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PURPOSE Anti–epidermal growth factor receptor (EGFR) antibodies are effective treatments for metastatic colorectal cancer. Improved understanding of acquired resistance mechanisms may facilitate circulating tumor DNA (ctDNA) monitoring, anti-EGFR rechallenge, and combinatorial strategies to delay resistance. METHODS Patients with treatment-refractory metastatic colorectal cancer (n = 169) enrolled on the CO.26 trial had pre–anti-EGFR tissue whole-exome sequencing (WES) compared with baseline and week 8 ctDNA assessments with the GuardantOMNI assay. Acquired alterations were compared between patients with prior anti-EGFR therapy (n = 66) and those without. Anti-EGFR therapy occurred a median of 111 days before ctDNA assessment. RESULTS ctDNA identified 12 genes with increased mutation frequency after anti-EGFR therapy, including EGFR ( P = .0007), KRAS ( P = .0017), LRP1B ( P = .0046), ZNF217 ( P = .0086), MAP2K1 ( P = .018), PIK3CG ( P = .018), BRAF ( P = .048), and NRAS ( P = .048). Acquired mutations appeared as multiple concurrent subclonal alterations, with most showing decay over time. Significant increases in copy-gain frequency were noted in 29 genes after anti-EGFR exposure, with notable alterations including EGFR ( P < .0001), SMO ( P < .0001), BRAF ( P < .0001), MET ( P = .0002), FLT3 ( P = .0002), NOTCH4 ( P = .0006), ERBB2 ( P = .004), and FGFR1 ( P = .006). Copy gains appeared stable without decay 8 weeks later. There were 13 gene fusions noted among 11 patients, all but one of which was associated with prior anti-EGFR therapy. Polyclonal resistance was common with acquisition of ≥ 10 resistance related alterations noted in 21% of patients with previous anti-EGFR therapy compared with 5% in those without ( P = .010). Although tumor mutation burden (TMB) did not differ pretreatment ( P = .63), anti-EGFR exposure increased TMB ( P = .028), whereas lack of anti-EGFR exposure resulted in declining TMB ( P = .014). CONCLUSION Paired tissue and ctDNA sequencing identified multiple novel mutations, copy gains, and fusions associated with anti-EGFR therapy that frequently co-occur as subclonal alterations in the same patient.

PURPOSE High pathologic complete response (pCR) rates and comparably good survival data were seen in a phase II trial combining perioperative fluorouracil, leucovorin, oxaliplatin, and docetaxel (FLOT) chemotherapy with trastuzumab for resectable, esophagogastric adenocarcinoma (EGA). The current trial evaluates the addition of trastuzumab and pertuzumab to FLOT as perioperative treatment for human epidermal growth factor receptor 2–positive resectable EGA. METHODS In this multicenter, randomized phase II/III trial, patients with human epidermal growth factor receptor 2–positive, resectable EGA (≥ clinical tumor 2 or clinical nodal–positive) were assigned to four pre- and postoperative cycles of either FLOT alone (arm A) or combined with trastuzumab and pertuzumab, followed by nine cycles of trastuzumab/pertuzumab (arm B). The primary end point for the phase II part was the rate of pCR. RESULTS The trial was closed prematurely, without transition into phase III, after results of the JACOB trial were reported. Eighty-one patients were randomly assigned (A: 41/B: 40) during the phase II part. The pCR rate was significantly improved with the trastuzumab/pertuzumab treatment (A: 12%/B: 35%; P = .02). Similarly, the rate of pathologic lymph node negativity was higher with trastuzumab/pertuzumab (A: 39%/B: 68%), whereas the R0 resection rate (A: 90%/B: 93%) and surgical morbidity (A: 43%/B: 44%) were comparable. Moreover, the inhouse mortality was equal in both arms (overall 2.5%). The median disease-free survival was 26 months in arm A and not yet reached in arm B (hazard ratio, 0.58; P = .14). After a median follow-up of 22 months, the median overall survival was not yet reached (hazard ratio, 0.56; P = .24). Disease-free survival and overall survival rates at 24 months were 54% (95% CI, 38 to 71) and 77% (95% CI, 63 to 90) in arm A and 70% (95% CI, 55 to 85) and 84% (95% CI, 72 to 96) in arm B, respectively. More ≥ grade 3 adverse events were reported with trastuzumab/pertuzumab, especially diarrhea (A: 5%/B: 41%) and leukopenia (A: 13%/B: 23%). CONCLUSION The addition of trastuzumab/pertuzumab to perioperative FLOT significantly improved pCR and nodal negativity rates at the price of higher rates of diarrhea and leukopenia.

180 Background: CCTG CO.28 evaluated 3 months of neoadjuvant CAPOX or FOLFOX followed by transanal excision surgery (TES) in patients (pts) with T1-T3, N0 rectal cancers with the goal of organ preservation. Total mesorectal excision (TME) was recommended for inadequate downstaging or high-risk features on TES pathology. Detection of ctDNA is associated with a high rate of recurrence in high risk resected colorectal cancer. However, the sensitivity of ctDNA in early-stage rectal cancer is unclear. Methods: Tissue CGP was performed retrospectively on pretreatment resected tumor using Foundation OneCDx (F1CDx), followed by MRD detection using FoundationOneTracker. Briefly, variants were selected from F1CDx using an algorithm that filters out non-tumor derived variants (e.g. germline). A tumor-informed personalized multiplex PCR-next generation sequencing assay, consisting of up to 16 variants, was used to detect ctDNA and quantify plasma mean variant allelic frequency (VAF) and mean tumor molecules per mL of plasma (MTM/mL). FoundationOneTracker was performed on blood samples collected pre/post-neoadjuvant chemotherapy. Buffy coat sequencing was performed at FMI to validate the monitoring variant selection algorithm. Results: 52 pts underwent F1CDx and FoundationOneTracker analysis. KRAS mutations were observed in 60% (n =31) of cases; MSI-H and BRAF V600E mutations were not detected. From 1,580 distinct variants detected in tissue and paired buffy coat sequencing, 985 had VAF >30% in the buffy coat specimen and were considered germline. The variant selection algorithm filtered 99.8% (n = 983) of germline variants; the remaining 2 were not selected for primer design. A median of 7 variants (range 2-16) were tracked per pt. Detectable ctDNA was found in 0% (0/6), 19% (6/32) and 55% (6/11) of treatment naïve T1, T2 and T3ab cases, respectively, and 83% of those which were initially positive (10/12) cleared ctDNA post chemotherapy. Three pts had detectable ctDNA pre-excision, including 1 who was negative pretreatment. From 42 pts with pre/post-treatment plasma, 19 pts were recommended for TME; 2 had detectable ctDNA, 5 had ctDNA clearance, and 12 had no ctDNA detection. Conclusions: Node-negative rectal cancer exhibited ctDNA shed in a minority of pts, with most clearing ctDNA on chemotherapy before their TES. The dynamic ctDNA signal we demonstrate could supplement clinical factors in informing organ preservation in very early-stage rectal cancer. Larger studies are needed to determine if this information can be used to personalize therapy. Clinical trial information: NCT03259035 . [Table: see text]

183 Background: In response to energetic stress, colorectal cancer cells secrete creatine kinase brain-type (CKB). CKB converts creatine and ATP from the extracellular matrix to PC, which is imported intracellularly to sustain survival and metastatic spread. In addition, PC modulates immune cell functions and may play a role in mediating responses to immune checkpoint inhibition. CO.26 was a phase II trial (NCT02870920) that randomized patients (pts) with refractory mCRC to durvalumab plus tremelimumab (D+T) versus best supportive care (BSC). In an exploratory, post-hoc analysis, we investigated the role of plasma PC as a predictive biomarker for response to D+T. Methods: PC concentrations were determined from pre-treatment blood samples with HPLC-tandem mass spectrometer. A minimum p-value approach was used to select an optimum cut-off value which dichotomized patients into low (< 95.6 ng/ml) versus high (≥ 95.6 ng/ml) groups predictive for benefit. Cox proportional hazard models were used to analyze predictive impacts of PC on progression free survival (PFS) and overall survival (OS). Results: Of 180 pts enrolled, pre-treatment blood samples were available for 162 pts (N =115 for D+T; 47 BSC). Pre-treatment PC was low in 15% (N=24) and high in 85% (N=138). There were no differences in baseline characteristics between pts included in this analysis and the total study pts, or PC low and high pts. D+T improved OS significantly in PC low pts (median OS 4.7 months vs 2.3 months; Hazard Ratio (HR) 0.32, 95% confidence interval (CI): 0.11 – 0.95, p = 0.03). There was no improvement in PC high pts with D+T (median OS 6.8 vs 5.2 months; HR 0.80, 95% CI: 0.55 – 1.17, p = 0.24. Interaction p < 0.0001). Plasma PC values had no impact on PFS and rates of disease control. Conclusions: Pts with low plasma PC derived more benefit from immune checkpoint inhibition with D+T in pts with refractory mCRC. Further prospective validation studies are needed. Predictive analysis for OS with pre-treatment PC levels dichotomized by minimum p approach. [Table: see text]

3600 Background: Immune checkpoint blockade has limited activity in microsatellite-stable (MSS) or mis-match repair proficient (pMMR) CRC. Recent findings suggest that immunotherapy efficacy may be modulated by the presence of liver metastases. We conducted a retrospective analysis of the Canadian Cancer Trials Group (CCTG) CO.26 study to investigate the relationship between the presence of liver metastases and activity of immune checkpoint blockade. Methods: The CCTG CO.26 study was a randomized phase II study (NCT02870920). Pts with treatment refractory CRC were randomized to durvalumab, tremelimumab and best supportive care (BSC) or BSC alone in a 2:1 fashion. Treatment consisted of durvalumab (1500 mg) q 28 days and tremelimumab (75 mg) q 28 days for the first 4 cycles. The primary endpoint was overall survival (OS) and a two-sided p-value

3551 Background: CO.26 was a phase 2 trial (2-sided α=0.1 and 80% power) that randomized 180 patients with refractory mCRC 2:1 to durvalumab + tremelimumab vs BSC with improved overall survival (OS) (HR 0.73, 90%CI 0.55-0.97, P=0.07). A Nanostring assay validated for use with FFPE was used to determine CMS for correlation with outcome. Methods: Archival FFPE from 163/180 (91%) of patients (pts) underwent RNA extraction and CMS subtyping. Cox proportional hazard models evaluated the prognostic and predictive impact of CMS on overall survival. Results: CMS distribution was skewed towards CMS4 (76%), with lower prevalence of CMS1 (2%), CMS2 (16%) and CMS3 (2%). There were 7/163 cases of indeterminate CMS (4%). Subgroup analysis was restricted to CMS2 and CMS4 based on sample size. With BSC alone, CMS2 showed trends to worse OS compared to all other patients pooled (HR 1.93, 90% CI 1.03-3.61, P=0.085), while CMS4 did not (HR 0.86, 90% CI 0.50-1.48, P=0.64). OS but not progression free survival (PFS) was improved with durvalumab + tremelimumab in the overall population. OS was improved with durvalumab + tremelimumab among patients with CMS2 tumors (HR 0.39, 90% CI 0.19-0.82, P=0.035) but not in patients with CMS4 tumors (HR 0.73, 90% CI 0.52-1.02, P=0.12) compared to BSC. Neither CMS2 (P-interaction=0.37) nor CMS4 (P-interaction=0.91) were predictive of OS benefit from durvalumab + tremelimumab compared to BSC. Disease control rate (DCR) trended to being better among CMS4 (24/85) than CMS2 cancers (1/15, OR 5.51, 90% CI 1.10-29.88, P=0.11) or CMS4 vs all non CMS4 cancers (1/21, OR 7.87, 90% CI 1.65-41.98, P=0.023) for patients on durvalumab + tremelimumab. PFS was not improved with durvalumab + tremelimumab in CMS2 (P=0.19) or CMS4 (P=0.29) cancers relative to BSC. Conclusions: In this trial of refractory colorectal cancer, we saw a shift in CMS subtype with more CMS4 than expected. Compared to CMS4, CMS2 showed stronger signals towards improved OS with durvalumab + tremelimumab but had a lower disease control rate. Differences in immune signaling by CMS may be important determinants of which component of immune regulation needs to be targeted in mCRC to improve outcomes. Clinical trial information: NCT02870920. [Table: see text]

2024 Background: Patients with RET fusion-positive NSCLC have an estimated 25% incidence of CNS metastasis at diagnosis, and up to 40% during disease progression. Effective anti-RET therapy that penetrates the blood-brain barrier is essential to extending survival. TAS0953/HM06 is a structurally distinct RET-specific inhibitor that exhibits a distinct binding mode to RET and is effective against RET solvent front (G810) and gatekeeper (V804) mutations. TAS0953/HM06 also inhibits growth of xenograft tumors established from RET fusion-driven tumors of multiple histologies. TAS0953/HM06, therefore, represents a potentially effective strategy to overcome the emergence of acquired resistance to first generation RET-selective inhibitors. Here, we compared the brain penetration and efficacy of TAS0953/HM06 to selpercatinib (FDA-approved RET inhibitor) in models of intracranial RET fusion-positive cancers, specifically NSCLC and sarcoma. Methods: We compared the brain: plasma ratio of unbound TAS0953/HM06 and selpercatinib in mice to determine the unbound partition coefficient, Kpuu, brain. We injected ECLC5 (NSCLC cell line, TRIM33-RET) and HMSC-RET (immortalized human mesenchymal stem cells in which SPECCL1-RET was introduced by CRISPR-Cas9 genomic engineering, sarcoma model) cells expressing luciferase into the cerebellum of mice. Tumor-bearing mice were treated with TAS0953/HM06 (50 mg/kg BID), selpercatinib (10 mg/kg BID) or vandetanib (multi-kinase RET inhibitor, 50 mg/kg QD), and assessed weekly for tumor growth via bioluminescence imaging. Results: Kpuu, brain, of TAS0953/HM06 and selpercatinib were 1.3 and 0.20, respectively. Substances with brain Kpuu > 0.3 in mice are regarded as brain-penetrable. TAS0953/HM06 was superior to selpercatinib at inhibiting growth of ECLC5 (p < 0.0001) and HMSC-RET (p = 0.0005) brain xenograft tumors, and increasing survival of tumor-bearing animals (ECLC5: TAS0953/HM06 139±0.5 days, selpercatinib 95+2.3 days, p = 0.002; HMSC-RET: TAS0953/HM06 41± 2.2 days, selpercatinib 20±3 days, p = 0.0001). Vandetanib, which is highly brain-penetrant, did not cause a significant decrease in growth of either brain tumor xenograft models. At the doses used, the 3 RET inhibitors induced similar regression in several peripheral subcutaneous xenograft tumor models. Conclusions: Our data in animal models suggest that TAS0953/HM06 penetrates the CNS more effectively than selpercatinib, and is superior at decreasing CNS disease and extending survival. TAS0953/HM06 represents a promising new therapeutic option for patients with RET fusions with acquired resistance mutations, including those with brain metastasis and those resistant to first-generation selective RET inhibitors. TAS0953/HM06 is currently undergoing a biomarker-driven phase 1/ 2 clinical trial for patients with solid tumors driven by RET alterations (NCT04683250).

343 Background: Esophageal adenocarcinoma (EAC) continues to have extremely poor prognosis despite advances in surgical and oncological regimens. Targeted and immune therapy hold promise to improve outcomes in solid tumors including EAC. At present HER-2 and anti-PD-L1 therapies are approved by the FDA for EAC. Additionally, off-label use of EGFR directed therapy has been reported. The aim of this study is to measure expression of 80+ oncoproteins in EAC using multiplexed mass spectrometry to elucidate expression of currently approved-drug targets and explore promising new targets for drug development. Methods: After Institutional Review Board approval (Mayo Clinic and KUMC), EAC patients whose tumors were resected via esophagectomy before chemotherapy and/or radiation were identified. Normal esophageal mucosa samples with no history of EAC were also retrieved for analysis. Tissue sections were microdissected to exclude stroma from adenocarcinoma cells. Isolated EAC cells were solubilized for mass spectrometry-based quantitation of more than 80 clinically relevant tumor markers. Results: 55 EAC tissues were analyzed. HER2, PD-L1 and EGFR were overexpressed only in 16.3%, 0%, and 0% of patients, respectively. New oncoproteins #2 and #6 were noted to have high (7,634.0 and 27,222.6 attomoles/ug) and consistent (98.1% and 94.5%) overexpression in EAC samples. Expression of HER2, oncoproteins #2 and #6 were significantly higher in EAC compared to normal esophageal mucosa. There was no statistical difference in the expression of PD-L1 and EGFR between EAC and normal samples. Conclusions: Mass spectrometry revealed a very low prevalence of target oncoproteins in EAC for currently used drugs. Our study potentially reveals alternate markers which are near universally present at high levels and should be further explored for targeted treatment.[Table: see text]

249 Background: Since 1975, Esophageal adenocarcinoma (EAC) incidence has increased more than any other cancer (̃700%). Pre-malignant metaplastic tissue, or Barrett's Esophagus (BE), is the only known precursor for EAC. Periodic histological assessment of endoscopic biopsy along dysplasia-carcinoma sequence is used to assess progression risk. Current surveillance protocols have clinical limitations as 80% of patients still present with stage II-IV EAC. The aim of this study is to compare novel proteomic expression patterns to distinguish stable BE patients from those who developed EAC using the STLA101 assay. Methods: After Institutional Review Board approval patients with BE/GERD were identified. Two cohorts were selected: progressors (progressed to EAC at subsequent interval of 0.5-3 years) and non–progressors (̃10 yrs of f/u surveillance without progression along dysplasia/carcinoma path). 24 normal esophageal mucosa samples with no h/o of BE were also retrieved for baseline expression levels. Tissue sections were microdissected to isolate pure BE cells from surrounding stromal cells. BE cells underwent a multi-step process where tissue was heated and digested to produce a liquid biopsy for mass spec. Isotopically labeled control peptides representing the STLA101 panel were spiked in with digested tissue samples to quantify all of the markers simultaneously using a Triple-quad mass spectrometer. Results: A total of 95 tissues were analyzed. 29 BE biopsies progressed to EAC within a mean of 380 days while the remaining 28 BE and 14 GERD specimens did not progress over a 10-yr period. All 8 biomarkers were expressed at a higher level in BE tissue that progressed to cancer when compared to BE/GERD tissue that remained stable over 10 years. The assay had a 100% technical success rate as no samples yielded “non-detectable” data points. Conclusions: The differential overexpression of the STLA101 panel on progressive and non-progressive BE tissues demonstrates the clinical utility of STLA101 as a predictive biomarker panel for early detection of esophageal carcinogenesis. Non-progressive BE tissues were similar to normal mucosa, therefore, patients whose BE tissue samples resemble this profile may receive a reduced screening cadence. On the other hand, patients with overexpression of the STLA101 panel may be eligible to receive more frequent screening or therapeutic intervention.

185 Background: The FOLFIRI regimen (5-fluorouracil, leucovorin and irinotecan) is a predominant treatment regimen for metastatic colorectal cancer. To optimize the benefit for the patients, it would be desirable to increase the efficacy of irinotecan by increasing the exposure of SN-38 (the active metabolite of irinotecan) to the cancer cells, while maintaining a good safety profile. The oral drug SCO-101, that is currently being developed by Scandion Oncology A/S, was tested in an early clinical trial (CORIST-trial, ClinicalTrials.gov identifier: NCT04247256). SCO-101 is an inhibitor of ABCG2, UGT1A1 and SRPK1. The CORIST trial was set up to address the safety, tolerability, and efficacy of SCO-101 given orally for 6 days followed by FOLFIRI at varying doses from day 5 to 7, in a biweekly schedule, in patients with metastatic colorectal cancer who have formerly been treated with FOLFIRI and afterwards progressed. The first part of the study was a dose-finding study, where the impact of SCO-101 on the pharmacokinetics (PK) of SN-38 was studied. Methods: 12 patients from the dose-finding part of the CORIST study received 150 mg SCO-101 for 6 days and 45 – 80% of the recommended dose of FOLFIRI. 6 of the patients had RAS wild-type tumors and these patients are the subject of the current analysis. Blood for PK analysis was sampled from the patients at 1, 2, 4, 8, 24, 48, and 96 hours after treatment with FOLFIRI and SCO-101. The blood samples were analyzed for Cmax, T½ and AUC (0-24h) of SCO-101, irinotecan and SN-38. Results: The PK of SN-38 from the patients in the study, normalized to a dose of irinotecan of 90 mg/m2 showed a T½ day 5 of 19 hours (SD 5,7) a Cmax of 60 ng/ml (SD 20,6) and an AUC_0-24h of 1415 hxng/ml (SD: 670). The results were compared to SN-38 data from treatment with irinotecan at 180 mg/m2, which is used in standard doses of FOLFIRI and the data is presented in the table. The data analysis showed an increased T½, increased Cmax and increased AUC of SN-38 when combining SCO-101 with 90 mg/m2 irinotecan, compared to SN-38 PK data from standard irinotecan treatment of 180 mg/m2. The toxicity profile of the patients treated with 90 mg irinotecan/m2 (50% FOLFIRI) showed only grade 1 and 2 adverse events. Conclusions: SCO-101 in combination with FOLFIRI has demonstrated the ability to modulate the PK profile of SN-38 in mCRC patients with RAS wild-type tumors, by significantly increasing the half-life, the peak plasma concentration, and area under the curve of SN-38. The combined treatment was well tolerated, and the drug is now being tested for efficacy in the CORIST trial.[Table: see text]

Purpose To compare pre-agreed health-related quality of life (HRQOL) domains in patients with esophageal or junctional cancer who received neoadjuvant chemoradiotherapy (nCRT) followed by surgery or surgery alone. Secondary aims were to examine the effect of nCRT on HRQOL before surgery and the effect of surgery on HRQOL. Patients and Methods Patients were randomly assigned to nCRT (carboplatin plus paclitaxel with concurrent 41.4-Gy radiotherapy) followed by surgery or surgery alone. HRQOL was measured using the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire–Core 30 (QLQ-C30) and –Oesophageal Cancer Module (QLQ-OES24) questionnaires pretreatment and at 3, 6, 9, and 12 months postoperatively. The nCRT group also received preoperative questionnaires. Physical functioning (PF; QLQ-C30) and eating problems (EA; QLQ-OES24) were chosen as predefined primary end points. Predefined secondary end points were global QOL (GQOL; QLQ-C30), fatigue (FA; QLQ-C30), and emotional problems (EM; QLQ-OES24). Results A total of 363 patients were analyzed. No statistically significant differences in postoperative HRQOL were found between treatment groups. In the nCRT group, PF, EA, GQOL, FA, and EM scores deteriorated 1 week after nCRT (Cohen’s d: −0.93, P < .001; 0.47, P < .001; −0.84, P < .001; 1.45, P < .001; and 0.32, P = .001, respectively). In both treatment groups, all end points declined 3 months postoperatively compared with baseline (Cohen’s d: −1.00, 0.33, −0.47, −0.34, and 0.33, respectively; all P < .001), followed by a continuous gradual improvement. EA, GQOL, and EM were restored to baseline levels during follow-up, whereas PF and FA remained impaired 1 year postoperatively (Cohen’s d: 0.52 and −0.53, respectively; both P < .001). Conclusion Although HRQOL declined during nCRT, no effect of nCRT was apparent on postoperative HRQOL compared with surgery alone. In addition to the improvement in survival, these findings support the view that nCRT according to the Chemoradiotherapy for Esophageal Cancer Followed by Surgery Study–regimen can be regarded as a standard of care.

3508 Background: CO.28 (NCT03259035) is a phase II study designed to determine if patients with cT1-T3a/bN0 rectal cancer can be treated with induction chemotherapy (FOLFOX/CAPOX) and organ-preserving surgery. Methods: Patients with MRI staged cT1-3a/bN0 tumors and no pathologic (p) high risk features received 6/4 cycles of FOLFOX/CAPOX, repeat sigmoidoscopy/pelvic MRI and subsequent Transanal Endoscopic Surgery (TES) in the absence of tumor progression. ypT0/T1N0 tumors were treated with observation while ypT2+ or ypN+ stage were recommended Total Mesorectal Excision (TME). The primary endpoint was protocol specified Organ Preservation Rate (psOPR = ypT0/T1N0, no p high risk features) and actual Organ Preservation Rate (aOPR = ypT0/T1N0 stage plus higher yp stage patients who declined TME surgery). The study would be considered negative with an psOPR of 50% or lower (H0) and as promising if it is 65% or higher (H1). Results: Between 08/2017 to 05/2020, 58 eligible patients were accrued in Canada and the United States, median age was 67 years, 71% male. All had well-moderately differentiated, non-mucinous rectal adenocarcinoma and median tumor height was 6 cm (range 0-18). Median follow-up was 15.4 months. Chemotherapy with FOLFOX (32) or CAPOX (26) was administered, 90% completed all planned cycles. A total of 56/58 (97%) proceeded to TES, while one patient was ineligible due to tumor progression (1.7%) and one declined. In the intention to treat analysis, the psOPR was 57% (95% CI 43-70%) while the aOPR was 79% (95% CI 67% to 89%) due to 13/23 declining recommended TME surgery. Of 10 patients who proceeded to recommended TME, a complete R0 TME was performed in 9/10, and no p residual carcinoma was found in 7/10. Crude loco-regional (LR) and distant recurrence rates were 3.5% (95% CI 0.4 to 12%) and 0%, respectively. A recurrence occurred in 1/13 patients who initially declined TME surgery. Conclusions: In select patients with early stage rectal cancer, three months of induction CAPOX/FOLFOX followed by TES resulted in a high OPR without the use of pelvic irradiation. The observed high rate of pathologic downstaging may point to high chemo-responsiveness in early rectal adenocarcinoma with no p high risk features. Further trials to evaluate this approach are justified and updated results will be presented. Clinical trial information: NCT03259035. [Table: see text]

61 Background: Pembrolizumab was recently granted tissue agnostic FDA accelerated approval for metastatic cancers with TMB≥10 mut/Mb. However, limited data supports immunotherapy in microsatellite stable (MSS) mCRC with TMB≥10 mut/Mb. We assessed tissue TMB and contrasted it to plasma derived TMB in the CO.26 trial. Methods: CO.26 was a phase 2 trial (2-sided ⍺ = 0.1 and 80% power) that randomized 180 patients (pts) 2:1 to D+T or BSC in refractory mCRC. Pre-treatment plasma was sequenced with the GuardantOMNI assay and archival tissue underwent exome sequencing with TMB assessed per the TMB harmonization project. MSI-H cases were excluded. For plasma TMB, we used a previously published cut point (≥28). Results: Overall survival (OS) but not progression free survival (PFS) was improved with D+T in the entire population. Of 180 pts, 163 were evaluable for plasma and 110 for tissue TMB. Median time between archival tissue and plasma collection was 3.1 yrs (IQR 1.9-5.1). Median tissue TMB was 6.6 muts/Mb (IQR 4.1-12.0), while median plasma TMB was 16.3 muts/Mb (IQR 9.4-25.9). Tissue and plasma TMB (r = -0.039, P = 0.69) were not correlated. Tissue TMB≥10 was not prognostic in the BSC arm (HR 1.01, 90%CI 0.52-1.92, P = 0.99) and OS was not improved in pts with tissue TMB≥10 (32/110 pts) following D+T vs BSC. A test of interaction suggested this threshold was not predictive (P = 0.85). Using a minimum P-value approach, no threshold supported high tissue TMB as predictive in MSS mCRC. In fact, the optimal cut point suggested low tissue TMB ( < 4.1 muts/Mb) had the greatest benefit from D+T (P-interaction = 0.048) and pts with TMB ≥4.1 mut/Mb (HR 0.50, 90%CI 0.26-0.96, P = 0.083) trended to better OS in the BSC arm. In contrast, 35/163 pts (21%) were identified in a high plasma TMB group associated with worse OS (HR 2.56, 90%CI 1.45-4.54, P = 0.007) in the BSC arm but improved OS following D+T compared to BSC with P-interaction = 0.082. Only 1 response was noted following D+T in a pt with tissue TMB = 16 mut/Mb and plasma TMB = 13 mut/Mb. Conclusions: Archival tissue TMB≥10 mut/Mb does not appear predictive of D+T benefit in MSS mCRC. Plasma derived TMB may better reflect evolutionary changes following intervening therapy than archival tissue. Clinical trial information: NCT02870920. [Table: see text]

32 Background: Trimodality therapy (TT) remains standard for stage 2/3 rectal cancer and emerging evidence supports better outcomes with total neoadjuvant therapy (TNT) than similar therapy given post-operatively (Post-op). Real-world practice patterns and sequence of TT have significant implications on rectal cancer quality of care, outcomes and clinical trial design, and are not well described. Methods: The National Cancer Database was used to identify patients with clinical (c) stage 2/3 rectal adenocarcinoma diagnosed between 2006-2016, while pathologic (p) stage 2/3 was used when c stage was missing. Patients were included if they received Chemo/Radiation (C/RT), single or multiagent (SA or MA) chemotherapy (CT) and surgery. TNT was defined as receipt of pre-op C/RT and MA CT and no post-op therapy. Survival analyses were performed using Kaplan-Meier (KM) estimates and Cox univariate and multivariate hazard ratios (HRs) adjusted for TNT group, sex, race, year of diagnosis, stage, facility type, and age of diagnosis. Five-year KM survival proportions were limited to years 2006-2012, while Cox HRs were estimated from 2006 to 2015. Results: Of 32,467 patients who received TT, 8883 (27%) received TNT and 23,584 were classified no-TNT. TNT and no-TNT cohorts were numerically similar in age, race and gender. A significantly higher proportion of pStages 0-2 patients were observed in the TNT vs the no-TNT cohorts (61% vs. 48%, p < .001). In analysis of annual treatment patterns of the whole cohort, a gradual reduction in Post-op C/RT was observed between 2006 (28%) and 2016 (8%, p < .001), while use of TNT and MA CT did not increase. A migration to lower pStages 0/1/2/3 was seen between 2006 (1/10/31/57%, respectively) and 2016 (3/22/29/45%) (2016) (p < .001). Five-year OS analysis showed superior OS for Pre-op vs Post-op C/RT (74 vs 69%, HR = .81, p < .001), and MA CT vs. SA CT (76 vs 71%, HR = 0.79, p < .001) but inferior 5-y OS for the TNT vs. no-TNT cohorts (74 vs 78%, HR = 1.17, p < .001). Conclusions: Between 2006-16, a minority of patients treated with TT for stage 2/3 rectal cancer received TNT which did not increase over time. Significant annual shifts from Post-op C/RT to Pre-op C/RT, and migration to lower pStages were seen during this time period. Pre-op C/RT and MA CT were associated with improved OS, while TNT patients experienced inferior OS vs. the no-TNT cohort, possibly related to confounding factors in this observational cohort; multivariate sub-group analysis to be presented. Emerging evidence in support of TNT may further change treatment patterns and outcomes. [Table: see text]

117 Background: Previous studies have identified MAPK and PIK3CA/AKT/mTOR pathways as common mechanisms of acquired resistance to anti-EGFR antibodies (EGFRab) in mCRC. However, such alterations do not account for all patients that become treatment resistant. Using paired whole-exome (WES; tissue) and circulating tumor DNA (ctDNA; plasma) sequencing, we performed characterization of the EGFRab resistance landscape in patients with mCRC. Methods: Post-treatment (ctDNA; plasma) sequencing was performed for 169 patients with mCRC, with 112 (66%) patients also receiving pre-treatment (WES; primary tumor) sequencing as part of the CO.26 trial. 66 (39%) patients received EGFRab previously at a median of 132.0 days prior to plasma sampling. Using bespoke bioinformatics pipelines (WES) coupled with the GuardantOMNI assay (plasma), we compared somatic mutation (SNV/indel, CNV and fusion) frequencies between pre- and post-EGFRab timepoints, and contrasted results between the two groups. Results: Significant increases in de novo acquisition of EGFR (p = 5.6e-4), KRAS (p = 0.011), ZNF217 (p = 0.0022), MAP2K1 (p = 0.0078) and LRP1B (p = 0.017) SNV/indels were unique to the EGFRab group and often occurred as multiple, low allele frequency events in the same patient. De novo copy number amplification of known resistance genes EGFR/ BRAF/ MET were observed in EGFRab-treated patients (p < 0.05), along with SMO (p = 6.8e-7), PTEN inhibitory gene PREX2 (p = 5.6e-4), FLT3 (p = 2.0e-5), NOTCH4 (p = 6.3e-5), ERBB2 (7.4e-4), KMT2A (p = 3.7e-4) and ARID1B (p = 0.0014). Genes impacted by fusion events in EGFRab-treated patients included BRAF-KIAA1549 (1 patient) and MET-CAV1 (1 patient), and these events were not detected in matched pre-treatment samples. EGFRab-treated patients were found to acquire a combination of multiple (≥5) mutation events (SNV/indel, CNV or fusion) at much higher frequency compared to non-EGFRab-treated patients (67% versus 25% of patients, p = 8.7e-8). Tumor mutation burden (TMB) was not significantly different (p = 0.71) between treatment groups prior to therapy initiation, while post-treatment TMB was significantly higher (p = 1.8e-7) in EGFRab-treated patients (median 25.4 versus 13.1 mut/mb). Conclusions: In addition to previously established resistance pathways, we identified acquired alterations in additional genes such as SMO, PREX2 and epigenetic modifiers KMT2A/ARID1B in EGFRab-treated patients . Moreover, we highlight the phenomenon by which EGFRab-treated tumors acquire multiple concurrent resistance mutations and heightened TMB. Our analysis provides novel insight into the landscape of resistance mechanisms to EGFRab in mCRC while highlighting the potential role for immunotherapy post-EGFRab. Clinical trial information: NCT02870920.

6078 Background: PARP inhibitors (PARPi) have been established as a targeted therapeutic approach not only in patients with high-grade serous ovarian cancer (HGSOC) that have genetic loss of function of BRCA1/2-associated DNA repair. However, treatment efficacy varies and neither BRCA mutation, nor homolog recombination deficiency (HRD) status seem to be optimal predictors. Moreover, mechanisms of treatment resistance are poorly understood and novel approaches are urgently required. Methods: Here we created gene expression data of HGSOC patients (n = 52) before PARPi treatment to elucidate key signaling pathways of resistance to increase their efficacy in combinatorial therapeutic strategies. We performed a comprehensive bioinformatics analysis of the differentially expressed genes between the 25% extreme responders (n = 26; 13 each group), including gene set enrichment analysis (GSEA) and causal inference analysis with the CARNIVAL pipeline to elucidate the underlying molecular and regulatory mechanisms governing treatment efficacy and resistance. Results: In accordance with recent publications, we found higher levels of MYC activity in non-responders and deregulation of the Wnt/ß-catenin signaling pathway resulting in PARPi treatment resistance. The pathway enrichment analysis also revealed specific pathways especially PDGFR, FGFR, PI3K/mTOR and MAPK signaling pathway associated with resistant phenotype. Furthermore, we have identified key kinases, particularly JAK1/2 and SRC that might mediate resistance to PARP inhibition. In addition, differential gene expression analysis revealed folate receptor 1 (FOLR1) to be significantly higher expressed in non-responders (logFC = 2.66; p < 0.0026) with the potential as a serum-based biomarker not only for ovarian cancer, as it correlates closely with CA125, but also PARPi treatment efficacy. Conclusions: In conclusion, these findings define a network of pathways, that are crucial to mediate mechanism of PARPi resistance and identified key signaling kinases as therapeutic targets in ovarian cancer.

e19222 Background: Use of EOL CTx is an established quality metric in patients with advanced malignancies but less is known about which types of CTx are most commonly used and association with ED and ICU utilization. We sought to describe the different types of EOL CTx and to quantify the frequency of EOL ED and ICU care associated with them. Methods: Patients in the cancer registry of an urban cancer center who died between January 1, 2018 and October 10, 2019, and ever received CTx were included. EOL CTx was defined as any CTx given within 30 days of death, while any ED visits or ICU admissions in the last 30 days of life were defined as EOL ED and ICU care, respectively. CTx was categorized by administration route (intravenous (IV), oral (PO), other), and by type (immunotherapy (IMT), non-immunotherapy biologics (NIB), other). We used Pearson’s chi-squared to measure associations between EOL CTx and EOL ED and ICU care, logistic regression to assess how CTx type modulates those associations, and Mood’s median test to compare median IMT doses between groups. Results: Among 390 eligible patients, 32% received EOL CTx, 30% EOL ED care, and 11% EOL ICU care. Most received IV CTx (78%), and 10% received IMT. Median age at diagnosis was 69 years (interquartile range (IQR) 62 - 77), and median days from diagnosis to death was 390 (IQR 185 - 665). Most common malignancies were pancreatobiliary (40%), other gastrointestinal (15%), lung (13%) and hematologic (6%). Patients treated with EOL CTx were significantly more likely to receive IMT (p = 0.03). Receipt of any EOL CTx was significantly associated with EOL ED care (p < 0.0001) and EOL ICU care (p < 0.0001). Subgroup analyses showed significant modulatory effect of IMT on association of EOL CTx with EOL ED care (b = -0.89, p = 0.046), but was not significant for ICU care (b = -0.67, p = 0.29). Median doses of IMT was 2.5 (IQR 2 - 3.8) among patients who were given EOL IMT and 4 doses (IQR 2 - 6) in those that discontinued IMT before EOL (p = 0.06). Conclusions: EOL CTx is associated with significantly increased rates of EOL ED and ICU care, which may indicate poorer quality of life. While rates of use of other CTx modalities did not significantly differ at EOL, patients were more likely to receive IMT within 30 days of death, which could be due to the belief that IMT is more tolerable or more effective than other CTx modalities at EOL. IMT at EOL is associated with a reduced risk of EOL ED care, but not ICU care. Further research on strategies to reduce EOL CTx and appropriateness of IMT at EOL is warranted.

e20505 Background: Immunoparesis (hypogammaglobinemia) has been shown to impact outcomes in patients with newly diagnosed multiple myeloma (MM). Its impact in the post-transplant setting and potential risk of both infections and immune dysregulation impacting relapse is less clear. We sought to assess the role of immunoparesis both pre and post autologous stem cell transplant (ASCT) in newly diagnosed MM patients. Our primary outcome was the impact of immunoparesis on progression free survival (PFS) post-ASCT controlling for maintenance therapy. Methods: We evaluated all MM patients between 2008 and 2017 at Loyola University who underwent ASCT. Inclusion criteria included first ASCT and age > 18 years. We evaluated both the presence of and number of impacted immunoglobulin (Ig) classes pre-transplant and day 100, 6-months, and 12 months post-ASCT. Results: The most common MM isotype was IgG Kappa (41.01%). Immunoparesis pre-ASCT and post-ASCT at day 100, 6 months, and 12 months was seen in 75%, 89.5%, 80%, and 58.5% respectively. Median time from diagnosis to transplant was 7.75 months (5.98 – 13.73). When adjusting for pre-transplant response, conditioning regimen, and post-transplant maintenance, neither the absolute presence of or number of Ig-classes impacting was predictive of hazard of disease progression or death (OS) while ISS-stage, best response post ASCT, and time from diagnosis to transplant were (table). Conclusions: Contrary to studies in the pre-transplant setting, our analysis indicates that immunoparesis is not predictive of relapse or survival in the post-transplant setting, suggesting modification of disease process with treatment and ASCT. When controlled for maintenance therapy, immunoparesis does not increase risk of progression or decrease overall survival alleviating the concern for increased morbidity/mortality secondary to infections in the face of immunoparesis in the era of maintenance therapy. [Table: see text]