Multimodal single-cell and whole-genome sequencing of small, frozen clinical specimens.

Single-cell genomics enables dissection of tumor heterogeneity and molecular underpinnings of drug response at an unprecedented resolution1-11. However, broad clinical application of these methods remains challenging, due to several practical and preanalytical challenges that are incompatible with typical clinical care workflows, namely the need for relatively large, fresh tissue inputs. In the present study, we show that multimodal, single-nucleus (sn)RNA/T cell receptor (TCR) sequencing, spatial transcriptomics and whole-genome sequencing (WGS) are feasible from small, frozen tissues that approximate routinely collected clinical specimens (for example, core needle biopsies). Compared with data from sample-matched fresh tissue, we find a similar quality in the biological outputs of snRNA/TCR-seq data, while reducing artifactual signals and compositional biases introduced by fresh tissue processing. Profiling sequentially collected melanoma samples from a patient treated in the KEYNOTE-001 trial12, we resolved cellular, genomic, spatial and clonotype dynamics that represent molecular patterns of heterogeneous intralesional evolution during anti-programmed cell death protein 1 therapy. To demonstrate applicability to banked biospecimens of rare diseases13, we generated a single-cell atlas of uveal melanoma liver metastasis with matched WGS data. These results show that single-cell genomics from archival, clinical specimens is feasible and provides a framework for translating these methods more broadly to the clinical arena.

Interplay of Immunosuppression and Immunotherapy Among Patients With Cancer and COVID-19.

Importance: Cytokine storm due to COVID-19 can cause high morbidity and mortality and may be more common in patients with cancer treated with immunotherapy (IO) due to immune system activation. Objective: To determine the association of baseline immunosuppression and/or IO-based therapies with COVID-19 severity and cytokine storm in patients with cancer. Design, Setting, and Participants: This registry-based retrospective cohort study included 12 046 patients reported to the COVID-19 and Cancer Consortium (CCC19) registry from March 2020 to May 2022. The CCC19 registry is a centralized international multi-institutional registry of patients with COVID-19 with a current or past diagnosis of cancer. Records analyzed included patients with active or previous cancer who had a laboratory-confirmed infection with SARS-CoV-2 by polymerase chain reaction and/or serologic findings. Exposures: Immunosuppression due to therapy; systemic anticancer therapy (IO or non-IO). Main Outcomes and Measures: The primary outcome was a 5-level ordinal scale of COVID-19 severity: no complications; hospitalized without requiring oxygen; hospitalized and required oxygen; intensive care unit admission and/or mechanical ventilation; death. The secondary outcome was the occurrence of cytokine storm. Results: The median age of the entire cohort was 65 years (interquartile range [IQR], 54-74) years and 6359 patients were female (52.8%) and 6598 (54.8%) were non-Hispanic White. A total of 599 (5.0%) patients received IO, whereas 4327 (35.9%) received non-IO systemic anticancer therapies, and 7120 (59.1%) did not receive any antineoplastic regimen within 3 months prior to COVID-19 diagnosis. Although no difference in COVID-19 severity and cytokine storm was found in the IO group compared with the untreated group in the total cohort (adjusted odds ratio [aOR], 0.80; 95% CI, 0.56-1.13, and aOR, 0.89; 95% CI, 0.41-1.93, respectively), patients with baseline immunosuppression treated with IO (vs untreated) had worse COVID-19 severity and cytokine storm (aOR, 3.33; 95% CI, 1.38-8.01, and aOR, 4.41; 95% CI, 1.71-11.38, respectively). Patients with immunosuppression receiving non-IO therapies (vs untreated) also had worse COVID-19 severity (aOR, 1.79; 95% CI, 1.36-2.35) and cytokine storm (aOR, 2.32; 95% CI, 1.42-3.79). Conclusions and Relevance: This cohort study found that in patients with cancer and COVID-19, administration of systemic anticancer therapies, especially IO, in the context of baseline immunosuppression was associated with severe clinical outcomes and the development of cytokine storm. Trial Registration: ClinicalTrials.gov Identifier: NCT04354701.

Characterizing metastatic uveal melanoma patients who develop symptomatic brain metastases.

Metastatic uveal melanoma (mUM) is an advanced ocular malignancy characterized by a hepatotropic pattern of spread. As the incidence of brain metastases (BM) in mUM patients has been thought to be low, routine CNS surveillance has not been recommended. Notably, no formal assessment of BM incidence in mUM has to date been published to support this clinical practice. We aimed to determine the true rate of BM in mUM and to clarify the clinical and genomic risk factors associated with BM patients through a collaborative multicenter, retrospective research effort. Data collected from 1,845 mUM patients in databases across four NCI-designated comprehensive cancer centers from 2006-2021 were retrospectively analyzed to identify patients with BM. Brain imaging in most cases were performed due to onset of neurological symptoms and not for routine surveillance. An analysis of demographics, therapies, gene expression profile, tumor next generation sequencing (NGS) data, time to metastasis (brain or other), and survival in the BM cohort was completed. 116/1,845 (6.3%) mUM patients were identified with BM. The median age at time of UM diagnosis was 54 years old (range: 18-77). The median time to any metastasis was 4.2 years (range: 0-30.8). The most common initial metastatic site was the liver (75.9%). 15/116 (12.9%) BM patients presented with BM at the time of initial metastatic diagnosis. Median survival after a diagnosis of BM was 7.6 months (range: 0.4-73.9). The median number of organs involved at time of BM diagnosis was 3 (range: 1-9). DecisionDX-UM profiling was completed on 13 patients: 10-Class 2, 2-Class 1B, and 1-Class 1A. NGS and cytogenetic data were available for 34 and 21 patients, respectively. BM was identified in 6.3% of mUM cases and was associated with high disease burden and a median survival of under 8 months once diagnosed. Since most patients in this cohort were symptomatic, the incidence of asymptomatic BM remains unknown. These data suggest the use of routine brain imaging in all mUM patients at risk for developing BM for early detection.

Dissecting the treatment-naive ecosystem of human melanoma brain metastasis.

Melanoma brain metastasis (MBM) frequently occurs in patients with advanced melanoma; yet, our understanding of the underlying salient biology is rudimentary. Here, we performed single-cell/nucleus RNA-seq in 22 treatment-naive MBMs and 10 extracranial melanoma metastases (ECMs) and matched spatial single-cell transcriptomics and T cell receptor (TCR)-seq. Cancer cells from MBM were more chromosomally unstable, adopted a neuronal-like cell state, and enriched for spatially variably expressed metabolic pathways. Key observations were validated in independent patient cohorts, patient-derived MBM/ECM xenograft models, RNA/ATAC-seq, proteomics, and multiplexed imaging. Integrated spatial analyses revealed distinct geography of putative cancer immune evasion and evidence for more abundant intra-tumoral B to plasma cell differentiation in lymphoid aggregates in MBM. MBM harbored larger fractions of monocyte-derived macrophages and dysfunctional TOX+CD8+ T cells with distinct expression of immune checkpoints. This work provides comprehensive insights into MBM biology and serves as a foundational resource for further discovery and therapeutic exploration.

Concurrent radiotherapy and ipilimumab immunotherapy for patients with melanoma.

Ipilimumab and radiotherapy are commonly used to treat unresectable and metastatic melanoma. Results from preclinical studies and case reports suggest a biologic interaction between these two treatments. To understand the clinical implications of the interaction, we carried out a retrospective study reviewing records of patients treated with ipilimumab and radiotherapy for melanoma at our institution between 2005 and 2011. The review included details of treatment, response, adverse events (AE), and overall survival (OS). Twenty-nine patients underwent 33 courses of non-brain radiotherapy between their first and last dose of ipilimumab. Immune-related AEs (ir-AEs) were observed in 43% of patients receiving ipilimumab at 10 mg/kg and in 22% of patients receiving 3 mg/kg; the frequency of ir-AEs was not significantly different compared with previous studies of ipilimumab alone. Radiotherapy-related AEs were significantly more common in patients receiving higher doses of radiation. Palliation of symptoms was reported by 77% of patients after radiotherapy. Median OS was 9 and 39 months in patients receiving radiotherapy during induction and maintenance with ipilimumab, respectively. In this retrospective study, concurrent ipilimumab and radiotherapy was neither associated with higher than expected rates of AEs nor did it abrogate palliative effects of radiotherapy or survival benefits of ipilimumab. Further studies to prospectively explore the efficacy of this therapeutic combination are warranted.