RAS Wt Patients Research Articles

Survival benefit with regorafenib and/or trifluridine/tipiracil sequencing to rechallenge with anti-EGFR-based third-line regimens in metastatic colorectal cancer: A multicenter retrospective real-world subgroups comparison.

e15553 Background: Rechallenge with anti-EGFR drugs has recently proven therapeutic activity in a subset of RAS wild type (wt) metastatic colorectal cancer (mCRC) patients who had previously benefited from cetuximab- or panitumumab-based first-line regimen. Regorafenib (R) and Trifluridine/tipiracil (T) have been found to improve survival in patients with refractory mCRC. This real-life subgroup survival analysis focused on treatment with R and T, sequential or not, in patients who had received prior third-line anti-EGFR therapy. Methods: Clinical data of patients diagnosed with mCRC who received R and/or T between July 2012 and March 2022, were retrospectively collected from 13 Italian cancer institutes. The objective of this subgroup analysis was to compare overall survival (OS) and progression-free survival (PFS) in patients who received late-line R and/or T according to prior third-line anti-EGFR-based treatment. Results: The entire study enrolled 866 patients. 146 (16.8%) of them received the T/R sequence (T/R), 116 (13.3%) the reverse sequence (R/T), 325 (37.5%) T, and 279 (32.5%) R. For the purpose of this subgroup analysisi we identified 338 RAS wt patients (39%) who were eligible. Of these, 290/338 patients (85.8%) did not receive the previous third-line anti-EGFR therapy, while 48 patients (14.2%) did it (12 T/R, 10 R/T, 13 T, 13 R). When comparing T/R to R/T in patients who did not previously received third-line anti-EGFR drug rechallenge, we observed a statistically significant and clinically meaningful improvement in PFS. In detail, the median PFS benefit was 67,5% when treating patients with T/R sequence vs. the reverse sequence (11,4 vs. 3,7 months, respectively, ∆ = 7,7 months; p = < 0,0001; HR = 0,17; 95% CI = 0,09-0,32). In patients who received T/R or R/T following rechallenge with anti-EGFR drug, we also observed a comparable, albeit smaller and non-statistically significant, improvement in PFS (8.1 vs. 7.8 months, respectively; p = 0.22; HR 0,51; 95% CI = 0,17-1,50). There were no statistically significant differences in OS between these groups. Anti-EGFR rechallenge data analysis showed that the non-sequential administration of R and T had no statistically significant impact on survival outcomes. Conclusions: With the limit of the sample size, our retrospective subgroup analysis failed to validate the promising beneficial effects of anti-EGFR rechallenge prior to R and/or T treatment. A statistically significant longer PFS was only observed in patients with refractory metastatic colorectal cancer receiving T/R sequence without prior cetuxumab- or panitumumab-based regimens.

Transcriptional Profiling and Consensus Molecular Subtype Assignment to Understand Response and Resistance to Anti-Epidermal Growth Factor Receptor Therapy in Colorectal Cancer.

Activating mutations in KRAS, NRAS, and BRAF are known to cause resistance to anti-epidermal growth factor receptor (EGFR) therapy; however, only approximately 40% of patients with colorectal cancer (CRC) with RASWT tumors respond to anti-EGFR treatment. We sought to discover novel biomarkers to predict response to anti-EGFR antibody treatment in CRC and to understand mechanisms of resistance to anti-EGFR therapy. Transcriptomic profiles from three clinical and two preclinical cohorts treated with cetuximab were used to assign consensus molecular subtypes (CMS) to each sample and correlated with outcomes. Restricting to RASWT patients, we observed that CMS2 tumors (canonical subtype) had significantly higher response rates relative to other CMS when treated with cetuximab combination with doublet chemotherapy (Okita et al cohort: 92% disease control rate (DCR) for CMS2, chi-square P = .04; CALGB/SWOG 80405 cohort: 90% objective response rate (ORR) for CMS2, chi-square P < .001) and with single-agent cetuximab (68%, chi-square P = .01). CMS2 tumors showed best response among right-sided (ORR = 80%) and left-sided (ORR = 92%) tumors in the CALGB/SWOG 80405 cohort. CMS2 cells lines were most likely to be sensitive to cetuximab (60%) and CMS2 patient-derived xenograft had the highest DCR (84%). We found Myc, E2F, and mammalian target of rapamycin pathways were consistently upregulated in resistant samples (enrichment score >1, false discovery rate <0.25). Inhibitors of these pathways in resistant cell lines exhibited additive effects with cetuximab. These data suggest that CRC transcriptional profiles, when used to assign CMS, provide additional ability to predict response to anti-EGFR therapy relative to using tumor sidedness alone. Notably both right-sided and left-sided CMS2 tumors had excellent response, suggesting that anti-EGFR therapy be included as a treatment option for right-sided CMS2 tumors.

Frequency of KRAS, NRAS, and BRAF mutations in colorectal cancer in an Argentinian population.

e15604 Background: Colorectal cancer (CRC) is one of the major causes of morbidity and mortality worldwide. At diagnosis, about 25% of patients are metastatic (mCRC); also 50% patients will develop metastases during the disease First line treatment (1L) for mCRC is guided by clinical and molecular features and includes the use of chemotherapy combined with monoclonal antibodies, like anti-epidermal growth factor receptor, such as cetuximab and panitumumab, or anti-vascular endothelial growth factor, such as bevacizumab. Treatment guidelines advise assessing the mutational status of some oncogenes (KRAS, NRAS and BRAF) prior to the choice of 1L The aim of this study is to analyze the population characteristics of patients with CRC in Argentina and to determine the incidence of mutations in KRAS, NRAS and BRAF in this population. Methods: Tissue (TB) or liquid biopsies (LB) were collected and analyzed from March 2018 to November 2022 from an adult patient population with CRC in Argentina. Mutations in KRAS, NRAS and BRAF were determined using real time PCR (cut-off values TB: KRAS/NRAS 1-5%; BRAF 5%; LB: KRAS 25-75 copies/mL in 64000 copies of normal DNA; RAS/BRAF 100-200 copies/mL in 160000 copies of normal DNA). Also population characteristics (age, sex, and geographic location) were considered for analysis. Only patients having results for the three genes were included. Results: A total of 9150 samples were analyzed, 91% (N = 8326) for TB and 9% (N = 824) for LB; from these, 56.6% came from male (N = 5181) and 43.4% female (N = 3969) patients. The median age was 64 years (range 22-89) Patients tested by TB mainly had stage IV 68.6% (N = 6129), 1.7% (N = 149) stage II and 5.9% (N = 523) stage III disease. For LB patients, also stage IV 79.7% (N = 610) disease was the most common followed by stage III 4.4% (N = 34), and stage II 0.7% (N = 5) For stage IV patients with TB, 81.6% (N = 5060) were tested prior 1L therapy, 10.9% (N = 674) before second line (2L) and 0.6% (N = 34) before third line (3L). For LB patients, 1L was the most common 48.5% (N = 361), followed by 2L 5.0% (N = 37), and 3L 19.1% (N = 142) In stage IV patients tested before 1L by TB, the frequency of mutations in the RAS family genes was 47% (N = 2257), KRAS mutations were found in 91.4% of cases (N = 2084) and 8.7% in NRAS (N = 173). RAS wt patients were 52.9% (N = 2537). Mutations in BRAF were found in 12.1% of cases For those patients tested using LB, mutations in RAS were found in 38.3% (N = 121) of cases, KRAS in 92.6% (N = 112) and NRAS in 7.4% (N = 9). RAS wt patients were 61.7% (N = 195) while BRAF mutated patients were 13.3%. Conclusions: This analysis is the largest cohort showing the molecular profile in CRC patients from Argentinean population. Mutations in RAS genes arose in 38-47% of mCRC patients before 1L. BRAF mutations were found in 12.1-13.3% slightly higher than in other series. Testing rates for RAS and BRAF before starting treatment was high, this reinforces the need of mutational screening before deciding any therapy in mCRC.

Phase III FIRE-4 study (AIO KRK-0114): Influence of baseline liquid biopsy results in first-line treatment efficacy of FOLFIRI/cetuximab in patients with tissue RAS-WT mCRC.

3507 Background: FIRE-4 (AIO KRK-0114) is performed in RAS wild-type (wt) mCRC patients. This randomized study tests the efficacy of early switch maintenance during 1st-line therapy (part 1) and re-challenge with cetuximab (part 2) in later-line treatment. In part 1, arm A patients continued FOLFIRI/Cet until progression or intolerable toxicity. In arm B, patients received FOLFIRI/Cet for 8-12 cycles, after which maintenance therapy with 5-FU/FA plus bevacizumab (5-FU/Bev) was applied. The first randomization evaluates the question if an early switch from cetuximab to bevacizumab may prolong PFS. Within the translational protocol, serial baseline liquid biopsy were taken to analyze RAS and BRAF mutations. Methods: Within this randomized, controlled, open-label phase-III study, patients received FOLFIRI (irinotecan plus 5-FU/FA) plus cetuximab at the standard dosing schedule. In arm A, FOLFIRI plus cetuximab was continued until progression or intolerable toxicity. In arm B, patients received 8 cycles of FOLFIRI plus cetuximab (in case of tumor response) or 12 cycles (in case of stable disease) followed by maintenance with 5-FU/FA plus bevacizumab (5mg/kg) until disease progression or intolerable toxicity. Overall survival after second randomization (part 2) is evaluated as a primary endpoint. Liquid Biopsies were analyzed by RAS BEAMing and BRAF ddPCR technology. Results: From August 2015 to January 2021, 673 patients were randomized, and liquid biopsies of 540 patients were evaluable at baseline. Of those, 70 (13%) were RAS mutant and 38 (7%) BRAFV600E mutant at baseline. Patients with a detectable RASmut had a significant shorter PFS and OS when compared to RASwt patients (PFS: 9.0mo vs. 11.5mo; p &lt; 0.001; OS: 22.1mo vs 33.6mo; p &lt; 0.001). Whereas, for RASwt patients no difference for both arms with respect to PFS and OS could be observed, RASmut patients (n = 70) had a clear trend towards shorter survival in the standard FOLFIRI cetuximab arm (PFS: 6.4mo vs. 10.1mo, p = 0.54; OS: 24.9mo vs. 16.3mo, p = 0.10). Patients with a BRAF mutation in liquid biopsy had median survival times as expected for BRAF mutant patients (PFS = 5.5mo; OS = 12.0mo). In the standard arm, with a continuous administration of cetuximab, the conversion rate from RASwt to RASmut was significantly higher at progression than in the switch maintenance arm. Conclusions: Liquid biopsy detected RAS mutation in 13% of patients deemed RASwt based on tissue analyses. These patients show outcome characteristics expected for RAS mutant patients (median PFS of 9.0 months and median OS of 22 months). The study thus shows the clinical relevance of liquid biopsy in the verification of RAS mutational status. Clinical trial information: NCT02934529 .

Association of Circulating Tumor Cells and Tumor Molecular Profile With Clinical Outcomes in Patients With Previously Untreated Metastatic Colorectal Cancer: A Pooled Analysis of the Phase III VISNÚ-1 and Phase II VISNÚ-2 Randomized Trials

BackgroundThe bCTC count is a well-established prognostic biomarker in mCRC, as well as in other tumor types. The aim of this analysis was to evaluate the prognostic/predictive role of the bCTC count (≥3 vs. <3) in previously untreated mCRC. Patients and MethodsThe study involved 589 untreated mCRC patients included in the intention-to-treat population of 2 randomized clinical trials (phase III VISNU-1 [NCT01640405] and phase II VISNU-2 [NCT01640444] studies). ResultsOf the 589 patients, 349 (59.2%) had bCTC≥3 and 240 (40.7%) had bCTC<3. Multivariate analysis showed that the bCTC count is an independent prognostic factor for overall survival (OS) (HR 0.59, 95% CI 0.48-0.72; P = 0.000) and potential for progression-free survival (PFS) (P = 0.0549). Median OS was 32.9 and 19.5 months in patients with bCTC<3 and bCTC≥3 (P <0.001), respectively. This effect was also observed comparing OS in RASwt patients from both studies. Other prognostic factors were: ECOG-PS, primary tumor site, number of metastatic sites and surgery of the primary tumor. Median OS was lower for patients treated with anti-VEGF versus anti-EGFR (22.3 vs. 33.3 months, P <0.0001) while there were no significant differences in PFS according to the targeted treatment received. ConclusionThis post-hoc analysis of 2 randomized studies confirms the poor prognosis of patients with bCTC≥3 but this is not associated with other adverse independent prognostic factors such as RAS/BRAF mutations.

Biomarkers and treatment characteristics in metastatic colorectal cancer RASwt patients in Latin America

Biomarkers and treatment characteristics in metastatic colorectal cancer RASwt patients in Latin America

403P First-line anti-EGFR therapy, patient characteristics and survival: Results from South Australian (SA) metastatic colorectal registry (mCRCR)

Treatment of mCRC is guided by clinical and molecular features which include side of primary, RAS, BRAF and MMR status. For left sided RAS WT mCRC survival is optimized by using first-line anti-EGFR anti-bodies combined with chemotherapy. We aim to assess the use of first-line anti-EGFR/chemotherapy (FaEC) combinations in patients with mCRC and assess for differences between cetuximab (C) and panitumumab (P) using the SA mCRCR. This real word registry has collected data from all patients diagnosed with mCRC in SA prospectively since 2/2006. We compared C and P in RAS WT patients, and those treated with bevacizumab (B) from 2006 and those treated since January 2015 when FaEC was funded in Australia. Survival was analysed using the Kaplan Meier method. Of the 5537 patients currently entered onto the registry, 1313 had RAS status recorded and 245 received FaEC (167/68% since 2015). 1068 patients received B (52% KRAS WT). Table summarises patient characteristics and median OS for FaEC (C or P) and B. Overall there was no statistical difference in survival for C v P (p=0.125). Patients entered from 2015 had mostly similar patient characteristics including significant use in right sided primary (24% v 20% respectively).Table: 403PChemo/C (139)Chemo/P (106)Chemo/B (1068)Median age (range)65.3 yrs (24-87)60.1 yrs (26-89)64.6 (20.5-93)Male66.2%64%61%Oxaliplatin15%43.4%70%Irinotecan59%46.2%15.5%Stage 4 at diagnosis47.5%64.2%67.5%Left primary65.5%77.4%58%Liver mets only38.1%42.5%37.2%Lung mets only5.8%9.4%8.7%BRAF MT7.9%2.8%7.3%Liver resection6.5%14.2%9.8%Median OS (95% CI)21.6 mths (16.7-26.4)25.3 mths (20.8-29.7)22.7 mths (21-23.9) Open table in a new tab When comparing C & P in first-line therapy, C was more often combined with irinotecan chemo. There were lower rates of liver resection and higher right primary and BRAF MT in patients treated with C which may explain the numerically lower median overall survival.

Embryologic Origin of the Primary Tumor and RAS Status Predict Survival after Resection of Colorectal Liver Metastases.

Background and objectives. In colorectal cancers, the embryologic origin of the primary tumor determines important molecular dissimilarities between right-sided (RS) and left-sided (LS) carcinomas. Although important prognostic differences have been revealed between RS- and LS-patients with resected colorectal liver metastases (CLMs), it is still unclear if this observation depends on the RAS mutational status. To refine the impact of primary tumor location (PTL) on the long-term outcomes of patients with resected CLMs, the rates of overall survival (OS), relapse-free survival (RFS) and survival after recurrence (SAR) were compared between RS- vs. LS-patients, according to their RAS status. Material and Methods. All patients with known RAS status, operated until December 2019, were selected from a prospectively maintained database, including all patients who underwent hepatectomy for histologically-proven CLMs. A log-rank test was used to compare survival rates between the RS- vs. LS-group, in RAS-mut and RAS-wt patients, respectively. A multivariate analysis was performed to assess if PTL was independently associated with OS, RFS or SAR. Results. In 53 patients with RAS-mut CLMs, the OS, RFS and SAR rates were not significantly different (p = 0.753, 0.945 and 0.973, respectively) between the RS and LS group. In 89 patients with RAS-wt CLMs, the OS and SAR rates were significantly higher (p = 0.007 and 0.001, respectively) in the LS group vs. RS group, while RFS rates were similar (p = 0.438). The multivariate analysis performed in RAS-wt patients revealed that RS primary (p = 0.009), extrahepatic metastases (p = 0.001), N-positive (p = 0.014), age higher than 65 (p = 0.002) and preoperative chemotherapy (p = 0.004) were independently associated with worse OS, while RS location (p < 0.001) and N-positive (p = 0.007) were independent prognostic factors for poor SAR. Conclusions. After resection of CLMs, PTL had no impact on long-term outcomes in RAS-mut patients, while in RAS-wt patients, the RS primary was independently associated with worse OS and SAR.

Abstract 1246: Consensus molecular subtypes (CMS) of colorectal cancer predict anti-EGFR response irrespective of tumor sidedness

Abstract Background: Activating mutations in KRAS, NRAS, and BRAF are known to cause resistance to anti-EGFR therapy. However, only ~40% of colorectal cancer (CRC) patients with RASWT tumors respond to that treatment. We aim to leverage tumor transcriptomes to predict response to anti-EGFR antibody (e.g., cetuximab) treatment and understand intrinsic resistance mechanisms in CRC. Experimental Design: Transcriptomic profiles with RAS mutation status from two clinical (Okita et al., n = 135; Khambata-Ford et al., n = 68), and preclinical cohorts of CRC cell lines (Medico et al., n = 146) and PDX models (Bertotti et al., n = 216) treated with cetuximab were downloaded from Gene Expression Omnibus (GEO). Each cohort was divided into RASWT and RASMut groups, and transcriptomic profiles were used to assign consensus molecular subtypes (CMS) to each sample. Gene Set Enrichment Analysis (GSEA) was performed to identify resistant pathways in each CMS RASWT group. Results: Restricting to RASWT patients (n = 80) in Okita et al. cohort, we observed that CMS2 tumors had significantly higher disease control rates (DCR) [92% (33/36); chi-square p = 0.03] with cetuximab in combination with doublet chemotherapy relative to CMS1 [50% (2/4)], CMS3 [59% (13/22)], and CMS4 [83% (15/18)] tumors. RASWT CMS2 tumors (n = 43) also showed significantly higher DCR [68% (15/22); chi-square p = 0.03] with single-agent cetuximab compared to CMS1 [0% (0/4)], and CMS4 [29% (5/17)] tumors in Khambata-Ford et al. cohort. In multivariate analysis including both CMS and tumor sidedness, only CMS2 (HR = 0.62, p = 0.03) was significant predictor of cetuximab response in Okita et al. cohort. CMS1 tumors showed worst treatment response irrespective of tumor sidedness [DCR, left: 50% (1/2) vs right: 50% (1/2)]. In contrast both left and right-sided CMS2 tumors responded well [DCR, left: 91% (31/34) vs 100% (2/2)] to cetuximab plus chemotherapy [PFS, 7.9 vs 7 months; log-rank test p = 0.7]. In microsatellite stable (MSS) RASWT cell lines (n = 27), CMS2 cells lines were most sensitive [59% (7/17)] relative to CMS4 [0% (0/10)]. In MSS RASWT PDX models (n = 60), CMS2 had highest DCR [75% (18/24)] than CMS1 [36% (4/11)], CMS3 [55% (5/9)], CMS4 [62% (10/16)]. We found Myc, E2F, and mTOR gene sets/pathways were consistently enriched in resistant patients, cell lines, and PDX models (Normalized enrichment score &amp;gt; 1, FDR &amp;lt; 0.25). Small molecule inhibitors of Myc (JQ1), E2F (AZD7762 &amp; MK-8776), and mTOR (Everolimus) pathways in resistant CRC cell line (HT55) exhibited additive, but not synergistic, effects with cetuximab. Conclusions: These data suggest that tumor transcriptional profiles are a better predictor of anti-EGFR response than tumor-sidedness. Use of CMS to predict anti-EGFR response should be validated in a larger, prospective study. Activation of Myc, E2F, and mTOR pathways are associated with cetuximab resistance in colorectal cancer cells. Citation Format: Saikat Chowdhury, Ria Gupta, Valsala Haridas, Mohammad A. Zeineddine, Scott Kopetz, John Paul Shen. Consensus molecular subtypes (CMS) of colorectal cancer predict anti-EGFR response irrespective of tumor sidedness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1246.

Patterns of care and survival of first line anti-EGFR therapy; results from South Australian (SA) metastatic colorectal (mCRC) registry.

e15577 Background: Treatment of mCRC has changed dramatically over the last decade with therapy guided by clinical and molecular features which include side of primary, RAS, BRAF and MMR status. For left sided RAS WT mCRC survival is optimized by using first line anti-EGFR anti-bodies combined with chemotherapy. This is reflected in modern guidelines. Methods: We aim to assess the uptake of first line anti-EGFR/chemotherapy combinations in patients with mCRC and assess for difference between cetuximab (C) and panitumumab (P) use from the SAmCRCR. The real word registry has collected data from all patients diagnosed with mCRC in SA prospectively since 2/2006. We compared RAS WT patients treated with chemo/bevacizumab (CB). Survival was analysed using the Kaplan Meier method. Results: Of the 5537 patients currently entered onto the registry, only 97 had RAS status recorded and had received first line anti-EGFR/chemotherapy (FaEC). 102 patients were RAS WT and received CB. Table summarises patient characteristics and median OS for FaEC (C or P) and CB. There was no statistical difference in survival for C v P (p = 0.055). Conclusions: When comparing C &amp; P choice in first line therapy, C was more often combined with irinotecan/chemo. C patients had higher rate of left sided primary. There were lower rates of liver resection in patients treated with C which may explain the numerically lower median overall survival. An updated cohort analysis will be included to assess changes in practice over time.[Table: see text]

Allogeneic Hematopoietic Cell Transplantation Outcomes of Patients with R/R AML or Higher-Risk MDS Treated with the TIM-3 Inhibitor MBG453 (Sabatolimab) and Hypomethylating Agents

Allogeneic Hematopoietic Cell Transplantation Outcomes of Patients with R/R AML or Higher-Risk MDS Treated with the TIM-3 Inhibitor MBG453 (Sabatolimab) and Hypomethylating Agents

Abstract 2373: Expression of exosomal let-7g in biofluids and outcome in colon cancer patient treated with anti-EGFR therapy

Abstract Introduction: Monoclonal antibodies against the Epidermal Growth Factor Receptor (EGFR) such as cetuximab or panitumumab are used for the treatment of metastatic colorectal cancer (mCRC) patients. Unfortunately, most patients develop resistance against these therapies within months. Several studies have shown that aberrations in the RAS pathway are responsible for resistance. However, even in metastases that are refractory to anti-EGFR treatment a significant fraction of RAS wild-type (wt) cells remain. These findings suggest a cross-talk between RAS mutant and wt cells in mediating resistance in the wt compartment. Methods: Mouse and patient-derived organoids from mCRC as well as CRC cell lines were used to test the contribution of extracellular vesicles in mediating resistance in RAS wt cells. Using conditioned media, transfection experiments and liquid biopsies (plasma and urine) from patients differential expression of the let-7g microRNA was demonstrated in microvesicles from cetuximab sensitive and resistant cells. Changes in expression of let-7g were further analyzed by in-situ hybridization in tissues. Results: Conditioned media from RAS mutant organoids rendered RAS wt organoids resistant against cetuximab treatment. Basal let-7g expression from pre-treatment plasma and urine samples of RAS wt patients correlated with clinical outcome and changes in let-7g circulating levels mirrored clinical behavior. In-situ hybridization in tissues confirmed changes in expression of the let-7g microRNA observed in plasma and urine samples. Conclusions: Our data suggest that let-7g microRNA might function as a paracrine mediator of anti-EGFR resistance and might be exploited as a non-invasive biomarker of resistance to cetuximab treatment. Further work is ongoing to characterize the molecular mechanisms underpinning let-7g mediated effect on anti-EGFR sensitivity in RAS wt CRC cells. Citation Format: Jens C. Hahne, Andrea Lampis, Michele Ghidini, Margherita Ratti, Chiara Senti, Rodolfo Passalacqua, Luciano Cascione, Chiara Braconi, Owen Sansom, Matteo Fassan, Nicola Valeri. Expression of exosomal let-7g in biofluids and outcome in colon cancer patient treated with anti-EGFR therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2373.

Artificial intelligence-assisted immunohistochemical (IHC) evaluation of tumor amphiregulin (AREG) and epiregulin (EREG) expression as a combined predictive biomarker for panitumumab (Pan) therapy benefit in RAS wild-type (wt) metastatic colorectal cancer (mCRC): Analysis within the phase III PICCOLO trial.

111 Background: High tumor mRNA levels of the EGFR ligands, AREG and EREG are associated with anti-EGFR agent response in patients (pts) with RAS-wt mCRC, regardless of tumor location. However, ligand RNA assays have not been adopted into routine clinical practice due to issues with analytical precision and practicality. Here we test whether AREG and EREG expression assessed by IHC can predict benefit from Pan. Methods: A retrospective biomarker study within the PICCOLO trial (NCT00389870; irinotecan [Ir] ± Pan in fluoropyrimidine-resistant RAS-wt mCRC). AREG and EREG positive tumor cells were assessed by IHC in all RAS-wt patients with available tumor tissue. Pathologists annotated tumor areas on digital images of glass slides. Artificial intelligence (AI) algorithms calculated the percentage of tumor cells staining positive for AREG and EREG within the tumor areas. More than 50% AREG and/or EREG tumor cell positivity was regarded as high ligand expression. The primary endpoint was progression-free survival (PFS) and secondary endpoints were RECIST response rate (RR) and overall survival (OS). Results: 274 RAS-wt pts had available tumor tissue. High ligand expression (n = 132) was associated with significant PFS benefit from IrPan compared with Ir (8.0 vs 3.2 months; HR 0.54 [0.37-0.79]; p = 0.001); whereas low ligand expression (n = 142) was not (3.4 vs 4.4 months; HR 1.05 [95% CI, 0.74-1.49]; p = 0.78). The ligand-treatment interaction was significant (p = 0.02) and independent of BRAF-mutation status and primary tumor location. Likewise RR was significantly improved in pts with high ligand expression (IrPan vs Ir: 48% vs 6%; risk ratio, 7.8 [2.90-20.69]; p &lt; 0.0001) but not those with low ligand expression (IrPan vs Ir: 25% vs 14%; risk ratio, 1.8 [95% CI, 0.89-3.65]; p = 0.10) (interaction p = 0.01). Lesser effect was seen on OS. Conclusions: IHC assessment of AREG and EREG identified pts who did or did not benefit from Pan, as has been previously demonstrated through mRNA quantification. IHC represents a more practicable technique as it can be provided at the point of care and is associated with shorter turn-around times. AREG and EREG IHC may be of use in routine practice to identify patients who would benefit from anti-EGFR therapy and those for whom alternative treatment strategies should be explored.

Combined PD-1 inhibition (Pembrolizumab) and CCR5 inhibition (Maraviroc) for the treatment of refractory microsatellite stable (MSS) metastatic colorectal cancer (mCRC): First results of the PICCASSO phase I trial.

3010 Background: Checkpoint inhibition using PD-1/PD-L1 inhibitors does not show clinically relevant activity in MSS/pMMR (Mismatch Repair Proficient) colorectal cancer. Previous work showed that inhibition of CCR5 (C-C chemokine receptor type 5) leads to a macrophage re-polarization towards M1 macrophages within the tumor microenvironment which directly affects immune cell infiltrates. The current phase I trial explores a combined modification of the innate immune system (by CCR 5 blockade) and the adaptive immune system (by PD-1 inhibition) in the treatment of MSS CRC. Methods: 20 patients with metastatic MSS/pMMR colorectal cancer with failure of fluoropyrimidines, oxaliplatin, irinotecan, VEGF antibodies and EGFR antibodies (in ras WT patients) received pembrolizumab 200 mg q21d and maraviroc 300 mg bid cont. for 8 cycles, followed by pembrolizumab monotherapy for a maximum of 24 additional cycles. Imaging was performed every nine weeks (RECIST and irRECIST criteria). Primary endpoint was the feasibility rate (rate of patients receiving the protocol treatment during the core treatment without special event: treatment-related Grade ≥ 3 immune-related abnormalities, treatment-related Grade ≥ 4 AEs or any toxicity-related premature withdrawal of treatment). Secondary endpoints included safety/toxicity, ORR, PFS and OS. Results: 20 patients were enrolled. The median number of applied cycles was 3.5 for pembrolizumab and 3.5 for maraviroc. Two patients completed the core treatment period with pembrolizumab and started maintenance treatment. The feasibility rate was 94.7% (90% CI 77.4 to 99.7%), with one patient experiencing a special event. Except this grade 4 event (hyperglycemia) no ≥ 3 treatment-related toxicities were observed. According to irRECIST criteria one patient showed a partial response and one a stable disease as best response, resulting in an irDCR of 10.5%. Median PFS according to irRECIST was 2 months (CI 95%, 2 to 3), median OS 9 months (CI 95%, 6 to 20). Conclusions: Therapy with pembrolizumab and maraviroc was feasible and showed a beneficial toxicity pattern. Clinical activity in MSS CRC patients was limited, however prolonged disease stabilizations were observed in single patients and overall survival was higher than expected in this heavily pretreated population. Clinical trial information: NCT03274804 .

Initial correlative studies from a trial of cetuximab and pembrolizumab in metastatic colorectal cancer (mCRC).

4062 Background: Cetuximab is an EGFR-targeting IgG1 mAb. Pre-clinical data suggests cetuximab induces CD8+ cytotoxic T-cell (CTL) infiltration of tumors. We hypothesized that augmentation of CTLs in the tumor microenvironment (TME) may provide the proper milieu for effective PD-1 inhibition in metastatic colorectal cancer (mCRC). We conducted a phase Ib/II study of cetuximab with the PD-1 antibody, pembrolizumab, in mCRC. Correlative blood and tissue samples were collected to assess the impact of this treatment on CTLs, as well as potential compensatory alterations in regulatory T-cells (Tregs) and suppressive MDSCs (NCT02713373). Methods: 3 week treatment cycles included pembrolizumab at 200 mg on day 1 and cetuximab 250 mg/m2 following the 400 mg/m2 loading dose. Tumor biopsies were obtained at baseline and at c4d1 (Day 64). Peripheral blood (PB) was drawn at baseline, c2d1 (day 22) and c4d1 (Day 64). Flow cytometry was performed within 24 hours with additional samples stored for future analysis. In the present analysis, we assessed changes in levels of the cellular populations of interest between cycle 4 and cycle 1. Results: Forty-two RAS-wt patients were enrolled through October 2019. Paired tumor tissues were successfully analyzed for 16 patients and PB for 38. Intratumoral CTLs (CD3+CD8+) increased significantly (+47%, p &lt; .05). In PB, there was a slight overall decrease in PB CTLs (-5%, p = NS) and a significant decrease in CD8+CD45RO+PD1+ cells (-42%, p &lt; .05). We saw simultaneous decreases in PD-1+ CTLs in the tumor and PB. There was a trend for increase in Tregs (CD4+ Cd25+ FoxP3+) in PB (+11%, p = NS), but an overall increase in the Teff:Treg ratio (+30%, p = NS). CD4+CTLA4+ cells significantly increased (+37%, p &lt; .05). Granulocytic MDSCs (CD11b+CD14−CD33+HLADR−) in PB decreased significantly on-treatment (-30%, p &lt; 0.5). The sample size and tissue limitations prohibited meaningful evaluation of tissue Tregs and MDSCs via the present methods. Conclusions: Cetuximab and pembrolizumab induced dynamic changes in the TME and PB. The treatment associated increase in intratumoral CTLs was particularly pronounced, consistent with their local expansion and/or influx. This was accompanied by a decrease in PB CTLs. Decreases in multiple PD-1+ lymphoid populations were observed in both tumor and PB, notably PD-1+ CTLs. Of note, we saw a synchronous increase in immunosuppressive CD4+CTLA4+ T-cells in PB. Patient outcomes are pending maturation. Further analyses are planned, coupled with integration of clinical data. Clinical trial information: NCT02713373 .

Anatomic Resection Is Not Required for Colorectal Liver Metastases with RAS Mutation

BackgroundNon-anatomic resection (NAR) has emerged as a safe and effective technique for resection of colorectal liver metastases (CRLM). More recently, RAS mutation has been identified as an important indicator of aggressive disease, which may require anatomic resection (AR). In this retrospective study, we compared the long-term outcomes of AR versus NAR in CRLM patients with and without RAS mutations. MethodsPatients with known RAS mutation status who underwent AR or NAR for CRLM between 2006 and 2016 were included. Differences in baseline characteristics were adjusted using 1:1 propensity score matching, including the most important factors that contributed to the decision to use the resection technique. Overall survival (OS), recurrence-free survival (RFS), and liver-specific recurrence-free survival (L-RFS) were compared between cohorts. ResultsAmong 622 total patients, 338 (54%) underwent AR and 284 (46%) NAR. There was no difference in OS or L-RFS between the AR and NAR groups, regardless of mutation status. There was increased RFS in the RAS WT patients with NAR (P = 0.034), but no difference in RFS in the whole cohort or RAS mutant group. After propensity score matching, 360 patients were analyzed, and no differences in OS, RFS, or L-RFS rates were seen between any groups. There was also no difference in margin recurrence. ConclusionsSimilar outcomes can be achieved with both AR and NAR, regardless of RAS mutation status. These data do not support a universal requirement for AR in RAS mutant CRLM when not necessary to achieve an R0 resection.

RAS Mutations Are Independently Associated with Decreased Overall Survival and Event-Free Survival in Patients with AML Receiving Induction Chemotherapy

Cai: Imago Biosciences, Inc.: Consultancy. Viny:Hematology News: Membership on an entity's Board of Directors or advisory committees; Mission Bio: Other: Sponsored travel. Goldberg:American Society of Clinical Oncology: Research Funding; Abbvie: Research Funding; ADC Therapeutics: Research Funding; American Society of Hematology: Research Funding; DAVA Oncology: Honoraria; Pfizer: Research Funding; Arog Pharmaceuticals: Research Funding; Abbvie: Consultancy; Daiichi-Sankyo: Consultancy, Research Funding; Celgene: Consultancy. Tallman:BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Biosight: Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Rigel: Consultancy; Cellerant: Research Funding; ADC Therapeutics: Research Funding; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees. Stein:Astellas Pharma US, Inc: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; PTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo, Inc.: Membership on an entity's Board of Directors or advisory committees; Bioline: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees.

Impact and Outcomes of RAS gene Mutations in Core Binding Factor Acute Myeloid Leukemia

Acute myeloid leukemia (AML) with t(8;21) or inv(16) chromosomal rearrangements are distinct heterogeneous disease entities within AML that are classified together as core binding factor AML (CBF-AML). Given the nature of the chromosomes involved, these rearrangements lead to the production of leukemogenic chimeric transcripts (RUNX1-RUNX1T1 and CBFB-MYH11) which disrupt the physiologic activity of the heterodimeric transcription factor CBF complex. Although CBF-AML patients have a favorable prognosis and good response to treatment compared to other AML subtypes, survival outcomes are not uniform. Indeed, 30-50% of patients with CBF-AML eventually relapse, and the 5-10 yr survival ranges between 55-61% for patients &lt; 60 yr in MRC/NCRI AML trials. Studies have analyzed the clonal architecture of CBF-AML patients and identified cooperating mutations independently of receptor tyrosine kinases (FLT3, KIT) mutations while others have found a 30% occurrence of KIT mutations. Studies of murine models of Runx1 and Cbfb have demonstrated that inactivation of both genes does not lead to leukemia, suggesting that other factors are necessary to recapitulate the leukemia phenotype fully. Although mutations in RAS family of genes (NRAS/KRAS) are among the most frequently observed mutations described in CBF-AML [54% in inv(16) and 30% in t(8;21)], no associations between those mutations and survival outcomes have been found. Because of the lack of association between RASMT and clinical outcomes, their role in CBF-AML is still unknown. Here, we focused on dissecting the impact of RAS mutations (NRAS/KRAS; RASMT) on the clinical characteristics, survival outcomes, and the molecular associations among CBF-AML patients by evaluating the clonal succession of RASMT. In total, 284 CBF-AML patients were identified, in whom inv(16) and t(8;21) represent 61% (n=173) and 39% (n=111) of the cases, respectively. Thirty-five % (99/284) of the patients carried RAS mutations (NRAS=78; KRAS=21) with 8 patients harboring 2 mutations comprising NRAS, KRAS, or both NRAS/KRAS genes. RAS mutations were point mutations affecting known hotspots in NRAS and KRAS. Both RASMT and RASWT had a median age &lt; 60 years (55 (14-83) vs. 49 (7-83) years, P=0.9) and sex was equally distributed among the two groups. Leukopenia, defined as white blood cell count &lt;3.5 x 109/L, was the only significant different hematological parameter between RASMT and RASWT. Fewer RASMT patients had leukopenia (3 vs. 12%; P=0.02). Compared to RASWT patients, RASMT patients had more often +8 (14 vs. 6%; P=0.02) and less -Y (7 vs. 16%; P=0.04) abnormalities. The analysis of 30 genes frequently mutated in myeloid malignancies revealed that RASMT patients had a significantly lower number of additional mutations compared to RASWT patients (n=59 vs. 197; P=0.03). Focusing on molecular associations, there were no differences in the frequency of FLT3 (TKD or ITD) mutations between RASMT and RASWT (15 vs. 18%). Although RASWT were more enriched with ASXL1, DNMT3A, IDH1/2, TET2, and WT1 mutations, no significant difference in frequencies was found when compared to RASMT. KIT mutations have been associated with poor outcomes in CBF-AML. In our cohort, KIT mutations (small deletions, points mutations), were significantly less associated with RASMT than RASWT (8 vs. 59%; P&lt;.0001). RASMT were ancestral events in 58% of the cases (with FLT3 mutations the most common secondary lesions), secondary in 38% (with TET2,ASXL1, RUNX1 and IDH2 mutations represented the most common dominant hits), and codominant in 11% (with KIT mutations). RASMT in CBF-AML was associated with worse OS (HR: 1.520; P=0.04) compared to RASWT. Clonal hierarchy and succession analysis showedno change in OS when RASMT were secondary vs. dominant hits in CBF-AML (HR: 0.8; P=0.5). In sum, our study of the frequency, clonal architecture and impact on survival of RASMT in CBF-AML patients points out the unique characteristics of this specific disease subgroup in which sole RASMT might represent, in addition to KITMT, a mutational lesion cooperating with inv(16) and t(8;21) rearrangements in driving leukemic evolution. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Advani:Glycomimetics: Consultancy, Research Funding; Macrogenics: Research Funding; Abbvie: Research Funding; Amgen: Research Funding; Pfizer: Honoraria, Research Funding; Kite Pharmaceuticals: Consultancy. Nazha:Abbvie: Consultancy; Incyte: Speakers Bureau; Jazz Pharmacutical: Research Funding; Tolero, Karyopharma: Honoraria; MEI: Other: Data monitoring Committee; Novartis: Speakers Bureau; Daiichi Sankyo: Consultancy. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Sekeres:Millenium: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion: Consultancy; Novartis: Consultancy.

595P - Quality of life (QoL) in patients with aflibercept (AFL) and FOLFIRI for metastatic colorectal cancer (mCRC): Interim analysis with focus on mutational status of the non-interventional study QoLiTrap (AIO-LQ-0113)

BackgroundThe anti-angiogenic fusion protein AFL targets VEGF-A, VEGF-B and PlGF. It is approved in combination with FOLFIRI for treatment of mCRC that is resistant to or has progressed after oxaliplatin-containing therapy. MethodsQoLiTrap is an ongoing non-interventional study conducted in the DACH region to evaluate Quality of Life in 1500 mCRC patients treated with AFL+FOLFIRI using the EORTC-QLQ C30 questionnaire at baseline and before every cycle. ResultsFor this interim analysis (cut-off: 02 Jan 2019) 839 patients (mean age: 64.7±9.8 years; 64.4% male, 50.9% with documented RAS mutation, ECOG 0-1: 86.0%) who completed the baseline and at least 2 post-baseline EORTC-QLQ C30 questionnaires were evaluated. AFL was administered for a median of 7 cycles to RAS-wild-type (wt) (range: 1-61) and RAS-mutated (mut) (range 1-44) patients. Of those 772 patients with a previous therapy, 56.7 % received bevacizumab (BEV), 15.3% anti-EGFR antibodies (cetuximab and/or panitumumab) and 14.6 % both agents. 42.1% of RAS-wt and 56.7% of RAS-mut patients received AFL in second line setting. Median global health score at baseline was 58.3 and decreased moderately (mean change -3.4%, p<0.0001) within the first 12 weeks of therapy. Reduction was greater in RAS-mut compared to RAS-wt (mean change -5.1% vs. -1.1%). Among evaluable patients receiving AFL in second line after anti-EGFR or BEV, 25.0% exhibited CR+PR and 51.6% had SD as best response. RAS mutational status had an impact on the rate of disease control in these patients (85.7% disease control in RAS-wt vs. 69.8% in RAS-mut; p=0.0242). Median PFS in these patients was 7.6 months (95% CI 6.3- 12.4) for RAS-wt patients and 8.2 months (95% CI 5.9- 9.0) for RAS-mut patients. Toxicity was in line with the known safety profile. ConclusionsThe current interim analysis suggests that FOLFIRI + AFL administered in routine conditions after either BEV or anti-EGFR is an active second-line therapy. Both RAS-wt and RAS-mut patients benefited from this combination. No clinically relevant decrease in global health status was observed during study treatment. Supported by Sanofi-Aventis Deutschland GmbH. Clinical trial identificationAIO-LQ-0113. Editorial acknowledgementSandra Baumgart (Alcedis GmbH Gießen, Germany), funded by Sanofi-Aventis Deutschland GmbH. Legal entity responsible for the studySanofi-Aventis Deutschland GmbH. FundingSanofi-Aventis Deutschland GmbH. DisclosureR. von Moos: Honoraria (self), Advisory / Consultancy: Amgen; Advisory / Consultancy: Bayer; Advisory / Consultancy: Eli Lilly; Advisory / Consultancy: Novartis; Advisory / Consultancy: Sanofi Aventis; Honoraria (self), Advisory / Consultancy: Roche; Advisory / Consultancy: Merck-Serono; Advisory / Consultancy, Travel / Accommodation / Expenses: Tesaro; Advisory / Consultancy: BMS; Advisory / Consultancy: MSD; Advisory / Consultancy, Travel / Accommodation / Expenses: Vifor; Travel / Accommodation / Expenses: Pfizer. F. Scholten: Honoraria (self), Travel / Accommodation / Expenses: Pharmamar; Travel / Accommodation / Expenses: Eli-Lilly; Travel / Accommodation / Expenses: Celgene; Travel / Accommodation / Expenses: Sanofi-Aventis. H. Derigs: Travel / Accommodation / Expenses: Celgene; Travel / Accommodation / Expenses: Janssen; Travel / Accommodation / Expenses: Roche. R.D. Hofheinz: Honoraria (self), Advisory / Consultancy, Research grant / Funding (self): Amgen; Honoraria (self), Advisory / Consultancy, Research grant / Funding (self): Roche; Honoraria (self), Advisory / Consultancy, Research grant / Funding (self): Merck; Honoraria (self), Advisory / Consultancy, Research grant / Funding (self): Sanofi; Honoraria (self), Advisory / Consultancy: Bayer; Honoraria (self), Advisory / Consultancy: Ipsen; Honoraria (self), Advisory / Consultancy: BMS; Honoraria (self), Advisory / Consultancy: MSD; Honoraria (self), Research grant / Funding (self): Medac; Honoraria (self): Boehringer; Honoraria (self): Celgene; Honoraria (self): Saladax; Honoraria (self): AstraZeneca; Speaker Bureau / Expert testimony: Deutsche Krebshilfe. All other authors have declared no conflicts of interest.

1953P - Detection of microRNAs as biomarker for anti-EGFR antibody resistance in colon cancer patients

BackgroundMonoclonal antibodies against the Epidermal Growth Factor Receptor (EGFR) such as cetuximab or panitumumab are used for the treatment of metastatic colorectal cancer (mCRC) patients. Unfortunately, most patients develop resistance against these therapies within months. Several studies have shown that aberrations in the RAS pathway are responsible for resistance. However, even in metastases that are refractory to anti-EGFR treatment a significant fraction of RAS wild-type (wt) cells remain. These findings suggest a cross-talk between RAS mutant and wt cells in mediating resistance in the wt compartment. MethodsWe used mouse and patient-derived organoids from mCRC as well as CRC cell lines to test the contribution of extracellular vesicles in mediating resistance in RAS wt cells. Using conditioned media, transfection experiments and liquid biopsies (plasma and urine) from patients differential expression of the let-7g microRNA was demonstrated in microvesicles from cetuximab sensitive and resistant cells. Changes in expression of the let-7g microRNA were further analysed by in-situ hybridization in tissues. ResultsBasal let-7g expression from pre-treatment plasma and urine samples of RAS wt patients correlated with clinical outcome and changes in let-7g circulating levels mirrored clinical behaviour. In-situ hybridization in tissues confirmed changes in expression of the let-7g microRNA observed in plasma and urine samples. ConclusionsOur data suggest that let-7g microRNA might function as a paracrine mediator of anti-EGFR resistance and might be exploited as a non-invasive biomarker of resistance to cetuximab treatment. Further work is ongoing to characterize the molecular mechanisms underpinning let-7g mediated effect on anti-EGFR sensitivity in RAS wt CRC cells. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureN. Valeri: Speaker Bureau / Expert testimony: Bayer; Speaker Bureau / Expert testimony: Eli Lilly; Speaker Bureau / Expert testimony: Pfizer; Speaker Bureau / Expert testimony: Merck. All other authors have declared no conflicts of interest.