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Background Trastuzumab deruxtecan (T-DXd) has shown durable antitumor activity in pretreated patients with HER2-positive advanced breast cancer (ABC), but its efficacy has not yet been evaluated in patients with active brain metastases (BMs). DEBBRAH aims to assess T-DXd in patients with HER2-positive or HER2-low ABC and central nervous system involvement. Methods This ongoing, five-cohort, phase II study (NCT04420598) enrolled patients with pretreated HER2-positive or HER2-low ABC with stable, untreated, or progressing BMs, and/or leptomeningeal carcinomatosis. Here, we report findings from HER2-positive ABC patients with non-progressing BMs after local therapy (n = 8; cohort 1), asymptomatic untreated BMs (n = 4; cohort 2), or progressing BMs after local therapy (n = 9; cohort 3). Patients received 5.4 mg/kg T-DXd intravenously once every 21 days. The primary endpoint was 16-week progression-free survival (PFS) for cohort 1 and intracranial objective response rate (ORR-IC) for cohorts 2 and 3. Results As of October 20, 2021, 21 patients received T-DXd. In cohort 1, 16-week PFS rate was 87.5% (95%CI, 47.3-99.7; P < .001). ORR-IC was 50.0% (95%CI, 6.7-93.2) in cohort 2 and 44.4% (95%CI, 13.7-78.8; P < .001) in cohort 3. Overall, the ORR-IC in patients with active BMs was 46.2% (95%CI, 19.2-74.9). Among patients with measurable intracranial or extracranial lesions at baseline, the ORR was 66.7% (12 out of 18 patients; 95%CI, 41.0-86.7), 80.0% (95%CI, 28.4-99.5) in cohort 1, 50.0% (95%CI, 6.7-93.2) in cohort 2, and 66.7% (95%CI, 29.9-92.5) in cohort 3. All responders had partial responses. The most common adverse events included fatigue (52.4%; 4.8% grade ≥3), nausea (42.9%; 0% grade ≥3), neutropenia (28.6%; 19% grade ≥3), and constipation (28.6%; 0% grade ≥3). Two (9.5%) patients suffered grade 1 interstitial lung disease/pneumonitis. Conclusions T-DXd showed intracranial activity with manageable toxicity and maintained the quality of life in pretreated HER2-positive ABC patients with stable, untreated, or progressing BMs. Further studies are needed to validate these results in larger cohorts.

Endometrial cancer (EC) is the most common gynecologic malignancy in developed countries. Although most patients are diagnosed at early stages, 15-20% will relapse despite local treatment. Presently, there are no reliable markers to identify patients with worse outcomes who may benefit from adjuvant treatments, such as chemotherapy, and liquid biopsies may be of use in this setting. Peritoneal lavages are systematically performed during endometrial surgery but little data are available about their potential as liquid biopsies. We analyzed KRAS and PIK3CA mutations in paired surgical biopsies, blood and cytology-negative peritoneal lavages in a cohort of 50 EC patients. Surgical biopsies were submitted to next-generation sequencing (NGS) while circulating-free DNA (cfDNA) purified from plasma and peritoneal lavages was analyzed for KRAS and PIK3CA hotspot mutations using a sensitive quantitative polymerase chain reaction (PCR) assay. NGS of biopsies revealed KRAS, PIK3CA or concomitant KRAS + PIK3CA mutations in 33/50 (66%) EC patients. Of those, 19 cases carried hotspot mutations. Quantitative PCR revealed KRAS and/or PIK3CA mutations in the lavages of 9/19 (47.4%) hotspot EC patients. In contrast, only 2/19 (10.5%) blood samples from hotspot EC patients were positive. Mutations found in cfDNA consistently matched those in paired biopsies. One of the two patients positive in plasma and lavage died in less than 6 months. In conclusion, mutational analysis in peritoneal lavages and blood from early stage EC is feasible. Further studies are warranted to determine if it might help to identify patients with worse prognosis. Human genes discussed: KRAS, KRAS proto-oncogene, GTPase; PIK3CA, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha.

Background: Luminal advanced breast cancer (ABC) patients eventually progress on endocrine therapy. REVERT aimed to explore whether eribulin could restore endocrine sensitivity in a randomized, non-comparative phase II trial. Methods: Aromatase inhibitor (AI)-resistant patients with luminal ABC were randomized 1:1 to receive eribulin +/− AI. Patients were stratified by prior cyclin-dependent kinases 4/6 inhibitor (CDK4/6i) treatment. The primary endpoint was an investigator-assessed overall response rate (ORR) according to RECIST version 1.1 in the eribulin + AI arm. An interim analysis was planned with 11 evaluable patients according to a two-stage Simon design. Results: Twenty-two patients were enrolled (15 eribulin + AI arm; 7 eribulin arm). The trial was terminated early in March 2021, with eight (36.4%) patients still on treatment. ORR was 26.7% in the eribulin + AI arm (95% CI, 7.8–55.1%; p = 0.0541). In the eribulin arm, two (28.6%) patients had an objective response (95% CI, 3.7–71.0%). The difference between the study arms was not significant (p = 0.918). The addition of AI to eribulin also failed to show improvement in other efficacy endpoints. A significant interaction between the treatment arm and previous CDK4/6i treatment was observed for ORR (p = 0.018) and progression-free survival (p = 0.084). Overall, the toxicity profile was consistent with the known safety profile of eribulin. No treatment-related deaths were reported. Conclusion: Eribulin + AI does not seem to improve outcomes compared with eribulin monotherapy in patients with AI-resistant luminal ABC. This chemo–endocrine approach deserves further investigation after progression to CDK4/6i-based therapy.

Somatic copy number variations (CNV, i.e., amplifications and deletions) have been implicated in the origin and development of multiple cancers and some of these aberrations are designated targets for therapies. Although FISH is still considered the gold standard for CNV detection, the increasing number of potentially druggable amplifications to be assessed makes a gene-by-gene approach time- and tissue-consuming. Here we investigated the potential of next generation sequencing (NGS) custom panels to simultaneously determine CNVs across FFPE solid tumor samples. DNA was purified from cell lines and FFPE samples and analyzed by NGS sequencing using a 20-gene custom panel in the GeneReader Platform®. CNVs were identified using an in-house algorithm based on the UMI read coverage. Retrospective validation of in-house algorithm to identify CNVs showed 97.1% concordance rate with the NGS custom panel. The prospective analysis was performed in a cohort of 243 FFPE samples from patients arriving at our hospital, which included 74 NSCLC tumors, 148 CRC tumors, and 21 other tumors. Of them, 33% presented CNVs by NGS and in 14 cases (5.9%) the CNV was the only alteration detected. We have identified CNV alterations in about one-third of our cohort, including FGFR1, CDK6, CDK4, EGFR, MET, ERBB2, BRAF, or KRAS. Our work highlights the need to include CNV testing as a part of routine NGS analysis in order to uncover clinically relevant gene amplifications that can guide the selection of therapies.

Background With the advent of precision oncology, liquid biopsies are quickly gaining acceptance in the clinical setting. However, in some cases, the amount of DNA isolated is insufficient for Next-Generation Sequencing (NGS) analysis. The nCounter platform could be an alternative, but it has never been explored for detection of clinically relevant alterations in fluids. Methods Circulating-free DNA (cfDNA) was purified from blood, cerebrospinal fluid, and ascites of patients with cancer and analyzed with the nCounter 3 D Single Nucleotide Variant (SNV) Solid Tumor Panel, which allows for detection of 97 driver mutations in 24 genes. Results Validation experiments revealed that the nCounter SNV panel could detect mutations at allelic fractions of 0.02–2% in samples with ≥5 pg mutant DNA/µL. In a retrospective analysis of 70 cfDNAs from patients with cancer, the panel successfully detected EGFR, KRAS, BRAF, PIK3CA, and NRAS mutations when compared with previous genotyping in the same liquid biopsies and paired tumor tissues [Cohen kappa of 0.96 (CI = 0.92–1.00) and 0.90 (CI = 0.74–1.00), respectively]. In a prospective study including 91 liquid biopsies from patients with different malignancies, 90 yielded valid results with the SNV panel and mutations in EGFR, KRAS, BRAF, PIK3CA, TP53, NFE2L2, CTNNB1, ALK, FBXW7, and PTEN were found. Finally, serial liquid biopsies from a patient with NSCLC revealed that the semiquantitative results of the mutation analysis by the SNV panel correlated with the evolution of the disease. Conclusions The nCounter platform requires less DNA than NGS and can be employed for routine mutation testing in liquid biopsies of patients with cancer.

1084 Background: A substantial benefit from adding an immune checkpoint inhibitor to chemotherapy (CT) was reported in mTNBC patients (pts) with PD-L1+ tumors. However, many pts still have a poor outcome. ATRACTIB is exploring the synergism between A (anti-PD-L1 antibody) and B (a VEGF-targeted antibody) with P in mTNBC irrespective of PD-L1 status. We report results from protocol-specified SIA. Methods: ATRACTIB is an open-label, single-arm, phase 2 trial (NCT04408118). Pts aged ≥18 years, with unresectable locally advanced or mTNBC, ECOG performance status of 0–1, who had received no prior systemic therapy or ≥12 months since (neo)adjuvant taxane-based CT are eligible. Pts receive A (840 mg IV, days 1, 15) with P (90 mg/m2 IV, days 1, 8, 15), and B (10 mg/kg IV, days 1, 15) on each 28-day cycle until disease progression, unacceptable toxicity, or patient withdrawal. Primary endpoint is investigator-assessed progression-free survival (PFS) as per RECIST v.1.1. Secondary endpoints include objective response and clinical benefit rates, overall survival, and safety. The trial was designed to detect a treatment effect in terms of median PFS (H0: ≤7 months; H1: ≥9.5 months) and 100 pts are needed to attain 80% power at a nominal one-sided α level of 5%. One SIA was planned for evaluating safety as per CTCAE v.5.0 on the first 20 pts who had completed a 3-month follow-up or reached the end of study. Results: From Oct 5, 2020, through Nov 21, 2021, 34 pts were enrolled at 13 sites in Spain and Germany and received at least 1 dose of study treatment. Median age was 57.5 (range 40–84) years, 23 (67.6%) pts had received prior CT for early disease, and 19 (56.0%) had visceral disease. At data cutoff (Sep 30, 2021), 25 (71.4%) pts were still receiving the drug regimen. Adverse events (AEs) led to drug discontinuation in 3 (8.8%) pts. Mean relative dose intensity was 90.2% for A, 96.5% for P, and 95.7% for B. P dose reduction was reported in 7 (20.6%) pts. Five (14.7) pts required a dose delay due to AEs (11.8% for A, 11.8% for P, and 8.8% for B). The most common AEs of any grade (G) were fatigue (47.1%; 8.8% G≥3), diarrhea (38.2%; 0% G≥3), and neurotoxicity (35.3%; 8.8% G≥3). Anemia (20.6%; 0% G≥3) and neutropenia (17.6%; 8.8% G≥3) were the most frequent hematological AEs. AEs of clinical interest (AECI) for B were hypertension (17.6%; 5.9% G≥3) and pulmonary embolism (2.9%; 0% G≥3). AECI for A were pneumonitis (2.9%; 0% G≥3), autoimmune hepatitis (2.9%; 2.9% G≥3), and alanine aminotransferase increased (2.9%; 2.9% G≥3). No treatment-related deaths were reported. Conclusions: The addition of A to P and B as 1L therapy for mTNBC shows a tolerable safety profile which is consistent with known safety profile of each agent without a significant synergistic toxicity. Based on the independent data monitoring committee recommendation, patient recruitment is ongoing. Clinical trial information: NCT04408118.

We studied MET alterations in 474 advanced non‐small‐cell lung cancer (NSCLC) patients by nCounter, an RNA‐based technique. We identified 3% with METΔex14 mRNA and 3.5% with very‐high MET mRNA expression, a surrogate of MET amplification. MET alterations identified by nCounter correlated with clinical benefit from MET inhibitors. Quantitative mRNA‐based techniques can improve the selection of patients for MET‐targeted therapies., MET inhibitors have shown activity in non‐small‐cell lung cancer patients (NSCLC) with MET amplification and exon 14 skipping (METΔex14). However, patient stratification is imperfect, and thus, response rates have varied widely. Here, we studied MET alterations in 474 advanced NSCLC patients by nCounter, an RNA‐based technique, together with next‐generation sequencing (NGS), fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), and reverse transcriptase polymerase chain reaction (RT–PCR), exploring correlation with clinical benefit. Of the 474 samples analyzed, 422 (89%) yielded valid results by nCounter, which identified 13 patients (3%) with METΔex14 and 15 patients (3.5%) with very‐high MET mRNA expression. These two subgroups were mutually exclusive, displayed distinct phenotypes and did not generally coexist with other drivers. For METΔex14, 3/8 (37.5%) samples positive by nCounter tested negative by NGS. Regarding patients with very‐high MET mRNA, 92% had MET amplification by FISH and/or NGS. However, FISH failed to identify three patients (30%) with very‐high MET RNA expression, among which one received MET tyrosine kinase inhibitor treatment deriving clinical benefit. Our results indicate that quantitative mRNA‐based techniques can improve the selection of patients for MET‐targeted therapies.

Background: 80% of non-small cell lung cancer (NSCLC) cases are diagnosed at stages IIIB-IV and have a dismal prognosis with a median life expectancy that does not exceed 2 years. In contrast, patients diagnosed at early and locally advanced stages (I-IIIA) can undergo surgery and have the potential to be totally cured. Imaging technologies often detect lung nodules of unknown significance that pose a diagnostic challenge; some patients with benign nodules are submitted to unnecessary surgical interventions while others with small tumors are just kept in observation, risking a significant delay for treatment. A diagnostic test that could differentiate between benign and malignant masses would be of great help in this setting. Methods: Circulating-free RNA (cfRNA) was isolated from the plasma of healthy individuals (N=21), early(I-II) stage (N=22) and stage IIIA (N=12) NSCLC patients, using an automatic extraction method(Qiasymphony, Qiagen). Purified cfRNA was quantified using Qubit, retrotranscribed and pre-amplified (14cycles) using the Low RNA Input Amplification kit (NanoString Technologies). Gene expression analysis was performed on the nCounter platform using the PanCancer IO360TM (NanoString Technologies), which can detect 770 transcripts related to tumor biology, micro-environment and the immune system. Results: Gene expression analysis revealed differential patterns for some cf-mRNAs from localized stage NSCLC patients versus healthy controls. A bioinformatics recursive feature elimination algorithm selected a 16-gene mRNA signature that was able to distinguish between localized NSCLC and control samples with an area under the ROC curve of 0.91 to 0.95. Furthermore, the signature scores derived from the algorithm were significantly different between the two cohorts. Conclusions: We have found an 16-gene signature that can differentiate between cfRNA of localized stages NSCLC patients and control individuals. Our results warrant validation studies in larger cohorts. Citation Format: Ana Giménez Capitán, Jillian Bracht, Nicolas Potie, María González-Cao, Santiago Viteri, Alejandro Martínez-Bueno, Carlos Cabrera-Gálvez, Pablo Rubinstein, Clara Mayo-de-las-Casas, Joselyn Valarezo, Chung-Ying Huang, Carlos Pedraz, Richard Boykind, Sarah Warren, Rafael Rosell, Miguel Ángel Molina-Vilaa, Andrés Aguilar-Hernández. A nCounter-Based mRNA signature in plasma associates with localized non-small cell lung cancer [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 2606.

Background: When tumour tissue is unavailable, cell-free DNA (cfDNA)can serve as a surrogate for genetic analyses. Because mutated alleles in cfDNA are usually below 1%, next-generation sequencing (NGS)must be narrowed to target only clinically relevant genes. In this proof-of-concept study, we developed a panel to use in ultra-deep sequencing to identify such mutations in cfDNA. Methods: Our panel (‘SiRe') covers 568 mutations in six genes (EGFR, KRAS, NRAS, BRAF, cKIT and PDGFRα)involved in non-small-cell lung cancer (NSCLC), gastrointestinal stromal tumour, colorectal carcinoma and melanoma. We evaluated the panel performance in three steps. First, we analysed its analytical sensitivity on cell line DNA and by using an artificial reference standard with multiple mutations in different genes. Second, we analysed cfDNA from cancer patients at presentation (n=42), treatment response (n=12) and tumour progression (n=11); all patients had paired tumour tissue and cfDNA previously genotyped with a Taqman-derived assay (TDA). Third, we tested blood samples prospectively collected from NSCLC patients (n=79) to assess the performance of SiRe in clinical practice. Results: SiRe had a high analytical performance and a 0.01% lower limit of detection. In the retrospective series, SiRe detected 40 EGFR, 11 KRAS, 1 NRAS and 5 BRAF mutations (96.8% concordance with TDA). In the baseline samples, SiRe had 100% specificity and 79% sensitivity relative to tumour tissue. Finally, in the prospective series, SiRe detected 8.7% (4/46) of EGFR mutations at baseline and 42.9% (9/21) of EGFR p.T790M in patients at tumour progression. Conclusions: SiRe is a feasible NGS panel for cfDNA analysis in clinical practice.

Many advanced cases of cancer show central nervous system, pleural, or peritoneal involvement. In this study, we prospectively analyzed if cerebrospinal fluid (CSF), pleural effusion (PE), and/or ascites (ASC) can be used to detect driver mutations and guide treatment decisions. We collected 42 CSF, PE, and ASC samples from advanced non‐small‐cell lung cancer and melanoma patients. Cell‐free DNA (cfDNA) was purified and driver mutations analyzed and quantified by PNA‐Q‐PCR or next‐generation sequencing. All 42 fluid samples were evaluable; clinically relevant mutations were detected in 41 (97.6%). Twenty‐three fluids had paired blood samples, 22 were mutation positive in fluid but only 14 in blood, and the abundance of the mutant alleles was significantly higher in fluids. Of the 34 fluids obtained at progression to different therapies, EGFR resistance mutations were detected in nine and ALK acquired mutations in two. The results of testing of CSF, PE, and ASC were used to guide treatment decisions, such as initiation of osimertinib treatment or selection of specific ALK tyrosine–kinase inhibitors. In conclusion, fluids close to metastatic sites are superior to blood for the detection of relevant mutations and can offer valuable clinical information, particularly in patients progressing to targeted therapies., Many advanced cases of cancer show central nervous system, pleural, or peritoneal involvement. Here, we present the results of prospective genetic testing of 42 cerebrospinal fluids, pleural effusions, and ascites, and we show that these fluids, which are closer to metastatic sites, are superior to plasma (P) for the detection of clinically relevant mutations.

Background: In a significant percentage of advanced non-small-cell lung cancer (NSCLC) patients, tumor tissue is unavailable or insufficient for genetic analyses. We prospectively analyzed if circulating-free DNA (cfDNA) purified from blood can be used as a surrogate in this setting to select patients for treatment with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). Patients and methods: Blood samples were collected in 119 hospitals from 1138 advanced NSCLC patients at presentation (n = 1033) or at progression to EGFR-TKIs (n = 105) with no biopsy or insufficient tumor tissue. Serum and plasma were sent to a central laboratory, cfDNA purified and EGFR mutations analyzed and quantified using a real-time PCR assay. Response data from a subset of patients (n = 18) were retrospectively collected. Results: Of 1033 NSCLC patients at presentation, 1026 were assessable; with a prevalence of males and former or current smokers. Sensitizing mutations were found in the cfDNA of 113 patients (11%); with a majority of females, never smokers and exon 19 deletions. Thirty-one patients were positive only in plasma and 11 in serum alone and mutation load was higher in plasma and in cases with exon 19 deletions. More than 50% of samples had

Background: A variety of alterations in MET have been described in advanced non-small cell lung cancer (NSCLC) patients, including gene amplification, protein overexpression, splicing variants and point mutations. MET alterations are receiving increasing attention as targets in precision medicine, and several clinical trials of anti-MET agents are ongoing in NSCLC. Methods: A cohort of 610 patients with stage IIIb-IV NSCLC from two institutions was retrospectively analyzed for MET alterations by next-generation sequencing (NGS) (Ion Torrent PGM® or GeneReader®), nCounter, reverse transcriptase polymerase chain reaction (RT-PCR) or immunohistochemistry (IHC) during a 2-year period. Patients positive for MET amplification by NGS or MET overexpression by nCounter and/or IHC were submitted to fluorescence in situ hybridization (FISH). Representative patients positive for MET exon 14-skipping (METΔex14) mutations by nCounter and/or RT-PCR were confirmed by sequencing exons 13-15 of the METgene. Results: Overall, MET alterations were found in 116/610 patients (19%). Some patients had ≥2 MET aberrations. The most frequent finding was MET overexpression (58/333; 17.6%), followed by METΔex14 (31/610; 5.1%) and MET point mutations (3/129; 2.3%). Regarding MET amplification, it was found in 24/157 patients (15.3%). MET positivity by IHC (3+, >50%) showed a 90.8% concordance with MET mRNA overexpression by nCounter, with a 0.768 Cohen’s kappa (confidence interval, CI 95% 0.575-0.961). A moderate agreement between RT-PCR and nCounter was found for METΔex14 (Cohen’s kappa 0.629; CI 95% 0.434-0.825). METamplification by FISH was found in the subset of MET-overexpressing patients with the highest mRNA levels by nCounter. Interestingly, a patient with a concomitant EGFR mutation and MET overexpression derived two years clinical benefit from crizotinib. Conclusions: MET aberrations are present in 19% of advanced NSCLC patients and represent one of the most frequent targetable alterations in this malignancy. A comprehensive testing of MET alterations in advanced NSCLC patients, including NGS and nCounter techniques, is needed to identify a broader number of patients’ candidates for targeted therapies. Citation Format: Cristina Aguado Esteban, Cristina Teixidó, Ruth Román, Ana María Gimenez Capitán, Carlos Cabrera, Mireia García, Clara D. Mayo De Las Casas, Ainara Arcocha, Sonia Rodriguez, Roxana Reyes, Elba Marín, Ana Perez Rosado, Niki Karachaliou, Aleix Prat, Rafael Rosell, Alejandro Martinez-Bueno, Miguel Angel Molina-Vila, Noemi Reguart. Comprehensive characterization of MET alterations in a large cohort of 610 advanced non-small cell lung cancer patients [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 4905.

Liquid biopsy analyses are already incorporated in the routine clinical practice in many hospitals and oncology departments worldwide, improving the selection of treatments and monitoring of lung cancer patients. Although they have not yet reached its full potential, liquid biopsy-based tests will soon be as widespread as “standard” biopsies and imaging techniques, offering invaluable diagnostic, prognostic, and predictive information. This review summarizes the techniques available for the isolation and analysis of circulating free DNA and RNA, exosomes, tumor-educated platelets, and circulating tumor cells from the blood of cancer patients, presents the methodological challenges associated with each of these materials, and discusses the clinical applications of liquid biopsy testing in lung cancer.

Background: Surgical resection remains the best treatment option for early-stage endometrial (CE), colorectal (CRC) and lung cancer (LC) patients. However, a proportion of patients (p) develop tumor recurrence, even after curative resection. Peritoneal or pleural lavages may be performed during surgery but little data is available about their potential as liquid biopsies with predictive value for recurrence. This proof-of concept study aimed to determine if somatic mutations can be detected in these lavages. Methods: We analyzed mutations in paired surgical biopsies, blood and peritoneal or pleural lavages supernatants in an early-stage cohort of 25 EC, 12 CRC and 19 LC p. Molecular characterization of surgical biopsies was performed using the GeneReader NGS platform (Qiagen) and the Actionable Insight Tumor Panel, which includes 12 genes (KRAS, NRAS, KIT, BRAF, PDGFRA, ALK, EGFR, ERBB2, PIK3CA, ERBB3, ESR1, and RAF1). cfDNA was purified from blood and lavages using an automatic extractor and the hotspot mutations identified in tumor tissue were tested using a sensitive Taqman assay for codons 12, 13, and 61 in KRAS, codons 542, 545 and 1047 in PIK3CA and codon 600 in BRAF. Results: NGS of tumor biopsies revealed hotspot KRAS, PIK3CA or BRAF mutations in a total of 11/25 (44%) early-stage EC p, 7/12 (58%) early-stage CRC p and 6/19 (32%) early-stage LC p. In peritoneal lavages, hotspot mutations were found in 6/11 (55%) EC p and 2/7 (29%) CRC p and, in pleural lavages, hotspot mutations appeared in 2/6 (33%) LC p. Furthermore, 2/11 (18%) blood samples from EC p, 1/7 (14%) blood samples from CRC p and 1/6 (17%) blood samples from LC p were positive. Conclusions: Our study reveals that mutational analysis in peritoneal or pleural lavages from early stage tumors is feasible and that mutations are more frequently detected in lavages than in blood. Further research will determine if positivity in blood or lavages is associated with early relapse. Citation Format: Núria Jordana Ariza, Mónica Garzón Ibañez, Alejandro Martinez-Bueno, Sonia Gatius Caldero, Clara Mayo de las casas, Ana Velasco, Maria Ruiz, Berta Roman Canal, Jordi Bertran Alamillo, Sonia Rodríguez Muñoz, Raquel Campos Fuentes, Beatriz García, Ariadna Balada Bel, Sergi Villatoro, Miguel Ángel Molina-Vila, Xavier Matias-Guiu. Utility of peritoneal and pleural lavages for detection of somatic alterations: A proof-of-concept study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3643.

Background: Mutations in the KRAS proto-oncogene, GTPase (KRAS) are driver alterations in several tumors, such as non-small-cell lung (NSCLC) and colorectal cancer (CRC). However, their role as a prognostic marker in liquid biopsies is unclear. We studied the status of KRAS mutations in the peripheral blood of advanced NSCLC and colorectal cancer patients visited in our hospital and evaluated its potential value as a monitoring marker in the clinical practice. Methods: We developed a sensitive TaqMan assay, in the presence of a PNA clamp, for the determination of KRAS mutations (codons 12, 13 and 61) in circulating-free DNA (cfDNA). The assay detected 2.5 pg mutated DNA/µL and a ratio of KRAS mutated versus wild type allele of 1:20000 and was validated in 80 cancer patients previously genotyped in tumor tissue showing a clinical sensitivity of 72.5% and specificity of 100%. cfDNA was isolated from serum and plasma specimens, using an automatic extractor and mutational analyses were performed in quadruplicates samples. Serum and plasma samples were drawn at diagnosis, during follow-up and progression. Results: 239 NSCLC and 11 colorectal cancer patients were screened for KRAS mutations in cfDNA at presentation in the period of 2013 to 2016. In addition, 160 cfDNA samples collected during disease monitoring of 61 patients were analyzed. Paired biopsies were available from 110/250 p at diagnosis (60 positive for KRAS in FFPE and 50 WT). The KRAS mutation was detected in the cfDNA of 76.6% (46/60) p positive in tumor tissue vs. only in 1/50 WT patients where a G12C was detected in cfDNA in a consistent manner, probably associated with a heterogeneity of the tumor. In patients with no biopsy available, we detected KRAS mutations in cfDNA in 14.3% (20/140) cases at diagnosis. The most prevalent mutations observed in cfDNA were G12C (44.7 %), G12V (22.4%) and G12D (10.4%) for NSCLC patients and G13D (5.9%) for colorectal patients. The Q61H mutation was detected in the cfDNA of one NSCLC and one CRC p. Of the 67 patient’s positive in cfDNA at presentation, KRAS mutations were detected in serum and plasma in 38 patients, only in plasma in 24 and only in serum in 5. Regarding the 61 patients with serial blood samples, we observed a correlation between the KRAS mutation load in cfDNA and the course of the disease, with a decrease in patients responding to chemotherapy and an increase during disease progression. Conclusions: Our results demonstrate the feasibility of KRAS mutation analysis in the cfDNA of advanced NSCLC and colorectal patients. Analysis of KRAS mutations in the blood of mutated patients can have a value in patients with no biopsy available and to monitor the course of the disease. Citation Format: Mónica Garzón Ibañez, Clara Mayo de las casas, Nùria Jordana Ariza, Ariadna Balada Bel, Beatriz Garcia, Sergio Villatoro, Jordi Bertrán-Alamillo, Alejandro Martinez-Bueno, Santiago Viteri Ramirez, Ana Pérez Rosado, Daniela Morales Espinosa, Maria José Catalán, Niki Karachaliou, Miguel Ángel Molina-Vila, Rafael Rosell. Routine testing for KRAS mutations in cfDNA from blood of advanced cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5688. doi:10.1158/1538-7445.AM2017-5688

e19085 Background: Repeat biopsies of cancer p are useful for monitoring molecular evolution of the disease and the possible appearance or disappearance of key somatic mutations, particularly those...

1042 Background: Neoadjuvant chemotherapy is generally used for treatment of both early stage and locally advanced breast cancer. However, robust prognostic or predictive biomarkers are needed in t...

e22119 Background: Although it is know that pCR following neoadjuvant chemotherapy is more frequent in some subtypes of breast cancer such as Triple Negative (TN) or erb2 tumors, the predictive role of gene expression and mutation status is not well defined in this setting. Methods: We analyzed samples from 41 patients (p) prospectively treated with neoadjuvant chemotherapy (sequential AC followed by weekly TXL, or inverse sequence, plus trastuzumab for erb2 positive p). Pathologic response (PR) was classified according to Miller-Payne and RCB criteria. Radiologic evaluation was performed by ultrasonography, dynamic MR and PET-TAC after each chemotherapy sequence. We performed expression analysis of AXL, BRCA1, RAP80, BIM, EZH2, ROR1, FGFR1, PTPN12, YAP, GAS6, beta-TRCP, HIF1 alpha and ZNF217 by RT-PCR, and mutational status of p53 and PI3K genes in pretreatment biopsies. Statistical analysis was performed using Mann-Whitney U and Pearson’s chi-squared tests. Results: pCR was detected in 5 p (3TN, 2 erb2) of 25 p (9 luminal A, 5 luminal B, 6 erb2 and 5 TN) evaluated for PR at time of submitting this abstract. TN tumors had lower levels of RAP80 (p=.0013), PTPN12 (p=.003), beta TRCP (p=.001), ZNF217 (p=.014) and YAP (p=.097). Luminal B tumors had low levels of YAP and the highest levels of FGFR1 (p=.09) and ZNF217 (p=.014). Luminal A tumors had high levels of beta-TRCP (p=.003). We found no differences in BRCA1, AXL, BIM, EZH2, ROR1, GAS6 and HIF1 levels by breast cancer subtype. P with low levels of FGFR1 (p=.087), HIF1alpha (p=.07) or EZH2 (p=.005) had higher probability of pCR. No pCR was observed in p with higher levels of AXL, EZH2, RAP80, GAS6, beta TRCP, HIF alpha. Four p had p53 mutations (1 luminal B, 1 erb2 and 2 TN) and 4 p had PI3K mutations (2 luminal A, 1 erb2, 1 luminal B). There was no correlation between p53 status and PR. P with PI3K mutations did not achieve pCR vs 46% of p with wild type PI3K (p=.23). Conclusions: Gene expression profile varies by breast cancer subtype. Chemosensitivity could be higher in tumors with lower levels of FGFR1, HIF1alpha or EZH2. Further results will be presented.

e18137 Background: Advanced NSCLC p with EGFR activating mutations show an impressive progression-free survival (PFS) to erlotinib. The co-existence of the EGFR T790M mutation, in conjunction with high BRCA1 mRNA levels, affected PFS to erlotinib (Rosell et al. CCR 2011). LMO4 is a negative regulator of BRCA1 function in sporadic breast cancers, and CtIP can bind to BRCA1 and LMO4. We have assessed the expression of CtIP, LMO4 and BRCA1 and examined the impact of CtIP and LMO4 levels on outcome. Methods: mRNA expression of LMO4 and CtIP was examined by RT-PCR in the original pretreatment tumor biopsies of 81 NSCLC p with sensitive EGFR mutations. Results: Expression of BRCA1 and LMO4 was successfully assessed in 55 p: median age, 68; 61.8% female; 98.2% Caucasian; 63.6% never-smokers; 81.8% ECOG PS

e18143 Background: Advanced NSCLC p with EGFR mutations have a median PFS of 14 months (m) and OS of 27 m when treated with erlotinib. In NSCLC cell lines, tyrosine kinase inhibitors (TKIs) induce p53 translocation from the cytoplasm to the nucleus and subsequent upregulation of Fas and caspase activation leading to apoptosis, but this mechanism was defective in p53-null cells. We tested whether TP53 mutations influence outcome to erlotinib in EGFR-mutated p. Expression levels of the p53 repressor MDM2 were also examined. Methods: We assessed p53 status in pretreatment paraffin-embedded tumor samples from 93 erlotinib-treated, EGFR-mutated advanced NSCLC p. Mutations in exons 5, 6, 7 and 8 were screened by High Resolution Melting analysis followed by sequencing of the amplified products with non-wild-type (wt) melt curves. All mutant samples were re-confirmed by standard PCR and sequencing. Expression levels of MDM2 mRNA were determined by quantitative RT-PCR. Results: Mutations in exons 5-8 of TP53 were detected in 26 of 93 p (28%). We found an unusually high frequency of in-frame and frameshift deletions (23% of mutations), indicating that the spectrum of p53 mutations might be different in EGFR-mutated NSCLC. Mutations were less frequent in p with ECOG PS >2 and more frequent in p with the T790M mutation. OS was 15 m in the 16 p with missense mutations 31 m in p with wt p53 and not reached in p with non-missense mutations (P=0.04). PFS was 9 m for 14 p with mutations in one of the p53 DNA binding motifs (DBMs), compared to 19 m for wt p and 27 m for p with non-DBM mutations. MDM2 mRNA levels were significantly lower in tumors with p53 mutations, especially in DBM mutations. In the case of wt p, high MDM2 expression correlated with longer PFS and OS in p with wt p53. Conclusions: TP53 mutations co-exist with EGFR mutations in a significant number of p; missense mutations correlate with shorter OS and mutations in DBMs correlate with shorter PFS. This finding paves the way for the possibility of combining erlotinib with a drug restoring p53 function in those p harboring certain types of mutations in the TP53 gene.