Advancing Genomic Cancer Medicine in Rural and Underserved States.

This Viewpoint presents an organizational and social template for medical communities to provide contemporary care for patients with cancer in rural and underserved states.

Genome-matched treatments and patient outcomes in the Maine Cancer Genomics Initiative (MCGI).

Genomic tumor testing (GTT) is an emerging technology aimed at identifying variants in tumors that can be targeted with genomically matched drugs. Due to limited resources, rural patients receiving care in community oncology settings may be less likely to benefit from GTT. We analyzed GTT results and observational clinical outcomes data from patients enrolled in the Maine Cancer Genomics Initiative (MCGI), which provided access to GTTs; clinician educational resources; and genomic tumor boards in community practices in a predominantly rural state. 1603 adult cancer patients completed enrollment; 1258 had at least one potentially actionable variant identified. 206 (16.4%) patients received a total of 240 genome matched treatments, of those treatments, 64% were FDA-approved in the tumor type, 27% FDA-approved in a different tumor type and 9% were given on a clinical trial. Using Inverse Probability of Treatment Weighting to adjust for baseline characteristics, a Cox proportional hazards model demonstrated that patients who received genome matched treatment were 31% less likely to die within 1 year compared to those who did not receive genome matched treatment (HR: 0.69; 95% CI: 0.52-0.90; p-value: 0.006). Overall, GTT through this initiative resulted in levels of genome matched treatment that were similar to other initiatives, however, clinical trials represented a smaller share of treatments than previously reported, and "off-label" treatments represented a greater share. Although this was an observational study, we found evidence for a potential 1-year survival benefit for patients who received genome matched treatments. These findings suggest that when disseminated and implemented with a supportive infrastructure, GTT may benefit cancer patients in rural community oncology settings, with further work remaining on providing genome-matched clinical trials.

High-throughput deconvolution of 3D organoid dynamics at cellular resolution for cancer pharmacology with Cellos.

Three-dimensional (3D) organoid cultures are flexible systems to interrogate cellular growth, morphology, multicellular spatial architecture, and cellular interactions in response to treatment. However, computational methods for analysis of 3D organoids with sufficiently high-throughput and cellular resolution are needed. Here we report Cellos, an accurate, high-throughput pipeline for 3D organoid segmentation using classical algorithms and nuclear segmentation using a trained Stardist-3D convolutional neural network. To evaluate Cellos, we analyze ~100,000 organoids with ~2.35 million cells from multiple treatment experiments. Cellos segments dye-stained or fluorescently-labeled nuclei and accurately distinguishes distinct labeled cell populations within organoids. Cellos can recapitulate traditional luminescence-based drug response of cells with complex drug sensitivities, while also quantifying changes in organoid and nuclear morphologies caused by treatment as well as cell-cell spatial relationships that reflect ecological affinity. Cellos provides powerful tools to perform high-throughput analysis for pharmacological testing and biological investigation of organoids based on 3D imaging.

The Maine Cancer Genomics Initiative: Implementing a Community Cancer Genomics Program Across an Entire Rural State.

PURPOSE: The Maine Cancer Genomics Initiative (MCGI) aimed to overcome patient- and provider-level barriers to using genomic tumor testing (GTT) in rural practices by providing genomic tumor boards (GTBs), clinician education, and access to comprehensive large-panel next-generation sequencing to all patients with cancer in Maine. This paper describes the successful implementation of the initiative and three key services made operative between 2016 and 2020. METHODS: A community-inclusive, hub-and-spoke approach was taken to implement the three program components: (1) a centralized GTB program; (2) a modular online education program, designed using an iterative approach with broad clinical stakeholders; and (3) GTT free of charge to clinicians and patients. Implementation timelines, participation metrics, and survey data were used to describe the rollout. RESULTS: The MCGI was launched over an 18-month period at all 19 oncology practices in the State. Seventy-nine physicians (66 medical oncologists, 5 gynecologic oncologists, 1 neuro-oncologist, and 7 pediatric oncologists) enrolled on the study, representing 100% of all practicing oncologists in Maine. Between July 2017 and September 2020, 1610 patients were enrolled. A total of 515 cases were discussed by 47 (73%) clinicians in 196 GTBs. Clinicians who participated in the GTBs enrolled significantly more patients on the study, stayed in Maine, and reported less time spent in clinical patient care. CONCLUSION: The MCGI was able to engage geographically and culturally disparate cancer care practices in a precision oncology program using a hub-and-spoke model. By facilitating access to GTT, structured education, and GTBs, we narrowed the gap in the implementation of precision oncology in one of the most rural states in the country.

Mouse models for immuno-oncology.

Realizing the clinical promise of cancer immunotherapy is hindered by gaps in our knowledge of in vivo mechanisms underlying treatment response as well as treatment limiting toxicity. Preclinical in vivo model systems and technologies are required to address these knowledge gaps and to surmount the challenges faced in the clinical application of immunotherapy. Mice are commonly used for basic and translational research to support development and testing of immune interventions, including for cancer. Here, we discuss the advantages and the limitations of current models as well as future developments.

Cellos: High-throughput deconvolution of 3D organoid dynamics at cellular resolution for cancer pharmacology.

Three-dimensional (3D) culture models, such as organoids, are flexible systems to interrogate cellular growth and morphology, multicellular spatial architecture, and cell interactions in response to drug treatment. However, new computational methods to segment and analyze 3D models at cellular resolution with sufficiently high throughput are needed to realize these possibilities. Here we report Cellos (Cell and Organoid Segmentation), an accurate, high throughput image analysis pipeline for 3D organoid and nuclear segmentation analysis. Cellos segments organoids in 3D using classical algorithms and segments nuclei using a Stardist-3D convolutional neural network which we trained on a manually annotated dataset of 3,862 cells from 36 organoids confocally imaged at 5 µm z-resolution. To evaluate the capabilities of Cellos we then analyzed 74,450 organoids with 1.65 million cells, from multiple experiments on triple negative breast cancer organoids containing clonal mixtures with complex cisplatin sensitivities. Cellos was able to accurately distinguish ratios of distinct fluorescently labelled cell populations in organoids, with ≤3% deviation from the seeding ratios in each well and was effective for both fluorescently labelled nuclei and independent DAPI stained datasets. Cellos was able to recapitulate traditional luminescence-based drug response quantifications by analyzing 3D images, including parallel analysis of multiple cancer clones in the same well. Moreover, Cellos was able to identify organoid and nuclear morphology feature changes associated with treatment. Finally, Cellos enables 3D analysis of cell spatial relationships, which we used to detect ecological affinity between cancer cells beyond what arises from local cell division or organoid composition. Cellos provides powerful tools to perform high throughput analysis for pharmacological testing and biological investigation of organoids based on 3D imaging.

Adaptive Sentinel Testing in Workplace for COVID-19 Pandemic.

Testing and isolation of infectious employees is one of the critical strategies to make the workplace safe during the pandemic for many organizations. Adaptive testing frequency reduces cost while keeping the pandemic under control at the workplace. However, most models aimed at estimating test frequencies were structured for municipalities or large organizations such as university campuses of highly mobile individuals. By contrast, the workplace exhibits distinct characteristics: employee positivity rate may be different from the local community because of rigorous protective measures at workplace, or self-selection of co-workers with common behavioral tendencies for adherence to pandemic mitigation guidelines. Moreover, dual exposure to COVID-19 occurs at work and home that complicates transmission modeling, as does transmission tracing at the workplace. Hence, we developed bi-modal SEIR (Susceptible, Exposed, Infectious, and Removed) model and R-shiny tool that accounts for these differentiating factors to adaptively estimate the testing frequency for workplace. Our tool uses easily measurable parameters: community incidence rate, risks of acquiring infection from community and workplace, workforce size, and sensitivity of testing. Our model is best suited for moderate-sized organizations with low internal transmission rates, no-outward facing employees whose position demands frequent in-person interactions with the public, and low to medium population positivity rates. Simulations revealed that employee behavior in adherence to protective measures at work and in their community, and the onsite workforce size have large effects on testing frequency. Reducing workplace transmission rate through workplace mitigation protocols and higher sensitivity of the test deployed, although to a lesser extent. Furthermore, our simulations showed that sentinel testing leads to only marginal increase in the number of infections even for high community incidence rates, suggesting that this may be a cost-effective approach in future pandemics. We used our model to accurately guide testing regimen for three campuses of the Jackson Laboratory.

Lipid-laden lung mesenchymal cells foster breast cancer metastasis via metabolic reprogramming of tumor cells and natural killer cells.

While the distant organ environment is known to support metastasis of primary tumors, its metabolic roles in this process remain underdetermined. Here, in breast cancer models, we found lung-resident mesenchymal cells (MCs) accumulating neutral lipids at the pre-metastatic stage. This was partially mediated by interleukin-1ß (IL-1ß)-induced hypoxia-inducible lipid droplet-associated (HILPDA) that subsequently represses adipose triglyceride lipase (ATGL) activity in lung MCs. MC-specific ablation of the ATGL or HILPDA genes in mice reinforced and reduced lung metastasis of breast cancer respectively, suggesting a metastasis-promoting effect of lipid-laden MCs. Mechanistically, lipid-laden MCs transported their lipids to tumor cells and natural killer (NK) cells via exosome-like vesicles, leading to heightened tumor cell survival and proliferation and NK cell dysfunction. Blockage of IL-1ß, which was effective singly, improved the efficacy of adoptive NK cell immunotherapy in mitigating lung metastasis. Collectively, lung MCs metabolically regulate tumor cells and anti-tumor immunity to facilitate breast cancer lung metastasis.

Animals, quality and the pursuit of relevance.

In 2021, the National Institutes of Health Advisory Committee to the Director (ACD) announced recommendations to improve the reproducibility of biomedical research using animals. In response, The Jackson Laboratory faculty and institutional leaders identified key strategies to further address this important issue. Taking inspiration from the evolution of clinical trials over recent decades in response to similar challenges, we identified opportunities for improvement, including establishment of common standards, use of genetically diverse populations, requirement for robust study design with appropriate statistical methods, and improvement in public databases to facilitate meta-analyses. In this Perspective, we share our response to ACD recommendations, with a specific focus on mouse models, with the aim of promoting continued active dialogue among researchers, using any animal system, worldwide. Such discussion will help to inform the biomedical community about these recommendations and further support their much-needed implementation.

A Genomically and Clinically Annotated Patient-Derived Xenograft Resource for Preclinical Research in Non-Small Cell Lung Cancer.

Patient-derived xenograft (PDX) models are an effective preclinical in vivo platform for testing the efficacy of novel drugs and drug combinations for cancer therapeutics. Here we describe a repository of 79 genomically and clinically annotated lung cancer PDXs available from The Jackson Laboratory that have been extensively characterized for histopathologic features, mutational profiles, gene expression, and copy-number aberrations. Most of the PDXs are models of non-small cell lung cancer (NSCLC), including 37 lung adenocarcinoma (LUAD) and 33 lung squamous cell carcinoma (LUSC) models. Other lung cancer models in the repository include four small cell carcinomas, two large cell neuroendocrine carcinomas, two adenosquamous carcinomas, and one pleomorphic carcinoma. Models with both de novo and acquired resistance to targeted therapies with tyrosine kinase inhibitors are available in the collection. The genomic profiles of the LUAD and LUSC PDX models are consistent with those observed in patient tumors from The Cancer Genome Atlas and previously characterized gene expression-based molecular subtypes. Clinically relevant mutations identified in the original patient tumors were confirmed in engrafted PDX tumors. Treatment studies performed in a subset of the models recapitulated the responses expected on the basis of the observed genomic profiles. These models therefore serve as a valuable preclinical platform for translational cancer research. SIGNIFICANCE: Patient-derived xenografts of lung cancer retain key features observed in the originating patient tumors and show expected responses to treatment with standard-of-care agents, providing experimentally tractable and reproducible models for preclinical investigations.

Genomic and epigenomic BRCA alterations predict adaptive resistance and response to platinum-based therapy in patients with triple-negative breast and ovarian carcinomas.

Triple-negative breast cancer (TNBC) and ovarian carcinomas (OvCas) with BRCA1 promoter methylation (BRCA1meth) respond more poorly to alkylating agents compared to those bearing mutations in BRCA1 and BRCA2 (BRCAmut). This is a conundrum given the biologically equivalent homologous recombination deficiency (HRD) induced by these genetic and epigenetic BRCA perturbations. We dissected this problem through detailed genomic analyses of TNBC and OvCa cohorts and experimentation with patient-derived xenografts and genetically engineered cell lines. We found that despite identical downstream genomic mutational signatures associated with BRCA1meth and BRCAmut states, BRCA1meth uniformly associates with poor outcomes. Exposure of BRCA1meth TNBCs to platinum chemotherapy, either as clinical treatment of a patient or as experimental in vivo exposure of preclinical patient derived xenografts, resulted in allelic loss of BRCA1 methylation and increased BRCA1 expression and platinum resistance. These data suggest that, unlike BRCAmut cancers, where BRCA loss is a genetically "fixed" deficiency state, BRCA1meth cancers are highly adaptive to genotoxin exposure and, through reversal of promoter methylation, recover BRCA1 expression and become resistant to therapy. We further found a specific augmented immune transcriptional signal associated with enhanced response to platinum chemotherapy but only in patients with BRCA-proficient cancers. We showed how integrating both this cancer immune signature and the presence of BRCA mutations results in more accurate predictions of patient response when compared to either HRD status or BRCA status alone. This underscores the importance of defining BRCA heterogeneity in optimizing the predictive precision of assigning response probabilities in TNBC and OvCa.

Employees' Views and Ethical, Legal, and Social Implications Assessment of Voluntary Workplace Genomic Testing.

Employers have begun to offer voluntary workplace genomic testing (wGT) as part of employee wellness benefit programs, but few empirical studies have examined the ethical, legal, and social implications (ELSI) of wGT. To better understand employee perspectives on wGT, employees were surveyed at a large biomedical research institution. Survey respondents were presented with three hypothetical scenarios for accessing health-related genomic testing: via (1) their doctor; (2) their workplace; and 3) a commercial direct-to-consumer (DTC) genetic testing company. Overall, 594 employees (28%) responded to the survey. Respondents indicated a preference for genomic testing in the workplace setting (70%; 95% CI 66-74%), followed by doctor's office (54%; 95% CI 50-58%), and DTC testing (20%; 95% CI 17-24%). Prior to participating in wGT, respondents wanted to know about confidentiality of test results (79%), existence of relevant laws and policies (70%), and privacy protection (64%). Across scenarios, 92% of respondents preferred to view the test results with a genetic counselor. These preliminary results suggest that many employees are interested and even prefer genetic testing in the workplace and would prefer testing with support from genetic health professionals. Confirmation in more diverse employer settings will be needed to generalize such findings.

FANCM regulates repair pathway choice at stalled replication forks.

Repair pathway "choice" at stalled mammalian replication forks is an important determinant of genome stability; however, the underlying mechanisms are poorly understood. FANCM encodes a multi-domain scaffolding and motor protein that interacts with several distinct repair protein complexes at stalled forks. Here, we use defined mutations engineered within endogenous Fancm in mouse embryonic stem cells to study how Fancm regulates stalled fork repair. We find that distinct FANCM repair functions are enacted by molecularly separable scaffolding domains. These findings define FANCM as a key mediator of repair pathway choice at stalled replication forks and reveal its molecular mechanism. Notably, mutations that inactivate FANCM ATPase function disable all its repair functions and "trap" FANCM at stalled forks. We find that Brca1 hypomorphic mutants are synthetic lethal with Fancm null or Fancm ATPase-defective mutants. The ATPase function of FANCM may therefore represent a promising "druggable" target for therapy of BRCA1-linked cancer.

Reduced subgenomic RNA expression is a molecular indicator of asymptomatic SARS-CoV-2 infection.

Background: It is estimated that up to 80% of infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are asymptomatic and asymptomatic patients can still effectively transmit the virus and cause disease. While much of the effort has been placed on decoding single nucleotide variation in SARS-CoV-2 genomes, considerably less is known about their transcript variation and any correlation with clinical severity in human hosts, as defined here by the presence or absence of symptoms. Methods: To assess viral genomic signatures of disease severity, we conducted a systematic characterization of SARS-CoV-2 transcripts and genetic variants in 81 clinical specimens collected from symptomatic and asymptomatic individuals using multi-scale transcriptomic analyses including amplicon-seq, short-read metatranscriptome and long-read Iso-seq. Results: Here we show a highly coordinated and consistent pattern of sgRNA expression from individuals with robust SARS-CoV-2 symptomatic infection and their expression is significantly repressed in the asymptomatic infections. We also observe widespread inter- and intra-patient variants in viral RNAs, known as quasispecies frequently found in many RNA viruses. We identify unique sets of deletions preferentially found primarily in symptomatic individuals, with many likely to confer changes in SARS-CoV-2 virulence and host responses. Moreover, these frequently occurring structural variants in SARS-CoV-2 genomes serve as a mechanism to further induce SARS-CoV-2 proteome complexity. Conclusions: Our results indicate that differential sgRNA expression and structural mutational burden are highly correlated with the clinical severity of SARS-CoV-2 infection. Longitudinally monitoring sgRNA expression and structural diversity could further guide treatment responses, testing strategies, and vaccine development.

Chromatin topology reorganization and transcription repression by PML-RARα in acute promyeloid leukemia.

BACKGROUND: Acute promyeloid leukemia (APL) is characterized by the oncogenic fusion protein PML-RARα, a major etiological agent in APL. However, the molecular mechanisms underlying the role of PML-RARα in leukemogenesis remain largely unknown. RESULTS: Using an inducible system, we comprehensively analyze the 3D genome organization in myeloid cells and its reorganization after PML-RARα induction and perform additional analyses in patient-derived APL cells with native PML-RARα. We discover that PML-RARα mediates extensive chromatin interactions genome-wide. Globally, it redefines the chromatin topology of the myeloid genome toward a more condensed configuration in APL cells; locally, it intrudes RNAPII-associated interaction domains, interrupts myeloid-specific transcription factors binding at enhancers and super-enhancers, and leads to transcriptional repression of genes critical for myeloid differentiation and maturation. CONCLUSIONS: Our results not only provide novel topological insights for the roles of PML-RARα in transforming myeloid cells into leukemia cells, but further uncover a topological framework of a molecular mechanism for oncogenic fusion proteins in cancers.

High-resolution deconstruction of evolution induced by chemotherapy treatments in breast cancer xenografts.

The processes by which tumors evolve are essential to the efficacy of treatment, but quantitative understanding of intratumoral dynamics has been limited. Although intratumoral heterogeneity is common, quantification of evolution is difficult from clinical samples because treatment replicates cannot be performed and because matched serial samples are infrequently available. To circumvent these problems we derived and assayed large sets of human triple-negative breast cancer xenografts and cell cultures from two patients, including 86 xenografts from cyclophosphamide, doxorubicin, cisplatin, docetaxel, or vehicle treatment cohorts as well as 45 related cell cultures. We assayed these samples via exome-seq and/or high-resolution droplet digital PCR, allowing us to distinguish complex therapy-induced selection and drift processes among endogenous cancer subclones with cellularity uncertainty <3%. For one patient, we discovered two predominant subclones that were granularly intermixed in all 48 co-derived xenograft samples. These two subclones exhibited differential chemotherapy sensitivity-when xenografts were treated with cisplatin for 3 weeks, the post-treatment volume change was proportional to the post-treatment ratio of subclones on a xenograft-to-xenograft basis. A subsequent cohort in which xenografts were treated with cisplatin, allowed a drug holiday, then treated a second time continued to exhibit this proportionality. In contrast, xenografts from other treatment cohorts, spatially dissected xenograft fragments, and cell cultures evolved in diverse ways but with substantial population bottlenecks. These results show that ecosystems susceptible to successive retreatment can arise spontaneously in breast cancer in spite of a background of irregular subclonal bottlenecks, and our work provides to our knowledge the first quantification of the population genetics of such a system. Intriguingly, in such an ecosystem the ratio of common subclones is predictive of the state of treatment susceptibility, showing how measurements of subclonal heterogeneity could guide treatment for some patients.

Mouse Models for Cancer Immunotherapy Research.

Immunotherapy has revolutionized cancer therapy, largely attributed to the success of immune-checkpoint blockade. However, there are subsets of patients across multiple cancers who have not shown robust responses to these agents. A major impediment to progress in the field is the availability of faithful mouse models that recapitulate the complexity of human malignancy and immune contexture within the tumor microenvironment. These models are urgently needed across all malignancies to interrogate and predict antitumor immune responses and therapeutic efficacy in clinical trials. Herein, we seek to review pros and cons of different cancer mouse models, and how they can be used as platforms to predict efficacy and resistance to cancer immunotherapies.Significance: Although immunotherapy has shown substantial benefit in the treatment of a variety of malignancies, a key hurdle toward the advancement of these therapies is the availability of immunocompetent preclinical mouse models that recapitulate human disease. Here, we review the evolution of preclinical mouse models and their utility as coclinical platforms for mechanistic interrogation of cancer immunotherapies. Cancer Discov; 8(11); 1358-65. ©2018 AACR.

Development, Function, and Clinical Significance of Plasmacytoid Dendritic Cells in Chronic Myeloid Leukemia.

Plasmacytoid dendritic cells (pDC) are the main producers of a key T-cell-stimulatory cytokine, IFNα, and critical regulators of antiviral immunity. Chronic myeloid leukemia (CML) is caused by BCR-ABL, which is an oncogenic tyrosine kinase that can be effectively inhibited with ABL-selective tyrosine kinase inhibitors (TKI). BCR-ABL-induced suppression of the transcription factor interferon regulatory factor 8 was previously proposed to block pDC development and compromise immune surveillance in CML. Here, we demonstrate that pDCs in newly diagnosed CML (CML-pDC) develop quantitatively normal and are frequently positive for the costimulatory antigen CD86. They originate from low-level BCR-ABL-expressing precursors. CML-pDCs also retain their competence to maturate and to secrete IFN. RNA sequencing reveals a strong inflammatory gene expression signature in CML-pDCs. Patients with high CML-pDC counts at diagnosis achieve inferior rates of deep molecular remission (MR) under nilotinib, unless nilotinib therapy is combined with IFN, which strongly suppresses circulating pDC counts. Although most pDCs are BCR-ABL-negative in MR, a substantial proportion of BCR-ABL + CML-pDCs persists under TKI treatment. This could be of relevance, because CML-pDCs elicit CD8+ T cells, which protect wild-type mice from CML. Together, pDCs are identified as novel functional DC population in CML, regulating antileukemic immunity and treatment outcome in CML.Significance: CML-pDC originates from low-level BCR-ABL expressing stem cells into a functional immunogenic DC-population regulating antileukemic immunity and treatment outcome in CML. Cancer Res; 78(21); 6223-34. ©2018 AACR.