A cellular and molecular spatial atlas of dystrophic muscle.

Asynchronous skeletal muscle degeneration/regeneration is a hallmark feature of Duchenne muscular dystrophy (DMD); however, traditional -omics technologies that lack spatial context make it difficult to study the biological mechanisms of how asynchronous regeneration contributes to disease progression. Here, using the severely dystrophic D2-mdx mouse model, we generated a high-resolution cellular and molecular spatial atlas of dystrophic muscle by integrating spatial transcriptomics and single-cell RNAseq datasets. Unbiased clustering revealed nonuniform distribution of unique cell populations throughout D2-mdx muscle that were associated with multiple regenerative timepoints, demonstrating that this model faithfully recapitulates the asynchronous regeneration observed in human DMD muscle. By probing spatiotemporal gene expression signatures, we found that propagation of inflammatory and fibrotic signals from locally damaged areas contributes to widespread pathology and that querying expression signatures within discrete microenvironments can identify targetable pathways for DMD therapy. Overall, this spatial atlas of dystrophic muscle provides a valuable resource for studying DMD disease biology and therapeutic target discovery.

A single-cell map of dynamic chromatin landscapes of immune cells in renal cell carcinoma.

A complete chart of the chromatin regulatory elements of immune cells in patients with cancer and their dynamic behavior is necessary to understand the developmental fates and guide therapeutic strategies. Here, we map the single-cell chromatin landscape of immune cells from blood, normal tumor-adjacent kidney tissue and malignant tissue from patients with early-stage clear cell renal cell carcinoma (ccRCC). We catalog the T cell states dictated by tissue-specific and developmental-stage-specific chromatin accessibility patterns, infer key chromatin regulators and observe rewiring of regulatory networks in the progression to dysfunction in CD8+ T cells. Unexpectedly, among the transcription factors orchestrating the path to dysfunction, NF-κB is associated with a pro-apoptotic program in late stages of dysfunction in tumor-infiltrating CD8+ T cells. Importantly, this epigenomic profiling stratified ccRCC patients based on a NF-κB-driven pro-apoptotic signature. This study provides a rich resource for understanding the functional states and regulatory dynamics of immune cells in ccRCC.

Simple mucinous cysts of the pancreas have heterogeneous somatic mutations.

Simple mucinous cysts of the pancreas have an epithelial lining resembling pancreatic intraepithelial neoplasia but may have a clinical presentation similar to premalignant mucinous neoplasms such as intraductal papillary mucinous neoplasms. Whether the epithelial lining shares genomic alterations with other pancreatic preinvasive neoplasms such as PanIN and intraductal papillary mucinous neoplasm has not been determined. We performed targeted sequencing analysis using a custom-designed MiSeq panel including the full coding regions of 18 pancreatic cancer genes on 13 clinically and pathologically well-characterized simple mucinous cysts. We detected 59 mutations in 15 genes in the cohort, with a median of 4 mutations per cyst (range = 0-16 mutations per cyst). The mutated genes and rate of detected mutations were as follows: KMT2C (MLL3) (62%), KRAS (15%), BRAF (8%), RNF43 (8%), CDKN2a (8%), TP53 (15%), and SMAD4 (8%). No GNAS mutations were detected. Four cases (31%) had no mutations detected. These findings place the majority of simple mucinous cysts of the pancreas in the spectrum of early, low-grade mucinous neoplasia, albeit with a different spectrum of genomic alterations compared with PanIN and intraductal papillary mucinous neoplasm.

The Evolutionary Origins of Recurrent Pancreatic Cancer.

Surgery is the only curative option for stage I/II pancreatic cancer; nonetheless, most patients will experience a recurrence after surgery and die of their disease. To identify novel opportunities for management of recurrent pancreatic cancer, we performed whole-exome or targeted sequencing of 10 resected primary cancers and matched intrapancreatic recurrences or distant metastases. We identified that recurrent disease after adjuvant or first-line platinum therapy corresponds to an increased mutational burden. Recurrent disease is enriched for genetic alterations predicted to activate MAPK/ERK and PI3K-AKT signaling and develops from a monophyletic or polyphyletic origin. Treatment-induced genetic bottlenecks lead to a modified genetic landscape and subclonal heterogeneity for driver gene alterations in part due to intermetastatic seeding. In 1 patient what was believed to be recurrent disease was an independent (second) primary tumor. These findings suggest routine post-treatment sampling may have value in the management of recurrent pancreatic cancer. SIGNIFICANCE: The biological features or clinical vulnerabilities of recurrent pancreatic cancer after pancreaticoduodenectomy are unknown. Using whole-exome sequencing we find that recurrent disease has a distinct genomic landscape, intermetastatic genetic heterogeneity, diverse clonal origins, and higher mutational burden than found for treatment-naïve disease.See related commentary by Bednar and Pasca di Magliano, p. 762.This article is highlighted in the In This Issue feature, p. 747.

A unifying paradigm for transcriptional heterogeneity and squamous features in pancreatic ductal adenocarcinoma.

Pancreatic cancer expression profiles largely reflect a classical or basal-like phenotype. The extent to which these profiles vary within a patient is unknown. We integrated evolutionary analysis and expression profiling in multiregion-sampled metastatic pancreatic cancers, finding that squamous features are the histologic correlate of an RNA-seq-defined basal-like subtype. In patients with coexisting basal and squamous and classical and glandular morphology, phylogenetic studies revealed that squamous morphology represented a subclonal population in an otherwise classical and glandular tumor. Cancers with squamous features were significantly more likely to have clonal mutations in chromatin modifiers, intercellular heterogeneity for MYC amplification and entosis. These data provide a unifying paradigm for integrating basal-type expression profiles, squamous histology and somatic mutations in chromatin modifier genes in the context of clonal evolution of pancreatic cancer.

Accelerated single cell seeding in relapsed multiple myeloma.

Multiple myeloma (MM) progression is characterized by the seeding of cancer cells in different anatomic sites. To characterize this evolutionary process, we interrogated, by whole genome sequencing, 25 samples collected at autopsy from 4 patients with relapsed MM and an additional set of 125 whole exomes collected from 51 patients. Mutational signatures analysis showed how cytotoxic agents introduce hundreds of unique mutations in each surviving cancer cell, detectable by bulk sequencing only in cases of clonal expansion of a single cancer cell bearing the mutational signature. Thus, a unique, single-cell genomic barcode can link chemotherapy exposure to a discrete time window in a patient's life. We leveraged this concept to show that MM systemic seeding is accelerated at relapse and appears to be driven by the survival and subsequent expansion of a single myeloma cell following treatment with high-dose melphalan therapy and autologous stem cell transplant.

Characterization of genetic subclonal evolution in pancreatic cancer mouse models.

The KPC mouse model, driven by the Kras and Trp53 transgenes, is well regarded for faithful recapitulation of human pancreatic cancer biology. However, the extent that this model recapitulates the subclonal evolution of this tumor type is unknown. Here we report evidence of continuing subclonal evolution after tumor initiation that largely reflect copy number alterations that target cellular processes of established significance in human pancreatic cancer. The evolutionary trajectories of the mouse tumors show both linear and branching patterns as well as clonal mixing. We propose the KPC model and derivatives have unexplored utility as a functional system to model the mechanisms and modifiers of tumor evolution.