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Myelofibrosis (MF) is a clonal stem cell neoplasm characterized by abnormal JAK-STAT signaling, chronic inflammation, cytopenias, and risk of transformation to acute leukemia. Despite improvements in the therapeutic options for patients with MF, allogeneic hematopoietic stem cell transplantation remains the only curative treatment. We previously demonstrated multiple immunosuppressive mechanisms in patients with MF, including increased expression of programmed cell death protein 1 (PD-1) on T cells compared with healthy controls. Therefore, we conducted a multicenter, open-label, phase 2, single-arm study of pembrolizumab in patients with Dynamic International Prognostic Scoring System category of intermediate-2 or greater primary, post-essential thrombocythemia or post-polycythemia vera myelofibrosis that were ineligible for or were previously treated with ruxolitinib. The study followed a Simon 2-stage design and enrolled a total of 10 patients, 5 of whom had JAK2V617mutation, 2 had CALR mutation, and 6 had additional mutations. Most patients were previously treated with ruxolitinib. Pembrolizumab treatment was well tolerated, but there were no objective clinical responses, so the study closed after the first stage was completed. However, immune profiling by flow cytometry, T-cell receptor sequencing, and plasma proteomics demonstrated changes in the immune milieu of patients, which suggested improved T-cell responses that can potentially favor antitumor immunity. The fact that these changes were not reflected in a clinical response strongly suggests that combination immunotherapeutic approaches rather than monotherapy may be necessary to reverse the multifactorial mechanisms of immune suppression in myeloproliferative neoplasms. This trial was registered at www.clinicaltrials.gov as #NCT03065400.

Purpose: Most ALK-positive lung cancers will develop ALK-independent resistance after treatment with next-generation ALK inhibitors. MET amplification has been described in patients progressing on ALK inhibitors, but frequency of this event has not been comprehensively assessed. Experimental Design: We performed FISH and/or next-generation sequencing on 207 posttreatment tissue (n = 101) or plasma (n = 106) specimens from patients with ALK-positive lung cancer to detect MET genetic alterations. We evaluated ALK inhibitor sensitivity in cell lines with MET alterations and assessed antitumor activity of ALK/MET blockade in ALK-positive cell lines and 2 patients with MET-driven resistance. Results: MET amplification was detected in 15% of tumor biopsies from patients relapsing on next-generation ALK inhibitors, including 12% and 22% of biopsies from patients progressing on second-generation inhibitors or lorlatinib, respectively. Patients treated with a second-generation ALK inhibitor in the first-line setting were more likely to develop MET amplification than those who had received next-generation ALK inhibitors after crizotinib (P = 0.019). Two tumor specimens harbored an identical ST7-MET rearrangement, one of which had concurrent MET amplification. Expressing ST7-MET in the sensitive H3122 ALK-positive cell line induced resistance to ALK inhibitors that was reversed with dual ALK/MET inhibition. MET inhibition resensitized a patient-derived cell line harboring both ST7-MET and MET amplification to ALK inhibitors. Two patients with ALK-positive lung cancer and acquired MET alterations achieved rapid responses to ALK/MET combination therapy. Conclusions: Treatment with next-generation ALK inhibitors, particularly in the first-line setting, may lead to MET-driven resistance. Patients with acquired MET alterations may derive clinical benefit from therapies that target both ALK and MET.

Cancer is a disease manifesting in abrogation of developmental programs, and malignancies are named based on their cell or tissue of origin. However, a systematic atlas of tumor origins is lacking. Here we map the single cell organogenesis of 56 developmental trajectories to the transcriptomes of over 10,000 tumors across 33 cancer types. We use this map to deconvolute individual tumors into their constituent developmental trajectories. Based on these deconvoluted developmental programs, we construct a Developmental Multilayer Perceptron (D-MLP) classifier that outputs cancer origin. The D-MLP classifier (ROC-AUC: 0.974 for top prediction) outperforms classification based on expression of either oncogenes or highly variable genes. We analyze tumors from patients with cancer of unknown primary (CUP), selecting the most difficult cases where extensive multimodal workup yielded no definitive tumor type. D-MLP revealed insights into developmental origins and diagnosis for most patient tumors. Our results provide a map of tumor developmental origins, provide a tool for diagnostic pathology, and suggest developmental classification may be a useful approach for otherwise unclassified patient tumors.

Developing and deploying new diagnostic tests is difficult, but the need to do so in response to a rapidly emerging pandemic such as COVID-19 is crucially important for an effective response. In the early stages of a pandemic, laboratories play a key role in helping health care providers and public health authorities detect active infection, a task most commonly achieved using nucleic acid-based assays. While the landscape of diagnostics is rapidly evolving, polymerase chain reaction (PCR) remains the gold-standard of nucleic acid-based diagnostic assays, in part due to its reliability, flexibility, and wide deployment. To address a critical local shortage of testing capacity persisting during the COVID-19 outbreak, our hospital set up a molecular based laboratory developed test (LDT) to accurately and safely diagnose SARS-CoV-2. We describe here the process of developing an emergency-use LDT, in the hope that our experience will be useful to other laboratories in future outbreaks and will help to lower barriers to fast and accurate diagnostic testing in crisis conditions.

Genetic diversity among metastases is poorly understood but contains important information about disease evolution at secondary sites. Here we investigate inter- and intra-lesion heterogeneity for two types of metastases that associate with different clinical outcomes: lymph node and distant organ metastases in human colorectal cancer. We develop a rigorous mathematical framework for quantifying metastatic phylogenetic diversity. Distant metastases are typically monophyletic and genetically similar to each other. Lymph node metastases, in contrast, display high levels of inter-lesion diversity. We validate these findings by analyzing 317 multi-region biopsies from an independent cohort of 20 patients. We further demonstrate higher levels of intra-lesion heterogeneity in lymph node than in distant metastases. Our results show that fewer primary tumor lineages seed distant metastases than lymph node metastases, indicating that the two sites are subject to different levels of selection. Thus, lymph node and distant metastases develop through fundamentally different evolutionary mechanisms. Analysis of metastases using a mathematical framework and multi-region sampling data shows that lymph node metastases have higher levels of intratumor heterogeneity than distant metastases, and that these form via different evolutionary mechanisms.