Microsatellite Stable Tumors Research Articles

Characterization and impact of canonical Wnt Signaling Pathway (WSP) alterations on outcomes of metastatic prostate cancer.

5053 Background: Recurrent alterations in the WSP have been identified in patients with advanced prostate cancer. Aberrant Wnt signaling has been implicated in disease progression and hormonal resistance in prostate cancer. We utilized a multi-institutional real-world dataset to characterize molecular alterations in the canonical WSP in men with prostate cancer, and correlate that with overall survival (OS). Methods: Prostate cancer patients who underwent tissue-based DNA and RNA sequencing utilizing a commercially available CLIA-certified assay (Caris Life Sciences) were investigated. Next generation sequencing (NGS)/ whole exome (WES) and transcriptome sequencing (WTS) was performed on prostate cancer tissue derived from prostatic and/or metastatic sites. Patients with somatic activating mutations in CTNNB, pathogenic fusions in RSPO2, or inactivating mutations in APC or RNF43 were characterized as having aberrant canonical Wnt signaling (WSP-MT). A subset analysis was conducted in metastatic prostate cancer (mPCa) samples with microsatellite stable (MSS) tumors excluding RNF43 (G569fs*) mutations. Comparative analyses were done using Fisher-Exact or X2 tests, and significance was determined by adjusted p value using Benjamini-Hochberg correction (q < 0.05). OS was obtained from insurance claims data and calculated using Kaplan-Meier estimates. Enriched mRNA transcripts were identified as those with an adjusted p value < 0.001, logFC > 1.5, and (-)log10 FDR > 20. Results: Overall, 21.8% (n = 715/3283) of samples were classified as WSP-MT, of which 297 (41.5%) were from primary tumor samples and 418 (58.5%) were from metastatic sites. WSP-MT were highest in metastases to CNS (54.5%, n = 12/22), liver (43.8%, n = 91/208) and lung (42.3%, n = 91/208). Compared to WSP wild-type, WSP-MT tumors had a greater frequency of dMMR/MSI-H status (18% vs. 2%, q < 0.001). In the subset of mPCa, MSS tumors (WSP-MT excluding RNF43 (G569fs*) mutations, n = 318; WSP wild-type n = 951), WSP-MT were enriched for mutations in SPOP (14.3% vs. 6.9%, q = 0.004). In these tumors, WSP-MT samples were most enriched for mRNA expression of CST1, NKD1, AXIN2, and ZNRF3 (all known canonical WSP activators). OS was inferior in mPCa WSP-MT patients compared to WSP wild-type (HR 0.49, 95% CI 0.37-0.64, p < 0.0001). Relative to WSP wild-type cases, OS was shorter from time of enzalutamide/abiraterone/apalutamide start (HR 0.25, 95% CI 0.15-0.43, p = 0.0001), and from first-line taxane chemotherapy (HR 0.47, 95% CI 0.21-1.02, p = 0.052) in pts with WSP-MT. Conclusions: Canonical WSP alterations are enriched in metastatic tumors, with highest prevalence in visceral (CNS, liver, lung) metastases. Clinical outcomes in WSP-mutant cancers are inferior with both hormonal and chemotherapies, heralding an urgent need to develop novel therapeutic strategies for such patients.

Microsatellite-stable tumors with high tumor mutational burden in association with tumor response to immune checkpoint inhibitor therapy across solid tumors and correlation with specific oncogenic alterations.

2634 Background: High tumor mutational burden (TMB-H) has shown promise as a predictive biomarker in certain tumors, but broad applicability across tumors is unclear. In 2020, pembrolizumab was approved for treatment refractory metastatic or unresectable solid tumors with TMB ≥10 mutations/megabase based on exploratory analysis of the KEYNOTE-158 study. Growing evidence suggest limitations of TMB-H across tumors. Our aim is to evaluate outcomes of patients (pts) with advanced microsatellite-stable (MSS) solid tumors with TMB ≥10 mutations/megabase treated with immune checkpoint inhibitors (ICIs) and identify genetic alterations that predict poor response to therapy. Methods: Pts treated between 01/2015 to 12/2020 at Robert H Lurie Cancer Center, Northwestern University, with ICIs and TMB ≥10 mutations/megabase on next generation sequencing by FoundationOne CDx, Tempus xT or Guardant360 CDx platforms were identified. Tumors other than carcinomas and microsatellite instability were excluded. Radiologic response was assessed using iRECIST criteria. Responders (CR/PR) were compared with non-responders (SD/PD). Subgroup analyses were conducted based on tumor type, genetic alterations within signaling pathways. Survival curves were calculated using Kaplan-Meier method. Multivariate analyses were performed to determine impact of clinical variables on response. Results: Of 119 pts, median age was 68 yrs, and 61% of pts were male. 15 tumor types were represented, most commonly melanoma (33%), NSCLC (28%), small cell lung carcinoma (5.9%), colorectal carcinoma (5%), and pancreatobiliary carcinoma (5%). When evaluating for efficacy (n = 106), ORR was 36% with CR 14%. Median PFS and OS overall were 10.9 mo and 29.9 mo. Lack of exposure to previous therapy was associated with response (p = 0.039). PD-L1 status, specific ICI regimen, age, and sex did not have prognostic significance. When comparing melanoma/NSCLC (n = 66) and other tumor types (n = 40) there was no significant difference in response (p = 0.3). When comparing response by specific gene mutations, TP53 (p = 0.026) and PIK3CA (p = 0.025) were associated with worse response. Similarly, ROS1 alteration trended to poorer response (p = 0.057). When evaluating genetic pathways, mutations within TP53 pathway were associated inferior response (p = 0.018). There was no significant difference in RTK/RAS, NRF2, PI3K, TGFβ, WNT, MYC, Cell Cycle, and Notch pathways. Conclusions: Growing evidence shows limitations of TMB-H as a predictive biomarker for ICI therapy in MSS tumors. Our data suggests alterations within TP53 and its signaling pathway, PIK3CA mutations, and likely ROS1 alterations are associated with non-responders in MSS TMB-H solid tumors. Further analysis of tumor-specific cohorts and genetic alterations will be presented.

P-49 GOBLET: A phase 1/2 multiple indication signal finding and biomarker study in advanced gastrointestinal cancers treated with pelareorep and atezolizumab – safety and preliminary response results

In GI cancers, checkpoint inhibitors are only effective in patients with high microsatellite instability (MSI-H) or deficient mismatch repair (dMMR) tumors. Oncolytic viruses may improve the susceptibility of microsatellite stable (MSS) tumors to immunotherapy by modifying the tumor microenvironment (TME). Pelareorep (pela) is an intravenously delivered, non-genetically modified, oncolytic reovirus that mediates cancer cell killing by activating innate and adaptive immune responses directed against the tumor as well as direct tumor oncolysis. Accordingly, treatment with pelareorep results in increased T cell infiltration and PD-L1 expression in tumors which primes the TME for responsiveness to checkpoint inhibitors. Pela has demonstrated activity in many cancers including colorectal and pancreatic cancer. The GOBLET study is designed to assess the efficacy of pela plus atezolizumab (atezo) with or without chemotherapy in multiple GI cancers. GOBLET is a phase 1/2, open-label, non-randomized study in patients with advanced or metastatic GI cancers and utilizes a Simon two-stage design. In the first stage of the study, four treatment groups are being enrolled: Cohort 1 – First-line metastatic/advanced pancreatic cancer treated with pela plus atezo and chemotherapy (gemcitabine and nab-paclitaxel) (N=12); Cohort 2 – First-line MSI-H/dMMR metastatic CRC treated with pela plus atezo (N=19); Cohort 3 – Third-line metastatic CRC treated with pela plus atezo and chemotherapy (trifluridine/tipiracil) (N=14); and Cohort 4 – Second-line or later advanced squamous cell carcinoma of the anal canal treated with pela plus atezo (N=10). In Cohorts 1 and 3 (chemotherapy-containing cohorts) the first 3-6 patients enrolled comprise a safety run-in. The primary objectives of GOBLET are safety and efficacy measured by the objective response rate (ORR) at week 16. Based on pre-specified response thresholds, any cohort showing a positive ORR signal in Stage 1 may advance to the 2nd Stage and enroll additional patients. The three safety run-in patients in Cohorts 1 and 3 have been enrolled and their safety data reviewed by the independent Data Safety Monitoring Board (DSMB). The DSMB identified no safety signal and recommended that enrollment into these cohort continue without modification. Enrollment into Cohorts 2 and 4 is ongoing as these cohorts do not include safety run-ins. Tumor response results to date indicate that two of the three Cohort 1 patients had a partial response at week 8, the third Cohort 1 patient had a partial response at week 16. No safety signal was observed in either the Cohort 1 (first-line pancreatic cancer) or Cohort 3 (third-line CRC) patients. This is consistent with the favorable safety profile observed in prior studies of pela in multiple cancer indications, and it supports the ability to safely treat patients with advanced GI cancers using pela in combination with checkpoint inhibitors and chemotherapy. Preliminary tumor responses to therapy in first-line pancreatic cancer patients are encouraging.

An Immune-metabolic signature (IMMETCOLS) identifies three clusters in mCRC with different immune-phenotype distribution and potential clinical implications.

e15534 Background: The overall response rate following immune check-point blockade (ICB) is < 5% in microsatellite stable (MSS) advanced colorectal cancer (mCRC), and between 30-35% in MSI mCRC. We here evaluate the relationship between our immune-metabolic signature (IMMETCOLS) distribution (cluster (IMC) 1; mesenchymal glycolytic, IMC 2; epithelial-non glycolytic and IMC 3; epithelial mitochondrial oxidative) with immune phenotypes (inflamed (IF), immune-excluded (IE) and immune-desert (ID)) in MSS and MSI mCRC patients (pts). Methods: We have analyzed IMMETCOLS in a retrospective cohort of 128 mCRC pts enriched with MSI-H and BRAF genotype using the nCounter platform (Nanostring Technologies, US). Immune phenotypes were evaluated by immunohistochemistry using 7 immune markers (CD68, CD163, CD8, CD3, FOXP3, PD-L1 and PD1) in a subset of 47 mCRC pts (27 MSS and 20 MSI), in TMAs containing 4 cores from the center of the tumor (CT) and invasive front (IF). The number of stained immune cells was counted using a computerized image analysis system, and PD-L1 was evaluated using CPS score (< 1 vs > 1). Patients having a CD3 cell count below 572 cells/mm2 and a CD8 density below 175 cells/mm2 in IF or CT were considered as ID. (Halama Nils, 2011). CD3 density was also semi-quantitatively estimated in peritumoral stroma and intra-tumoral (range 1-4), defining as IE those cases with a difference > 1 between the two regions in both IF and CT (Brooks JM, 2019). Results: Cluster distribution was similar between MSI (IMC1-40%, IMC2-20% and IMC-40%) and MSS (IMC1-34%, IMC2-24%, IMC3-43%) pts. Patients with high LDH levels (> 1.5ULN) were enriched with IMC3 phenotype (p < 0.0001). MSI have increased CD3 (p = 0.002), CD68 (p = 0.002) and CD163 (p = 0.001) in CT than MSS patients. IMC1 pts had increased CD163/CD68 ratio in CT (p = 0.004). 75% of MSI pts were IE and 25% IF. 55% of MSS pts were IE, 38% ID and 7% IF. IMMETCOLS/ immune-phenotype distribution was: IMC1 (16% ID/ 84% IE), IMC2 (12% ID/ 63% IE/ 25% IF) and IMC3 (35%ID/ 47% IE/ 18% IF). PD1 immuno-expression > 1% (IF and CT) and PD-L1 positivity was observed in 38%, 30% and 28% of pts respectively. There were no differences in PD-L1 (CPS) and PD1 expression between MSS and MSI tumors. Inflamed tumors show higher PD-L1 expression compared with IE and ID immune-phenotypes (p = 0.014). PD1 > 1% expression at IF was significatively lower in IMC1 vs IMC2 and IMC3 tumors (p = 0.032). Conclusions: IMC1 subtype is associated with low PD1 expression, high CD163/CD68 ratio and IE phenotype even in MSI-H mCRC patients. Therefore, IMMETCOLS testing would be of value to identify MSI mCRC pts that do not benefit to ICB therapy.

The frequency of second primary malignancies and colonic polyps in Lynch syndrome with MSI tumors following immune checkpoint blockade.

10505 Background: Lynch syndrome (LS) is caused by germline mutations in DNA mismatch repair genes and is characterized by microsatellite instability (MSI) status of associated tumors. MSI sensitizes tumors to immune checkpoint inhibition (ICI). The incidence of second primary malignancies and colonic polyps in LS patients with MSI tumors following ICI has not been evaluated. Methods: The Memorial Sloan Kettering LS database was queried for all patients with cancer who received ≥1 cycle of ICI using an IRB approved protocol. LS was confirmed by presence of a germline pathogenic/likely pathogenic alteration in a DNA mismatch repair gene ( MLH1, MSH2, MSH6, PMS2, EPCAM). Tumor and matched normal DNA sequencing was performed via MSK-IMPACT, an IRB approved protocol (NCT01775072). MSI status was assessed using MSIsensor. Results: At our center, 131 patients with LS received ICI mostly (73.3%) in the context of metastatic cancer. While 108 patients received ICI for an MSI tumor, 23 LS patients received ICI for a MSS (Microsatellite Stable) tumor (4 hepatobiliary, 3 colorectal, 3 CNS, 2 endometrial, 2 gastroesophageal, 2 renal, 2 sarcoma, 5 other). Five patients (3.8%) developed ≥1 second primary malignancy while on or following ICI comprised of an MSI small bowel adenocarcinoma, MSI upper tract urothelial cancer, 2 MSS prostate cancers, MSS HCC and MSI sebaceous neoplasm; 1 patient developed both MSS prostate cancer and MSI urothelial cancer on ICI. All five patients’ primary malignancy was MSI. Only a minority of patients underwent surveillance colonoscopies post completion of ICI (31.3%, (41/131)). Of these 41, nine (22%, (9/41)) were identified to have polyps including 8 tubular adenomas and 1 tubulo-villous adenoma. Median time to development of a polyp was 22 months (95% CI 16.82-27.18) from last colonoscopy and 13 months (95% CI 4-25 months) from last ICI. Notably amongst the 23 LS patients whose tumors were MSS, 14 had progression on ICI. Conclusions: Herein, we demonstrate that in LS-patients in receipt of ICI for cancer treatment, the risk of a second primary cancer and polyps remain high following treatment with ICI. Biological mechanisms underlying immune escape warrant further investigation. Surveillance strategies should be continued for LS patients post ICI.

Phase II study of ceralasertib (AZD6738) in combination with durvalumab in patients with advanced gastric cancer.

4045 Background: Alterations in DNA damage response (DDR) and repair are associated with genomic instability and increased somatic tumor mutational burden, and modulating DNA repair using specific inhibitors is a promising strategy to boost the efficacy of cancer immunotherapy. Ceralasertib is an oral inhibitor of the serine/threonine protein kinase Ataxia Telangiectasia and Rad3 Related (ATR), which is crucial to the cell’s response to replication stress. Methods: This phase 2 trial was designed to evaluate the efficacy and safety of ceralasertib in combination with durvalumab in patients with advanced gastric cancer (AGC). The study drug regimen was ceralasertib 240 mg BD days 15 to 28 in a 28-day cycle in combination with durvalumab at 1500 mg day 1 every 4 weeks. The primary end point was overall response rate (ORR) by RECIST (v1.1). Exploratory biomarker analysis was performed using fresh tumor biopsies in all enrolled patients. Results: 31 patients (median no. of prior lines, 2; range, 2-5) were enrolled between Jul 2019 and Mar 2020. All enrolled patients had confirmed microsatellite stable tumors, 5 patients were EBV positive, and 24 patients were PD-L1 positive (CPS≥1). Two patients had received prior anti-PD-1 treatment. At the time of data cut-off (Dec 2020), 30 patients were evaluable for response: 7 partial responses (one patient with prior anti-PD-1 treatment), 11 stable disease, and 12 disease progression were observed. The ORR was 22.6%, DCR 58.1 %, median PFS 3.0 months (95% confidence interval (CI), 2.1-3.9), median duration of response 5.7 months (95% CI, 4.9-6.5), and median OS was 6.7 months (95% CI, 3.8-9.6). A subgroup of patients (n = 11) who with loss of ATM expression and/or high proportion of mutational signature attributable to homologous repair deficiency (sig. HRD) demonstrated significantly longer PFS than those (n = 12) who had intact ATM and low sig. HRD (5.60 vs 1.65 months, hazard ratio 0.13, 95% CI 0.045-0.39, long-rank P < 0.001). The most common adverse events of any grade were fatigue (n = 22, 71.0%), nausea (n = 20, 64.5%) and anorexia (n = 19, 61.3%), and the most common adverse events of grade 3 or more were anemia and thrombocytopenia (n = 11, 35.5% each). Conclusions: Ceralasertib in combination with durvalumab demonstrated promising anti-tumor activity with durable responses in refractory AGC. Clinical trial information: NCT03780608.

PARP Inhibitor Insensitivity to BRCA1/2 Monoallelic Mutations in Microsatellite Instability-High Cancers.

PURPOSETo examine the overlap of homologous recombination deficiency (HRD) and microsatellite instability high (MSI-H) status, and to dissect driver versus bystander status of BRCA1/2 mutations (BRCAm) in this context.METHODSA pan-cancer comprehensive genomic profiling cohort (n = 213,199) was examined for overlap between BRCAm and MSI-H status. BRCA1/2 variant zygosity was examined and correlated with MSI-H status, tumor mutational burden, and genome-wide loss of heterozygosity (gLOH). Clinical histories of two patients with prostate cancer with co-occurring BRCAm and MSI-H are described.RESULTSHRD and MSI-H phenotypes were generally mutually exclusive events (P < .001). BRCAm that co-occurred together with high tumor mutational burden or MSI-H were predominantly monoallelic bystander alterations. In breast, ovarian, and pancreatic cancers, very few BRCAm occurred in the context of MSI-H; however, in prostate cancer, 12.8% of BRCA1 and 3.4% of BRCA2 alterations co-occurred with MSI-H. In these BRCA-associated cancers, co-occurring BRCAm were generally monoallelic and were not associated with elevated gLOH. Two patients with prostate cancer with co-occurring BRCAm and MSI-H showed resistance to poly (ADP-ribose) polymerase inhibition but sensitivity to subsequent anti–programmed cell death protein 1 therapy.CONCLUSIONMSI-H status and HRD are generally mutually exclusive phenomena across cancer types, but may rarely co-occur, especially in prostate cancer. Although MSI-H samples had a higher BRCAm prevalence relative to microsatellite-stable tumors, these BRCA1/2 mutations were generally monoallelic and were not associated with elevated gLOH. Our findings suggest that most BRCAm coexisting with microsatellite instability are likely bystander events that may not result in sensitivity to poly (ADP-ribose) polymerase inhibitors.

Impact of microsatellite status in early-onset colonic cancer.

The molecular profile of early-onset colonic cancer is undefined. This study evaluated clinicopathological features and oncological outcomes of young patients with colonic cancer according to microsatellite status. Anonymized data from an international collaboration were analysed. Criteria for inclusion were patients younger than 50 years diagnosed with stage I-III colonic cancer that was surgically resected. Clinicopathological features, microsatellite status, and disease-specific outcomes were evaluated. A total of 650 patients fulfilled the criteria for inclusion. Microsatellite instability (MSI) was identified in 170 (26.2 per cent), whereas 480 had microsatellite-stable (MSS) tumours (relative risk of MSI 2.5 compared with older patients). MSI was associated with a family history of colorectal cancer and lesions in the proximal colon. The proportions with pathological node-positive disease (45.9 versus 45.6 per cent; P = 1.000) and tumour budding (20.3 versus 20.5 per cent; P = 1.000) were similar in the two groups. Patients with MSI tumours were more likely to have BRAF (22.5 versus 6.9 per cent; P < 0.001) and KRAS (40.0 versus 24.2 per cent; P = 0.006) mutations, and a hereditary cancer syndrome (30.0 versus 5.0 per cent; P < 0.001; relative risk 6). Five-year disease-free survival rates in the MSI group were 95.0, 92.0, and 80.0 per cent for patients with stage I, II, and III tumours, compared with 88.0, 88.0, and 65.0 per cent in the MSS group (P = 0.753, P = 0.487, and P = 0.105 respectively). Patients with early-onset colonic cancer have a high risk of MSI and defined genetic conditions. Those with MSI tumours have more adverse pathology (budding, KRAS/BRAF mutations, and nodal metastases) than older patients with MSI cancers.

Immunopeptidomic Analyses of Colorectal Cancers With and Without Microsatellite Instability

Colorectal cancer is the second leading cause of cancer death worldwide, and the incidence of this disease is expected to increase as global socioeconomic changes occur. Immune checkpoint inhibition therapy is effective in treating a minority of colorectal cancer tumors; however, microsatellite stable tumors do not respond well to this treatment. Emerging cancer immunotherapeutic strategies aim to activate a cytotoxic T cell response against tumor-specific antigens, presented exclusively at the cell surface of cancer cells. These antigens are rare and are most effectively identified with a mass spectrometry–based approach, which allows the direct sampling and sequencing of these peptides. Although the few tumor-specific antigens identified to date are derived from coding regions of the genome, recent findings indicate that a large proportion of tumor-specific antigens originate from allegedly noncoding regions. Here, we employed a novel proteogenomic approach to identify tumor antigens in a collection of colorectal cancer–derived cell lines and biopsy samples consisting of matched tumor and normal adjacent tissue. The generation of personalized cancer databases paired with mass spectrometry analyses permitted the identification of more than 30,000 unique MHC I–associated peptides. We identified 19 tumor-specific antigens in both microsatellite stable and unstable tumors, over two-thirds of which were derived from noncoding regions. Many of these peptides were derived from source genes known to be involved in colorectal cancer progression, suggesting that antigens from these genes could have therapeutic potential in a wide range of tumors. These findings could benefit the development of T cell–based vaccines, in which T cells are primed against these antigens to target and eradicate tumors. Such a vaccine could be used in tandem with existing immune checkpoint inhibition therapies, to bridge the gap in treatment efficacy across subtypes of colorectal cancer with varying prognoses. Data are available via ProteomeXchange with identifier PXD028309.

Synergistic antitumor activity of 5-fluorouracil and atosiban against microsatellite stable colorectal cancer through restoring GATA3

Clinically, 5-fluorouracil (5-Fu) is a first-line drug for the treatment of patients with colorectal cancer (CRC). However, chemoresistance to 5-Fu-based chemotherapy is a leading obstacle in achieving effective treatment for CRC, especially microsatellite stable (MSS) CRC. Since the cytotoxicity of 5-Fu is negatively correlated with oxytocin receptor (OXTR) expression in MSS CRC cell lines, our current study aimed to investigate the synergistic antitumor activity of 5-Fu combined with atosiban, an antagonist of OXTR. Our results suggested that atosiban remarkably potentiated the inhibitory effect of 5-Fu on the growth of MSS-type CRC cells in vitro and in vivo. Moreover, 5-Fu induced GATA3 in MSS CRC cells and tumors, which were eradicated by atosiban. Further investigation showed that atosiban strengthened the antitumor activity of 5-Fu through eradiation of 5-Fu-induced GATA3 in MSS-type CRC cells. Taken together, our findings suggest that atosiban potentiates the antitumor effect of 5-Fu by abolishing 5-Fu-induced GATA3, which provides a novel therapeutic strategy for MSS-type CRC via the combination of atosiban and 5-Fu.

Transfer learning based classification of MSI and MSS gastrointestinal cancer

Gastrointestinal and Colorectal cancers are treated with chemotherapy and its other forms which are not able to provide higher survival rates [1]. Immunotherapy is increasingly becoming popular due to its promising response especially to mutated tumors such as MicroSatellite Instability (MSI) cancers with deficient DNA Mismatch-Repair system (dMMR). Generally, 85% of all the cases related to gastrointestinal and colorectal cancers have proficient DNA Mismatch-Repair system (pMMR) which are also called MicroSatellite Stability (MSS). Only about 15% of the gastrointestinal and colorectal cancer patients have deficient DNA Mismatch-Repair system (dMMR) causing MicroSatellite Instability (MSI) in their tumors. While Immunotherapy responds well to patients with MSI tumors, it is resistant to MSS tumors [2]. Hence, it’s important to classify MSI vs. MSS tumors so that appropriate treatment can be given to the patients. Clinically MSI cancers are difficult to be detected after stage III due to their sensitivity to pembrolizumab inhibitors [3] [4]. In this work, deep learning based transfer learning approach is detailed that can accurately classify MSI vs. MSS cancers using histological images which are derived from formalin-fixed paraffin-embedded (FFPE).

Associations of HER2 Mutation With Immune-Related Features and Immunotherapy Outcomes in Solid Tumors.

BackgroundHER2 is one of the most extensively studied oncogenes in solid tumors. However, the association between tumor microenvironment (TME) and HER2 mutation remains elusive, and there are no specific therapies for HER2-mutated tumors. Immune checkpoint inhibitors (ICIs) have been approved for some tumor subgroups that lack targeted therapies, while their effects are still unclear in HER2-mutated tumors. We examined whether HER2 mutation impacts treatment outcomes of ICIs in solid tumors via its association with anticancer immunity.MethodsMulti-omics data of solid tumors from The Cancer Genome Atlas (TCGA), the Asian Cancer Research Group and the Affiliated Hospital of Jiangsu University were used to analyze the association between HER2 mutations and tumor features. Data of patients with multiple microsatellite-stable solid tumors, who were treated by ICIs including antibodies against programmed cell death-1 (PD-1), programmed cell death ligand-1 (PD-L1), or cytotoxic T lymphocyte-associated protein 4 (CTLA-4) in eight studies, were collected to investigate the effects of HER2 mutations on immunotherapy outcomes.ResultsThe mutation rate of HER2 varied in solid tumors of TCGA, with an overall incidence of 3.13%, ranged from 0.39% to 12.2%. Concurrent HER2 mutations and amplifications were rare (0.26%). HER2 mutation was not associated with HER2 protein expression but was positively associated with microsatellite instability, tumor mutation and neoantigen burdens, infiltrating antitumor immune cells, and signal activities of antitumor immunity. Of 321 ICI-treated patients, 18 carried HER2 mutations (5.6%) and showed improved objective response rates compared with those with HER2 wild-type (44.4% vs. 25.7%, p=0.081), especially in the anti-PD-1/anti-PD-L1 subgroup (62.5% vs. 28.4%, p=0.04). Heterogeneity was observed among tumor types. Patients with HER2 mutations also had superior overall survival than those with HER2 wild-type (HR=0.47, 95%CI: 0.23-0.97, p=0.04), especially in the presence of co-mutations in ABCA1 (HR = 0.23, 95% CI: 0.07-0.73, p=0.013), CELSR1 (HR = 0.24, 95% CI: 0.08-0.77, p=0.016), LRP2 (HR = 0.24, 95% CI: 0.07-0.74, p=0.014), or PKHD1L1 (HR = 0.2, 95% CI: 0.05-0.8, p=0.023).ConclusionsHER2 mutations may improve the TME to favor immunotherapy. A prospective basket trial is needed to further investigate the impacts of HER2 mutations on immunotherapy outcomes in solid tumors.

Assessment of Capecitabine and Bevacizumab With or Without Atezolizumab for the Treatment of Refractory Metastatic Colorectal Cancer

Cotargeting vascular endothelial growth factor and programmed cell death 1 or programmed cell death ligand 1 may produce anticancer activity in refractory metastatic colorectal cancer (mCRC). The clinical benefit of atezolizumab combined with chemotherapy and bevacizumab remains unclear for the treatment of mCRC. To assess whether the addition of atezolizumab to capecitabine and bevacizumab therapy improves progression-free survival (PFS) among patients with refractory mCRC and to perform exploratory analyses among patients with microsatellite-stable (MSS) disease and liver metastasis. This double-blind phase 2 randomized clinical trial enrolled 133 patients between September 25, 2017, and June 28, 2018 (median duration of follow-up for PFS, 20.9 months), with data cutoff on May 4, 2020. The study was conducted at multiple centers through the Academic and Community Cancer Research United network. Adult patients with mCRC who experienced disease progression while receiving fluoropyrimidine, oxaliplatin, irinotecan, bevacizumab, and anti-epidermal growth factor receptor antibody therapy (if the patient had a RAS wild-type tumor) were included. Patients were randomized (2:1) to receive capecitabine (850 or 1000 mg/m2) twice daily on days 1 to 14 and bevacizumab (7.5 mg/kg) on day 1 plus either atezolizumab (1200 mg; investigational group) or placebo (placebo group) on day 1 of each 21-day cycle. The primary end point was PFS; 110 events were required to detect a hazard ratio (HR) of 0.65 with 80% power (1-sided α = .10). Secondary end points were objective response rate, overall survival (OS), and toxic effects. Of 133 randomized patients, 128 individuals (median age, 58.0 years [IQR, 51.0-65.0 years]; 77 men [60.2%]) were assessed for efficacy (82 in the investigational group and 46 in the placebo group). Overall, 15 patients (11.7%) self-identified as African American or Black, 8 (6.3%) as Asian, 1 (0.8%) as Pacific Islander, 101 (78.9%) as White, 1 (0.8%) as multiple races (Asian, Native Hawaiian/Pacific Islander, and White), and 2 (1.6%) as unknown race or unsure of race. Microsatellite-stable disease was present in 110 patients (69 in the investigational group and 41 in the placebo group). Median PFS was 4.4 months (95% CI, 4.1-6.4 months) in the investigational group and 3.6 months (95% CI, 2.2-6.2 months) in the placebo group (1-sided log-rank P = .07, a statistically significant result; HR, 0.75; 95% CI, 0.52-1.09). Among patients with MSS and proficient mismatch repair, the HR for PFS was 0.66 (95% CI, 0.44-0.99). The most common grade 3 or higher treatment-related adverse events in the investigational vs placebo groups were hypertension (6 patients [7.0%] vs 2 patients [4.3%]), diarrhea (6 patients [7.0%] vs 2 patients [4.3%]), and hand-foot syndrome (6 patients [7.0%] vs 2 patients [4.3%]). One treatment-related death occurred in the investigational group. In the investigational group, the response rate was higher among patients without liver metastasis (3 of 13 individuals [23.1%]) vs with liver metastasis (4 of 69 individuals [5.8%]). The benefit of atezolizumab for PFS and OS was greater among patients without vs with liver metastasis (primary analysis of PFS: HR, 0.63 [95% CI, 0.27-1.47] vs 0.77 [95% CI, 0.51-1.17]; OS: HR, 0.33 [95% CI, 0.11-1.02] vs 1.14 [95% CI, 0.72-1.81]). In this randomized clinical trial, the addition of atezolizumab to capecitabine and bevacizumab therapy provided limited (ie, not clinically meaningful) clinical benefit. Patients with MSS and proficient mismatch repair tumors and those without liver metastasis benefited more from dual inhibition of the vascular endothelial growth factor and programmed cell death 1 or programmed cell death ligand 1 pathways. ClinicalTrials.gov Identifier: NCT02873195.

Expression of CD10 and CD15 in colorectal mucinous and signet ring adenocarcinomas and its relation to clinicopathological features and prognosis.

CD10 and CD15 expression has been reported in several tumors. Whether CD10 and CD15 have a role in colorectal mucinous and signet ring adenocarcinoma (MSA) tumorigenesis is not yet known. We aimed to investigate the role of CD10 and CD15 expression in mucinous colorectal adenoma-carcinoma sequence (ACS) and determine if there is any clinical and prognostic significance associated with their expression. Seventy-five cases of colorectal MSA, and 9 cases of adenoma samples were collected. Manual TMA blocks were constructed and immunohistochemistry for CD10 and CD15 was done. Compared to adenomas, CD15 expression was significantly higher in MSA (p= 0.002), in contrast to CD10 expression. CD15 positivity was significantly associated with microsatellite stable (MSS) tumors (p= 0.018). The association between CD10 positivity and fungating tumor growth showed marginal significance. Unlike CD10, CD15 positivity showed significant association with overall survival of colorectal MSA patients. CD15 expression seems to have a role in mucinous colorectal ACS, with significant impact on the survival of MSA patients. Further studies are suggested to identify any genetic alterations that may underlie a potential association with disease progression.

Identification and characterization of recurrent neoantigens in upper gastrointestinal (GI) cancers.

246 Background: Neoantigens are short peptides derived from tumor-specific somatic mutations that can bind to HLA molecules and be presented on the cell surface to activate the immune system. Recognition of neoantigens by autologous T cells promotes sensitivity to immune checkpoint blockade of mismatch repair deficient (MMRd)/microsatellite instability high (MSI-H) tumors. Neoantigen-targeted reactivity by autologous T cells has also been reported in microsatellite stable (MSS) tumors. We aimed to comprehensively assess the spectrum of neoantigens in upper GI cancers and identify recurrent immunogenic candidate neoantigens. Methods: 600 tumor specimens including 268 esophageal (EC), 211 gastric (GC), and 121 gastroesophageal junction (GEJ) cancers tested at Caris Life Sciences (Phoenix, AZ) with NextGen Sequencing (NGS) on DNA (whole exome) and RNA (whole transcriptome) were analyzed. MSI status was determined by immunohistochemistry of MMR protein and/or NGS. Immune epitope prediction was performed on translated peptide sequences harboring detected mutations using the NetMHCpan v4.0 method in the Immune Epitope Database, with HLA genotyping performed using arcasHLA. Immune/stromal cell abundance was quantified using the Microenvironment Cell Populations Counter method. Gene expression profiles were analyzed for a transcriptional signature predictive of response to immunotherapy (T cell-inflamed signature, TIS). Results: Overall, the median patient age was 66 years (range 22-90), 72.8% were male, and 33.7% of samples were metastatic site biopsies. MMRd/MSI-H rate was 2.2% in EC, 12.4% in GC, and 2.5% in GEJ. A total of 1172 unique recurrent neoantigens with predicted binding-level affinity for patient specific HLA alleles were identified (317 in EC, 786 in GC, 157 in GEJ), with 442 and 552 neoantigens exclusively associated with MSS and MSI-H tumors, respectively. Across each cancer type, a higher positive TIS score correlated with both immune and stromal cell population abundance in the tumor microenvironment, most notably cytotoxic lymphocyte and monocytic cell types (r &gt; 0.80, P &lt; 0.0001). Recurrent peptides associated with highest average TIS scores resulted from mutation of TP53 (R248W, 2 peptides: 2.8%/3.2% of samples, respectively) and CUX1 (L162F, 1.6%) in MSS EC; REL (D318A, 2.3%) and ERBB2 (V842I, 1.2%) in MSS GC; MSH3 (K383fs, 36%) and ASXL1 (G645fs, 24%) in MMRd/MSI-H GC; and HOXD12 (A70-A71dup , 2.8%) and TP53 (R248Q, 3.7%) in MSS GEJ. Conclusions: This is one of the largest studies to investigate the landscape of recurrent neoantigens in upper GI cancers. We were able to identify candidate recurrent peptides with high HLA binding affinity and an association with a positive TIS signature in both MSI and MSS tumors, supporting the role of recurrent neoantigens as potential cancer immunotherapy targets.

Transcriptomic profiling to identify subsets of immune hot locally advanced rectal adenocarcinomas with favorable outcomes after neoadjuvant treatment.

155 Background: Understanding the role of the tumor microenvironment in the response to chemotherapy and radiation in patients with locally advanced rectal cancer (LARC, stage II-III) can lead to the identification of novel immunologic biomarkers to preselect patients who can avoid surgery and benefit from watch-and-wait strategies. Methods: We performed DNA and RNA sequencing of pre-treatment biopsies from 89 LARC patients who received neoadjuvant therapy, including 5 microsatellite unstable (MSI) and 84 microsatellite stable (MSS) patients. We computed single-sample gene set enrichment analysis (ssGSEA) scores for immune infiltrates and signaling pathways implicated in tumor progression. Immunofluorescence and hematoxylin-eosin staining of tumor slides was performed to confirm significant correlations with RNA-Seq estimates of immune markers. Other genomic variables were also included in the analysis, such as tumor mutational burden (TMB), fraction of genome altered by copy number changes, whole genome duplication events and somatic mutations in rectal cancer driver genes and pathways. Results were largely replicated using an independent cohort of 42 LARC samples with publicly available data from The Cancer Genome Atlas (TCGA). Results: Since MSI tumors are known to have a distinct immunologic profile, we separated them into their own group and performed unsupervised hierarchical clustering on the MSS tumors. We identified a set of immune hot MSS tumors (n = 7) with extensive immune infiltration. These tumors had low TMB and were predominantly classified as CMS4 (5/7). None of the 12 patients in the combined MSI and immune hot MSS groups recurred during the length of our study and they had response rates &gt; 50% (vs. &lt; 25% in the rest of MSS patients). MSI and immune hot MSS tumors had lower frequency of TP53 and APC mutations, and they exhibited increased levels of T cell infiltration. In particular, we observed overexpression of markers for Th1 cells, which produce inflammatory cytokines (e.g., IFN-gamma) and are associated with antitumor immunity. Genes encoding protein targets of immune checkpoint blockade, such as PDCD1 (PD-1), CD274 (PD-L1), CTLA4, HAVCR2 (TIM3) and LAG3, were also overexpressed in the immune hot MSS and - to a lesser extent – the MSI tumors, suggesting that these patients might benefit from the use of immune checkpoint inhibitors. Conclusions: Our results uncover a unique LARC tumor immune profile evident in the pre-treatment setting that could be used to better prognosticate rectal cancer patients and develop novel therapeutic strategies.

GOBLET: A phase 1/2 multiple-indication biomarker, safety, and efficacy study in advanced or metastatic gastrointestinal cancers exploring treatment combinations with pelareorep and atezolizumab.

TPS216 Background: Checkpoint blockade therapy only benefits a small subset of GI cancer patients (approximately 4%) with microsatellite instability-high (MSI-H) tumors, which are characterized as immunologically ‘hot’ (Bonneville et al., 2017). Most GI cancers, however, have microsatellite stable (MSS) tumors, which have an immunologically “cold” phenotype with fewer genetic mutations, reduced immune cell infiltration, and downregulated immune checkpoint proteins. These attributes make MSS tumors resistant to conventional immunotherapy including checkpoint blockade therapy (Ooki et al., 2021). Pelareorep is a naturally occurring, non-genetically modified reovirus. Upon intravenous administration, pelareorep selectively kills tumor cells and promotes several immunologic effects that prime tumors to respond to checkpoint blockade. These include the stimulation of tumor-directed innate and adaptive immune responses, increased T cell infiltration, expansion of new T cell clones, and increased PD-L1 expression in tumors (Samson et al., 2018, Manso et al. 2021 AACR). Given its expected synergy with checkpoint blockade, as well as its encouraging efficacy in prior GI cancer studies (Mahalingam et al. 2020), the GOBLET study is designed to evaluate pelareorep plus atezolizumab in multiple GI cancer indications. Methods: GOBLET is an open-label, non-randomized, multiple-cohort, phase 1/2 study in patients with advanced or metastatic GI cancers. This study employs a Simon two-stage design. Stage 1 comprises four treatment groups: Cohort 1 – First-line pancreatic cancer treated with pelareorep plus atezolizumab and chemotherapy (gemcitabine and nab-paclitaxel) (N = 12); Cohort 2 – First-line MSI-H colorectal cancer (CRC) treated with pelareorep plus atezolizumab (N = 19); Cohort 3 – Third-line CRC treated with pelareorep plus atezolizumab and chemotherapy (trifluridine/tipiracil) (N = 14); and Cohort 4 – Second-line or later squamous cell carcinoma of the anal canal treated with pelareorep plus atezolizumab (N = 10). The first 3-6 patients enrolled into the chemotherapy-containing cohorts (Cohorts 1 and 3) comprise a safety run-in that must be successfully concluded prior to enrolling additional patients into these cohorts. The primary objectives are safety and efficacy based on objective response rate (ORR) at week 16. Any cohort showing a promising ORR in Stage 1, based on pre-specified criteria, may be advanced to Stage 2 and enroll additional patients. Clinical trial information: 2020-003996-16.

Poorly differentiated histology as a predictive biomarker in patients with advanced gastrointestinal (GI) cancers treated with immunotherapy.

664 Background: Checkpoint inhibitors (CPI) have significantly improved survival among patients with various cancer types. Prior studies have showed a correlation between immune cell infiltration and poorly differentiated cancers. We evaluated the impact of poorly differentiated histology on survival in patients with advanced GI malignancies treated with immunotherapy. Methods: We performed a retrospective, single institution analysis of patients with poorly differentiated GI tumors treated with CPI between 2016 and 2021. Univariate (UVA) and multivariable analyses (MVA) were performed to identify factors associated with patient outcome. Kaplan-Meier analysis and Cox proportional hazards models were used to assess the association between tumor/patient characteristics, progression free survival (PFS) and overall survival (OS). Results: A total of 123 patients were included. Median age was 66years. Majority had stage IV disease (88.2%), were White (65.5%) and male (64.5%). Most common diagnoses were HCC (28.5%), gastric (15.4%) and esophageal (14.6%) adenocarcinomas. About 32% were MSI-H, with 10% BRAF mutation rate. About 25% received CPI as initial treatment and 35.5% received 2 or more prior lines of therapy. Compared with well and moderately differentiated histology, poorly differentiated tumors had shorter mPFS (3.4 vs 2.5 vs. 2.2months; p = 0.29) and mOS (not reached [NR] vs. 20.9 vs. 14.2 months; p = 0.14) respectively. On UVA, the degree of tumor differentiation did not correlate with mPFS or mOS. Female patients (HR: 0.55, CI: 0.34-0.90, p = 0.018) and ECOG of 1 vs. ≥2 had significantly longer mPFS (HR: 0.58, CI: 0.35-0.97, p = 0.036). ECOG 1 correlated with better mOS (HR: 0.47, CI: 0.23-0.94, p = 0.034). Microsatellite stable (MSS) tumors had significantly shorter mPFS (HR: 5.74, CI: 2.41-13.63, p &lt; 0.001) and mOS (HR: 5.45, CI: 1.64-18.12, p = 0.006). Number of prior systemic therapies was also associated with shorter mPFS (HR: 1.19, CI: 1.03-1.39, p = 0.022) and mOS (HR: 1.23, CI: 1.01-1.50, p = 0.045). On MVA, ECOG status of 0/1 vs. ≥2 and MSI-H vs. MSS remained significantly associated with longer mPFS and mOS. There was no correlation with race or mutations such as BRAF or KRAS. Conclusions: Poorly differentiated histology did not predict for PFS or OS. Treatment naïve patients, female gender, ECOG 1 and MSI-H were most likely to benefit from CPI.

Tumor board presentation of a woman with metastatic, hormone receptor‐positive, mismatch repair‐deficient endometrial cancer

Tumor board presentation of a woman with metastatic, hormone receptor‐positive, mismatch repair‐deficient endometrial cancer

Abstract P037: Interleukin-10 is a dominant and reversible mechanism of immune evasion in human colorectal cancer liver metastasis

Abstract Background: Colorectal cancer liver metastasis (CRLM) causes major morbidity and mortality. Improved systemic therapies are crucial. We previously reported that macrophages infiltrated CRLM and expressed the immunosuppressive cytokine interleukin-10 (IL-10). In patient-derived CRLM tumor slice cultures, we found that a neutralizing antibody against IL-10 (anti-IL-10) caused tumor apoptosis. Here, we investigated the efficacy and immune-dependent mechanisms of IL-10 blockade in a larger cohort of CRLM patients. Methods: Tumor specimens were obtained from consenting patients at the time of surgery and cut into 250mm-thick tumor slice cultures as previously described. Tumor slices were treated with control or neutralizing antibodies against programmed cell death protein 1 (PD-1, EH12.1), IL-10 (JES3-9D7), IL-10 receptor alpha (IL-10RA, 3F9), major histocompatibility complex (MHC) class I (G46-2.6) or class II (Tu39). After 4-6 days of treatment, tumor apoptosis was evaluated by cleaved caspase-3 (CC3) immunohistochemistry (IHC) or terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). Tumors were evaluated by IHC and in situ hybridization (ISH) for immune markers. Student's t-test, paired t-test, or 1-way ANOVA was used as indicated. p &amp;lt; 0.05 was defined as significant. Results: We generated tumor slice cultures from 34 unique CRLM patients, 76% (26/34) of whom received preoperative chemotherapy, and all of whom had either microsatellite stable tumors (76%, 26/34) or unknown microsatellite status. Anti-PD-1 did not generate tumor apoptosis (n = 4 patients' tumors). In contrast, anti-IL-10 caused nearly 2-fold increased tumor apoptosis compared with control, in the majority of 34 CRLM patients' tumors evaluated (median 50.1% versus 27.4% apoptotic cells, p &amp;lt; 0.0001). IL-10 receptor blockade generated a similar but non-significant increase in apoptosis. Tumors treated with anti-IL-10 demonstrated increased frequency of CD8+ T cells (median 17.8% versus 7.0% of total cells, n = 5 patients' tumors, p = 0.02) and a non-significant increase in activated PD-1+CD3+ T cells. IL-10 blockade also increased tumor IFNG expression (median 2.9 versus 2.3 counts per total cell number, n = 6 patients' tumors, p &amp;lt; 0.05). Macrophage frequency did not increase, but MHC class II expression nearly doubled (median 19.5% versus 11.5% of total cells, n = 5 patients' tumors, p = 0.02). To test which immune cells were required for anti-IL-10 effect, we treated human CRLM slices with anti-IL-10 +/- blocking antibodies against MHC class I or II. In the 3 of 4 patients' tumors that responded to anti-IL-10, the effect was nearly completely reversed by aMHC-I or aMHC-II (p = 0.0005). Conclusion: IL-10 is a dominant and reversible mechanism of immune evasion in human CRLM. IL-10 blockade nearly doubled tumor apoptosis in a heterogeneous cohort of CRLM patients' tumors, through a mechanism that required intact CD8+ T cell and antigen-presenting cell function. IL-10 is a compelling immunotherapeutic target for CRLM. Citation Format: Teresa S. Kim, Kevin Sullivan, Xiuyun Jiang, Cynthia Hsu, Kevin Labadie, Karan Kohli, Heidi Kenerson, Sara Daniel, Arezou Abbasi, Raymond Yeung, Venu Pillarisetty. Interleukin-10 is a dominant and reversible mechanism of immune evasion in human colorectal cancer liver metastasis [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P037.