EGFR Inhibitors Cetuximab Research Articles

Peptostreptococcus stomatis promotes colonic tumorigenesis and receptor tyrosine kinase inhibitor resistance by activating ERBB2-MAPK

Peptostreptococcus stomatis (P.stomatis) is enriched in colorectal cancer (CRC), but its causality and translational implications in CRC are unknown. Here, we show that P.stomatis accelerates colonic tumorigenesis in ApcMin/+ and azoxymethane/dextran sodium sulfate (AOM-DSS) models by inducing cell proliferation, suppressing apoptosis, and impairing gut barrier function. P.stomatis adheres to CRC cells through its surface protein fructose-1,6-bisphosphate aldolase (FBA) that binds to the integrin α6/β4 receptor on CRC cells, leading to the activation of ERBB2 and the downstream MEK-ERK-p90 cascade. Blockade of the FBA-integrin α6/β4 abolishes ERBB2-mitogen-activated protein kinase (MAPK) activation and the protumorigenic effect of P.stomatis. P.stomatis-driven ERBB2 activation bypasses receptor tyrosine kinase (RTK) blockade by EGFR inhibitors (cetuximab, erlotinib), leading to drug resistance in xenograft and spontaneous CRC models of KRAS-wild-type CRC. P.stomatis also abrogates BRAF inhibitor (vemurafenib) efficacy in BRAFV600E-mutant CRC xenografts. Thus, we identify P.stomatis as an oncogenic bacterium and a contributory factor for non-responsiveness to RTK inhibitors in CRC.

Palbociclib inhibits the progression of head and neck cancer and improves the Cetuximab response under specific condition in vitro and in vivo.

In head and neck squamous cell carcinoma (HNSCC) with human papillomavirus (HPV)-negative, deregulation of cell cycle is partly due to inactivation of p16INK4 and overexpression of cyclin D1. Here we investigated the in vitro and in vivo effects of the CDK4/6 inhibitor Palbociclib alone or combined with EGFR inhibitor Cetuximab in HNSCC. CCK-8, soft agar assay, colony formation assay, wound healing assay and transwell assay, β-galactosidase assay, western blotting, and cell-derived xenografts were used to investigated the in vitro and in vivo activity of drugs. Cell viability and colony formation decreased in a dose-dependent manner in Tu686, AMC-HN8, and Fadu cells under Palbociclib treatment. Palbociclib remarkably inhibited migration, invasion and the expression MMP-9 in HNSCC cells. Palbociclib also induced senescence. Palbociclib caused the dephosphorylation of RB but increased the cyclin D1 level in a dose-dependent manner. Moreover, combination with Cetuximab could significantly prevent the induction of cyclin D1 and activation of EGFR signals from Palbociclib treatment. Nevertheless, only within the range of certain concentrations, a synergistic inhibitory effect on cell growth was observed when combined with Palbociclib and Cetuximab. Although the synergistic effect in Palbociclib/Cetuximab combined therapy was comparable to that in traditional chemotherapeutic regimens (cisplatin/Cetuximab) in Fadu tumor xenograft, the combination therapy was less active than Cetuximab monotherapy in Tu686 tumor xenograft. In HPV-negative HNSCC, CDK4/6 inhibitor shows promising anti-tumor effects, which exhibits a synergistic effect when combined with EGFR inhibitor only in specific drug concentration and mouse model.

Abstract 1995: DUSP4 loss enhances resistance to encorafenib plus cetuximab in BRAF mutant colorectal cancer

Abstract The 5-year survival rate for metastatic colorectal cancer (CRC) is less than 14%. Hyperactivation of the ERK/MAPK pathway occurs at a high frequency in CRC, due to mutations (RAS, BRAF) or overexpression/amplification (EGFR) of its signalling components. Aberrant activation of this pathway drives tumour growth and survival and several inhibitors of this pathway are now clinically utilized. The BRAF V600E activating mutation occurs in ~10% of CRC patients and is correlated with an extremely poor prognosis. Use of the small molecule BRAF inhibitor encorafenib in combination with the EGFR inhibitor cetuximab is now approved for BRAF V600E mutant patients, however, the objective response rate is only ~20% and the treatment is rarely curative. Therefore, there is an urgent need to identify predictive biomarkers of response to encorafenib+cetuximab treatment. Dual-specificity phosphatase 4 (DUSP4) is rapidly induced upon activation of ERK/MAPK signaling upon which it acts as a critical negative regulator of signaling output by directly de-phosphorylating ERK. We have observed that DUSP4 is highly expressed in BRAF-mutant CRC cell lines and primary tumours, although there is a range of expression. Whether DUSP4 expression status impacts on response to encorafenib+cetuximab therapy is unknown. The aim of this study therefore was to determine the role of DUSP4 on sensitivity of BRAF-mutant CRC cells to encorafenib+cetuximab treatment in vitro. To examine this, we determined the impact of DUSP4 knock-down, knock-out and re-expression on sensitivity of BRAF-mutant CRC cancer cell lines to encorafenib+cetuximab treatment. Screening a panel of BRAF mutant CRC cell lines for sensitivity to encorafenib+cetuximab revealed a subset of resistant cell lines had either low or undetectable DUSP4 protein expression, while all sensitive cell lines had high DUSP4 expression. siRNA-mediated knockdown of DUSP4 in DUSP4 expressing BRAF-mutant CRC cell lines increased pERK and pFRA1, suggesting activation of ERK/MAPK signalling. DUSP4 knockdown also reduced sensitivity of BRAF mutant CRC cell lines to encorafenib+cetuximab compared to control. These findings were further validated using DUSP4 knock-out with CRISPR-Cas9. Conversely, DUSP4 re-expression in BRAF-mutant CRC cell lines lacking DUSP4 expression, decreased pERK and increased sensitivity to encorafenib+cetuximab treatment. These results demonstrate that DUSP4 regulates ERK/MAPK signalling in BRAF mutant CRC cell lines and that loss of DUSP4 promotes resistance to encorafenib plus cetuximab treatment. These findings warrant investigation of DUSP4 expression status as a predictive biomarker of encorafenib+cetuximab response in patient samples. Citation Format: Kristen Needham, Fiona Chionh, Stan Kaczmarczyk, Connor Kearney, Laura Jenkins, Ian Luk, John M. Mariadason. DUSP4 loss enhances resistance to encorafenib plus cetuximab in BRAF mutant colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1995.

BCL-XL inhibitors enhance the apoptotic efficacy of BRAF inhibitors in BRAFV600E colorectal cancer

Metastatic BRAFV600E colorectal cancer (CRC) carries an extremely poor prognosis and is in urgent need of effective new treatments. While the BRAFV600E inhibitor encorafenib in combination with the EGFR inhibitor cetuximab (Enc+Cet) was recently approved for this indication, overall survival is only increased by 3.6 months and objective responses are observed in only 20% of patients. We have found that a limitation of Enc+Cet treatment is the failure to efficiently induce apoptosis in BRAFV600E CRCs, despite inducing expression of the pro-apoptotic protein BIM and repressing expression of the pro-survival protein MCL-1. Here, we show that BRAFV600E CRCs express high basal levels of the pro-survival proteins MCL-1 and BCL-XL, and that combining encorafenib with a BCL-XL inhibitor significantly enhances apoptosis in BRAFV600E CRC cell lines. This effect was partially dependent on the induction of BIM, as BIM deletion markedly attenuated BRAF plus BCL-XL inhibitor-induced apoptosis. As thrombocytopenia is an established on-target toxicity of BCL-XL inhibition, we also examined the effect of combining encorafenib with the BCL-XL -targeting PROTAC DT2216, and the novel BCL-2/BCL-XL inhibitor dendrimer conjugate AZD0466. Combining encorafenib with DT2216 significantly increased apoptosis induction in vitro, while combining encorafenib with AZD0466 was well tolerated in mice and further reduced growth of BRAFV600E CRC xenografts compared to either agent alone. Collectively, these findings demonstrate that combined BRAF and BCL-XL inhibition significantly enhances apoptosis in pre-clinical models of BRAFV600E CRC and is a combination regimen worthy of clinical investigation to improve outcomes for these patients.

Unveiling the role of MGMT and DAPK hypermethylation in response to anti-EGFR agents: Molecular insights for advancing HNSCC treatment.

Epidermal growth factor receptor (EGFR) is frequently activated in head and neck squamous cell carcinoma (HNSCC) and serves as a valuable target for therapy. Despite the availability of the EGFR inhibitors Cetuximab, Afatinib, and Allitinib, there are limited predictive markers for their response. Understanding molecular aberrations in HNSCC could facilitate the identification of new strategies for patient clinical and biological classification, offering novel therapeutic avenues. We assessed CCNA1, DCC, MGMT, CDKN2A/p16, and DAPK methylation status in HNSCC cell lines and their association with anti-EGFR treatment response. MGMT methylation status displayed high sensitivity and specificity in distinguishing sensitive and resistant HNSCC cell lines to Afatinib (AUC = 0.955) and Allitinib (AUC = 0.935). Moreover, DAPK methylation status predicted response to Allitinib with high accuracy (AUC = 0.852), indicating their putative predictive biomarker roles. These findings hold promise for the development of more personalized and effective treatment approaches for HNSCC patients.

Mutant PIK3CA as a negative predictive biomarker for treatment with a highly selective PIM1 inhibitor in human colon cancer

ABSTRACT Significant improvement in targeted therapy for colorectal cancer (CRC) has occurred over the past few decades since the approval of the EGFR inhibitor cetuximab. However, cetuximab is used only for patients possessing the wild-type oncogene KRAS, NRAS, and BRAF, and even most of these eventually acquire therapeutic resistance, via activation of parallel oncogenic pathways such as RAS-MAPK or PI3K/Akt/mTOR. The two aforementioned pathways also contribute to the development of therapeutic resistance in CRC patients, due to compensatory and feedback mechanisms. Therefore, combination drug therapies (versus monotherapy) targeting these multiple pathways may be necessary for further efficacy against CRC. In this study, we identified PIK3CA mutant (PIK3CA MT) as a determinant of resistance to SMI-4a, a highly selective PIM1 kinase inhibitor, in CRC cell lines. In CRC cell lines, SMI-4a showed its effect only in PIK3CA wild type (PIK3CA WT) cell lines, while PIK3CA MT cells did not respond to SMI-4a in cell death assays. In vivo xenograft and PDX experiments confirmed that PIK3CA MT is responsible for the resistance to SMI-4a. Inhibition of PIK3CA MT by PI3K inhibitors restored SMI-4a sensitivity in PIK3CA MT CRC cell lines. Taken together, these results demonstrate that sensitivity to SMI-4a is determined by the PIK3CA genotype and that co-targeting of PI3K and PIM1 in PIK3CA MT CRC patients could be a promising and novel therapeutic approach for refractory CRC patients.

A randomized trial of consolidation-targeted adjuvant therapy with encorafenib and cetuximab versus usual care for patients with stage II/III BRAF V600E colon cancer: Alliance for Clinical Trials in Oncology A022004.

TPS3641 Background: Patients with mismatch repair proficient (pMMR) BRAF V600E mutant high-risk stage II (T4) or stage III colon cancer have a substantial risk of recurrence despite standard adjuvant therapy. In patients with metastatic BRAF V600E mutant colorectal cancer, the combination of the RAF inhibitor encorafenib and the EGFR inhibitor cetuximab has been shown to improve overall and progression-free survival compared to standard therapy after at least one prior line of therapy. This study will evaluate if this combination improves disease-free survival (DFS) in patients with resected BRAF V600E mutant pMMR/microsatellite stable (MSS) high-risk stage II (T4) or stage III colon cancer after standard adjuvant therapy. Methods: The study has a seamless phase II/III design, enrolling a maximum of 394 patients. BRAF mutation status can be determined locally or submitted for central testing in trial pre-screening. Eligible patients will be randomized 1:1 to usual surveillance versus 6 months of treatment with encorafenib (300mg oral daily) plus cetuximab (500mg/m2 intravenous every 14 days). Stratification factors are adjuvant chemotherapy regimen (FOLFOX versus CAPOX), duration of adjuvant therapy (3 months versus > 3 months), tumor stage (T4N0 versus T1-3N1 versus T4N1/TanyN2), and ctDNA marker status (detectable versus undetectable) at randomization. Adjuvant therapy must be completed at most 8 weeks prior to registration. For the phase II component, in the ctDNA detectable cohort, the primary endpoint will be ctDNA clearance rate at 6 months, and, in the ctDNA undetectable cohort, the primary endpoint will be 6-month ctDNA recurrence-free survival, where events consist of death, recurrence, or ctDNA positivity. The primary endpoint of the phase III component is DFS, and secondary endpoints are overall survival, adverse events, and alternative DFS calculated from the date of primary resection, rather than date of study randomization. Patient reported symptoms (PRO-CTCAE) will be collected and analyzed as an exploratory endpoint, and serial blood banking will support future correlative studies. The trial will proceed to the phase III portion if either of the two endpoints in the phase II cohorts is statistically superior in patients receiving encorafenib plus cetuximab. The phase III study sample size will provide 80% power at a one-sided α of 0.05 to detect an approximately 10% improvement in the three-year DFS rate, assuming 68% in the control arm. Two interim analyses will be performed for DFS endpoint. Support: U10CA180821, U10CA180882; https://acknowledgments.alliancefound.org . U10CA180820 (ECOG-ACRIN); U10CA180868 (NRG); U10CA180888 (SWOG); Pfizer, Eli Lilly; Clinicaltrials.gov ID: NCT05710406. Clinical trial information: NCT05710406 .

The five-year KRAS, NRAS and BRAF analysis results and treatment patterns in daily clinical practice in Slovenia in 1st line treatment of metastatic colorectal (mCRC) patients with RAS wild-type tumour (wtRAS) - a real- life data report 2013-2018.

We preformed a Phase IV non-interventional study to assess KRAS, NRAS and BRAF status in metastatic colorectal cancer (mCRC) patients suitable for 1st line treatment and to evaluate the decisions for 1st line treatment considering the treatment goals in the RAS wild type (wt) patients. The aim of our study was also to evaluate the influence of a waiting period for biomarkers analysis on the start of first-line treatment. Patients with histologically confirmed mCRC adenocarcinoma suitable for first-line treatment fulfilling all inclusion criteria were included in the study. The KRAS, NRAS and BRAF analysis was performed from tissue samples of primary tumor site or metastatic site. All included patients have given consent to participate in the study by signing the informed consent form. From April 2013 to March 2018 at the Institute of Oncology Ljubljana 650 patients were included, 637 of them were treated with first- line systemic treatment according to RAS and BRAF status. Remaining 13 patients with mCRC did not receive systemic first-line treatment. The distribution of patients with KRAS mutated and wild-type tumors, was almost equal, 48.8% and 47.9% respectively, 89 % of the patients had wt NRAS tumours and 86.1% had wt BRAF tumours. The most frequently prescribed treatment was bevacizumab-based therapy (53.1%), either in combination with doublet chemotherapy or with mono-chemotherapy. EGFR inhibitors cetuximab and panitumumab were prescribed in wt RAS mCRC patients (30.9%). The waiting period for biomarkers analysis was two weeks. Our real-world data, single centre 5-year analysis showed that the distribution between wild type and mutated type tumors of the patients with mCRC was approximately the same, as worldwide, so the Slovenian population with mCRC has the same ratio distribution of KRAS, NRAS and BRAF wild and mutated genes. We concluded that a two-week waiting period for biomarkers analysis did not influence the first line treatment decision, so it was in the accordance with the worldwide treatment guidelines based on evidence-based medicine.

A phase I study of avelumab, palbociclib, and cetuximab in patients with recurrent or metastatic head and neck squamous cell carcinoma

ObjectivesWe aimed to test the safety of the CDK4/6 inhibitor palbociclib in combination with the EGFR inhibitor cetuximab and the PD-L1 inhibitor avelumab in recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC). Materials and MethodsThis phase I study enrolled eligible adult patients with R/M HNSCC into three sequential single dose-escalation cohorts of palbociclib (75, 100, and 125 mg) PO daily on days 1 to 21 of a 28-day cycle in combination with avelumab 10 mg/kg IV every 2 weeks and cetuximab 400 mg/m2IV on day 1, then 250 mg/m2weekly thereafter. The study followed a 3 + 3 design with no intra-patient escalation. The primary objective was to identify the recommended phase II dose (RP2D); secondary objectives included overall response rate (ORR), duration of response (DOR), progression free survival (PFS), and overall survival (OS). ResultsPalbociclib in combination with avelumab and cetuximab was well tolerated, with rash and fatigue being the most common adverse events. A single dose-limiting toxicity was observed at the 125 mg dose of palbociclib: a grade 3 infusion reaction related to cetuximab. The RP2D of palbociclib is 125 mg, with avelumab and cetuximab at standard doses. The ORR by RECIST v1.1 was 42 %, the median DOR and OS have not been reached. Median PFS was 6.5 months. ConclusionsThe combination of avelumab, cetuximab, and palbociclib was well tolerated and supports further evaluation in patients with R/M HNSCC.Clinical Trial Registration Number: NCT03498378.

38 (PB028) - Establishment and characterization of a panel of BRAFV600E colorectal XPDX models from patients with acquired resistance to targeted therapies

Background: The BRAF V600E mutation occurs in up to 20% of patients with metastatic colorectal cancer (CRC). Combination of the BRAF inhibitor encorafenib with the EGFR inhibitor cetuximab was recently approved for V600E-mutated metastatic CRC cancer as second-line therapy. While this treatment provides needed benefit to BRAFV600E-mutated CRC patients, some patients will progress on therapy with few viable options. To this end we have established four XenoSTART Patient-Derived Xenograft (XPDX) models from CRC BRAFV600E patients clinically refractory to targeted therapies. These models, designated ST5111, ST5986, STM054 and STM110C, were characterized for receptor expression, genomic alterations, and in vivo drug sensitivities toward multiple chemotherapies and targeted agents. Methods: ST5111 was established from a liver biopsy collected from a 69-year-old Caucasian female following treatment with FOLFOX (2 m), dabrafenib/trametinib (21 m), encorafenib /binimetinib (14 m) and radiation. ST5986 was established from a vaginal biopsy collected from a 72-year-old Hispanic female following treatment with capecitabine (9 m), pembrolizumab (6 m), and encorafenib/panitumumab (9 m). STM054 was established from fluid collected from a 53-year-old female following treatment with FOLFOX/ beva (3 m), FOLFOXIRI (4 m) and encorafenib/binimetinib/cetuximab (10 m). STM110C was established from fluid collected from a 52-year-old male following treatment with FOLFOX/beva (3 m), FOLFIRI (3 m), encorafenib/cetuximab (2 m) and an investigation therapy plus encorafenib/cetuximab (7 m). The resulting models were passaged and characterized using genomic analysis including WES and RNAseq. In vivo studies were performed testing therapies; study endpoints included tumor volume and time from treatment initiation with %T/C values and tumor regression reported at study completion; a %T/C of £20 versus control was considered sensitive. Tumor regression (%T/C < 0) versus Day 0 tumor volume was also reported. Results: Genomic analysis of models confirmed BRAFV600E in all models as well as several co-mutations including BRCA2T2685fs in ST5986, MCL1E110del in STM054 and PIK3CAQ546E in STM110C. In vivo models were resistant to encorafenib plus binimetinib but adding cetuximab significantly improved response. Single agent trametinib but not binimetinib was active in two of four models and dabrafenib alone or in combination did not provide benefit in any model. Sacituzumab and alpelisib alone reported some activity towards STM110C but HER2 therapies T-DM1 or DS-8201a were not effective. Conclusion: We established and characterized a panel of four CRC BRAFV600E models from patients with acquired resistance to BRAF/MEK/ EGFR targeted therapies. These models are useful for evaluating next generation therapies and elucidating mechanisms of resistance to targeted therapies in V600E CRC. No conflict of interest.

Antiproliferative Effects of EGFR inhibitor Cetuximab and PARP Inhibitor Combination on Non-Small Cell Lung Cancer Cell Line A549 and Cervical Cancer Cell Line HeLa.

In this study, the efficacy of Cetuximab and Parp inhibitor (Parp 1 inhibitor) used in targeted therapies, alone or in combination, on non-small cell lung cancer cell line A549 and cervical cancer cell line HeLa cells were evaluated. For this purpose different cell kinetic parameters were used. Cell viability, mitotic index, BrdU labelling index and apoptotic index were evaluated in experiments. In single applications Cetuximab at concentrations ranging from 1 mg/ml to 10 mg/ml and Parp inhibitor at concentrations 5 µM -7 µM - 10 µM were applied. IC50 concentration of Cetuximab for A549 was 1 mg/ml, the IC50 concentration of Cetuximab for HeLa was 2 mg/ml, the IC50 concentration of Parp inhibitor for A549 was 5 µM, and the IC50 concentration of Parp inhibitor for HeLa was 7 µM. In both single and combinations, there was a significant decrease in cell viability, mitotic index, BrdU labelling index and there was a significant increase in apoptotic index. A comparison of cetuximab, PARPi and combination applications showed the superiority of combined applications over single applications in all cell kinetic parameters used.

BREAKWATER safety lead-in (SLI): Encorafenib + cetuximab (EC) ± chemotherapy for first-line (1L) treatment (tx) of BRAF V600E-mutant (BRAFV600E) metastatic colorectal cancer (mCRC).

134 Background: Currently, there are no 1L tx options indicated specifically for patients (pts) with BRAFV600E mCRC. Based on results of BEACON CRC (NCT02928224), BRAF inhibitor encorafenib + EGFR inhibitor cetuximab was approved for tx of previously treated pts with BRAFV600E mCRC. BREAKWATER (NCT04607421), an ongoing, open-label, global, multicenter, randomized phase 3 study, evaluates 1L EC ± chemotherapy for tx of pts with BRAFV600E mCRC. Here we present preliminary data on safety and pharmacokinetics (PK) from the BREAKWATER SLI, which aimed to identify the chemotherapy backbone for EC for the phase 3 portion of BREAKWATER. Methods: SLI inclusion criteria were BRAFV600E mCRC (determined using tumor tissue or blood); evaluable disease (RECIST v1.1); ≤1 prior systemic tx for mCRC; European Cooperative Oncology Group performance status (ECOG PS) 0/1; and adequate bone marrow, hepatic, and renal function. Pts previously treated with BRAF/EGFR inhibitors or both oxaliplatin and irinotecan were excluded. Pts received encorafenib 300 mg daily + cetuximab 500 mg/m2 every 2 weeks (Q2W) + either FOLFIRI Q2W or mFOLFOX6 Q2W in 28-day cycles. The primary endpoint was frequency of dose-limiting toxicities (DLTs). PK were a secondary endpoint. Data cutoff date: Sep 13, 2021. Results: 57 pts were enrolled (EC + FOLFIRI, n = 30; EC + mFOLFOX6, n = 27). Median (range) age was 57 (28–78) years; 25% were Asian; 65% had ECOG PS 0; 37% had ≥3 organs involved; 58% were treatment naive. At cutoff date, tx was ongoing in 45 (79%) pts. Median (range) duration of tx for encorafenib in EC + FOLFIRI and EC + mFOLFOX6 was 15 (0–31) and 14 (0–27) weeks, respectively. One DLT was observed: grade 4 neutropenia in 1 pt in EC + FOLFIRI. Tx-emergent all-cause serious adverse events (AEs) occurred in 20% and 19% and grade ≥3 AEs in 33% and 56% of pts in EC + FOLFIRI and EC + mFOLFOX6, respectively. The table shows frequent (all grade in ≥30% pts or grade ≥3 in ≥10% with either tx) tx-emergent all-cause AEs. One pt died due to disease progression. In EC + FOLFIRI, in the presence of steady-state encorafenib, AUCinf of irinotecan and its active metabolite, SN-38, significantly decreased ̃25% and ̃40%, respectively, compared with values in the absence of encorafenib. In EC + mFOLFOX6, oxaliplatin PK was not significantly altered by steady-state encorafenib. Conclusions: Based on these data, BREAKWATER phase 3 will compare EC ± mFOLFOX6 with mFOLFOX6/FOLFOXIRI/CAPOX ± bevacizumab. Clinical trial information: NCT04607421. [Table: see text]

EVI1 Promotes the Proliferation and Invasive Properties of Human Head and Neck Squamous Cell Carcinoma Cells.

Head and neck squamous cell carcinoma (HNSCC) is a frequent malignancy with a poor prognosis. So far, the EGFR inhibitor cetuximab is the only approved targeted therapy. A deeper understanding of the molecular and genetic basis of HNSCC is needed to identify additional targets for rationally designed, personalized therapeutics. The transcription factor EVI1, the major product of the MECOM locus, is an oncoprotein with roles in both hematological and solid tumors. In HNSCC, high EVI1 expression was associated with an increased propensity to form lymph node metastases, but its effects in this tumor entity have not yet been determined experimentally. We therefore overexpressed or knocked down EVI1 in several HNSCC cell lines and determined the impact of these manipulations on parameters relevant to tumor growth and invasiveness, and on gene expression patterns. Our results revealed that EVI1 promoted the proliferation and migration of HNSCC cells. Furthermore, it augmented tumor spheroid formation and the ability of tumor spheroids to displace an endothelial cell layer. Finally, EVI1 altered the expression of numerous genes in HNSCC cells, which were enriched for Gene Ontology terms related to its cellular functions. In summary, EVI1 represents a novel oncogene in HNSCC that contributes to cellular proliferation and invasiveness.

THE IMPORTANCE OF CIRCULATING TUMOR DNA IN THE ASSESSMENT OF METASTATIC COLORECTAL CANCER TREATMENT EFFECTIVNE

Challenges in cancer detection, prognosis and management are currently being solved by determining circulating tumor DNA (ctDNA). The assessment of this marker has acquired particular importance in metastatic colorectal cancer (mCRC), the systemic treatment of which depends on the RAS gene status, which has prognostic and predictive value. However, the possibilities of taking samples from the primary or metastatic lesion for pathomorphological and molecular analysis in CRC are often limited. The determination of ctDNA using liquid biopsy has an advantage over standard biopsy due to its low invasiveness and high availability of the method. Analysis of mutations using ctDNA as well as changes in the level of this marker is a criterion for the effectiveness of systemic treatment, as well as a factor that determines the risk of disease progression. Currently, the potential of using ctDNA to monitor effectiveness of first-and second-line chemotherapy, as well as to predict the development of secondary resistance to EGFR inhibitors (cetuximab and panitumumab) in the first-line treatment and assessment of RAS status for returning to therapy with EGFR inhibitors in the third-line treatment of mCRC is being studied. Several pilot studies have provided evidence of the efficacy of EGFR re-treatment. The modern literature data published in leading peer-reviewed journals in Russian and international scientific citation databases, such as Medline, Elibrary, and PubMed were analyzed. Of the 138 analyzed publications, 56 were used to write this review.

APC and TP53 Mutations Predict Cetuximab Sensitivity across Consensus Molecular Subtypes.

Simple SummaryColorectal cancer (CRC) is a major cause of cancer deaths. Cetuximab is an FDA-approved, underutilized therapeutic targeting the epidermal growth factor receptor (EGFR) in metastatic CRC. To date, despite selection of patients with wild-type RAS, it is still difficult to identify patients who may benefit from EGFR inhibitor (e.g., cetuximab) therapy. Our aim is to molecularly classify CRC patients to better identify subpopulations sensitive to EGFR targeted therapy. APC and TP53 are two major tumor suppressor genes in CRC whose mutations contribute to tumor initiation and progression and may identify cetuximab-sensitive tumors. Recently, it has been suggested that the consensus molecular subtype (CMS) classification may be used to help identify cetuximab-sensitive patients. Here, we report an analysis of multiple CRC tumor/PDX/cell line datasets using combined APC and TP53 mutations to refine the CMS classification to better predict responses to cetuximab to improve patient outcomes.Recently, it was suggested that consensus molecular subtyping (CMS) may aide in predicting response to EGFR inhibitor (cetuximab) therapies. We recently identified that APC and TP53 as two tumor suppressor genes, when mutated, may enhance cetuximab sensitivity and may represent easily measured biomarkers in tumors or blood. Our study aimed to use APC and TP53 mutations (AP) to refine the CMS classification to better predict responses to cetuximab. In total, 433 CRC tumors were classified into CMS1-4 subtypes. The cetuximab sensitivity (CTX-S) signature scores of AP vs. non-AP tumors were determined across each of the CMS classes. Tumors harboring combined AP mutations were predominantly enriched in the CMS2 class, and to a lesser degree, in the CMS4 class. On the other hand, AP mutated CRCs had significantly higher CTX-S scores compared to non-AP CRCs across all CMS classes. Similar results were also obtained in independent TCGA tumor collections (n = 531) and in PDMR PDX/PDO/PDC models (n = 477). In addition, the in vitro cetuximab growth inhibition was preferentially associated with the CMS2 cell lines harboring A/P genotypes. In conclusion, the AP mutation signature represents a convenient biomarker that refines the CMS classification to identify CRC subpopulations predicted to be sensitive to EGFR targeted therapies.

Tipifarnib enhances anti-EGFR activity of cetuximab in non-HRas mutated head and neck squamous cell carcinoma cancer (HNSCC)

ObjectiveTo test the potential ability of tipifarnib to impair proliferation and to enhance the activity of the EGFR inhibitor cetuximab in wild-type H-Ras HNSCC, which accounts for the majority of HNSCC. Materials and MethodsCell growth, apoptosis and signaling changes in HNSCC cells following tipifarnib exposure in vitro were assessed by SRB, colony formation assay, annexin V staining and Western blot, respectively. A patient-derived xenograft (PDX) animal model was adopted to evaluate the efficacy of tipifarnib in vivo with and without cetuximab. ResultsTreatment of wild-type H-Ras HNSCC cell lines in vitro with tipifarnib reduced cell growth and increased levels of defarnesylated H-Ras in a dose-dependent manner. In a PDX mouse model, treatment with single-agent tipifarnib led to only near-significant growth inhibition. The addition of cetuximab resulted in increased anti-proliferative effect both in culture and in PDX models, which was also mirrored by Western blot and apoptosis assay results. ConclusionTipifarnib has only a moderate ability to slow tumor growth as a single agent in HNSCC with wild type H-Ras, despite specifically inhibiting the farnesyltransferase upon which the function of H-Ras depends. The combination of cetuximab and tipifarnib appears to enhance the anti-proliferative effect of single-agent tipifarnib and marginally enhance that of single agent cetuximab. These findings deserve further evaluation.

KRAS, NRAS and BRAF mutational status and first line treatment patterns in Slovenian population with metastatic colorectal carcinoma: Five year results.

e15546 Background: A phase IV non-interventional study was performed from 2013 till 2018 including 650 patients with primary aim to assess KRAS, NRAS and BRAF mutational status in Slovenian population with metastatic colorectal carcinoma (mCRC) suitable for first-line treatment. The evaluation of decisions for first-line treatment regarding the biomarkers status and assessing the possible impact of the time period of the biomarker status analysis report on the treatment decision were also incorporated in the analysis. The molecular analyses for KRAS and NRAS gene mutations were performed on exons 2, 3 and 4, and for BRAF gene mutations on exon 15. The first line systemic treatment options for RAS (KRAS/NRAS) wild type (wt) and mutated type (mt) mCRC subjects were as follows: chemotherapy - Fluoropyrimidine based systemic therapy combined with oxaliplatin and/or irinotecan with/without VEGF inhibitor bevacizumab and for RAS wt subjects, with/without EGFR inhibitors, cetuximab or panitumumab. Methods: To indicate the degree of certainty of KRAS, NRAS and BRAF status frequency as being wild type or mutant type 95% confidence interval was calculated. Results: The KRAS/NRAS/BRAF mutation rates were as follows - The distribution of subjects with KRAS mutated and wild-type tumors, was almost equal, 48.8% and 47.9% respectively. Eighty nine percent of the subjects had NRAS wild type tumours and 86.1% had BRAF wild type tumours. The most frequently used treatment regardless the biomarkers status and in accordance with the treatment guidelines was bevacizumab based combination therapy (53.1%). The EGFR inhibitor (cetuximab or panitumumab) based combination therapy was used in one third of mCRC subjects (30.9%), all with mCRC RAS wt. The time period from the initial presentation of the patient until the biomarker status analysis report was two weeks. Conclusions: With this study, we have proven that the distribution of the mutations in exons 2-4 of KRAS and NRAS genes and exon 15 in the BRAF gene in the Slovenian population with metastatic colorectal cancer matches historical data. Based on this, we conclude that the treatment decision in Slovenian population with metastatic colorectal carcinoma should be in the accordance with international treatment guidelines and on evidence based medicine. The molecular analysis performed at the Institute of Oncology Ljubljana was providing necessary biomarkers status report in an acceptable time that didn’t affect the treatment decision or delay the needed cancer treatment.

Penile Cancer-Derived Cells Molecularly Characterized as Models to Guide Targeted Therapies.

Penile cancer (PeCa) is a common disease in poor and developing countries, showing high morbidity rates. Despite the recent progress in understanding the molecular events involved in PeCa, the lack of well-characterized in vitro models precludes new advances in anticancer drug development. Here we describe the establishment of five human primary penile cancer-derived cell cultures, including two epithelial and three cancer-associated fibroblast (CAF) cells. Using high-throughput genomic approaches, we found that the epithelial PeCa derived- cells recapitulate the molecular alterations of their primary tumors and present the same deregulated signaling pathways. The differentially expressed genes and proteins identified are components of key oncogenic pathways, including EGFR and PI3K/AKT/mTOR. We showed that epithelial PeCa derived cells presented a good response to cisplatin, a common therapeutic approach used in PeCa patients. The growth of a PeCa-derived cell overexpressing EGFR was inhibited by EGFR inhibitors (cetuximab, gefitinib, and erlotinib). We also identified CAF signature markers in three PeCa-derived cells with fibroblast-like morphology, indicating that those cells are suitable models for PeCa microenvironment studies. We thus demonstrate the utility of PeCa cell models to dissect mechanisms that promote penile carcinogenesis, which are useful models to evaluate therapeutic approaches for the disease.

Abstract 37: Computational analysis of the KRAS G13D colorectal cancer response to EGFR inhibition with an alternatively parameterized model

Abstract Controversially, KRAS G13D colorectal cancer patients have been reported to benefit from treatment with the EGFR inhibitor cetuximab. That EGFR inhibitors could somehow reduce signaling through a constitutively active KRAS mutant has been taken as inconsistent with known mechanisms of RAS biology. Through a combination of computational modeling and experimentation, we recently demonstrated that EGFR inhibition results in a reduction of wild-type (HRAS and NRAS) signaling in KRAS G13D colon cancer cells, but not in colon cancer cells with the other common KRAS mutants, G12D and G12V. We demonstrated this in multiple colon cancer cell lines, and we developed the hypothesis originally from our mathematical model of oncogenic RAS signal regulation adapted to biochemical rate constants for the KRAS G13D mutant. Here, we develop and analyze an alternatively parameterized version of the computational model. Analysis of this alternative model similarly suggests that EGFR inhibition causes a greater reduction of wild-type RAS signal in KRAS G13D cancers. This further substantiates our findings, analogous to demonstration in an additional cell line or an additional mouse model. In addition to reinforcing our proposed mechanism, this work also further demonstrates the power of mechanism-based computational models of protein network biochemistry in personalized cancer medicine. Note: This abstract was not presented at the conference. Citation Format: Thomas McFall, Edward C. Stites. Computational analysis of the KRAS G13D colorectal cancer response to EGFR inhibition with an alternatively parameterized model [abstract]. In: Proceedings of the AACR Special Conference on Advancing Precision Medicine Drug Development: Incorporation of Real-World Data and Other Novel Strategies; Jan 9-12, 2020; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_1):Abstract nr 37.

Preclinical Evaluation of Cetuximab and Benzoporphyrin Derivative-Mediated Intraperitoneal Photodynamic Therapy in a Canine Model.

Peritoneal carcinomatosis (PC) can occur as an advanced consequence of multiple primary malignancies. Surgical resection, radiation or systemic interventions alone have proven inadequate for this aggressive cancer presentation, since PC still has a poor survival profile. Photodynamic therapy (PDT), in which photosensitive drugs are exposed to light to generate cytotoxic reactive oxygen species, may be an ideal treatment for PC because of its ability to deliver treatment to a depth appropriate for peritoneal surface tumors. Additionally, epidermal growth factor receptor (EGFR) signaling plays a variety of roles in cancer progression and survival as well as PDT-mediated cytotoxicity, so EGFR inhibitors may be valuable in enhancing the therapeutic index of intraperitoneal PDT. This study examines escalating doses of benzoporphyrin derivative (BPD)-mediated intraperitoneal PDT combined with the EGFR-inhibitor cetuximab in a canine model. In the presence or absence of small bowel resection (SBR) and cetuximab, we observed a tolerable safety and toxicity profile related to the light dose received. Additionally, our findings that BPD levels are higher in the small bowel compared with other anatomical regions, and that the risk of anastomotic failure decreases at lower light doses will help to inform the design of similar PC treatments in humans.