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Purpose: To create an in vivo model of PDGFRA D842V-mutant gastrointestinal stromal tumor (GIST) and identify the mechanism of tumor persistence following avapritinib therapy. Experimental Design: We created a patient-derived xenograft (PDX) of PDGFRA D842V-mutant GIST and tested the effects of imatinib, avapritinib, and ML-7, an inhibitor of myosin light-chain kinase (MYLK). Bulk tumor RNA sequencing and oncogenic signaling were evaluated. Apoptosis, survival, and actin cytoskeleton were evaluated in GIST T1 cells and isolated PDX cells in vitro. Human GIST specimens were analyzed for MYLK expression. Results: The PDX was minimally responsive to imatinib but sensitive to avapritinib. Avapritinib therapy increased tumor expression of genes related to the actin cytoskeleton, including MYLK. ML-7 induced apoptosis and disrupted actin filaments in short-term cultures of PDX cells and decreased survival in GIST T1 cells in combination with imatinib or avapritinib. Combined therapy with ML-7 improved the antitumor effects of low-dose avapritinib in vivo. Furthermore, MYLK was expressed in human GIST specimens. Conclusions: MYLK upregulation is a novel mechanism of tumor persistence after tyrosine kinase inhibition. Concomitant MYLK inhibition may enable the use of a lower dose of avapritinib, which is associated with dose-dependent cognitive side effects.

The COVID-19 pandemic resulted in sweeping shutdowns of surgical operations to increase hospital capacity and conserve resources. Our institution, following national and state guidelines, suspended nonessential surgeries from March 16 to May 4, 2020. This study examines the financial impact of this decision on our institution's health system by comparing 2 waves of COVID-19 cases.The total revenue was obtained for surgical cases occurring during the first wave of the pandemic between March 1, 2020 and July 31, 2020 and the second wave between October 1, 2020 and February 29, 2021 for all surgical departments. During the same time intervals, in the prepandemic year 2019, total revenue was also obtained for comparison. Net revenue and work relative value units per month were compared to each respective month for all surgical divisions within the department of surgery.Comparing the 5-month first wave period in 2020 to prepandemic 2019 for all surgical departments, there was a net revenue loss of $99,674,376, which reflected 42% of the health system's revenue loss during this period. The department of surgery contributed to a net revenue loss of $58,368,951, which was 24.9% of the health system's revenue loss. Within the department of surgery, there was a significant difference between the net revenue loss per month per division of the first and second wave: first wave median -$636,952 [interquartile range: -1,432,627; 26,111] and second wave median -$274,626 [-781,124; 396,570] (P = .04). A similar difference was detected when comparing percent change in work relative value units between the 2 waves (wave 1: median -13.2% [interquartile range: -41.3%, -1.8%], wave 2: median -7.8% [interquartile range: -13.0%, 1.8%], P = .003).Stopping elective surgeries significantly decreased revenue for a health system. Losses for the health system totaled $234,839,990 during the first wave, with lost surgical revenue comprising 42% of that amount. With elective surgeries continuing during the second wave of COVID-19 cases, the health system losses were substantially lower. The contribution surgery has to a hospital's cash flow is essential in maintaining financial solvency. It is important for hospital systems to develop innovative and alternative solutions to increase capacity, offer comprehensive care to medical and surgical patients, and prevent shutdowns of surgical activity through a pandemic to maintain financial security.

The American College of Surgeons National Surgical Quality Improvement Program was established to help participating hospitals track and report surgical complications with the goal of improving surgical care. We sought to determine whether this has led to improvements in surgical outcomes for pancreatic malignancies.Patients with pancreatic malignancies who underwent surgical resection were identified from the American College of Surgeons National Surgical Quality Improvement Program database (2006-2019). Thirty-day postoperative major morbidity and mortality were analyzed by year. Major morbidity included organ and deep surgical site infection, venous thromboembolism, cardiac event, pneumonia, acute renal failure, sepsis, and respiratory failure.Of the 28,888 patients identified, 51% were male, the median age was 68, 74.3% underwent a pancreaticoduodenectomy, and 25.7% underwent a distal pancreatectomy. Among patients who underwent a pancreaticoduodenectomy, there was a significant increase in major morbidity (annual percent change 0.77, P = .012) driven by increases in organ space surgical site infection (annual percent change 3.52, P.001) and venous thromboembolism (annual percent change 4.72, P = .005). However, there was a decrease in postoperative mortality (annual percent change -4.58, P = .001). For distal pancreatectomy patients, there was no change in rates of overall major morbidity (annual percent change -1.35, P = .08) or mortality (annual percent change -3.21, P = .25).Although major morbidity and mortality have not significantly changed for distal pancreatectomy patients, mortality has steadily decreased for patients undergoing pancreaticoduodenectomy, despite an increase in major morbidity. Whether this trend reflects a change in patient selection, an increase in detection of postoperative morbidities and/or an improvement in mitigation of these morbidities warrants further study.

Supplemental Figure 5.Short term culture of cells derived from PDGFRA D842V PDXs are tumor cells.

Supplemental Figure 1.

Supplemental Figure 8.Relative MYLK mRNA expression by qPCR in GIST PDGFRA D842V PDXs treated with vehicle(Veh) or imatinib (IM) for 2 weeks.

Supplemental Figure 3. GIST PDGFRA D842V PDXs are not sensitive to crenolanib.

Supplemental Figure 7.

Supplemental Figure 4.

Purpose:To create an in vivo model of PDGFRA D842V-mutant gastrointestinal stromal tumor (GIST) and identify the mechanism of tumor persistence following avapritinib therapy.Experimental Design:We created a patient-derived xenograft (PDX) of PDGFRA D842V-mutant GIST and tested the effects of imatinib, avapritinib, and ML-7, an inhibitor of myosin light-chain kinase (MYLK). Bulk tumor RNA sequencing and oncogenic signaling were evaluated. Apoptosis, survival, and actin cytoskeleton were evaluated in GIST T1 cells and isolated PDX cells in vitro. Human GIST specimens were analyzed for MYLK expression.Results:The PDX was minimally responsive to imatinib but sensitive to avapritinib. Avapritinib therapy increased tumor expression of genes related to the actin cytoskeleton, including MYLK. ML-7 induced apoptosis and disrupted actin filaments in short-term cultures of PDX cells and decreased survival in GIST T1 cells in combination with imatinib or avapritinib. Combined therapy with ML-7 improved the antitumor effects of low-dose avapritinib in vivo. Furthermore, MYLK was expressed in human GIST specimens.Conclusions:MYLK upregulation is a novel mechanism of tumor persistence after tyrosine kinase inhibition. Concomitant MYLK inhibition may enable the use of a lower dose of avapritinib, which is associated with dose-dependent cognitive side effects.

Supplemental Figure 6.Cell survival of GIST T1 cells treated with imatinib or avapritinib for 72h in complete media.Each data point represents an average of 10 wells.

Supp to Fig. 4

Supplementary Figure from Tyrosine Kinase Inhibition Alters Intratumoral CD8+ T-cell Subtype Composition and Activity

Supplementary Table from Tyrosine Kinase Inhibition Alters Intratumoral CD8+ T-cell Subtype Composition and Activity

Targeted therapy with a tyrosine kinase inhibitor (TKI) such as imatinib is effective in treating gastrointestinal stromal tumor (GIST), but it is rarely curative. Despite the presence of a robust immune CD8+ T-cell infiltrate, combining a TKI with immune-checkpoint blockade (ICB) in advanced GIST has achieved only modest effects. To identify limitations imposed by imatinib on the antitumor immune response, we performed bulk RNA sequencing (RNA-seq), single-cell RNA-seq, and flow cytometry to phenotype CD8+ T-cell subsets in a genetically engineered mouse model of GIST. Imatinib reduced the frequency of effector CD8+ T cells and increased the frequency of naïve CD8+ T cells within mouse GIST, which coincided with altered tumor chemokine production, CD8+ T-cell recruitment, and reduced CD8+ T-cell intracellular PI3K signaling. Imatinib also failed to induce intratumoral T-cell receptor (TCR) clonal expansion. Consistent with these findings, human GISTs sensitive to imatinib harbored fewer effector CD8+ T cells but more naïve CD8+ T cells. Combining an IL15 superagonist (IL15SA) with imatinib restored intratumoral effector CD8+ T-cell function and CD8+ T-cell intracellular PI3K signaling, resulting in greater tumor destruction. Combination therapy with IL15SA and ICB resulted in the greatest tumor killing and maintained an effector CD8+ T-cell population in the presence of imatinib. Our findings highlight the impact of oncogene inhibition on intratumoral CD8+ T cells and support the use of agonistic T-cell therapy during TKI and/or ICB administration.

AFS98 depletion

Figure S1. The relative mRNA expression of DKK4 and CTNNB1 in human KIT+ cells.

Human GIST characteristics

Primary untreated KIT+ vs. Metastatic resistant KIT+, pathways with P< 0.05.

PDF file - 182K, Table S1 shows the correlation between all immune markers expression tested; Table S2 shows the lack of correlation between the favorable immune score and clinicopathologic characteristics. Figure S1 illustrates the quantification method for MHC Class I and Beta 2-microglobulin; Figure S2 shows the association between the immune score and the Clinical Risk Score.

Supplementary Table 2: Primary untreated (Prim/UT) KIT+ ( n = 8 ) vs. Metastatic resistant (Met/Res) KIT+ ( n = 4). Significant genes with a False Discovery Rate of < 0.05 and a fold change > 2.0

Tumor-infiltrating lymphocytes (TIL) in colorectal cancer liver metastases (CLM) have been associated with more favorable patient outcomes, but whether MHC class I (MHC-I) expression on cancer cells affects prognosis is uncertain. Immunohistochemistry was performed on a tissue microarray of 158 patients with CLM, who underwent partial hepatectomy with curative intent. Using the antibody HC-10, which detects HLA-B and HLA-C antigens and a minority of HLA-A antigens, MHC-I expression was correlated with β-2 microglobulin (β2m; r = 0.7; P < 0.001), but not with T-cell density (r < 0.32). The median follow-up for survivors was 9.7 years. High levels of MHC-I expression in tumors concomitant with high T-cell infiltration (CD3, CD4, or CD8) best identified patients with favorable outcomes, compared with patients with one or none of these immune features. The median overall survival (OS) of patients with MHC-IhiCD3hi tumors (n = 31) was 116 months compared with 40 months for the others (P = 0.001), and the median time to recurrence (TTR) was not reached compared with 17 months (P = 0.008). By multivariate analysis, MHChiCD3hi was associated with OS and TTR independent of the standard clinicopathologic variables. An immune score that combines MHC-I expression and TIL density may be a valuable prognostic tool in the treatment of patients with CLM. Cancer Immunol Res; 2(6); 530–7. ©2014 AACR.

Tyrosine kinase inhibition of gastrointestinal stromal tumors (GIST) is effective but typically culminates in resistance and is rarely curative. Immunotherapy has potential application to GIST, as we previously showed that T-cell checkpoint blockade increases the antitumor effects of imatinib. Here, we showed that ligation of CD40 using an agonistic antibody (anti-CD40) activated tumor-associated macrophages (TAMs) in vivo in a knock-in mouse model of GIST harboring a germline mutation in Kit exon 11. Activated TAMs had greater TNFα production and NFκB signaling and directly inhibited tumor cells in vitro. Anti-CD40 required concomitant therapy with imatinib for efficacy and depended on TAMs, and to a lesser extent CD8+ T cells, but not on CD4+ T cells or B cells. In an analysis of 50 human GIST specimens by flow cytometry, we found that CD40 was expressed on human TAMs and tumor cells yet was downregulated after response to imatinib. CD40 ligation did not have a direct inhibitory effect on human GIST cells. Our findings provide the rationale for combining anti-CD40 and tyrosine kinase inhibition to treat human GIST. Cancer Immunol Res; 6(4); 434–47. ©2018 AACR.

Purpose This guideline provides evidence-based recommendations for the indications and technique-dose of external beam radiation therapy (EBRT) in hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (IHC). Methods The American Society for Radiation Oncology convened a task force to address 5 key questions focused on the indications, techniques, and outcomes of EBRT in HCC and IHC. It is intended to cover the definitive, consolidative, salvage, preoperative (including bridge to transplant), and adjuvant settings as well as palliative EBRT for symptomatic primary lesions. Recommendations were based on a systematic literature review and created using a predefined consensus-building methodology and system for grading evidence quality and recommendation strength. Results Strong recommendations are made for using EBRT as a potential first-line treatment in patients with liver-confined HCC who are not candidates for curative therapy, consolidative therapy after incomplete response to liver-directed therapies, and as a salvage option for local recurrences. The guideline conditionally recommends EBRT for patients with liver-confined multifocal or unresectable HCC, or those with macrovascular invasion, sequenced with systemic or catheter-based therapies. Palliative EBRT is conditionally recommended for symptomatic primary HCC and/or macrovascular tumor thrombi. EBRT is conditionally recommended as a bridge to transplant or prior to surgery in carefully selected patients. For patients with unresectable IHC, consolidative EBRT with or without chemotherapy should be considered, typically after systemic therapy. Adjuvant EBRT is conditionally recommended for resected IHC with high-risk features. Selection of dose-fractionation regimen and technique should be based on disease extent, disease location, underlying liver function, and available technologies. Conclusions The task force has proposed recommendations to inform best clinical practices on the use of EBRT for HCC and IHC with strong emphasis on multidisciplinary care. Future studies should focus on further defining the role of EBRT in the context of liver-directed and systemic therapies and refining optimal regimens and techniques.

Serum metabolic chemistries and splenic immune cell frequencies in VLX600-treated mice

PDF file - 121KB

A. Overall survival in derivation (MSKCC) and validation (European) cohorts. B. Recurrence free survival in derivation (MSKCC) and validation (European) cohorts.

Real-time changes in OCR and ECAR in GIST-T1 cells treated with varying doses of VLX600 + imatinib

PDF file - 198KB, Basal expression levels of c-Fms and c-Kit in sarcoma cell lines and Effect of siRNA mediated knockdown on cell proliferation.

PDF file - 188KB, Effect of PLX5622, a c-Fms specific inhibitor, on MPNST tumor growth.

PDF file - 168KB, Effect of PLX3397 on Ki67, a cellular proliferation marker and CD31, an endothelial cell marker in MPNST xenografts.

mRNA expression and hierarchical clustering of molecular risk score (MRS) genes in colorectal liver metastases in derivation cohort. Genes are indicated on the x-axis, patients on the y-axis.

PDF file - 76KB, Effect of PLX3397 on phosphorylation of PDGFRbeta.

Purpose: Imatinib dramatically reduces gastrointestinal stromal tumor (GIST) 18F-FDG uptake, providing an early indicator of treatment response. Despite decreased glucose internalization, many GIST cells persist, suggesting that alternative metabolic pathways are used for survival. The role of mitochondria in imatinib-treated GIST is largely unknown.Experimental Design: We quantified the metabolic activity of several human GIST cell lines. We treated human GIST xenografts and genetically engineered KitV558del/+ mice with the mitochondrial oxidative phosphorylation inhibitor VLX600 in combination with imatinib and analyzed tumor volume, weight, histology, molecular signaling, and cell cycle activity. In vitro assays on human GIST cell lines were also performed.Results: Imatinib therapy decreased glucose uptake and downstream glycolytic activity in GIST-T1 and HG129 cells by approximately half and upregulated mitochondrial enzymes and improved mitochondrial respiratory capacity. Mitochondrial inhibition with VLX600 had a direct antitumor effect in vitro while appearing to promote glycolysis through increased AKT signaling and glucose transporter expression. When combined with imatinib, VLX600 prevented imatinib-induced cell cycle escape and reduced p27 expression, leading to increased apoptosis when compared to imatinib alone. In KitV558del/+ mice, VLX600 alone did not induce tumor cell death, but had a profound antitumor effect when combined with imatinib.Conclusions: Our findings show that imatinib alters the metabolic phenotype of GIST, and this may contribute to imatinib resistance. Our work offers preclinical proof of concept of metabolic targeting as an effective strategy for the treatment of GIST. Clin Cancer Res; 24(4); 972–84. ©2017 AACR.

Purpose: Risk stratification after surgery for colorectal cancer liver metastases (CRLM) is achieved using clinicopathologic variables, however, is of limited accuracy. We sought to derive and externally validate a multigene expression assay prognostic of overall survival (OS) that is superior to clinicopathologic variables in patients with surgically resected CRLM.Experimental Design: We measured mRNA expression in prospectively collected frozen tumor from 96 patients with surgically resected CRLM at Memorial Sloan Kettering Cancer Center (MSKCC, New York, NY). We retrospectively generated a 20-gene molecular risk score (MRS) and compared its prognostic utility for OS and recurrence-free survival (RFS) with three common clinical risk scores (CRS). We then tested the prognostic ability of the MRS in an external validation cohort (European) of 119 patients with surgically resected CRLM at the University Medical Center Utrecht (Utrecht, the Netherlands) and Paul Brousse Hospital (Villejuif, France).Results: For OS in the MSKCC cohort, MRS was the strongest independent prognosticator (HR, 3.7–4.9; P < 0.001) followed by adjuvant chemotherapy (HR, 0.3; P ≤ 0.001). For OS in the European cohort, MRS was the only independent prognosticator (HR, 3.5; P = 0.007). For RFS, MRS was also independently prognostic in the MSKCC cohort (HR, 2.4–2.6; P ≤ 0.001) and the European cohort (HR, 1.6–2.5; P ≤ 0.05).Conclusions: Compared with CRSs, the MRS is more accurate, broadly applicable, and an independent prognostic biomarker of OS in resected CRLM. This MRS is the first externally validated prognostic multigene expression assay after metastasectomy for CRLM and warrants prospective validation. Clin Cancer Res; 22(10); 2575–82. ©2016 AACR.

PDF file - 35KB, Supplemental Figure S2. Biodistribution of 99mTc-Sestamibi in KitV558del/+ mice.

Purpose: Tyrosine kinase inhibitors are effective in gastrointestinal stromal tumors (GISTs) but often are of transient benefit as resistance commonly develops. Immunotherapy, particularly blockade of the inhibitory receptor programmed death 1 (PD-1) or the ligand programmed death ligand 1 (PD-L1), has shown effectiveness in a variety of cancers. The functional effects of PD-1/PD-L1 blockade are unknown in GISTs.Experimental Design: We analyzed tumor and matched blood samples from 85 patients with GISTs and determined the expression of immune checkpoint molecules using flow cytometry. We investigated the combination of imatinib with PD-1/PD-L1 blockade in KitV558Δ/+ mice that develop GISTs.Results: The inhibitory receptors PD-1, lymphocyte activation gene 3, and T-cell immunoglobulin mucin-3 were upregulated on tumor-infiltrating T cells compared with T cells from matched blood. PD-1 expression on T cells was highest in imatinib-treated human GISTs. Meanwhile, intratumoral PD-L1 expression was variable. In human GIST cell lines, treatment with imatinib abrogated the IFNγ-induced upregulation of PD-L1 via STAT1 inhibition. In KitV558Δ/+ mice, imatinib downregulated IFNγ-related genes and reduced PD-L1 expression on tumor cells. PD-1 and PD-L1 blockade in vivo each had no efficacy alone but enhanced the antitumor effects of imatinib by increasing T-cell effector function in the presence of KIT and IDO inhibition.Conclusions: PD-1/PD-L1 blockade is a promising strategy to improve the effects of targeted therapy in GISTs. Collectively, our results provide the rationale to combine these agents in human GISTs. Clin Cancer Res; 23(2); 454–65. ©2016 AACR.

Clinicopathological features of the patients with GIST in this study.

72h cell viability assays of GIST cell lines treated with VLX600

PDF file - 119KB, Supplemental Figure S1. Additional PLX3397-mediated immune effects.

Purpose: Malignant peripheral nerve sheath tumor (MPNST) is a highly aggressive tumor type that is resistant to chemotherapy and there are no effective therapies. MPNSTs have been shown to have gene amplification for receptor tyrosine kinases (RTK), PDGFR and c-Kit. We tested the c-Kit inhibitor, imatinib, and PLX3397, a selective c-Fms and c-Kit inhibitor, to evaluate their efficacy against MPNST cells in vitro and in vivo.Experimental Design: We tested the efficacy of imatinib or PLX3397 either alone or in combination with TORC1 inhibitor rapamycin in a cell proliferation assay in vitro and by immunoblotting to determine target inhibition. Immunoblotting and immunohistochemical analysis was further carried out using xenograft samples in vivo.Results: Our in vitro studies show that imatinib and PLX3397 similarly inhibit cell growth and this can be enhanced with rapamycin with comparable target specificity. However, in vivo studies clearly demonstrate that compared with imatinib, PLX3397 results in sustained blockade of c-Kit, c-Fms, and PDGFRβ, resulting in significant suppression of tumor growth. Moreover, staining for Iba-1, a marker for macrophages, indicates that PLX3397 results in significant depletion of macrophages in the growing tumors. The combination of PLX3397 and rapamycin results in even greater macrophage depletion with continued growth suppression, even when the drug treatment is discontinued.Conclusions: Taken together, our data strongly suggest that PLX3397 is superior to imatinib in the treatment of MPNSTs, and the combination of PLX3397 with a TORC1 inhibitor could provide a new therapeutic approach for the treatment of this disease. Clin Cancer Res; 20(12); 3146–58. ©2014 AACR.

Univariate and multivariate analyses of recurrence free survival in derivation (Table 1) and validation (Table 2) cohorts.

Synergy of dasatinib and anti-CTLA-4. Using murine GIST mouse, it was observed that treatment with the KIT-targeted dasatinib for 7 days alone or with chronic anti-CTLA-4 alone decreased tumor volume initially; however tumors rapidly re-grew. Mice treated with KIT-targeted TKI for 7 days and chronic anti-CTLA-4 blockade showed augmentation and durability of the initial response. KITV5880+ mice received dasatinib 15mg/kg i.p. bid (Bristol Myers Squibb) on days 1 through 7, with or without anti-CTLA-5 (BioXcell.) Mice were imaged at baseline and serially during therapy using MRI and volumetric analysis was done using Image K software. Anti-CTLA-4 dosing regimen 200 microgram of clone 9H10 i.p. on day 3, then 100 microgram on day 6, 9 and 12, followed by clone 9D (100 micrograms i.p. every 3 days thereafter.)

Genes selected for molecular risk score (MRS).

PDF file - 48KB, Effect of AFS98, a c-Fms specific antibody, on MPNST tumor growth.