Transcription factor-mediated intestinal metaplasia and the role of a shadow enhancer.

Barrett's esophagus (BE) and gastric intestinal metaplasia are related premalignant conditions in which areas of human stomach epithelium express mixed gastric and intestinal features. Intestinal transcription factors (TFs) are expressed in both conditions, with unclear causal roles and cis-regulatory mechanisms. Ectopic CDX2 reprogrammed isogenic mouse stomach organoid lines to a hybrid stomach-intestinal state transcriptionally similar to clinical metaplasia; squamous esophageal organoids resisted this CDX2-mediated effect. Reprogramming was associated with induced activity at thousands of previously inaccessible intestine-restricted enhancers, where CDX2 occupied DNA directly. HNF4A, a TF recently implicated in BE pathogenesis, induced weaker intestinalization by binding a novel shadow Cdx2 enhancer and hence activating Cdx2 expression. CRISPR/Cas9-mediated germline deletion of that cis-element demonstrated its requirement in Cdx2 induction and in the resulting activation of intestinal genes in stomach cells. dCas9-conjugated KRAB repression mapped this activity to the shadow enhancer's HNF4A binding site. Altogether, we show extensive but selective recruitment of intestinal enhancers by CDX2 in gastric cells and that HNF4A-mediated ectopic CDX2 expression in the stomach occurs through a conserved shadow cis-element. These findings identify mechanisms for TF-driven intestinal metaplasia and a likely pathogenic TF hierarchy.

AKT/FOXO signaling enforces reversible differentiation blockade in myeloid leukemias.

AKT activation is associated with many malignancies, where AKT acts, in part, by inhibiting FOXO tumor suppressors. We show a converse role for AKT/FOXOs in acute myeloid leukemia (AML). Rather than decreased FOXO activity, we observed that FOXOs are active in ∼40% of AML patient samples regardless of genetic subtype. We also observe this activity in human MLL-AF9 leukemia allele-induced AML in mice, where either activation of Akt or compound deletion of FoxO1/3/4 reduced leukemic cell growth, with the latter markedly diminishing leukemia-initiating cell (LIC) function in vivo and improving animal survival. FOXO inhibition resulted in myeloid maturation and subsequent AML cell death. FOXO activation inversely correlated with JNK/c-JUN signaling, and leukemic cells resistant to FOXO inhibition responded to JNK inhibition. These data reveal a molecular role for AKT/FOXO and JNK/c-JUN in maintaining a differentiation blockade that can be targeted to inhibit leukemias with a range of genetic lesions.

An Enhancer-Driven Stem Cell-Like Program Mediated by SOX9 Blocks Intestinal Differentiation in Colorectal Cancer.

BACKGROUND AND AIMS: Genomic alterations that encourage stem cell activity and hinder proper maturation are central to the development of colorectal cancer (CRC). Key molecular mediators that promote these malignant properties require further elucidation to galvanize translational advances. We therefore aimed to characterize a key factor that blocks intestinal differentiation, define its transcriptional and epigenetic program, and provide preclinical evidence for therapeutic targeting in CRC. METHODS: Intestinal tissue from transgenic mice and patients were analyzed by means of histopathology and immunostaining. Human CRC cells and neoplastic murine organoids were genetically manipulated for functional studies. Gene expression profiling was obtained through RNA sequencing. Histone modifications and transcription factor binding were determined with the use of chromatin immunoprecipitation sequencing. RESULTS: We demonstrate that SRY-box transcription factor 9 (SOX9) promotes CRC by activating a stem cell-like program that hinders intestinal differentiation. Intestinal adenomas and colorectal adenocarcinomas from mouse models and patients demonstrate ectopic and elevated expression of SOX9. Functional experiments indicate a requirement for SOX9 in human CRC cell lines and engineered neoplastic organoids. Disrupting SOX9 activity impairs primary CRC tumor growth by inducing intestinal differentiation. By binding to genome wide enhancers, SOX9 directly activates genes associated with Paneth and stem cell activity, including prominin 1 (PROM1). SOX9 up-regulates PROM1 via a Wnt-responsive intronic enhancer. A pentaspan transmembrane protein, PROM1 uses its first intracellular domain to support stem cell signaling, at least in part through SOX9, reinforcing a PROM1-SOX9 positive feedback loop. CONCLUSIONS: These studies establish SOX9 as a central regulator of an enhancer-driven stem cell-like program and carry important implications for developing therapeutics directed at overcoming differentiation defects in CRC.

Hybrid Stomach-Intestinal Chromatin States Underlie Human Barrett's Metaplasia.

BACKGROUND & AIMS: Tissue metaplasia is uncommon in adults because established cis-element programs resist rewiring. In Barrett's esophagus, the distal esophageal mucosa acquires a predominantly intestinal character, with notable gastric features, and is predisposed to developing invasive cancers. We sought to understand the chromatin underpinnings of Barrett's metaplasia and why it commonly displays simultaneous gastric and intestinal properties. METHODS: We profiled cis-regulatory elements with active histone modifications in primary human biopsy materials using chromatin immunoprecipitation followed by DNA sequencing. Mutations in Barrett's esophagus were examined in relation to tissue-specific enhancer landscapes using a random forest machine-learning algorithm. We also profiled open chromatin at single-cell resolution in primary Barrett's biopsy specimens using the assay for transposase-accessible chromatin. We used 1- and 2-color immunohistochemistry to examine protein expression of tissue-restricted genes. RESULTS: Barrett's esophagus bears epigenome fingerprints of human stomach and intestinal columnar, but not esophageal squamous, epithelia. Mutational patterns were best explained as arising on the epigenome background of active gastric cis-elements, supporting the view that adjoining stomach epithelium is a likely tissue source. Individual cells in Barrett's metaplasia coexpress gastric and intestinal genes, reflecting concomitant chromatin access at enhancers ordinarily restricted to one or the other epithelium. Protein expression of stomach-specific mucins; CLDN18; and a novel gastric marker, ANXA10, showed extensive tissue and subclonal heterogeneity of dual stomach-intestinal cell states. CONCLUSIONS: These findings reveal mixed and dynamic tissue-restricted chromatin states and phenotypic heterogeneity in Barrett's esophagus. Pervasive intragland variation argues against stem-cell governance of this phenotype.

Outcomes and management strategies for graft failure after umbilical cord blood transplantation.

Graft failure is a life-threatening complication after allogeneic hematopoietic stem cell transplantation (HSCT). Graft failure is more prevalent after umbilical cord blood transplantation (UCBT) compared with conventional adult stem cell sources. We identified 21 consecutive patients who experienced graft failure after UCBT at our center between 2004 and 2013 and describe their treatment strategies and outcomes. Two patients experienced early death. Seven patients had return of autologous hematopoiesis including 1 patient who was given previously collected autologous stem cells. Twelve patients received a second early HSCT, six from separate UCB units and six from a haploidentical donor. With a median follow-up of 33.2 months for surviving patients, 3-year PFS is 23% and 3-year OS is 37%. Of the six long-term survivors without relapse, four received a second HSCT from a haploidentical donor with post-HSCT high-dose cyclophosphamide based GVHD prophylaxis. This strategy appears safe and merits further investigation in this setting.

Bridging the Divide: From Universal Germline Testing Guidance to Real-World Implementation in Pancreatic Cancer Care.

In this editorial accompanying manuscript the by Klatte and colleagues, Drs Singh, and Nipp review the data behind universal germline testing in pancreatic adenocarcinoma, and review possible reasons for and solutions continued inadequate rates of germline testing in our community. Making germline testing for all patients with pancreatic adenocarcinoma is critical and will require cross-disciplinary collaboration and innovation.

Systematic literature review and meta-analysis of HER2 amplification, overexpression, and positivity in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is the second most common cause of cancer death globally. Recent clinical trials suggest an emerging role for HER2 as a potential clinically relevant biomarker in CRC. Testing for HER2 in CRC is not standard practice; consequently, the prevalence of HER2 positivity (HER2+) in patients with CRC remains uncertain. METHODS: A systematic literature review and meta-analysis were conducted to generate estimates of proportions of patients with CRC with HER2 overexpression or HER2 amplification and HER2+ (either overexpression or amplification), overall and in patients with rat sarcoma virus (RAS) wild-type cancer. HER2+ was defined as 1) immunohistochemistry with a score of 3+, 2) immunohistochemistry with a score of 2+ and in situ hybridization+, or 3) next-generation sequencing positive. RESULTS: Of 224 studies identified with information on HER2 in CRC, 52 studies used a US Food and Drug Administration-approved assay and were selected for further analysis. Estimated HER2+ rate was 4.1% (95% confidence interval [CI] = 3.4% to 5.0%) overall (n = 17 589). HER2+ rates were statistically higher in RAS wild-type (6.1%, 95% CI = 5.4% to 6.9%) vs RAS mutant CRC (1.1%, 95% CI = 0.3% to 4.4%; P < .0001). Despite limited clinical information, we confirmed enrichment of HER2+ CRC in patients with microsatellite stable and left-sided CRC. CONCLUSION: This meta-analysis provides an estimate of HER2+ CRC and confirms enrichment of HER2 in microsatellite stable, left-sided, RAS wild-type CRC tumors. Our work is important given the recently described clinical efficacy of HER2-targeted therapies in HER2+ CRC and informs strategies for incorporation of HER2 testing into standard of care.

Mutational signature-based identification of DNA repair deficient gastroesophageal adenocarcinomas for therapeutic targeting.

Homologous recombination (HR) and nucleotide excision repair (NER) are the two most frequently disabled DNA repair pathways in cancer. HR-deficient breast, ovarian, pancreatic and prostate cancers respond well to platinum chemotherapy and PARP inhibitors. However, the frequency of HR deficiency in gastric and esophageal adenocarcinoma (GEA) still lacks diagnostic and functional validation. Using whole exome and genome sequencing data, we found that a significant subset of GEA, but very few colorectal adenocarcinomas, show evidence of HR deficiency by mutational signature analysis (HRD score). High HRD gastric cancer cell lines demonstrated functional HR deficiency by RAD51 foci assay and increased sensitivity to platinum chemotherapy and PARP inhibitors. Of clinical relevance, analysis of three different GEA patient cohorts demonstrated that platinum treated HR deficient cancers had better outcomes. A gastric cancer cell line with strong sensitivity to cisplatin showed HR proficiency but exhibited NER deficiency by two photoproduct repair assays. Single-cell RNA-sequencing revealed that, in addition to inducing apoptosis, cisplatin treatment triggered ferroptosis in a NER-deficient gastric cancer, validated by intracellular GSH assay. Overall, our study provides preclinical evidence that a subset of GEAs harbor genomic features of HR and NER deficiency and may therefore benefit from platinum chemotherapy and PARP inhibitors.

Clinical outcomes and ctDNA correlates for CAPOX BETR: a phase II trial of capecitabine, oxaliplatin, bevacizumab, trastuzumab in previously untreated advanced HER2+ gastroesophageal adenocarcinoma.

Preclinical studies suggest that simultaneous HER2/VEGF blockade may have cooperative effects in gastroesophageal adenocarcinomas. In a single-arm investigator initiated clinical trial for patients with untreated advanced HER2+ gastroesophageal adenocarcinoma, bevacizumab was added to standard of care capecitabine, oxaliplatin, and trastuzumab in 36 patients (NCT01191697). Primary endpoint was objective response rate and secondary endpoints included safety, duration of response, progression free survival, and overall survival. The study met its primary endpoint with an objective response rate of 81% (95% CI 65-92%). Median progression free and overall survival were 14.0 (95% CI, 11.3-36.4) and 23.2 months (95% CI, 16.6-36.4), respectively. The median duration of response was 14.9 months. The regimen was well tolerated without unexpected or severe toxicities. In post-hoc ctDNA analysis, baseline ctDNA features were prognostic: Higher tumor fraction and alternative MAPK drivers portended worse outcomes. ctDNA at resistance identified oncogenic mutations and these were detectable 2-8 cycles prior to radiographic progression. Capecitabine, oxaliplatin, trastuzumab and bevacizumab shows robust clinical activity in HER2+ gastroesophageal adenocarcinoma. Combination of VEGF inhibitors with chemoimmunotherapy and anti-PD1 regimens is warranted.

Clinical and genomic features of classical and basal transcriptional subtypes in pancreatic cancer.

PURPOSE: Transcriptional profiling of pancreatic cancers (PC) has defined two main transcriptional subtypes, classical and basal. Initial data suggest shorter survival for patients with basal tumors and differing treatment sensitivity to FOLFIRINOX (FFX) and gemcitabine nab-Paclitaxel (GnP) by transcriptional subtype. EXPERIMENTAL DESIGN: We examined 8,743 patients with RNA sequencing from PCs performed at Caris Life Sciences (Phoenix, AZ). Classical and basal subtypes were identified using PurIST algorithm on RNA-sequencing and two cohorts were analyzed: (1) Biomarker cohort included patients with complete molecular profiling data (n = 7,250); (2) Outcomes cohort included patients with metastatic disease with available survival outcomes (n=5,335). RESULTS: In the biomarker cohort, 3,063 tumors (42.2%) were strongly classical (SC), and 2,015 tumors (27.8%) were strongly basal (SB). SC and SB tumors showed strong associations with histologic phenotypes and biopsy site. SB tumors had higher rates of KRAS, TP53, and ARID1A mutations, lower rates of SMAD4 mutation, and transcriptional evidence of epithelial mesenchymal transition. Sixty of 77 cases (78%) maintained their transcriptional subtype between temporally and/or spatially disparate lesions. In the outcomes cohort, SB subtype was associated with shorter overall survival time, regardless of whether they received FFX or GnP as first line chemotherapy. Mutant KRAS allele type was prognostic of outcomes, however this impact was restricted to SC tumors, whereas all mutant KRAS alleles had similarly poor outcomes in SB tumors. CONCLUSIONS: SB subtype is a strong independent predictor of worse outcomes, irrespective of upfront chemotherapy regimen. Clinical trials should investigate PC transcriptional subtypes as a biomarker.

Biomarkers of pembrolizumab efficacy in advanced anal squamous cell carcinoma: analysis of a phase II clinical trial and a cohort of long-term responders.

BACKGROUND: Recent trials suggest that programmed cell death 1 (PD-1)-directed immunotherapy may be beneficial for some patients with anal squamous cell carcinoma and biomarkers predictive of response are greatly needed. METHODS: This multicenter phase II clinical trial (NCT02919969) enrolled patients with metastatic or locally advanced incurable anal squamous cell carcinoma (n=32). Patients received pembrolizumab 200 mg every 3 weeks. The primary endpoint of the trial was objective response rate (ORR). Exploratory objectives included analysis of potential predictive biomarkers including assessment of tumor-associated immune cell populations with multichannel immunofluorescence and analysis of circulating tumor tissue modified viral-human papillomavirus DNA (TTMV-HPV DNA) using serially collected blood samples. To characterize the clinical features of long-term responders, we combined data from our prospective trial with a retrospective cohort of patients with anal cancer treated with anti-PD-1 immunotherapy (n=18). RESULTS: In the phase II study, the ORR to pembrolizumab monotherapy was 9.4% and the median progression-free survival was 2.2 months. Despite the high level of HPV positivity observed with circulating TTMV-HPV DNA testing, the majority of patients had low levels of tumor-associated CD8+PD-1+ T cells on pretreatment biopsy. Patients who benefited from pembrolizumab had decreasing TTMV-HPV DNA scores and a complete responder's TTMV-HPV DNA became undetectable. Long-term pembrolizumab responses were observed in one patient from the trial (5.3 years) and three patients (2.5, 6, and 8 years) from the retrospective cohort. Long-term responders had HPV-positive tumors, lacked liver metastases, and achieved a radiological complete response. CONCLUSIONS: Pembrolizumab has durable efficacy in a rare subset of anal cancers. However, despite persistence of HPV infection, indicated by circulating HPV DNA, most advanced anal cancers have low numbers of tumor-associated CD8+PD-1+ T cells and are resistant to pembrolizumab.

RAS/RAF Comutation and ERBB2 Copy Number Modulates HER2 Heterogeneity and Responsiveness to HER2-directed Therapy in Colorectal Cancer.

PURPOSE: ERBB2-amplified colorectal cancer is a distinct molecular subtype with expanding treatments. Implications of concurrent oncogenic RAS/RAF alterations are not known. EXPERIMENTAL DESIGN: Dana-Farber and Foundation Medicine Inc. Colorectal cancer cohorts with genomic profiling were used to identify ERBB2-amplified cases [Dana-Farber, n = 47/2,729 (1.7%); FMI, n = 1857/49,839 (3.7%)]. Outcomes of patients receiving HER2-directed therapies are reported (Dana-Farber, n = 9; Flatiron Health-Foundation Medicine clinicogenomic database, FH-FMI CGDB, n = 38). Multisite HER2 IHC and genomic profiling were performed to understand HER2 intratumoral and interlesional heterogeneity. The impact of concurrent RAS comutations on the effectiveness of HER2-directed therapies were studied in isogenic colorectal cancer cell lines and xenografts. RESULTS: ERBB2 amplifications are enriched in left-sided colorectal cancer. Twenty percent of ERBB2-amplified colorectal cancers have co-occurring oncogenic RAS/RAF alterations. While RAS/RAF WT colorectal cancers typically have clonal ERBB2 amplification, colorectal cancers with co-occurring RAS/RAF alterations have lower level ERRB2 amplification, higher intratumoral heterogeneity, and interlesional ERBB2 discordance. These distinct genomic patterns lead to differential responsiveness and patterns of resistance to HER2-directed therapy. ERBB2-amplified colorectal cancer with RAS/RAF alterations are resistant to trastuzumab-based combinations, such as trastuzumab/tucatinib, but retain sensitivity to trastuzumab deruxtecan in in vitro and murine models. Trastuzumab deruxtecan shows clinical efficacy in cases with high-level ERBB2-amplified RAS/RAF coaltered colorectal cancer. CONCLUSIONS: Co-occurring RAS/RAF alterations define a unique subtype of ERBB2-amplified colorectal cancer that has increased intratumoral heterogeneity, interlesional discordance, and resistance to trastuzumab-based combinations. Further examination of trastuzumab deruxtecan in this previously understudied cohort of ERBB2-amplified colorectal cancer is warranted.

Blockade of IL1ß and PD1 with Combination Chemotherapy Reduces Systemic Myeloid Suppression in Metastatic Pancreatic Cancer with Heterogeneous Effects in the Tumor.

Innate inflammation promotes tumor development, although the role of innate inflammatory cytokines in established human tumors is unclear. Herein, we report clinical and translational results from a phase Ib trial testing whether IL1ß blockade in human pancreatic cancer would alleviate myeloid immunosuppression and reveal antitumor T-cell responses to PD1 blockade. Patients with treatment-naïve advanced pancreatic ductal adenocarcinoma (n = 10) were treated with canakinumab, a high-affinity monoclonal human antiinterleukin-1ß (IL1ß), the PD1 blocking antibody spartalizumab, and gemcitabine/n(ab)paclitaxel. Analysis of paired peripheral blood from patients in the trial versus patients receiving multiagent chemotherapy showed a modest increase in HLA-DR+CD38+ activated CD8+ T cells and a decrease in circulating monocytic myeloid-derived suppressor cells (MDSC) by flow cytometry for patients in the trial but not in controls. Similarly, we used patient serum to differentiate monocytic MDSCs in vitro and showed that functional inhibition of T-cell proliferation was reduced when using on-treatment serum samples from patients in the trial but not when using serum from patients treated with chemotherapy alone. Within the tumor, we observed few changes in suppressive myeloid-cell populations or activated T cells as assessed by single-cell transcriptional profiling or multiplex immunofluorescence, although increases in CD8+ T cells suggest that improvements in the tumor immune microenvironment might be revealed by a larger study. Overall, the data indicate that exposure to PD1 and IL1ß blockade induced a modest reactivation of peripheral CD8+ T cells and decreased circulating monocytic MDSCs; however, these changes did not lead to similarly uniform alterations in the tumor microenvironment.

Replicative stress in gastroesophageal cancer is associated with chromosomal instability and sensitivity to DNA damage response inhibitors.

Gastroesophageal adenocarcinoma (GEA) is an aggressive malignancy with chromosomal instability (CIN). To understand adaptive responses enabling DNA damage response (DDR) and CIN, we analyzed matched normal, premalignant, and malignant gastric lesions from human specimens and a carcinogen-induced mouse model, observing activation of replication stress, DDR, and p21 in neoplastic progression. In GEA cell lines, expression of DDR markers correlated with ploidy abnormalities, such as number of high-level focal amplifications and whole-genome duplication (WGD). Integrating TP53 status, ploidy abnormalities, and DDR markers into a compositive score helped predict GEA cell lines with enhanced sensitivity to Chk1/2 and Wee1 inhibition, either alone or combined with irinotecan (SN38). We demonstrate that Chk1/2 or Wee1 inhibition combined with SN38/irinotecan shows greater anti-tumor activity in human gastric cancer organoids and an in vivo xenograft mouse model. These findings indicate that specific DDR biomarkers and ploidy abnormalities may predict premalignant progression and response to DDR pathway inhibitors.

Replicative stress in gastroesophageal adenocarcinoma is associated with chromosomal instability and sensitivity to DNA damage response inhibitors.

Gastroesophageal adenocarcinoma (GEA) is an aggressive, often lethal, malignancy that displays marked chromosomal instability (CIN). To understand adaptive responses that enable CIN, we analyzed paired normal, premalignant, and malignant gastric lesions from human specimens and a carcinogen-induced mouse model, observing activation of replication stress, DNA damage response (DDR), and cell cycle regulator p21 in neoplastic progression. In GEA cell lines, expression of DDR markers correlated with ploidy abnormalities, including high-level focal amplifications and whole-genome duplication (WGD). Moreover, high expression of DNA damage marker H2AX correlated with CIN, WGD, and inferior patient survival. By developing and implementing a composite diagnostic score that incorporates TP53 mutation status, ploidy abnormalities, and H2AX expression, among other genomic information, we can identify GEA cell lines with enhanced sensitivity to DDR pathway inhibitors targeting Chk1/2 and Wee1. Anti-tumor properties were further augmented in combination with irinotecan (SN38) but not gemcitabine chemotherapy. These results implicate specific DDR biomarkers and ploidy abnormalities as diagnostic proxy that may predict premalignant progression and response to DDR pathway inhibitors.

Highly Sensitive Circulating Tumor DNA Assay Aids Clinical Management of Radiographically Occult Isolated Peritoneal Metastases in Patients With GI Cancer.

PURPOSE: GI cancers commonly spread to the peritoneal cavity, particularly from primary adenocarcinomas of the stomach and appendix. Peritoneal metastases are difficult to visualize on cross-sectional imaging and cause substantial morbidity and mortality. The purpose of this study was to determine whether serial highly sensitive tumor-informed circulating tumor DNA (ctDNA) measurements could longitudinally track changes in disease burden and inform clinical care. METHODS: This was a retrospective case series of patients with gastric or appendiceal adenocarcinoma and isolated peritoneal disease that was radiographically occult. Patients underwent quantitative tumor-informed ctDNA testing (Signatera) as part of routine clinical care. No interventions were prespecified based on ctDNA results. RESULTS: Of 13 patients studied, the median age was 65 (range, 45-75) years, with 7 (54%) women, 5 (38%) patients with gastric, and 8 (62%) patients with appendiceal adenocarcinoma. Eight (62%) patients had detectable ctDNA at baseline measurement, with median value 0.13 MTM/mL (range, 0.06-11.68), and assay was technically unsuccessful in two cases with appendiceal cancer because of limited tumor tissue. Five (100%) patients with gastric cancer and 3 (50%) patients with appendiceal cancer had detectable ctDNA at baseline. Although baseline levels of ctDNA were low, longitudinal assessment tracked with changes in disease burden among patients undergoing chemotherapy for metastatic disease. In two patients undergoing surveillance after definitive surgical management of gastric adenocarcinoma, detection of ctDNA prompted diagnosis of isolated peritoneal disease. CONCLUSION: Quantitative tumor-informed serial ctDNA testing aids clinical management of patients with isolated peritoneal disease. Low levels of baseline ctDNA suggest a role for highly sensitive ctDNA approaches over panel-based testing. Further exploration of this approach should be considered in patients with isolated peritoneal malignant disease.

G-CSF rescue of FOLFIRINOX-induced neutropenia leads to systemic immune suppression in mice and humans.

BACKGROUND: Recombinant granulocyte colony-stimulating factor (G-CSF) is routinely administered for prophylaxis or treatment of chemotherapy-induced neutropenia. Chronic myelopoiesis and granulopoiesis in patients with cancer has been shown to induce immature monocytes and neutrophils that contribute to both systemic and local immunosuppression in the tumor microenvironment. The effect of recombinant G-CSF (pegfilgrastim or filgrastim) on the production of myeloid-derived suppressive cells is unknown. Here we examined patients with pancreatic cancer, a disease known to induce myeloid-derived suppressor cells (MDSCs), and for which pegfilgrastim is routinely administered concurrently with FOLFIRINOX but not with gemcitabine-based chemotherapy regimens. METHODS: Serial blood was collected from patients with pancreatic ductal adenocarcinoma newly starting on FOLFIRINOX or gemcitabine/n(ab)paclitaxel combination chemotherapy regimens. Neutrophil and monocyte frequencies were determined by flow cytometry from whole blood and peripheral blood mononuclear cell fractions. Serum cytokines were evaluated pretreatment and on-treatment. Patient serum was used in vitro to differentiate healthy donor monocytes to MDSCs as measured by downregulation of major histocompatibility complex II (HLA-DR) and the ability to suppress T-cell proliferation in vitro. C57BL/6 female mice with pancreatic tumors were treated with FOLFIRINOX with or without recombinant G-CSF to directly assess the role of G-CSF on induction of immunosuppressive neutrophils. RESULTS: Patients receiving FOLFIRINOX with pegfilgrastim had increased serum G-CSF that correlated with an induction of granulocytic MDSCs. This increase was not observed in patients receiving gemcitabine/n(ab)paclitaxel without pegfilgrastim. Interleukin-18 also significantly increased in serum on FOLFIRINOX treatment. Patient serum could induce MDSCs as determined by in vitro functional assays, and this suppressive effect increased with on-treatment serum. Induction of MDSCs in vitro could be recapitulated by addition of recombinant G-CSF to healthy serum, indicating that G-CSF is sufficient for MDSC differentiation. In mice, neutrophils isolated from spleen of G-CSF-treated mice were significantly more capable of suppressing T-cell proliferation. CONCLUSIONS: Pegfilgrastim use contributes to immune suppression in both humans and mice with pancreatic cancer. These results suggest that use of recombinant G-CSF as supportive care, while critically important for mitigating neutropenia, may complicate efforts to induce antitumor immunity.

Oncogenic Drivers and Therapeutic Vulnerabilities in KRAS Wild-Type Pancreatic Cancer.

PURPOSE: Approximately 8% to 10% of pancreatic ductal adenocarcinomas (PDAC) do not harbor mutations in KRAS. Understanding the unique molecular and clinical features of this subset of pancreatic cancer is important to guide patient stratification for clinical trials of molecularly targeted agents. EXPERIMENTAL DESIGN: We analyzed a single-institution cohort of 795 exocrine pancreatic cancer cases (including 785 PDAC cases) with a targeted multigene sequencing panel and identified 73 patients (9.2%) with KRAS wild-type (WT) pancreatic cancer. RESULTS: Overall, 43.8% (32/73) of KRAS WT cases had evidence of an alternative driver of the MAPK pathway, including BRAF mutations and in-frame deletions and receptor tyrosine kinase fusions. Conversely, 56.2% of cases did not harbor a clear MAPK driver alteration, but 29.3% of these MAPK-negative KRAS WT cases (12/41) demonstrated activating alterations in other oncogenic drivers, such as GNAS, MYC, PIK3CA, and CTNNB1. We demonstrate potent efficacy of pan-RAF and MEK inhibition in patient-derived organoid models carrying BRAF in-frame deletions. Moreover, we demonstrate durable clinical benefit of targeted therapy in a patient harboring a KRAS WT tumor with a ROS1 fusion. Clinically, patients with KRAS WT tumors were significantly younger in age of onset (median age: 62.6 vs. 65.7 years; P = 0.037). SMAD4 mutations were associated with a particularly poor prognosis in KRAS WT cases. CONCLUSIONS: This study defines the genomic underpinnings of KRAS WT pancreatic cancer and highlights potential therapeutic avenues for future investigation in molecularly directed clinical trials. See related commentary by Kato et al., p. 4527.

A Phase I Expansion Cohort Study Evaluating the Safety and Efficacy of the CHK1 Inhibitor LY2880070 with Low-dose Gemcitabine in Patients with Metastatic Pancreatic Adenocarcinoma.

PURPOSE: Combining gemcitabine with CHK1 inhibition has shown promise in preclinical models of pancreatic ductal adenocarcinoma (PDAC). Here, we report the findings from a phase I expansion cohort study (NCT02632448) investigating low-dose gemcitabine combined with the CHK1 inhibitor LY2880070 in patients with previously treated advanced PDAC. PATIENTS AND METHODS: Patients with metastatic PDAC were treated with gemcitabine intravenously at 100 mg/m2 on days 1, 8, and 15, and LY2880070 50 mg orally twice daily on days 2-6, 9-13, and 16-20 of each 21-day cycle. Pretreatment tumor biopsies were obtained from each patient for correlative studies and generation of organoid cultures for drug sensitivity testing and biomarker analyses. RESULTS: Eleven patients with PDAC were enrolled in the expansion cohort between August 27, 2020 and July 30, 2021. Four patients (36%) experienced drug-related grade 3 adverse events. No objective radiologic responses were observed, and all patients discontinued the trial by 3.2 months. In contrast to the lack of efficacy observed in patients, organoid cultures derived from biopsies procured from two patients demonstrated strong sensitivity to the gemcitabine/LY2880070 combination and showed treatment-induced upregulation of replication stress and DNA damage biomarkers, including pKAP1, pRPA32, and γH2AX, as well as induction of replication fork instability. CONCLUSIONS: No evidence of clinical activity was observed for combined low-dose gemcitabine and LY2880070 in this treatment-refractory PDAC cohort. However, the gemcitabine/LY2880070 combination showed in vitro efficacy, suggesting that drug sensitivity for this combination in organoid cultures may not predict clinical benefit in patients.