Muscle Loss Is Associated with Overall Survival in Patients with Metastatic Colorectal Cancer Independent of Tumor Mutational Status and Weight Loss

Background: Survival in patients with metastatic colorectal cancer (mCRC) has been associated with tumor mutational status, muscle loss, and weight loss. We sought to explore the combined effects of these variables on overall survival. Materials and methods: We performed an observational cohort study, prospectively enrolling patients receiving chemotherapy for mCRC. We retrospectively assessed changes in muscle (using computed tomography) and weight, each dichotomized as >5% or ≤5% loss, at 3, 6, and 12 months after diagnosis of mCRC. We used regression models to assess relationships between tumor mutational status, muscle loss, weight loss, and overall survival. Additionally, we evaluated associations between muscle loss, weight loss, and tumor mutational status. Results: We included 226 patients (mean age 59 ± 13 years, 53% male). Tumor mutational status included 44% wild type, 42% RAS-mutant, and 14% BRAF-mutant. Patients with >5% muscle loss at 3 and 12 months experienced worse survival controlling for mutational status and weight (3 months hazard ratio, 2.66; p 5% muscle loss with BRAF-mutational status at 6 and 12 months. Weight loss was not associated with survival nor mutational status. Conclusion: Increased muscle loss at 3 and 12 months may identify patients with mCRC at risk for decreased overall survival, independent of tumor mutational status. Specifically, >5% muscle loss identifies patients within each category of tumor mutational status with decreased overall survival in our sample. Our findings suggest that quantifying muscle loss on serial computed tomography scans may refine survival estimates in patients with mCRC. Implications for practice: In this study of 226 patients with metastatic colorectal cancer, it was found that losing >5% skeletal muscle at 3 and 12 months after the diagnosis of metastatic disease was associated with worse overall survival, independent of tumor mutational status and weight loss. Interestingly, results did not show a significant association between weight loss and overall survival. These findings suggest that muscle quantification on serial computed tomography may refine survival estimates in patients with metastatic colorectal cancer beyond mutational status

Resistance to checkpoint blockade therapy through inactivation of antigen presentation

Treatment with immune checkpoint blockade (CPB) therapies often leads to prolonged responses in patients with metastatic melanoma, but the common mechanisms of primary and acquired resistance to these agents remain incompletely characterized and have yet to be validated in large cohorts. By analyzing longitudinal tumor biopsies from 17 metastatic melanoma patients treated with CPB therapies, we observed point mutations, deletions or loss of heterozygosity (LOH) in beta-2-microglobulin (B2M), an essential component of MHC class I antigen presentation, in 29.4% of patients with progressing disease. In two independent cohorts of melanoma patients treated with anti-CTLA4 and anti-PD1, respectively, we find that B2M LOH is enriched threefold in non-responders (~30%) compared to responders (~10%) and associated with poorer overall survival. Loss of both copies of B2M is found only in non-responders. B2M loss is likely a common mechanism of resistance to therapies targeting CTLA4 or PD1

Mycobacteria induce TPL-2 mediated IL-10 in IL-4-generated alternatively activated macrophages.

IL-4 drives expansion of Th2 cells that cause generation of alternatively activated macrophages (AAMs). Filarial infections are established early in life, induce increased IL-4 production are co-endemic with tuberculosis (TB). We sought to understand, therefore, how mycobacteria are handled in the context of IL-4-induced AAM. Comparing IL-4 generated in vitro monocyte derived human AAMs to LPS and IFN-γ generated classically macrophages (CAMs), both infected with mycobacteria (BCG), we demonstrated increased early BCG uptake and increased IL-10 production in AAMs compared to CAMs. We further demonstrated that increased IL-10 production is mediated by upregulation of tumor progression locus 2 (TPL-2), an upstream activator of extracellular signal related kinases (ERKs) in AAMs but not in CAMs, both at the transcript as well as the protein level. Pharmacologic inhibition of TPL-2 significantly diminished IL-10 production only in BCG-infected AAMs. Finally, we validated our findings in an in vivo C57Bl/6 model of filarial infection, where an exaggerated Th2 induced lung-specific alternative activation led to TPL-2 and IL-10 upregulation on subsequent TB infection. These data show that in response to mycobacterial infection, IL-4 generated AAMs in chronic filarial infections have impaired immune responses to TB infection by increasing IL-10 production in a TPL-2 mediated manner

AAMs compared to CAMs show decreased CCL13 expression and increased IL-10 production post BCG infection.

<p><b>Increased post-BCG IL-10 production was not seen in <i>B</i>. <i>malayi</i> microfilaria (mf) polarized macrophages. Panel a</b> shows change in relative mRNA expression of CCL13 from baseline to 24 hrs. post BCG infection expressed as 1/ΔCt in AAMs on the left panel and CAMs on the right panel. Individual lines represent each subject. <b>Panel b</b>: Net cytokine production (IL-6, IL-10, IL-12p40, IL-1α, IL-1β, and TNF-α) post BCG infection in pg/ml is compared between AAM and CAM conditions. Box and whisker plots represent median with 95% confidence intervals and individual dots representing each subject. <b>Panel c</b>: Net IL-10 production post BCG infection (measured in pg/ml) compared between mf polarized, mf+IL-4 polarized and CAMs. Box and whisker plots represent median with 95% confidence intervals and individual dots representing each subject.</p

AAMs show increased BCG uptake compared to CAMs using ImageStream^X.

<p><b>Panel a</b> shows sequential gating strategy for assessing bacterial internalization within the cell. Cells that were in the field of focus were defined using Gradient RMS vs. Area features; next, plotting symmetry vs. circularity distinguished macrophages from free bacteria. Gating was then performed on RFP positive cells to identify macrophages associated with bacteria. Delta Centroid feature was used to assess the distance between the center of the cell and the RFP labeled bacteria to assess internalization. <b>Panel b</b>: representative images showing reproducibility and accuracy of internalization assessment using the above gating strategy. Three different examples of RFP labeled bacteria internalized (left panel) within macrophages vs. remaining external (right panel) are shown. <b>Panel c</b>: Graph comparing median percentage of cells internalizing bacteria (for n = 12 donors) using above strategy in AAMs (grey column) vs. CAMs (black column).</p

Increased IL-10 production in AAMs but not CAMs in is mediated by TPL-2.

<p><b>Panel a</b>: Left panel (top and bottom) shows kinetics of net IL-10 production in AAMs and CAMs (in pg/ml) starting from pre BCG infection time point (pre-Inf) to 1, 4,6,18 and 24 hrs. post BCG infection. Middle panel shows relative mRNA expression of TPL-2 at the same time points, expressed as 1/ΔCt in AAMs and CAMs. Right panel shows relative mRNA expression of STAT3, expressed as 1/ΔCt in AAMs and CAMs. Vertical bars represent median relative expression Panel <b>b</b>: Whole cell extracts of AAMs and CAMs, generated at pre BCG infection and at 24hrs post infection were analyzed by immunoblotting with anti-TPL-2 antibodies utilizing anti-GAPDH as control (top panel). Bottom pane shows change in relative TPL-2 band density in AAMs vs. CAMs as assessed by ImageJ software comparing pre BCG infection time point (<b>Preinf</b>) with 24 hrs. post BCG infection (<b>24hr</b>). <b>Panel c</b>: Net IL-10 production (in pg/ml) is shown in AAMs and CAMs cultured with BCG alone for 24 hrs. or BCG and TPL-2 inhibitor (C₂₁H₁₄ClFN₆) at 500nM (IC⁵⁰). Individual dots representing each subject. A total of 12 subjects were analyzed for experiments in 4a, b and c.</p

At baseline AAMs have increased CD206 (Mannose receptor) expression compared to CAMs.

<p><b>Panel a</b>: Relative mRNA expression of previously defined AAM-specific marker (CCL13) expressed as 1/ΔCt, was compared between alternatively activated macrophages (AAMs) and classically activated macrophages (CAMs). Individual dots representing each subject. Horizontal bars represent median. <b>Panel b</b>: Representative plots showing CD206 (Mannose Receptor) expression (y-axis) compared on CD14+ cells (x-axis) between M-CSF,IL-4 (AAM) and LPS/IFN-γ (CAM) by flow cytometry <b>Panel c</b>:total cytokine production (IL-6, IL-10, IL-12p40, IL-1α, IL-1β, and tumor necrosis factor alpha [TNF-α]) in pg/ml is compared between AAM and CAM conditions. Cells were cultured with recombinant human IL-4 (rhIL-4; 50 ng/ml) for AAMs) or with LPS (1 μg/ml)/IFN-γ (20 ng/ml) for CAMs for 48 hours. Individual dots representing each subject. Horizontal bars represent median.</p

Polyclonal secondary FGFR2 mutations drive acquired resistance to FGFR inhibition in FGFR2 fusion-positive cholangiocarcinoma patients

Genetic alterations in the fibroblast growth factor receptor (FGFR) pathway are promising therapeutic targets in many cancers, including intrahepatic cholangiocarcinoma (ICC). The FGFR inhibitor BGJ398 displayed encouraging efficacy in patients with FGFR2 fusion-positive ICC in a phase II trial, but the durability of response was limited in some patients. Here, we report the molecular basis for acquired resistance to BGJ398 in three patients via integrative genomic characterization of cell-free circulating tumor DNA (cfDNA), primary tumors, and metastases. Serial analysis of cfDNA demonstrated multiple recurrent point mutations in the FGFR2 kinase domain at progression. Accordingly, biopsy of post-progression lesions and rapid autopsy revealed marked inter- and intra-lesional heterogeneity, with different FGFR2 mutations in individual resistant clones. Molecular modeling and in vitro studies indicated that each mutation lead to BGJ398 resistance that was surmountable by structurally distinct FGFR inhibitors. Thus, polyclonal secondary FGFR2 mutations represent an important clinical resistance mechanism that may guide development of future therapeutic strategies