (cfDNA) could enable comprehensive profiling of tumors from blood but the genome-wide concordance between cfDNA and tumor biopsies is uncertain. Here we report ...ichorCNA, software that quantifies tumor content in cfDNA from 0.1× coverage whole-genome sequencing data without prior knowledge of tumor mutations. We apply ichorCNA to 1439 blood samples from 520 patients with metastatic prostate or breast cancers. In the earliest tested sample for each patient, 34% of patients have ≥10% tumor-derived cfDNA, sufficient for standard coverage whole-exome sequencing. Using whole-exome sequencing, we validate the concordance of clonal somatic mutations (88%), copy number alterations (80%), mutational signatures, and neoantigens between cfDNA and matched tumor biopsies from 41 patients with ≥10% cfDNA tumor content. In summary, we provide methods to identify patients eligible for comprehensive cfDNA profiling, revealing its applicability to many patients, and demonstrate high concordance of cfDNA and metastatic tumor whole-exome sequencing.
A detailed understanding of the mechanisms by which tumors acquire resistance to targeted anticancer agents should speed the development of treatment strategies with lasting clinical efficacy. RAF ...inhibition in BRAF-mutant melanoma exemplifies the promise and challenge of many targeted drugs; although response rates are high, resistance invariably develops. Here, we articulate overarching principles of resistance to kinase inhibitors, as well as a translational approach to characterize resistance in the clinical setting through tumor mutation profiling. As a proof of principle, we performed targeted, massively parallel sequencing of 138 cancer genes in a tumor obtained from a patient with melanoma who developed resistance to PLX4032 after an initial dramatic response. The resulting profile identified an activating mutation at codon 121 in the downstream kinase MEK1 that was absent in the corresponding pretreatment tumor. The MEK1(C121S) mutation was shown to increase kinase activity and confer robust resistance to both RAF and MEK inhibition in vitro. Thus, MEK1(C121S) or functionally similar mutations are predicted to confer resistance to combined MEK/RAF inhibition. These results provide an instructive framework for assessing mechanisms of acquired resistance to kinase inhibition and illustrate the use of emerging technologies in a manner that may accelerate personalized cancer medicine.
Few patients with metastatic triple-negative breast cancer (mTNBC) benefit from immune checkpoint inhibitors (ICI). On the basis of immunotherapy response correlates in other cancers, we evaluated ...whether high tumor mutational burden (TMB) ≥10 nonsynonymous mutations/megabase and
alterations, defined as nonsynonymous mutations or 1 or 2 copy deletions, were associated with clinical benefit to anti-PD-1/L1 therapy in mTNBC.
We identified patients with mTNBC, who consented to targeted DNA sequencing and were treated with ICIs on clinical trials between April 2014 and January 2019 at Dana-Farber Cancer Institute (Boston, MA). Objective response rate (ORR), progression-free survival (PFS), and overall survival (OS) were correlated with tumor genomic features.
Sixty-two women received anti-PD-1/L1 inhibitors alone (23%) or combined with targeted therapy (19%) or chemotherapy (58%). High TMB (18%) was associated with significantly longer PFS (12.5 vs. 3.7 months;
= 0.04), while
alterations (29%) were associated with significantly lower ORR (6% vs. 48%;
= 0.01), shorter PFS (2.3 vs. 6.1 months;
= 0.01), and shorter OS (9.7 vs. 20.5 months;
= 0.02). Multivariate analyses confirmed that these associations were independent of performance status, prior lines of therapy, therapy regimen, and visceral metastases. The survival associations were additionally independent of PD-L1 in patients with known PD-L1 and were not found in mTNBC cohorts treated with chemotherapy (
= 90) and non-ICI regimens (
= 169).
Among patients with mTNBC treated with anti-PD-1/L1 therapies, high TMB and
alterations were associated with longer and shorter survival, respectively. These observations warrant validation in larger datasets.
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