anti-EGFR Drugs Research Articles

Anti-VEGFR therapy is one of the healing inhibitors of antiresorptive-related osteonecrosis of the jaw

Antiresorptive-related osteonecrosis of the jaw (ARONJ) is a rare but serious adverse event associated with bone-modifying agents (BMAs) and affects patients in the terminal stages of cancer. Molecular targeting drugs (MTDs), anti-vascular endothelial growth factor receptor (VEGFR), and anti-epidermal growth factor receptor (EGFR) drugs are essential in various cancer treatments, although MTDs are risk factors for ARONJ. However, the mechanism through which MTDs affect treatment prognosis of ARONJ remains unclear. Therefore, we investigated the potential inhibitory factors for healing in the conservative therapy of ARONJ with a focus on MTDs. Sixty patients who were administered BMAs for the treatment of malignancies and who underwent conservative treatment for ARONJ were assessed. The healing rate of ARONJ for each risk factor was retrospectively evaluated. Among the 60 patients, 27 were male and 33 were female. The median age was 67years, and the median follow-up period was 292 (range 91-1758) days. The healing rate was lower in those treated with both zoledronic acid (Za) and denosumab (Dmab) than in those treated with Za or Dmab alone (0% vs. 28.8%, p = 0.03). Regarding the administration of MTDs, the treatment rate with anti-VEGFR drugs was 7.1% (p = 0.04), anti-EGFR drugs was 12.5% (p = 0.18), and without MTDs was 36.8%. In the conservative treatment of ARONJ, the administration of several BMAs and anti-VEGFR drugs was the factor contributing to the inhibition of healing.

AXL is a predictor of poor survival and of resistance to anti-EGFR therapy in RAS wild-type metastatic colorectal cancer

BackgroundRAS mutations are the only validated biomarkers in metastatic colorectal cancer (mCRC) for anti-epidermal growth factor receptor (EGFR) therapy. Limited clinical information is available on AXL expression, marker of epithelial to mesenchymal transition, in mCRC. MethodsAXL was retrospectively assessed by immunohistochemistry in 307 patients. RAS wild-type (WT) patients (N = 136) received first-line anti-EGFR–based therapy; RAS mutant patients (N = 171) received anti-angiogenic–based regimens.Preclinical experiments were performed using human RAS WT CRC cell lines and xenograft models. AXL RNA levels were assessed in a cohort of patients with available samples at baseline and at progression to anti-EGFR treatment and in the GSE5851 dataset. ResultsAXL was expressed in 55/307 tumour tissues, correlating with worse survival in the overall population (AXL-positive, 23.7 months; AXL-negative, 30.8 months; HR, 1.455, P = 0.032) and in RAS WT patients (AXL-positive, 23.0 months; AXL-negative, 35.8 months; HR,1.780, P = 0.032). Progression-free survival (PFS) in the RAS WT cohort was shorter in the AXL-positive cohort (6.2 months versus 12.1 months; HR, 1.796, P = 0.013). Three-dimensional cultures obtained from a patient following anti-EGFR therapy resulted AXL-positive, showing resistance to anti-EGFR drugs and sensitivity to AXL inhibition. AXL transfection in CRC cell lines induced AXL overexpression and resistance to the EGFR blockade. At progression to cetuximab, 2/10 SW48-tumour xenograft mice showed AXL expression. Consistently, AXL RNA levels increased in 5/7 patients following anti-EGFR therapy. Moreover, in the GSE5851 dataset higher AXL RNA levels correlated with worse PFS with cetuximab in KRAS-exon2 WT chemorefractory patients. ConclusionsAXL is a marker of poor prognosis in mCRC with consistent clinical and preclinical evidences of involvement in primary and acquired resistance to anti-EGFR drugs in RAS WT patients.

Abstract 1873: Drivers of secondary resistance to anti-EGFR therapy in metastatic colorectal cancer

Abstract Secondary Resistance (SR) evolves in virtually all metastatic colorectal cancers (mCRC) treated with anti-EGFR targeting drugs such as Cetuximab (Cmab). Yet, SR patient-derived xenograft (pdx) models are largely lacking. Here, we established eight SR pdx models through chronic Cmab treatment of drug-sensitive mCRC pdx. All pdx models were initially wild-type for KRAS, NRAS, PI3K and BRAF, as required for Cmab treatment. Post SR establishment, two models showed KRAS mutations and were excluded from further studies. Whole exome sequencing of the remaining six SR pdx models did not reveal any known driver mutations which could confer SR. However, global gene expression analyses identified widespread transcriptional reprograming with many genes able to activate KRAS and/or PI3K signaling pathways. Among the highly dysregulated genes, fibroblast growth factors FGF13, FGF19, FGF20, and insulin-like growth factor IGF2 stood out as potential drivers of SR through upregulation of RTK signaling. Functional molecular analyses of these factors were performed using lentiviral vector mediating gene transduction. Cell viability, proliferation assays and western blot (WB) analyses were applied to assess the molecular functionality of FGFs and IGF2-ligands in vitro. Finally, transgenic cell lines were injected into nude mice to test their therapeutic response towards Cmab treatment following upregulation or downregulation of the chosen candidates. Stable lentiviral overexpression of FGF13 and IGF2 in drug-sensitive cell lines and pdx models triggered SR both in vitro and in vivo. WB analyses showed that FGF13 and IGF2 overexpression induced phosphorylation of pAKT, pS6, and pERK. Conversely, knockdown of IGF2 in a cell line with high endogenous IGF2 expression improved Cmab sensitivity. Lastly, when subjecting a Cmab SR pdx model with high endogenous overexpression of FGF13 and FGF19 to a Pan-FGF inhibitor (LY2874455) in combination with Cmab, we observed a conversion of the SR into partial response. Taken together, these results support a role of IGF2 and FGFs as candidate proteins conferring SR in an autocrine fashion in the setting of KRAS wild-type mCRC under chronic anti-EGFR treatment. Importantly, these results also suggest that potential combination therapies of Cmab and an IGF2 or FGF inhibitor could prevent establishment of SR through these pathways and offer new treatment opportunities for patients with SR mCRC. Citation Format: Soha Hussein Noseir, Stephan Hahn, Abdelouahid Maghnouj, Swetlana Ladigan. Drivers of secondary resistance to anti-EGFR therapy in metastatic colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1873.

Abstract 1935: The NEDD8 and EGFR pathways are independent therapeutic targets in colorectal cancer

Abstract Colorectal cancer (CRC) develops and progresses through an accumulation of genetic and epigenetic alterations in multiple molecular pathways. Currently, targeted therapies combined with standard chemotherapy are the first-line therapy for metastatic CRC; nevertheless, the effectiveness of these drugs is limited by primary and acquired resistance. Since pathways are highly interconnected, the development of rational combinations of targeted therapies can be exploited to circumvent resistance mechanisms. As a possible alternative pathway for CRC treatment, an attractive option is the blockade of the NEDD8-ubiquitin like protein conjugation pathway. We previously found that the NEDD8-inhibitor pevonedistat has significant in vitro and in vivo activity on a subset of CRCs. However, the in vivo response is limited to tumor stabilization rather than regression, highlighting the need for therapeutic combinations. When the optimal drug combination cannot be readily predicted, functional genetic screens represent a powerful tool for unbiased exploration. We therefore carried out a synthetic lethality screening with an shRNA library covering 200 genes associated with FDA-approved drugs. The screening was aimed at identifying shRNAs that would be depleted only in the presence of pevonedistat: such constructs were expected to target genes that could harbor synergistic effects with NEDD8 inhibition. After shRNA library transduction, CRC cell lines were grown in the absence or presence of low-dose pevonedistat; Next Generation Sequencing and bioinformatics analysis allowed comparing the transduced library repertoire with or without pevonedistat, and identifying candidate synthetic lethal genes. The screening results revealed a strong, pevonedistat-dependent depletion of constructs targeting different tyrosine kinases (i.e. EGFR, BRAF, FYN and FLT4) in specific CRC cell lines (CAR1, WIDR, LIM2099 and DIFI, respectively). Firstly, we focused on the remarkable drop-out of EGFR-targeting shRNAs in CAR1 cells, which represent a subgroup of aggressive tumors refractory to cetuximab treatment, despite the lack of currently known markers of resistance. We discovered an independent and additive effect of anti-EGFR drugs (cetuximab and lapatinib) combined with pevonedistat, which was then validated in vitro on further cell lines (HCA7 and HROC69) and in vivo on HCA7-transplanted mice. Furthermore, we considered the depletion of constructs targeting BRAF, a member of EGFR pathway, observed in WIDR BRAF-mutant cells: we found an additive effect of BRAF inhibitor vemurafenib and pevonedistat in reducing in vitro cell growth. Altogether our results suggest that the concomitant pharmacological inhibition of NEDD8 and EGFR-pathway could be a novel effective approach to treat clinically aggressive CRCs, worthy of further characterization. Citation Format: Federica Invrea, Alessandro Carugo, Consalvo Petti, Cristopher Bristow, Michael Peoples, Carlotta Cancelliere, Alessia Corrado, Alberto Bardelli, Claudio Isella, Giulio F. Draetta, Enzo Medico. The NEDD8 and EGFR pathways are independent therapeutic targets in colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1935.

Novel Somatic Genetic Variants as Predictors of Resistance to EGFR-Targeted Therapies in Metastatic Colorectal Cancer Patients.

Background: About 40% of RAS/BRAF wild-type metastatic colorectal cancer (mCRC) patients undergoing anti-EGFR-based therapy have poor outcomes. Treatment failure is not only associated with poorer prognosis but higher healthcare costs. Our aim was to identify novel somatic genetic variants in the primary tumor and assess their effect on anti-EGFR response. Patients and Methods: Tumor (somatic) and blood (germline) DNA samples were obtained from two well-defined cohorts of mCRC patients, those sensitive and those resistant to EGFR blockade. Genetic variant screening of 43 EGFR-related genes was performed using targeted next-generation sequencing (NGS). Relevant clinical data were collected through chart review to assess genetic results. Results: Among 61 patients, 38 were sensitive and 23 were resistant to treatment. We identified eight somatic variants that predicted non-response. Three were located in insulin-related genes (I668N and E1218K in IGF1R, T1156M in IRS2) and three in genes belonging to the LRIG family (T152T in LRIG1, S697L in LRIG2 and V812M in LRIG3). The remaining two variants were found in NRAS (G115Efs*46) and PDGFRA (T301T). We did not identify any somatic variants related to good response. Conclusions: This study provides evidence that novel somatic genetic variants along the EGFR-triggered pathway could modulate the response to anti-EGFR drugs in mCRC patients. It also highlights the influence of insulin-related genes and LRIG genes on anti-EGFR efficacy. Our findings could help characterize patients who are resistant to anti-EGFR blockade despite harboring RAS/BRAF wild-type tumors.

Metformin selectively inhibits metastatic colorectal cancer with the KRAS mutation by intracellular accumulation through silencing MATE1

Metastatic colorectal cancer (mCRC) patients have poor overall survival despite using irinotecan- or oxaliplatin-based chemotherapy combined with anti-EGFR (epidermal growth factor receptor) drugs, especially those with the oncogene mutation of KRAS Metformin has been reported as a potentially novel antitumor agent in many experiments, but its therapeutic activity is discrepant and controversial so far. Inspiringly, the median survival time for KRAS-mutation mCRC patients with diabetes on metformin is 37.8 mo longer than those treated with other hypoglycemic drugs in combination with standard systemic therapy. In contrast, metformin could not improve the survival of mCRC patients with wild-type KRAS Interestingly, metformin is preferentially accumulated in KRAS-mutation mCRC cells, but not wild-type ones, in both primary cell cultures and patient-derived xenografts, which is in agreement with its tremendous effect in KRAS-mutation mCRC. Mechanistically, the mutated KRAS oncoprotein hypermethylates and silences the expression of multidrug and toxic compound extrusion 1 (MATE1), a specific pump that expels metformin from the tumor cells by up-regulating DNA methyltransferase 1 (DNMT1). Our findings provide evidence that KRAS-mutation mCRC patients benefit from metformin treatment and targeting MATE1 may provide a strategy to improve the anticancer response of metformin.

Gene editing particle system as a therapeutic approach for drug-resistant colorectal cancer

The epidermal growth factor receptor (EGFR) pathway plays an important role in the progression of colorectal cancer (CRC). Anti-EGFR drugs based on antibodies have been widely used for treating CRC through inducing the cell death pathway. However, the majority of CRC patients will inevitably develop drug-resistance during anti-EGFR drug treatment, which is mainly caused by a point mutation in the KRAS oncogene. We developed a nanoliposomal (NL) particle containing the Cas9 protein and a single-guide RNA (sgRNA) complex (Cas9-RNP), for genomic editing of the KRAS mutation. The NL particle is composed of bio-compatible lipid compounds that can effectively encapsulate Cas9-RNP. By modifying the NL particle to include the appropriate antibody, it can specifically recognize EGFR expressing CRC and effectively deliver the gene-editing complexes. The conditions of NL treatment were optimized using a KRAS mutated CRC in vivo mouse model. Mice with KRAS-mutated CRC showed drug resistance against cetuximab, a therapeutic antibody drug. After treating the mice with the KRAS gene-editing NL particles, the implanted tumors showed a dramatic decrease in size. Our results demonstrated that this genomic editing complex has great potential as a therapeutic carrier system for the treatment of drug-resistant cancer caused by a point mutation.

Is There an Optimal Choice in Refractory Colorectal Cancer? A Network Meta-Analysis

BackgroundIn the absence of head-to-head comparison studies, the present network meta-analysis evaluated and compared the efficacy of 4 therapeutic alternatives for refractory colorectal cancer. Materials and MethodsThe search focused on results from phase III randomized controlled trials. Separate (subgroup) network meta-analyses were conducted to obtain drug comparisons stratified by various patient characteristics. The principal outcome of interest was overall survival (OS). ResultsNo difference in OS was found between regorafenib and TAS-102. For a rectal primary location, TAS-102 conferred benefit versus placebo (hazard ratio [HR], 0.671), but regorafenib did not (HR, 0.950). For patients aged > 65 years, TAS-102 showed benefit versus placebo (HR, 0.579) but regorafenib did not (HR, 0.816). For patients with an Eastern Cooperative Oncology Group (ECOG) performance status (PS) of 0 in the indirect comparison, regorafenib showed benefit versus placebo (HR, 0.687), as did TAS-102 (HR, 0.756) but with a lower advantage. For patients with RAS wild type not previously treated with anti-EGFR antibodies, panitumumab was the optimal choice for OS. ConclusionsNo differences in OS were found between regorafenib and TAS-102. Possible greater efficacy was found for TAS-102 compared with regorafenib for patients with a rectal primary location, ECOG PS > 0, and age > 65 years. In contrast, regorafenib showed possible greater effectiveness for patients with ECOG PS 0 and age < 65 years. In the RAS WT population, the anti-EGFR drug showed superiority with respect to TAS-102 and regorafenib. These results should be viewed as only exploratory, and further prospective studies are warranted to validate these data.

Ivermectin inhibits HSP27 and potentiates efficacy of oncogene targeting in tumor models.

HSP27 is highly expressed in, and supports oncogene addiction of, many cancers. HSP27 phosphorylation is a limiting step for activation of this protein and a target for inhibition, but its highly disordered structure challenges rational structure-guided drug discovery. We performed multistep biochemical, structural, and computational experiments to define a spherical 24-monomer complex composed of 12 HSP27 dimers with a phosphorylation pocket flanked by serine residues between their N-terminal domains. Ivermectin directly binds this pocket to inhibit MAPKAP2-mediated HSP27 phosphorylation and depolymerization, thereby blocking HSP27-regulated survival signaling and client-oncoprotein interactions. Ivermectin potentiated activity of anti-androgen receptor and anti-EGFR drugs in prostate and EGFR/HER2-driven tumor models, respectively, identifying a repurposing approach for cotargeting stress-adaptive responses to overcome resistance to inhibitors of oncogenic pathway signaling.

ACT-26 ABT-414 (DEPATUX-M) IN NEWLY DIAGNOSED AND RECURRENT GLIOBLASTOMA: WHERE DO WE STAND?

Epithelial growth factor receptor (EGFR) amplifications are found in approximately half of glioblastoma cases and targeting of the EGFR axis is an attractive treatment paradigm in this tumor type. However, several anti-EGFR drugs have failed to achieve significant and meaningful improvements in clincial trials. ABT-414 (Depatux-M) is an antibody-drug conjugate (ADC) that combines a cytotoxic agent with an antibody targeting EGFR, thus aiming at specific tummor cell killing through intracellular toxin delivery. The activity of ABT-414 has been evaluated in two large clinical trials enrolling glioblastoma patients. Intellance-2 enrolled 260 patients with first recurrence of EGFR-amplified glioblasoma into a chemotherapy control arm (temozolomide or lomustine) or one of two experimental arms (ABT-414 monotherapy or ABT-414 combined with temozolomide). Depatux-M in combination with temozolomide showed a trend towards improved survival times compared to temozolomide/lomustine alone, with patients relapsing more than 4 months after the last adjuvant temozolomide cycle deriving the greatest benefit. Intellance-1 was a randomized, placebo-controlled Phase 3 study and was designed to evaluate the efficacy and safety of Depatux-M versus placebo when administered with concurrent radiation and temozolomide and with adjuvant temozolomide in subjects with newly diagnosed EGFR-amplified glioblastoma. The primary endpoint was overall survival. Recently, it was announced that a preplaned interim analysis based on data from 639 patients showed the lack of a survival benefit for patients exposed to Depatux-M. In summary, the currently available data do not support routine use of Depatux-M in glioblastoma patients and further studies are needed to understand resistance mechanisms limiting therapeutic efficacy of EGFR-targeting in glioblastoma.

Abstract A088: Novel anti-EGFR antibody-drug conjugate AVID100: A phase 2a trial in patients with EGFR-overexpressing advanced solid tumors

Abstract Background: Currently, no therapies are approved specifically for EGFR-overexpressing tumors. AVID100 is a novel anti-EGFR antibody drug conjugate (ADC, DM1 payload) that was rationally designed for anti-tumor efficacy without accompanying on-target, off-tumor toxicity by employing an EGFR function-blocking antibody moiety. Although EGFR is overexpressed in a multitude of malignancies including squamous non-small cell lung cancer (sqNSCLC), head and neck carcinoma (HNSCC) and triple-negative breast cancer (TNBC), historically limited activity has been reported for EGFR monoclonal antibodies or EGFR TKIs in unselected patients. This emphasizes the need to develop novel and more efficacious agents targeting EGFR, and for biomarker- directed approaches to enrich for patients likely to respond. In pre-clinical models, AVID100 showed potent anti-tumour efficacy and a Phase 1 dose-escalation study in patients with advanced solid malignancies concluded that AVID100 was well tolerated at doses expected to be therapeutically active. An RP2D of 220 mg/m2 (~6 mg/kg) was established. Methods: This open-label, multicenter, multicohort, prospective Phase 2a study (NCT03094169) is designed to further evaluate the safety, tolerability, and preliminary efficacy of AVID100 when administered at 6 mg/kg Q3W per iv infusion in patients with locally advanced/unresectable or metastatic solid tumor malignancies of epithelial origin, and with documented EGFR over-expression. Additional endpoints include PK profiling and exploratory biomarkers. sqNSCLC and HNSCC patients with EGFR IHC of 3+ as well as mTNBC patients whose EGFR IHC shows 3+ intensity in ≥ 50% of tumor cells, or ≥ 2+ intensity in ≥ 75% of tumor cells, are eligible to enroll. Patients must have measurable disease according to RECIST v1.1 at enrolment and disease status is assessed radiographically every 6 weeks. EGFR over-expression positivity is established by a validated IHC assay (DAKO EGFRpharmDX) conducted by a central laboratory. Initial validation of the EGFR IHC was carried out retrospectively on archival tissue microarrays (US Biomax, Derwood, MD) from advanced sqNSCLC, HNSCC and TNBC patients. Results Initial validation of the EGFR IHC assay conducted with archival tissue samples yielded EGFR 3+ positivity in ≥ 50% of tumor cells for 30% (32 out of 105 samples tested) of sqNSCLC and 22% (31 out of 140) of HNSCC samples. Among TNBC samples tested, 6% were EGFR 3+ in ≥ 50% of tumor cells and 25% EGFR 2 or 3+ in ≥ 50% of tumor cells (16 and 62 out of 249, respectively). The prospective Phase 2a trial assessing AVID100 in patients selected for EGFR overexpression was is actively accruing and one sqNSCLC, four SCCHN and three TNBC patients had been enrolled at time of writing. Conclusions A significant proportion of retrospectively assessed sqNSCLC, HNSCC and TNBC patient samples were found to overexpress the EGFR. The prospective Phase 2a trial assessing AVID100, a novel growth factor receptor function-blocking anti-EGFR ADC, remains ongoing and updated results will be presented at the meeting. Citation Format: Jason Melear, Nehal Lakhani, Joyce A O'Shaughnessy, Sharon T Wilks, Saad Khan, Sree R Chandana, Anthony W Tolcher, Kyri P Papadopoulos, Yvette Cole, Karla Rivas, Ria Ghosh, Sandra Sinclair, Robert Lutz, Paul I Nadler, Debra L Wood, Barbara Burtness. Novel anti-EGFR antibody-drug conjugate AVID100: A phase 2a trial in patients with EGFR-overexpressing advanced solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A088. doi:10.1158/1535-7163.TARG-19-A088

Metaplastic carcinoma of the breast

Metaplastic carcinoma of the breast is a rare tumor which accounts for less than 1% of all the primary breast malignancies. It commonly occurs in women more than 50 years of age and are usually triple negative tumors with basal like subtype. Metaplastic carcinomas of breast have a very aggressive behaviour resulting in worse prognosis and a dismal disease free survival. However most metaplastic carcinomas express EGFR, therefore early and correct diagnosis benefits the patient to receive anti-EGFR drugs like cetuximab and geftinib. We are reporting two cases of metaplastic carcinomas, one with spindle cell differentiation and the other with squamous differentiation.

The Pharmacoeconomic Analysis of Cetuximab and Panitumumab in the 1st Line Treatment of mCRC in Real Clinical Practice in the Czech Republic.

The anti-epidermal growth factor receptor (EGFR) drugs cetuximab and panitumumab are currently reimbursed when administered during the first and subsequent lines of treatment of patients in the Czech Republic with metastatic colorectal cancer (mCRC). Because cetuximab and panitumumab do not show significant differences in efficacy, their choice may be dependent on cost. This retrospective study analyzed the costs of first-line treatment with cetuximab and panitumumab of patients with mCRC and wild type KRAS, as well as evaluated the correlations between costs and effectiveness, as determined by progression-free survival (PFS) and overall survival (OS). This analysis included 51 patients with mCRC and confirmed wild type KRAS treated at the comprehensive cancer centre in the Czech Republic between November 2011 and April 2018. Of these 51 patients, 22 were treated with cetuximab and 29 with panitumumab. Direct medical costs (medications, clinical examinations and procedures, and hospitalization) were evaluated from the initiation of treatment with anti-EGFR drug to disease progression and death. Mean follow-up was 21 months in the cetuximab group and 19 months in the panitumumab group. Reimbursement for anti-EGFR drugs until disease progression accounted for 71% (mean, 964,288 CZK per patient) of total costs in the cetuximab group and 77% (mean, 1,003,229 CZK per patient) of total costs in the panitumumab group, with median PFS in these two groups being 10.7 months and 8.1 months, respectively. Reimbursement of expensive center drugs from the start of anti-EGFR treatment to patient death accounted for 55% of total costs in the cetuximab group (mean, 1,752,702 CZK per patient) and 63% of total costs in the panitumumab group (mean, 1,596,919 CZK per patient), with median OS in these two groups being 20.2 months and 19.8 months, respectively. No significant between-group differences in clinical effectiveness and costs of treatment were observed (p &gt; 0.05 each). Reimbursement for biological agents is the most expensive item in the first-line treatment of mCRC patients with wild type KRAS, both to disease progression and death. The clinical effectiveness and costs of cetuximab and panitumumab did not differ significantly. Supported by CZECRIN (identification code LM2015090); CZECRIN_4 PACIENTY (No. CZ.02.1.01/0.0/0.0/16_013/0001826). The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 30. 4. 2019 Accepted: 17. 6. 2019.

Abstract 1364: A Non-Small Cell Lung Cancer Reference Material

Abstract Lung cancer is the leading cause of cancer death in the United States as well as globally. The major diagnosis (~85%) of lung cancer patients is non-small cell lung carcinoma (NSCLC). About 10-50% of NSCLCs have several activating EGFR driver mutations which have been clinically validated as therapeutic biomarkers for anti-EGFR drugs. Identifying these driver mutations through liquid biopsies is a promising alternative to traditional biopsies; however, the precision of mutation analysis for some of these targets, e.g., T790M, especially in advanced-stage metastatic NSCLC patients remains a challenge. To address this, we developed a platform of single and low complexity, highly patient-like circulating tumor DNA (ctDNA) EGFR reference standards designed for PCR assays that are used for EGFR-based patient testing. We mixed genomic DNA from the highly characterized human cell line (GM24385) with four synthetic EGFR variant-containing DNA sequences: T790M, L858R, G719S, and an ex19 deletion, in both individual and combined format. Using digital PCR we were able to precisely blend the material at the defined variant allele frequencies of 1% and 0.1%. In addition, wild-type reference material without any synthetic EGFR variants was also created as a comparable negative control. The materials were size-selected (160-170bp) after fragmentation for more patient-like ctDNA size characteristics. After size-selection the material is further refined to improve conversion efficiency. In order to make the material comparable to native ctDNA, such as that isolated from cancer patients, we encapsulated the DNA fragments into a synthetic plasma matrix. The process of DNA size selection followed by encapsulation and formulation into a synthetic plasma matrix enhances the commutability of these reference standards and improves the long-term stability of the samples to at least two years. The allele frequencies of the reference materials were confirmed by both dPCR and NGS. The Archer Reveal ctDNA 28 Kit was used for NGS library preparation while an Illumina MiSeq, using v2 (2x150 bp) PE chemistry reagents, was used for sequencing. Additional evaluation of these materials was conducted by laboratories that provide EGFR RT-PCR tests for patient anti-EGFR therapy stratification. The data from the dPCR, NGS and RT-PCR assays were comparable and highlight the broad compatibility of these EGFR ctDNA reference standards on a variety of assay testing platforms for NSCLC. Citation Format: Farol L. Tomson, Catherine Huang, Omo Clement, Russell Garlick, Bharathi Anekella. A Non-Small Cell Lung Cancer Reference Material [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1364.

Preferential Response of Basal-Like Head and Neck Squamous Cell Carcinoma Cell Lines to EGFR-Targeted Therapy Depending on EREG-Driven Oncogenic Addiction.

The management of locally advanced head and neck squamous cell carcinoma (HNSCC) with Cetuximab, a monoclonal antibody targeting the epidermal growth factor receptor (EGFR), achieves only moderate response rates, and clinical trials that evaluated EGFR-blockade with tyrosine kinase inhibitors (TKI) yielded disappointing results. Inter-tumor heterogeneity may hinder the therapeutic efficiency of anti-EGFR treatments. HNSCC heterogeneity was addressed in several studies, which all converged towards the definition of molecular subgroups. They include the basal subgroup, defined by the deregulated expression of factors involved in the EGFR signaling pathway, including the epiregulin EGFR ligand encoded by the EREG gene. These observations indicate that basal tumors could be more sensitive to anti-EGFR treatments. To test this hypothesis, we performed a screen of a representative collection of basal versus non-basal HNSCC cell lines for their sensitivity to several anti-EGFR drugs (Cetuximab, Afatinib, and Gefitinib), tested as monotherapy or in combination with drugs that target closely-linked pathways [Mitogen-activated protein kinase kinase/extracellular signal–regulated kinases (MEK), mammalian Target of Rapamycine (mTOR) or Human Epidermal growth factor Receptor 2 (HER2)]. Basal-like cell lines were found to be more sensitive to EGFR blockade alone or in combination with treatments that target MEK, mTOR, or HER2. Strikingly, the basal-like status was found to be a better predictor of cell response to EGFR blockade than clinically relevant mutations [e.g., cyclin-dependent kinase Inhibitor 2A (CDKN2A)]. Interestingly, we show that EGFR blockade inhibits EREG expression, and that EREG knock-down decreases basal cell clonogenic survival, suggesting that EREG expression could be a predictive functional marker of sensitivity to EGFR blockade in basal-like HNSCC.

Clinical, pathological and prognostic features of rare BRAF mutations (MTs) in metastatic colorectal cancer (mCRC): A bi-institutional retrospective analysis (REBUS study).

3554 Background: Recently, 3 classes of BRAF MTs have been described. BRAF V600 MTs, which identify mCRC with poor prognosis and not benefitting from anti-EGFR drugs, belong to class 1. Class 2 and 3 include BRAF non-V600 MTs, which occur in about 1-2% mCRC and are associated to favourable prognosis and specific clinicopathologic features. Class 2 and 3 differ in kinase activity and sensitivity to anti-EGFR: class 2 are activated and RAS-independent MTs; class 3 are kinase-dead and sensitive to inhibition of This study aims to retrospectively evaluate features and prognostic role of rare BRAF non-V600 compared to BRAF V600E MTs in mCRC pts treated at 2 Italian Institutions. Methods: mCRC pts harboring BRAF MTs, assessed by means of NGS, pyrosequencing or RT-PCR, treated between Jan-13 and Dec-18 at 2 Italian Institutions, were retrospectively analyzed. Clinico-pathological and treatment characteristics and survival data were collected. Results: 55 pts bearing BRAF MTs were identified. Of those, 46 (84%) harbored a V600E and 9 (16%) a non-V600 MT. Within the non-V600 group, 3 MTs (K601E, G469A, G469R) belonged to class 2, while 5 MTs (G466E, G466A, 2 D594G, D594N), belonged to class 3. One pt harboured a T599I MT, whose kinase activity is unknown. Compared to BRAF V600E mCRC, BRAF non-V600 mCRC were more frequently left-sided ( p .017) and displayed a lower grade ( p .045). In addition, non-V600 mCRC pts had a lower tumor burden (involving one metastatic site) ( p .026) and underwent more frequently to resection of metastases with radical intent (77.7 vs 18%; p .000175). mOS was significantly longer in the non-V600 compared to the V600E group (61.3 vs 20.4 m; HR 0.41, 95%CI 0.18-0.93; p .05). No difference in activity and efficacy of anti-EGFR agents was observed between class 2 and 3. Conclusions: Despite the small size of our retrospective analysis, the results were consistent with previous evidences. BRAF non-V600 MTs identified a subgroup of mCRC, differing both in terms of clinicopathologic characteristics and prognosis from BRAF V600 mCRC. Interestingly, the better prognostic features allowed more frequently radical resection of metastases, positively impacting on survival.

A retrospective analysis of elderly population with metastatic colorectal cancer treated with raltitrexed in the target therapy era.

e23022 Background: Raltitrexed is a antifolate chemotherapy agent with activity against metastatic colorectal cancer (mCRC). Most clinical trials with raltitrexed were performed before approval of anti-EGFR and anti-VEGF therapies. Its toxity profile suggests that might be a useful drug to treat elder patients, but there are no specific data in this population group. Methods: A retrospective review of elderly patients (70 years or older) with mCRC treated with raltitrexed monotherapy in our hospital between 2008 and 2017 was conducted. All of them had received previously oxaliplatin, irinotecan, fluoropyrimidines, and bevacizumab as standard therapy. All RAS native patients had received also anti-EGFR drugs. Results: Eighteen elder patients were reviewed, 11 (61%) male. Age ranged 70-85 years (median 78). All patients had ECOG PS 0 or 1. Locations were rectum (4), right colon (8), and left colon (6). Number of treatment lines ranged 2-5 (median 3). Three pts (16%) had received regorafenib previously. KRAS and BRAF were mutated in 61% and 5% respectively. Median number of cycles was 3 (1-13). Response rate was 11% (2 partial responses). Stable disease was achieved in 33%. Median progression-free and global survival were 2.8 (95% CI 1.2-10) and 5.9 (95% CI 1.6-18) months respectively. Grade 3-4 hematologic and non-hematologic toxicities were present in 22% and 5.5% respectively. Dose reduction was needed only in 11%. Sex, location, RAS status were not related to response or survival. Conclusions: Raltitrexed is a well tolerated option por elderly patients after standard treatment of mCRC.

Molecular profiling as predictor of outcomes in a Brazilian cohort of stage IV lung cancer.

e20668 Background: Lung cancer is the leading cause of cancer deaths globally. In stage IV non-small cell lung cancer (NSCLC), identification of a tumor driver mutation is critical, since it tailors treatment. We aimed to analyze clinical outcomes according to molecular profiling in patients (pts) with stage IV NSCLC. Methods: In this retrospective cohort study, we enrolled 57 pts with stage IV NSCLC treated at a public hospital in Southern Brazil between 2016 and 2018. Results: Median follow-up was 20.3 months, 53% were men, mean age was 65 years, 86% had smoked, 84% had de novo metastatic NSCLC and 96% had non-squamous carcinoma. Regarding molecular profiling, somatic mutations in KRAS, EGFR and BRAF were identified in 33%, 16% and 4% of pts, respectively. None of the pts had ALK mutations, and in 47% no identifiable driver mutation was found. In the EGFR-mutated subgroup, 67% had a deletion of exon 19, 22% had the exon 21 L858R mutation and 11% had exon 20 mutations. Palliative systemic therapy (PST) was delivered to 60% of the pts. Two or more lines of PST were delivered to 23%. The main reason for upfront best supportive care was ECOG PS 3-4 (poor). In the subgroup of pts with sensitizing EGFR mutations (8 pts), 75% received Gefitinib, the only anti-EGFR drug available in our public health system; the other pts died before recognition of the mutation. Median Progression Free-Survival was 6.3 months overall, 10.3 months for EGFR-mutated pts, 7.6 months for KRAS-mutated, 7.5 months for BRAF-mutated, and 5.2 months for pts with no mutation identified. Median Overall Survival (OS) was 7.7 months overall, 13.2 months for EGFR-mutated pts, 7.4 months for KRAS-mutated, 12.9 months for BRAF-mutated, and 5.3 months for pts with no mutation identified. In the Cox regression multivariate analysis, poor PS (HR 3.80, P &lt; 0.0001) and second-line PST (HR 0.23, P = 0.002) were independent predictors of OS. No driver mutations were predictors of OS, although we did find a tendency towards better OS in pts with EGFR mutations and worse OS in pts with KRAS mutations or no identifiable mutation. Conclusions: In this cohort, genotyping results did not predict survival outcomes. This is probably due to the small number of pts studied, and data should be reanalyzed in larger cohorts.

Landscape of lung cancer molecular biology in a NON-selected population.

e20648 Background: The molecular study of non-small cell lung cancer (NSCLC) allows for targeted treatment. EGFR and ALK alterations have been observed in 14% and 5% of Caucasian patients (p) respectively. These alterations are mainly detected in women with adenocarcinoma (ADC) and non-smokers. In a non-selected (all comers) NSCLC population from Ávila (Spain) we found a different profile. Methods: We collected data from 185 consecutive p the Pathology Department database of Hospital Nuestra Señora de Sonsoles from 2012 to 2017. All p were tested for EGFR and ALK. Analyses were done with Cobas or Therascreen tests. Results: EGFR mutations were found in 11 p out of 185 (6.7%). Histology was ADC in 8 p (72.7%) and squamous cell carcinoma (SC) in 3 p (27.3%). Male (m) &amp; smokers: 4/11 (36,4%). The type of mutation was: Exon 20: 3 p (27.3%). 2 m and 1 female (f). 2 smokers and 1 former smoker. 2 ADC and 1 SC; Del 19: 4 p (36.4%). 1 m and 3 f. 1 non-smoker, 2 former smokers and 1 smoker. 3 ADC and 1 SC; Exon 21: 4 p (36,4%). 2 m and 2 f. 3 non-smokers and 1 smoker. 3 ADC and 1 SC. All p. received treatment with 1st generation anti-EGFR drugs for a median 6.4 months (m). Progression Free Survival was 6.39 m (exon 20: 5.1 m; Del 19: 6.5 m; exon 21: 7.2 m). Overall survival was 18.0 m (exon 20: 26.1 m; Del 19: 18.2 m; exon 21: 11.8 m). ALK translocation was found in 5 p out of 158 (3.2%). Histology was ADC in 3 p (60%) (all f, 2 non-smokers and 1 former smoker) and SC in 2 p (40%) (2 smoker m.) Conclusions: Although the small sample size prevents definitive conclusions, EGFR mutations in exon 20 are more frequent in smokers and mutations in exon 21 in non-smoking patients with ADC. ALK translocation was observed independent of sex, tobacco or histologic type. For EGFR and ALK alterations, screening based on sex or pathology would have missed findings with therapeutic implications. We conclude it is important to extend the analysis of molecular alterations to all patients with NSCLC since more of them could benefit from targeted drugs.[Table: see text]

Protein Chemical Synthesis Combined with Mirror-Image Phage Display Yields D-Peptide EGF Ligands that Block the EGF-EGFR Interaction

EGFR, the receptor for epidermal growth factor (EGF), is arguably considered the most important therapeutic target in contemporary anticancer treatments. However, 20 years after the introduction of anti-EGFR drugs into the clinical setting, a number of severe drawbacks have been observed, namely low tumour penetration of monoclonal antibodies, acquisition of resistance to tyrosine-kinase inhibitors, and complete lack of efficacy in several types of cancer. Here, we report the design of a strategy to obtain all-D peptides directed to EGF instead of EGFR and we demonstrate their ability to block efficiently the EGF-EGFR interaction. We report an efficient chemical synthesis of the enantiomeric version of human EGF. Combined with the use of mirror-image phage display, this strategy has allowed us to discovery several protease-resistant peptides able to bind to EGF. Using alpha-screen technology we have demonstrated the capacity of the best peptide candidates to disrupt the EGF-EGFR interaction.