Tyrosine Kinase Inhibition Activates Intratumoral γδ T Cells in Gastrointestinal Stromal Tumor.

γδ T cells are a rare but potent subset of T cells with pleiotropic functions. They commonly reside within tumors but the response of γδ T cells to tyrosine kinase inhibition is unknown. To address this, we studied a genetically engineered mouse model of gastrointestinal stromal tumor (GIST) driven by oncogenic Kit signaling that responds to the Kit inhibitor imatinib. At baseline, γδ T cells were antitumoral, as blockade of either γδ T-cell receptor or IL17A increased tumor weight and decreased antitumor immunity. However, imatinib therapy further stimulated intratumoral γδ T cells, as determined by flow cytometry and single-cell RNA sequencing (scRNA-seq). Imatinib expanded a highly activated γδ T-cell subset with increased IL17A production and higher expression of immune checkpoints and cytolytic effector molecules. Consistent with the mouse model, γδ T cells produced IL17A in fresh human GIST specimens, and imatinib treatment increased γδ T-cell gene signatures, as measured by bulk tumor RNA-seq. Furthermore, tumor γδ T cells correlated with survival in patients with GIST. Our findings highlight the interplay between tumor cell oncogene signaling and antitumor immune responses and identify γδ T cells as targets for immunotherapy in GIST.

Multiple intratumoral sources of kit ligand promote gastrointestinal stromal tumor.

Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and is typically driven by a single mutation in the Kit or PDGFRA receptor. While highly effective, tyrosine kinase inhibitors (TKIs) are not curative. The natural ligand for the Kit receptor is Kit ligand (KitL), which exists in both soluble and membrane-bound forms. While KitL is known to stimulate human GIST cell lines in vitro, we used a genetically engineered mouse model of GIST containing a common human KIT mutation to investigate the intratumoral sources of KitL, importance of KitL during GIST oncogenesis, and contribution of soluble KitL to tumor growth in vivo. We discovered that in addition to tumor cells, endothelia and smooth muscle cells produced KitL in KitV558Δ/+ tumors, even after imatinib therapy. Genetic reduction of total KitL in tumor cells of KitV558Δ/+ mice impaired tumor growth in vivo. Similarly, genetic reduction of tumor cell soluble KitL in KitV558Δ/+ mice decreased tumor size. By RNA sequencing, quantitative PCR, and immunohistochemistry, KitL expression was heterogeneous in human GIST specimens. In particular, PDGFRA-mutant tumors had much higher KitL expression than Kit-mutant tumors, suggesting the benefit of Kit activation in the absence of mutant KIT. Serum KitL was higher in GIST patients with tumors resistant to imatinib and in those with tumors expressing more KitL RNA. Overall, KitL supports the growth of GIST at baseline and after imatinib therapy and remains a potential biomarker and therapeutic target.

Immunotherapy for GI Malignancies, Ready for Prime Time?

Gastrointestinal (GI) cancers include esophageal, gastric, pancreatic, hepatobiliary, and colorectal malignancies. Immunotherapy has proved to be an important treatment method for cancer, and its use for a wide range of GI malignancies has been evaluated recently. This article discusses the type and mechanism of various immunotherapies under investigation in GI cancer. The study also reviews recent clinical trials, with a particular focus on overall survival, and discusses their achievements and limitations. Immunotherapy has shown efficacy for microsatellite instability high colorectal cancer and some promise in some gastroesophageal junction and gastric cancers. Meanwhile, it has not been effective for pancreatic or neuroendocrine tumors. The identification of novel biomarkers likely will guide selection of therapy for individual patients. Nevertheless, immunotherapy for GI cancers is in its infancy, and many other classes of immune therapies are being developed besides anti-PD1 and anti-PDL1. Thus, although immunotherapy has not seen a dramatic advance to date, it still has great potential.

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.

Molecular and Immunologic Techniques in a Genetically Engineered Mouse Model of Gastrointestinal Stromal Tumor.

Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and is typically driven by a single mutation in the KIT receptor. Across tumor types, numerous mouse models have been developed in order to investigate the next generation of cancer therapies. However, in GIST, most in vivo studies use xenograft mouse models which have inherent limitations. Here, we describe an immunocompetent, genetically engineered mouse model of gastrointestinal stromal tumor harboring a KitV558Δ/+ mutation. In this model, mutant KIT, the oncogene responsible for most GISTs, is driven by its endogenous promoter leading to a GIST which mimics the histological appearance and immune infiltrate seen in human GISTs. Furthermore, this model has been used successfully to investigate both targeted molecular and immune therapies. Here, we describe the breeding and maintenance of a KitV558Δ/+ mouse colony. Additionally, this paper details the treatment and procurement of GIST, draining mesenteric lymph node, and adjacent cecum in KitV558Δ/+ mice, as well as sample preparation for molecular and immunologic analyses.

Correction to: Characteristics Associated with Pathologic Nodal Burden in Patients Presenting with Clinical Melanoma Nodal Metastasis.

In the original article, there is an error in the funding information. The correct funding information is as follows.

Characteristics Associated with Pathologic Nodal Burden in Patients Presenting with Clinical Melanoma Nodal Metastasis.

BACKGROUND: Nodal observation is safe for patients with microscopic melanoma metastasis in a sentinel lymph node (LN). Complete LN dissection (CLND) remains the standard of care for patients with clinically evident LN (cLN) metastases, even though about 40% have only one pathologic LN (pLN). We sought to identify clinical features associated with having one pLN among patients with cLNs. METHODS: Patients at three melanoma centers who underwent CLND for cLNs were identified. Clinicopathologic and imaging characteristics associated with one pLN were determined by multivariable logistic regression and classification tree analysis. RESULTS: Of 190 patients, 90 (47.4%) had one pLN and 100 (52.6%) had more than one pLN. By multivariable logistic regression, extremity versus truncal primary (odds ratio [OR] 2.15, p = 0.012), axillary versus superficial inguinal location (OR 3.89, p = 0.009), and preoperative cross-sectional imaging demonstrating more than one versus one cLN (OR 17.1, p < 0.001) were associated with more than one pLN. The negative predictive value for additional pathologic nodal disease of preoperative imaging was 70.9%, increasing to 74.4% for positron emission tomography/computed tomography. In the subgroup of patients with one cLN, the classification tree identified two groups with < 10% risk of additional pLNs: (1) Breslow thickness > 6.55 mm (n = 17); and (2) if unknown primary or Breslow thickness ≤ 6.55 mm, then LN diameter > 1.8 cm in the inguinal location (n = 22). CONCLUSION: The majority of patients with one cLN from melanoma by preoperative imaging will harbor no additional pathologic nodes on CLND. Safety of nodal observation after clinical nodal excision in these patients, particularly in an era of effective adjuvant therapies, deserves prospective evaluation.

Survival Outcomes of Patients with Clinical Stage III Melanoma in the Era of Novel Systemic Therapies.

BACKGROUND: Immune checkpoint and BRAF-targeted inhibitors have demonstrated significant survival benefits for advanced melanoma patients within the context of clinical trials. We sought to determine their impact on overall survival (OS) at a population level in order to better understand the current landscape for patients diagnosed with clinical stage III melanoma. METHODS: A retrospective study was performed using the National Cancer Database. Patients diagnosed with clinical stage III melanoma were categorized by diagnosis year into two cohorts preceding the advent of novel therapies (P1: 2004-2005, P2: 2008-2009) and a contemporary group (P3: 2012-2013). OS was estimated using standard time-to-event statistical methods. RESULTS: Of 3720 patients, 525 (14%) were diagnosed in P1, 1375 (37%) in P2, and 1820 (49%) in P3. Median age at diagnosis increased over time (58, 59, and 61 years in P1, P2, and P3, respectively, P = 0.004). OS increased between P2 (median 49.3 months) and P3 (median 58.2 months, Bonferroni-corrected log-rank P < 0.001) but did not differ between P1 (median 50.5 months) and P2 (Bonferroni-corrected log-rank P > 0.99). These differences persisted on multivariable analysis. OS improved for patients diagnosed in P3 compared with P1 [hazard ratio (HR) 0.76, P < 0.001] but not P2 compared with P1 (HR 0.96, P = 0.52). CONCLUSIONS: OS has significantly improved nationally for patients newly diagnosed with clinical stage III melanoma in the era of novel melanoma therapies. OS outcomes will likely continue to evolve as these agents are increasingly utilized in the adjuvant setting. These data may help to better inform affected patients with respect to prognosis.

National trends in centralization and perioperative outcomes of complex operations for cancer.

BACKGROUND: Complex cancer operations performed at high-volume and teaching hospitals have been associated with better outcomes. The purpose of this study was to determine the national trends in the performance of these operations at large teaching hospitals. METHODS: Patients who underwent elective esophagectomies, gastrectomies, pancreatectomies, and hepatectomies for cancer (2003-2015) were identified using the National Inpatient Sample. We determined average annual percent change (AAPC) in the proportion of operations at large teaching hospitals, inpatient complications, length of stay (LOS), and inpatient mortality. RESULTS: Between 2003 and 2015, 38,932 esophageal, 104,941 gastric, 96,098 hepatic, and 137,440 pancreatic cancer resections were performed. The proportion at large teaching hospitals increased with an AAPC of 2.5 for esophagectomies (P < .001), 3.6 for gastrectomies (P < .001), and 1.5 for pancreatectomies (P = .039), but did not change for hepatectomies (AAPC 0.48, P = .50). During the study period, mean LOS and inpatient mortality rates at large teaching hospitals decreased across hospital types. By 2013 to 2015, the operations at large hospitals were associated with decreased mortality only for pancreatectomies (odds ratio, 0.62, 95% confidence interval, 0.43-0.91, P = .015). CONCLUSIONS: Complex cancer operations are performed increasingly at large teaching hospitals, but perioperative outcomes have improved nationally across hospital types. Further studies should identify actionable areas for improvement to ensure accessible quality cancer care.

Predictors and outcomes of jejunostomy tube placement at the time of pancreatoduodenectomy.

BACKGROUND: Clinically relevant postoperative pancreatic fistula and delayed gastric emptying cause substantial morbidity after pancreatoduodenectomy. Per international guidelines, the placement of jejunostomy tubes may be considered for patients at risk for malnutrition, such as those with a high risk for clinically relevant postoperative pancreatic fistula and related complications. This study determined predictors and postoperative outcomes of jejunostomy tube placement. METHODS: Patients undergoing pancreatoduodenectomy in 2014 to 2015 were identified using the American College of Surgeons National Surgical Quality Improvement Program and Procedure-Targeted Pancreatectomy Participant Use Files. Multivariable logistic regressions were used to identify factors associated with concurrent jejunostomy tube placement and postoperative outcomes. RESULTS: Of 3,600 patients, 8.9% underwent jejunostomy tube placement. Patients given a jejunostomy tube were more likely white (odds ratio 1.46, P = .016), to have low preoperative serum albumin levels (odds ratio 2.13, P < .001), to have received neoadjuvant radiotherapy (odds ratio 2.14, P < .001), and to have received an intraoperative transfusion (odds ratio 1.50, P = .004). We observed no association between jejunostomy tube placement and an increasing number of risk factors for clinically relevant postoperative pancreatic fistula (P = .96) or delayed gastric emptying (P = .54). Overall, jejunostomy tube placement was associated with increased morbidity (odds ratio 1.34, P = .020) and duration of stay (P < .001), but not mortality (P = .12). Among patients with low serum albumin or those who developed clinically relevant postoperative pancreatic fistula or delayed gastric emptying, jejunostomy tube utilization was not associated with morbidity or mortality. CONCLUSION: Jejunostomy tube placement during pancreatoduodenectomy was not driven by risk factors for clinically relevant postoperative pancreatic fistula or delayed gastric emptying, suggesting that practice patterns play a role. Among patients with at-risk preoperative albumin or who developed these complications, jejunostomy tube placement was not associated with worse outcomes, supporting selective utilization per guideline recommendations.

Protein extraction from methanol fixed paraffin embedded tissue blocks: A new possibility using cell blocks.

BACKGROUND: Methanol fixed and paraffin embedded (MFPE) cellblocks are an essential cytology preparation. However, MFPE cellblocks often contain limited material and their relatively small size has caused them to be overlooked in biomarker discovery. Advances in the field of molecular biotechnology have made it possible to extract proteins from formalin fixed and paraffin embedded (FFPE) tissue blocks. In contrast, there are no established methods for extracting proteins from MFPE cellblocks. We investigated commonly available CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate) buffer, as well as two commercially available Qiagen(®) kits and compared their effectiveness on MFPE tissue for protein yields. MATERIALS AND METHODS: MFPE blocks were made by Cellient™ automated system using human tissue specimens from normal and malignant specimens collected in ThinPrep™ Vials. Protein was extracted from Cellient-methanol fixed and paraffin embedded blocks with CHAPS buffer method as well as FFPE and Mammalian Qiagen(®) kits. RESULTS: Comparison of protein yields demonstrated the effectiveness of various protein extraction methods on MFPE cellblocks. CONCLUSION: In the current era of minimally invasive techniques to obtain minimal amount of tissue for diagnostic and prognostic purposes, the use of commercial and lab made buffer on low weight MFPE scrapings obtained by Cellient(®) processor opens new possibilities for protein biomarker research.