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Phenotypic Cell Classification Tree

Direct interactions between tumor and immune cells mediate the antitumor effect of all modern cancer immunotherapeutic agents. Simultaneously, tumor cells have evolved mechanisms of evasion, including the downregulation of HLA-I, potentially disrupting the mechanism of action employed by many immune checkpoint inhibitors. And yet, the in situ interplay between these cells within the tumor immune microenvironment (TIME) remains elusive. Recent advances in histologic multiplex bioimaging platforms have enabled in-depth molecular characterization of single cells within spatially preserved and clinically archived tumor tissues. Herein, we applied multiplex immunofluorescence to excisional lymph node biopsies from 14 patients with metastatic melanoma who experienced clear objective responses to immunotherapy (7 complete response; 7 progressive disease) to determine distinguishing features of the TIME in the pretreatment setting. Distinct regions of the TIME were evaluated using 35 proteins probing tumor, immune, and vasculature components across 323 fields of view. Single-cell compositional analysis confirmed established prognostic immune cell types including increased prevalence of cytotoxic T cells within the tumor core fields of view of responders. Integrating single-cell quantification with the spatial arrangement of cellular neighborhoods surrounding tumor cells revealed novel, spatial immune signatures capable of stratifying TIME based on clinical response. Our analysis revealed dynamic cellular composition of the tumor-centric cellular neighborhood (TCCN) based on anatomic subregion, functional expression of HLA-I by the index tumor cell and ultimately clinical response to immunotherapy. Overall, this study provides an analytic framework to resolve the cellular complexity of the TIME, increasingly relevant to the outcomes of modern cancer immunotherapy.Significance:Findings from this work propose a novel approach to resolving clinical heterogeneity of the TIME by objectively quantifying the cellular interactions occurring in metastatic melanoma lymph node tissue utilizing multiplex immunofluorescence. This study provides an analytic and biologically derived unit of measure, the TCCN which is customizable for studying critical paracrine interactions within spatially preserved tissue of various cancers and across the spectrum of multiplex imaging modalities.

Antibody reagents and validation for MxIF panel design

Summary data of FOV distribution, single cells segmented, classified and clinical response to therapy

Virtual H&E images for whole excisional lymph node biopsies from each clinical case

Direct interactions between tumor and immune cells mediate the antitumor effect of all modern cancer immunotherapeutic agents. Simultaneously, tumor cells have evolved mechanisms of evasion, including the downregulation of HLA-I, potentially disrupting the mechanism of action employed by many immune checkpoint inhibitors. And yet, the in situ interplay between these cells within the tumor immune microenvironment (TIME) remains elusive. Recent advances in histologic multiplex bioimaging platforms have enabled in-depth molecular characterization of single cells within spatially preserved and clinically archived tumor tissues. Herein, we applied multiplex immunofluorescence to excisional lymph node biopsies from 14 patients with metastatic melanoma who experienced clear objective responses to immunotherapy (7 complete response; 7 progressive disease) to determine distinguishing features of the TIME in the pretreatment setting. Distinct regions of the TIME were evaluated using 35 proteins probing tumor, immune, and vasculature components across 323 fields of view. Single-cell compositional analysis confirmed established prognostic immune cell types including increased prevalence of cytotoxic T cells within the tumor core fields of view of responders. Integrating single-cell quantification with the spatial arrangement of cellular neighborhoods surrounding tumor cells revealed novel, spatial immune signatures capable of stratifying TIME based on clinical response. Our analysis revealed dynamic cellular composition of the tumor-centric cellular neighborhood (TCCN) based on anatomic subregion, functional expression of HLA-I by the index tumor cell and ultimately clinical response to immunotherapy. Overall, this study provides an analytic framework to resolve the cellular complexity of the TIME, increasingly relevant to the outcomes of modern cancer immunotherapy. Significance: Findings from this work propose a novel approach to resolving clinical heterogeneity of the TIME by objectively quantifying the cellular interactions occurring in metastatic melanoma lymph node tissue utilizing multiplex immunofluorescence. This study provides an analytic and biologically derived unit of measure, the TCCN which is customizable for studying critical paracrine interactions within spatially preserved tissue of various cancers and across the spectrum of multiplex imaging modalities.

Introduction Neutropenic fever following high-dose chemotherapy and autologous stem cell transplantation (ASCT) is a common (incidence 63-100%) and potentially life-threatening complication. Recommended time to antibiotic (TTA) administration is within 1 hr of fever onset with delays associated with significant morbidity, prolonged hospitalization, and mortality. Standard of care guidelines emphasize patient self-monitoring for fever, with instructions to seek immediate medical attention if body temperature (temp) reaches 100.4°F or higher. In this study, we evaluated if a novel wearable, continuous temp monitor, tPatch, could reliably estimate core body temp and detect fever in an outpatient setting following ASCT. Additionally, we gathered preliminary data to explore early detection and prediction of clinically relevant temp rise in this clinical setting. Methods Patients (N = 86) with hematologic malignancies (62% multiple myeloma) who underwent high-dose chemotherapy followed by ASCT at Mayo Clinic, MN were prospectively enrolled between June 2018 and March 2019. Patients (82% male) wore an axilla-placed tPatch continuously for 7 days in an outpatient setting during the post ASCT period and were asked to record self-measured oral temp in 3-4 hr intervals daily using a standardized thermometer after appropriate training . Patients followed standard of care procedures with daily clinic assessment of temp, blood counts, and vital signs. An optional patient questionnaire was given at end-of-study. A model was trained using both patient- and clinic-assessed oral temp measures to estimate core temp from 2 sensors on the tPatch device. Core temp estimates and trends were then compared to patient- and clinic-assessed measurements. Fever was defined as a temperature ≥100.4°F for at least 1 hr. Results When compared to all oral temp reads, the tPatch estimated core temp within 0.03 ± 0.7℉. Among the 86 patients, clinic-assessed fever incidence was 29.4% while tPatch-assessed incidence was 58.8%. Using all clinic-recorded temp readings as "ground truth," the sensitivity and specificity of the tPatch algorithm in detecting fevers were 88% and 86%, respectively, while patient self fever detection sensitivity was 62% and specificity 93%. With "fever episode" defined as a temp ≥100.4°F for at least 1 hr, tPatch detected 9.6 times the number of fever episodes vs. clinic reads. The average lead time of tPatch detection of clinic-recorded fevers was 3.7 hours. In 25% of all intervals between clinic temp readings, either tPatch or patients detected at least 1 fever episode. The tPatch was well-tolerated, the only adverse events reported were grade 1 skin irritation and discomfort in 4 (5%) patients. Of 65 patients who completed the survey, 95% reported the tPatch as "quite" or "somewhat" comfortable and 94% stated no difficulty in using the tPatch. Exploration of tPatch temp trends over various time intervals for use in fever prediction is ongoing. Conclusions Patient self-monitoring of temp has low sensitivity and is not feasible for long intervals of time (e.g., overnight). Continuous temperature monitoring by a wearable device overcomes these challenges and has the potential to improve early detection and consequently shorten time to antibiotic initiation. A follow-up randomized study is planned to assess the clinical benefits of continuous temp monitoring through patient and clinician alerts triggering early clinical intervention for febrile neutropenia. Figure Disclosures Vera-Aguilera: Verily Life Sciences: Research Funding. Haji-Abolhassani:Verily Life Sciences: Employment. Kulig:Verily Life Sciences: Employment. Heitz:Verily Life Sciences: Employment. Paludo:Verily Life Sciences: Research Funding; Verily Life Sciences: Research Funding; Celgene: Research Funding; Celgene: Research Funding. Ghoreyshi:Verily Life Sciences: Employment. Scheevel:Verily Life Sciences: Research Funding. Schimke:Verily Life Sciences: Research Funding. Markovic:Verily Life Sciences: Research Funding.