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Purpose: Chemotherapy plus anti-EGFR is standard first-line therapy in RAS wild-type (wt) metastatic colorectal cancer (mCRC), but biomarkers of early response are clinically needed. We aimed to define the utility of ctDNA to assess early response in patients with mCRC receiving first-line anti-EGFR therapy. Experimental Design: Prospective multicentric study of tissue patients with RAS wt mCRC treated with first-line chemotherapy plus cetuximab undergoing sequential liquid biopsies. Baseline and early (C3) ctDNA were analyzed by NGS. Trunk mutations were assessed as surrogate marker of total tumor burden. RAS/BRAF/MEK/EGFR-ECD were considered mutations of resistance. ctDNA results were correlated with clinical outcome. Results: One hundred patients were included. ctDNA was detected in 72% of patients at baseline and 34% at C3. Decrease in ctDNA trunk mutations correlated with progression-free survival (PFS; HR, 0.23; P = 0.001). RAS/BRAF were the only resistant mutations detected at C3. An increase in the relative fraction of RAS/BRAF at C3 was followed by an expansion of the RAS clone until PD, and was associated with shorter PFS (HR, 10.5; P < 0.001). The best predictor of response was the combined analysis of trunk and resistant mutations at C3. Accordingly, patients with “early molecular response” (decrease in trunk and decrease in resistant mutations) had better response (77.5% vs. 25%, P = 0.008) and longer PFS (HR, 0.18; P < 0.001) compared with patients with “early molecular progression” (increase in trunk and/or increase in resistant mutations). Conclusions: ctDNA detects early molecular response and predicts benefit to chemotherapy plus cetuximab. A comprehensive NGS-based approach is recommended to integrate information on total disease burden and resistant mutations. See related commentary by Eluri et al., p. 302

Supplementary Figure from mTOR Inhibition and T-DM1 in HER2-Positive Breast Cancer

Purpose:Chemotherapy plus anti-EGFR is standard first-line therapy in RAS wild-type (wt) metastatic colorectal cancer (mCRC), but biomarkers of early response are clinically needed. We aimed to define the utility of ctDNA to assess early response in patients with mCRC receiving first-line anti-EGFR therapy.Experimental Design:Prospective multicentric study of tissue patients with RAS wt mCRC treated with first-line chemotherapy plus cetuximab undergoing sequential liquid biopsies. Baseline and early (C3) ctDNA were analyzed by NGS. Trunk mutations were assessed as surrogate marker of total tumor burden. RAS/BRAF/MEK/EGFR-ECD were considered mutations of resistance. ctDNA results were correlated with clinical outcome.Results:One hundred patients were included. ctDNA was detected in 72% of patients at baseline and 34% at C3. Decrease in ctDNA trunk mutations correlated with progression-free survival (PFS; HR, 0.23; P = 0.001). RAS/BRAF were the only resistant mutations detected at C3. An increase in the relative fraction of RAS/BRAF at C3 was followed by an expansion of the RAS clone until PD, and was associated with shorter PFS (HR, 10.5; P < 0.001). The best predictor of response was the combined analysis of trunk and resistant mutations at C3. Accordingly, patients with “early molecular response” (decrease in trunk and decrease in resistant mutations) had better response (77.5% vs. 25%, P = 0.008) and longer PFS (HR, 0.18; P < 0.001) compared with patients with “early molecular progression” (increase in trunk and/or increase in resistant mutations).Conclusions:ctDNA detects early molecular response and predicts benefit to chemotherapy plus cetuximab. A comprehensive NGS-based approach is recommended to integrate information on total disease burden and resistant mutations.See related commentary by Eluri et al., p. 302

Supplementary Tables 1-8

Supplementary Figure 1 shows generation and cell-lineage characterization of primary CAF cultures derived from HER2-positive breast cancer patient biopsies. Moreover, it also shows NRG1 staining in two FFPE-associated patient samples from which primary CAFs were derived.

Baseline clinico-pathological characteristics from patients included versus not included in the ctDNA analysis

Supplementary Figure 3 shows how trastuzumab plus pertuzumab, but not trastuzumab alone, overcome NRG1 enhanced pro-survival signaling in HER2-positive breast cancer cells and in ex vivo tumors.

Study protocol

Supplementary Table from Comprehensive Analysis of Metabolic Isozyme Targets in Cancer

Metabolic reprogramming is a hallmark of malignant transformation, and loss of isozyme diversity (LID) contributes to this process. Isozymes are distinct proteins that catalyze the same enzymatic reaction but can have different kinetic characteristics, subcellular localization, and tissue specificity. Cancer-dominant isozymes that catalyze rate-limiting reactions in critical metabolic processes represent potential therapeutic targets. Here, we examined the isozyme expression patterns of 1,319 enzymatic reactions in 14 cancer types and their matching normal tissues using The Cancer Genome Atlas mRNA expression data to identify isozymes that become cancer-dominant. Of the reactions analyzed, 357 demonstrated LID in at least one cancer type. Assessment of the expression patterns in over 600 cell lines in the Cancer Cell Line Encyclopedia showed that these reactions reflect cellular changes instead of differences in tissue composition; 50% of the LID-affected isozymes showed cancer-dominant expression in the corresponding cell lines. The functional importance of the cancer-dominant isozymes was assessed in genome-wide CRISPR and RNAi loss-of-function screens: 17% were critical for cell proliferation, indicating their potential as therapeutic targets. Lists of prioritized novel metabolic targets were developed for 14 cancer types; the most broadly shared and functionally validated target was acetyl-CoA carboxylase 1 (ACC1). Small molecule inhibition of ACC reduced breast cancer viability in vitro and suppressed tumor growth in cell line– and patient-derived xenografts in vivo. Evaluation of the effects of drug treatment revealed significant metabolic and transcriptional perturbations. Overall, this systematic analysis of isozyme expression patterns elucidates an important aspect of cancer metabolic plasticity and reveals putative metabolic vulnerabilities.Significance:This study exploits the loss of metabolic isozyme diversity common in cancer and reveals a rich pool of potential therapeutic targets that will allow the repurposing of existing inhibitors for anticancer therapy.See related commentary by Kehinde and Parker, p. 1695

Calculation of the neoadjuvant rectal (NAR) score.

Kaplan-Meier estimates of disease-free survival and overall survival according to paired CEA and ctDNA baseline and pre-surgery A. Disease-free survival according to baseline ctDNA and baseline CEA B. Overall survival according to baseline ctDNA and baseline CEA C. Disease-free survival according to pre-surgery ctDNA and pre-surgery CEA D. Overall survival according to pre-surgery ctDNA and pre-surgery CEA pos: positive; neg: negative

Supplementary Figure from Comprehensive Analysis of Metabolic Isozyme Targets in Cancer

Kaplan-Meier estimates of disease-free survival and overall survival according to pCR and NAR score in the sub-study cohort A. Disease-free survival according to pCR B. Overall survival according pCR C. Disease-free survival according to NAR score D. Overall survival according NAR score

Supplementary Table 1 shows main clinical-pathological characteristics of HER2-positive breast cancer patients whom primary CAF cultures were derived from.

Supplementary Data from Comprehensive Analysis of Metabolic Isozyme Targets in Cancer

Supplementary Figure 2 shows how NRG1/CAFs, but not normal mammary fibroblasts, mediate trastuzumab resistance in HER2-positive breast cancer cells.

Supplementary Figure 4 shows that NRG1 enhances pro-migratory capacity of breast cancer cells as well as the induction of early markers from the EMT process. Moreover, it also shows that trastuzumab plus pertuzumab, but not trastuzumab alone, are able to prevent NRG1-derived effects.

Supplementary Table 2 shows main clinical-pathological characteristics of HER2-positive breast cancer patient's cohort treated with neoadjuvant-based therapy and the association of NRG1 protein expression with the diverse clinical parameters analysed.

BACKGROUND A cancer diagnosis is a source of psychological and emotional stress, which are often maintained for sustained periods of time that may lead to depressive disorders. Depression is one of the most common psychological conditions in patients with cancer. According to the Global Cancer Observatory, breast and colorectal cancers are the most prevalent cancers in both sexes and across all age groups in Spain. OBJECTIVE This study aimed to compare the prevalence of depression in patients before and after the diagnosis of breast or colorectal cancer, as well as to assess the usefulness of the analysis of free-text clinical notes in 2 languages (Spanish or Catalan) for detecting depression in combination with encoded diagnoses. METHODS We carried out an analysis of the electronic health records from a general hospital by considering the different sources of clinical information related to depression in patients with breast and colorectal cancer. This analysis included ICD-9-CM (International Classification of Diseases, Ninth Revision, Clinical Modification) diagnosis codes and unstructured information extracted by mining free-text clinical notes via natural language processing tools based on Systematized Nomenclature of Medicine Clinical Terms that mentions symptoms and drugs used for the treatment of depression. RESULTS We observed that the percentage of patients diagnosed with depressive disorders significantly increased after cancer diagnosis in the 2 types of cancer considered—breast and colorectal cancers. We managed to identify a higher number of patients with depression by mining free-text clinical notes than the group selected exclusively on ICD-9-CM codes, increasing the number of patients diagnosed with depression by 34.8% (441/1269). In addition, the number of patients with depression who received chemotherapy was higher than those who did not receive this treatment, with significant differences (P CONCLUSIONS This study provides new clinical evidence of the depression-cancer comorbidity and supports the use of natural language processing for extracting and analyzing free-text clinical notes from electronic health records, contributing to the identification of additional clinical data that complements those provided by coded data to improve the management of these patients.

BACKGROUND: Palbociclib in combination with endocrine therapy (ET) is the first- or second-line standard of care for patients (pts) with hormone receptor (HR)-positive (+)/human epidermal growth factor receptor 2 (HER2)-negative (-) metastatic breast cancer (MBC). No clinically validated markers of long-term benefit from palbociclib have been established and the clinically relevant resistance mechanisms to cyclin-dependent kinases 4 and 6 inhibitors remain undefined. In the present study, we explored candidate circulating exosomal microRNAs (miRNAs) as putative predictors of benefit and/or resistance to palbociclib in combination with ET in pts included in the PARSIFAL-1 trial (ClinicalTrials.gov identifier: NCT02491983). MATERIALS AND METHODS: PARSIFAL-1 was a randomized, open-label, phase II trial aimed at evaluating the efficacy and safety of palbociclib plus either fulvestrant or letrozole in HR+/HER2- MBC pts. For the study of exosomal miRNAs, forty-five consenting pts were selected based on primary endocrine resistance according to the ABC-4 criteria. Nine pts who progressed within the first six months after treatment initiation in the absence of an objective response were considered Resistant and thirty-six pts who progressed more than six months after starting therapy were classified as Sensitive. Exosomes were isolated from plasma samples collected at study entry (baseline) and after 12 weeks of treatment initiation. Differences in miRNA expression between Resistant and Sensitive pts as well as miRNA pharmacodynamic changes between baseline and 12-week samples were assessed. Ribonucleic acid (RNA) was isolated using the miRNeasy plasma kit and the library preparation was done using the QIAseq miRNA library kit according to manufacturer’s instructions. All experiments were conducted at QIAGEN Genomic Services in Germany. Differential expression of miRNA between different conditions was studied using EdgeR statistical software package from Bioconductor. Estimated p-values for significantly differentially expressed miRNAs were adjusted using Benjamini-Hochberg’s False Discovery Rate (FDR). Differences in expression of miRNA with an FDR below 0.05 were considered significant. RESULTS: Sequencing of exosomal RNA and preparation of miRNA libraries were successful for all included samples, with good technical performance. On average, 2.8 million Unique Molecular Index-corrected reads were obtained for each sample and the average percentage of mappable reads was 34.5%. Overall, expression of miRNAs was higher in Resistant compared with Sensitive pts. Eight miRNAs were called as differentially expressed between the two groups. Four miRNAs (miR-1246; miR-375; miR-193a-5p; miR-181d-5p) were differentially expressed at baseline, three (miR-196a-5p, miR-200a-3p, miR-320d) were differentially expressed at 12 weeks, and expression of one miRNA (miR-141-3p) was consistently higher across both time points in Resistant pts. Significant pharmacodynamic changes in miRNA expression were observed both in Resistant and Sensitive pts. Seven miRNAs (miR-224-5p, miR-16-5p, let-7a-5p, miR-381-3p, miR-200c-3p, miR-493-3p, let-7b-5p) in Sensitive pts and three miRNAs (miR-223-3p, miR-126-3p, miR-320b) in Resistant pts were differentially expressed between baseline and 12-week samples. CONCLUSIONS: Circulating exosomal miRNA profiling is feasible in liquid biopsies from MBC pts. Differential expression of selected miRNAs at baseline or their pharmacodynamic modulation may predict benefit from palbociclib combined with ET in pts with HR+/HER2- MBC. Validation of the most promising miRNAs by custom quantitative PCR is warranted. Citation Format: David Casadevall, Joan Albanell Mestres, Federico Rojo, Beatríz Bellosillo, Abel González, Violeta Serra, Meritxell Bellet, Miguel Angel Gil, Miquel Angel Pujana, Angelo Gámez, Enrique Espinosa, Peter Schmid, Joseph Gligorov, Frederik Marmé, Carlos L. Arteaga, Leonardo Mina, Andrea Malfettone, Miguel Sampayo, José Manuel Pérez-García, Javier Cortés, Antonio Llombart-Cussac. Baseline and pharmacodynamic changes of circulating exosomal microRNAs predict early versus late progression to palbociclib plus endocrine therapy in patients with metastatic breast cancer. A sub-analysis of the PARSIFAL-1 trial [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-10-17.

Background Several observational studies demonstrated the passage of postvaccine antibodies through breast milk in women vaccinated against coronavirus disease 2019 (COVID-19), mostly with messenger RNA (mRNA)–based vaccines, but lacked long-term data. Methods A 6-month prospective cohort study was performed to determine severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine–induced antibody levels in the breast milk of 33 lactating healthcare workers at different timepoints after mRNA BNT162b2 Pfizer-BioNTech COVID-19 vaccination. Moreover, we examined the correlation of SARS-CoV-2 antibody levels between serum and breast milk, adverse events related to vaccination, and rate of SARS-CoV-2 infections. Results Mothers’ median age was 38 (interquartile range [IQR], 36–39) years and 15 (IQR, 10–22) months for infants. Median (IQR) SARS-CoV-2 immunoglobulin G (IgG) spike protein subunit S1 (S1) vaccine–induced levels at different timepoints for serum–milk pairs were 519 (234–937) to 1 (0–2.9) arbitrary units (AU)/mL at 2 weeks after first dose and 18 644 (9923–29 264) to 78 (33.7–128), 12 478 (6870–20 801) to 50.4 (24.3–104), 4094 (2413–8480) to 19.9 (10.8–51.9), 1350 (831–2298) to 8.9 (7.8–31.5) AU/mL at 2, 4, 12 and 24 weeks after second dose, respectively. We observed a positive correlation of antibody levels between serum and breast milk, no serious adverse events related to vaccination, and 2 (6%) COVID-19 vaccine breakthrough infections. Conclusions Women vaccinated with Pfizer-BioNTech transmit antibodies into breast milk with a positive correlation with serum levels. Both decreased over time in a 6-month follow-up.

In patients with trastuzumab-resistant HER2-positive breast cancer, the combination of everolimus (mTORC1 inhibitor) with trastuzumab failed to show a clinically significant benefit. However, the combination of mTOR inhibition and the antibody-drug conjugate (ADC) trastuzumab-emtansine (T-DM1) remains unexplored. We tested T-DM1 plus everolimus in a broad panel of HER2-positive breast cancer cell lines. The combination was superior to T-DM1 alone in four cell lines (HCC1954, SKBR3, EFM192A, and MDA-MB-36) and in two cultures from primary tumor cells derived from HER2-positive patient-derived xenografts (PDX), but not in BT474 cells. In the trastuzumab-resistant HCC1954 cell line, we characterized the effects of the combination using TAK-228 (mTORC1 and -2 inhibitor) and knockdown of the different mTOR complex components. T-DM1 did not affect mTOR downstream signaling nor induct autophagy. Importantly, mTOR inhibition increased intracellular T-DM1levels, leading to increased lysosomal accumulation of the compound. The increased efficacy of mTOR inhibition plus T-DM1 was abrogated by lysosome inhibitors (chloroquine and bafilomycin A1). Our experiments suggest that BT474 are less sensitive to T-DM1 due to lack of optimal lysosomal processing and intrinsic resistance to the DM1 moiety. Finally, we performed several in vivo experiments that corroborated the superior activity of T-DM1 and everolimus in HCC1954 and PDXderived mouse models. In summary, everolimus in combination with T-DM1 showed strong antitumor effects in HER2-positive breast cancer, both in vitro and in vivo. This effect might be related, at least partially, to mTOR-dependent lysosomal processing of T-DM1, a finding that might apply to other ADCs that require lysosomal processing., This work was supported by ISCIII (CIBERONC CB16/12/00481, CB16/12/00241, PI18/00382, PI18/00006, PI18/01219), Generalitat de Catalunya (2017 SGR 507). MINECO through gBFU2015-71371-R grant and the CRIS Cancer Foundation supported work in AP lab. D. Casadevall was supported by ISCIII (Rio Hortega Research Contract CM16/00023 and Juan Rodés Research Contract JR18/00003). F.J. Sánchez-Martín and S. Menéndez were supported by Department de Salut Generalitat de Catalunya (PERIS SLT002/16/00008 and PERIS SLT006/17/00040). M. Qin received financial support from the China Scholarship Council (CSC) for her doctoral fellowship. Work carried out in our laboratories receives support from the European Community through the Regional Development Funding Program (FEDER).

ImportanceTo our knowledge, this is the first study to analyze long–term passage (6 months after immunization) of specific antibodies induced by BNT162b2 COVID-19 vaccine through breast milk.ObjectivesMain objective: to determine SARS-CoV-2 vaccine induced antibody levels in the breast milk of lactating women 4 weeks after mRNA BNT162b2 Pfizer-BioNTech COVID-19 complete vaccination.Secondary objectives: to analyze SARS-CoV-2 antibody levels (breast milk and serum) at different time-points after vaccination, examine the correlation of SARS-CoV-2 antibody levels between serum and breast milk, describe adverse events related to vaccination (AErV) in both mothers and infants and determine the rate of COVID-19 infections.DesignProspective cohort study between February and September 2021.SettingParc Sanitari Sant Joan de Déu, an urban hospital in Spain.ParticipantsDuring our health worker vaccination campaign at our hospital between January and March 2, we recruited 33 lactating women vaccinated with BNT162b2 Pfizer-BioNTech COVID-19.ResultsA total of 33 volunteers were included in the study. The median (IQR) age of mothers was 38 (36-39) years and 15 (10-22) months for the infants.Primary end-point: at 4 w after second dose median (IQR) IgG-S1 levels for serum–milk pairs were 12,478 (6,870-20,801) to 50.4 (24.3-104) arbitrary units (AU) per mL.Secondary end-pointsSARS-CoV-2 antibody levels at different time-points were (serum-milk): 519 (234-937) to 1 (0-2.9) AU/mL at 2w after first dose, 18,644 (9,923-29,264) to 78 (33.7-128) AU/mL at 2w, 4,094 (2,413-8,480) to 19.9 (10.8-51.9) AU/mL at 12w, and 1,350 (831-2,298) to 8.9 (7.8-31.5) at 24w after second dose.We found a positive correlation of SARS-CoV-2 antibody levels between serum and breast milk (Pearson correlation coefficient 0.68).No serious AErV were observed.We found two (6%) COVID-19 vaccine breakthrough infections.ConclusionsPfizer-BioNTech COVID-19 vaccination is safe during breastfeeding and it transmits antibodies into breast milk with a positive correlation with serum levels, and both decrease over time in a 6-month follow-up. Infants of breastfeeding vaccinated women could be protected for at least six months after vaccination and serum determination of SARS-CoV-2 IgG-S1 could indicate the breastmilk levels of antibodies during this period.

The COVID-19 pandemic has raised questions among individuals who are breastfeeding, both because of the possibility of viral transmission to infants during breastfeeding and, more recently, of the potential risks and benefits of vaccination in this specific population. Previous studies have reported the presence of anti–SARS-CoV-2 antibodies in breast milk of COVID-19–infected lactating women,1 and recently several studies have demonstrated the passage of postvaccine antibodies through breast milk in women vaccinated with novel mRNA-based vaccines.1,2 In the present study, conducted between February and March 2021 at Parc Sanitari Sant Joan de Deu, an urban hospital in Spain, we sought to characterize the levels of specific SARS-CoV-2 antibodies in the breast milk of mRNA-vaccinated women across time, as well as their correlation with serum antibody levels.

Background: Pancreatic cancer (PCa) and biliary tract carcinomas (BTCa) have high morbidity and mortality rates. Bile duct obstruction (BDO) develops in approximately 65–75% of PCa at diagnosis, delaying the administration of an optimal treatment. In patients not candidates for surgery, BDO is usually treated through endoscopy-guided placement of self-expanding stents in the bile duct. Methods: In this retrospective study, we sought to describe clinical characteristics and outcomes of a cohort of patients with BDO of malignant origin who underwent biliary stent placement (BSP), with a special focus regarding complications developed after the procedure. Patients with PCa or BTCa who underwent BSP from 2014 to April 2019 at our institution were included in this cohort. Demographic and clinicopathologic characteristics were collected. Statistical analysis stratified according to ECOG performance status. Specific information regarding stent material (metallic vs. plastic), as well as incidence and type of complications derived from BSP was also recorded. Results: One-hundred fifteen patients were included. The median age was 72 years. Sixty-six patients (57%) had PCa. All patients presented hyperbilirrubinemia, which decreased after the procedure in 111 (96%) patients. Complications were observed in 44 (38%) patients, most of which 33 (75%) were infections. Median time to complication was one month. The mean overall survival (OS) in our study was 20.3 weeks. Conclusion: BSP effectively decreased hyperbilirubinemia in patients with BDO, however, the procedure associated a significant rate of infectious complications, which can further compromise an effective anti-cancer therapy as well as optimal palliative strategies.

Pancreatic cancer (PCa) and biliary tract carcinomas (BTCa) have high morbidity and mortality rates. Bile duct obstruction (BDO) develops in approximately 65-75% of PCa at diagnosis, delaying the administration of optimal treatment. In patients not candidates for surgery, BDO is usually treated through the endoscopy-guided placement of self-expanding stents in the bile duct.In this retrospective study, we sought to describe clinical characteristics and outcomes of a cohort of patients with BDO of malignant origin who underwent biliary stent placement (BSP), with a special focus regarding complications developed after the procedure. Patients with PCa, BTCa, colon cancer, lung cancer, gastric cancer, and ovarian cancer who underwent BSP from 2014 to April 2019 at our institution were included in this cohort. Demographic and clinicopathologic characteristics were collected. Statistical analysis stratified according to ECOG performance status. Specific information regarding stent material (metallic vs. plastic), as well as incidence and type of complications derived from BSP, was also recorded.One hundred fifteen patients were included. The median age was 72 years. Sixty-six patients (57%) had PCa. All patients presented hyperbilirubinemia, which decreased after the procedure in 111 (96%) patients. Complications were observed in 44 (38%) patients, most of which 33 (75%) were infections. The median time to the complication was 1 month. The mean overall survival (OS) in our study was 20.3 weeks.BSP effectively decreased hyperbilirubinemia in patients with BDO; however, the procedure associated a significant rate of infectious complications, which can further compromise an effective anti-cancer therapy as well as optimal palliative strategies.

PURPOSE: African-American (AA) patients with triple-negative breast cancer (TNBC) are less likely to achieve pathologic complete response from neoadjuvant chemotherapy and have poorer prognosis than Caucasian patients with TNBC, suggesting potential biological differences by race. Immune infiltration is the most consistent predictive marker for chemotherapy response and improved prognosis in TNBC. In this study, we test the hypothesis that the immune microenvironment differs between AA and Caucasian patients. METHODS: RNA-seq expression data were obtained from The Cancer Genome Atlas (TCGA) database for 162 AA and 697 Caucasian breast cancers. Estrogen receptor (ER)-positive, human epidermal growth factor receptor-2 (HER2)-positive, and TNBC subtypes were included in the analyses. Tumor infiltrating lymphocyte (TIL) counts, immunomodulatory scores, and molecular subtypes were obtained from prior publications for a subset of the TNBC cases. Differences in immune cell distributions and immune functions, measured through gene expression and TIL counts, as well as neoantigen, somatic mutation, amplification, and deletion loads, were compared by race and tumor subtype. RESULTS: Immune metagene analysis demonstrated marginal immune attenuation in AA TNBC relative to Caucasian TNBC that did not reach statistical significance. The distributions of immune cell populations, lymphocyte infiltration, molecular subtypes, and genomic aberrations between AA and Caucasian subtypes were also not significantly different. The MHC1 metagene demonstrated increased expression in AA ER-positive cancers relative to Caucasian ER-positive cancers. CONCLUSIONS: This study suggests that the immunological differences between AA and Caucasian breast cancers represented by TCGA data are subtle, if they exist at all. We observed no consistent racial differences in immune gene expression or TIL counts in TNBC by race. However, this study cannot rule out small differences in immune cell subtype distribution and activity status that may not be apparent in bulk RNA analysis.

Ligand-dependent corepressor (LCOR) mediates normal and malignant breast stem cell differentiation. Cancer stem cells (CSCs) generate phenotypic heterogeneity and drive therapy resistance, yet their role in immunotherapy is poorly understood. Here we show that immune-checkpoint blockade (ICB) therapy selects for LCORlow CSCs with reduced antigen processing/presentation machinery (APM) driving immune escape and ICB resistance in triple-negative breast cancer (TNBC). We unveil an unexpected function of LCOR as a master transcriptional activator of APM genes binding to IFN-stimulated response elements (ISREs) in an IFN signaling-independent manner. Through genetic modification of LCOR expression, we demonstrate its central role in modulation of tumor immunogenicity and ICB responsiveness. In TNBC, LCOR associates with ICB clinical response. Importantly, extracellular vesicle (EV) Lcor-messenger RNA therapy in combination with anti-PD-L1 overcame resistance and eradicated breast cancer metastasis in preclinical models. Collectively, these data support LCOR as a promising target for enhancement of ICB efficacy in TNBC, by boosting of tumor APM independently of IFN. This work was supported by the Instituto de Salud Carlos III-FSE (nos. MS17/00037 and PI18/00014) and the Cancer Research Institute, Clinic and Laboratory Integration Program (grant no. CRI2477 to T.C.-T). We also thank the AECC LAB (grant no. LABAE19007CELI) and the FERO foundation (to T.C.-T). This work was also supported by ISCIII (CIBERONC nos. CB16/12/00481, CB16/12/00241, PI18/00006 and PI21/00002), Generalitat de Catalunya (no. 2017 SGR 507) and the European Community through the Regional Development Funding Program to J. Albanell. Y.K. is supported by the Brewster Foundation, the Breast Cancer Research Foundation, the Susan G. Komen Foundation and the American Cancer Society. J. Arribas is funded by the Breast Cancer Research Foundation (no. BCRF-20-08), Instituto de Salud Carlos III (no. PI19/01181), Asociación Española Contra el Cáncer (no. GCAEC19017ARRI) and Fundación BBVA (no. CAIMI VHIO-FBBVA 2018-2021). M.M.A. is funded by MICINN and the Spanish Government (no. PGC2018-094091-B-I00). D.C. was funded by Instituto de Salud Carlos III (no. JR1800003). S.M. is funded by PERIS (no. SLT006/17/00040). The wotk of R.R.G. is funded by MICINN, the Spanish Government (no. PID2019-104948RB-I00) and the BBVA Foundation. S.B and J.P. acknowledge support from the Spanish Ministry of Science, the EMBL partnership and the CESO and CERCA Program.

[Purpose]: To characterize expression of neuregulin-1 (NRG1), an HER3 ligand, in HER2-positive breast cancer and its relation with the efficacy of trastuzumab with or without pertuzumab., [Experimental Design]: Characterization of NRG1 expression in tumor cell lines, in tumor specimens, and in cancer-associated fibroblasts (CAFs). Patient-derived CAFs were used to investigate NRG1 impact on the activity of trastuzumab with or without pertuzumab in HER2-positive breast cancer cells. The relationship between NRG1 expression and pathologic response to anti-HER2–based neoadjuvant therapy was assessed in a retrospective patient cohort and in the NeoSphere trial., [Results]: NRG1 was expressed in HER2-positive breast cancer–derived fibroblasts at significantly higher levels than in cancer cells. NRG1 and the conditioned media (CM) from CAFs phosphorylated HER3 and AKT in cancer cells and mediated trastuzumab resistance. Stable genetic depletion of NRG1 from CAFs overcame trastuzumab resistance. Pertuzumab effectively suppressed trastuzumab resistance mediated by either NRG1 or CAF's CM. NRG1 engaged an epithelial-to-mesenchymal transition that was prevented by trastuzumab and pertuzumab. In clinical samples, stromal and/or tumor cell expression of NRG1 determined by immunohistochemistry was uncommon (13.2%) yet significantly linked with residual disease following trastuzumab-based neoadjuvant therapy. In the NeoSphere trial, the magnitude of the difference of pathologic complete response rates favoring the pertuzumab arm was higher in the NRG1-high group., [Conclusions]: CAF-derived NRG1 mediates trastuzumab resistance through HER3/AKT, which might be reverted by pertuzumab. In patients with HER2-positive breast cancer, high expression of NRG1 was associated to poor response to trastuzumab, but not in combination with pertuzumab., This work is supported by ISCIII (CIBERONC CB16/12/00481, CB16/12/00241, PI18/00382, PI18/00006, PI18/01219 and by Generalitat de Catalunya (2017 SGR 507). S. Menendez is supported by Department de Salut, Generalitat de Catalunya (PERIS SLT006/17/00040). MARBiobanc is supported by ISCiii/FEDER (PT17/0015/0011) and by “Xarxa de Bancs de tumors” sponsored by Pla Director d’ Oncologia de Catalunya (XBTC) and Fundacion Jimenez Díaz Biobanks Platform by PT13/0010/0012 grant. Ministry of Economy and Competitiveness of Spain (BFU2015-71371-R) and the CRIS Cancer Foundation provides support to A. Pandiella. Work carried out in our laboratories receive support from the European Community through the Regional Development Funding Program (FEDER). J.C. Montero is funded by the ISCIII through a Miguel Servet program (CPII17/00015) and receives research support from the same institution (PI18/00796). J. Albanell is supported by Breast Cancer Research Foundation (BCRF20-08), Instituto de Salud Carlos III Project Reference number AC15/00062 and the EC under the framework of the ERA-NET TRANSCAN-2 initiative co-financed by FEDER, Instituto de Salud Carlos III (CB16/12/00449 and PI19/01181), and Asociacion Espanola Contra el Cáncer (AECC).

e18742 Background: The clinical predictors of venous thromboembolism (VTE) recurrence in patients with cancer are not well known. Our objective was to develop a predictive model for risk of VTE recurrence in anticoagulant-treated cancer patients within the first 6 months following VTE diagnosis. Methods: Using the EHRead technology, based on Natural Language Processing (NLP) and machine learning (ML), the unstructured clinical data in EHRs from 9 Spanish hospitals between 2014 and 2018 was extracted and analyzed. The study population, comprising all adult anticoagulated cancer patients with VTE, was downsampled to prevent bias and class imbalance. A total of 94 patient characteristics were explored, and Random Forest (RF) feature selection was performed to identify the most relevant predictors for VTE recurrence. Multiple algorithms were used to train different prediction models, which were subsequently validated in a hold-out dataset. The model with the best performance metrics (i.e., ROC-AUC) was selected as the final model. Results: From a source population of 2,893,208 patients, 21,227 anticoagulant-treated patients with VTE and active cancer (53.9% male, median age of 70 years) were identified. Across the study period, yearly incidence of VTE remained relatively stable, ranging from 2.7 to 3.9%. The most common type of VTE was deep vein thrombosis (68.2% of patients), followed by pulmonary embolism (28.4%). Regarding primary cancer locations, the most frequent were colorectal (10.1%) and lung cancer (8.5%). Of all trained and validated models, the RF approach yielded the best performance, with a ROC-AUC = 0.72. The following predictors of VTE recurrence were identified: pulmonary embolism, deep vein thrombosis, metastasis, adenocarcinoma, hemoglobin values, serum creatinine values, platelet count, leukocyte count, family history of VTE, and patients’ age. Conclusions: Using NLP and ML, we were able to use the real-world data in EHRs to build a predictive model of VTE recurrence in cancer patients based on individual clinical features. These results may improve the clinical management of VTE recurrence in this population.

2551 Background: Inflammation and neutrophils play a central role in severe Covid-19 disease. In previous data, we showed that the FLARE score, combining both tumor and Covid-19-induced proinflammatory status (proinflam-status), predicts early mortality in cancer patients (pts) with Covid-19 infection. We aimed to assess the impact of this score in a larger cohort and characterize the immunophenotype (IF) of circulating neutrophils. Methods: Multicenter retrospective cohort (RC) of pts with cancer and Covid-19 infection across 14 international centers. Circulating inflammatory markers were collected at two timepoints: baseline (-15 to -45d before Covid-19 diagnosis) and Covid-19 diagnosis. Tumor-induced proinflam-status was defined by high dNLR (neutrophils/(leucocytes-neutrophils)>3) at baseline. Covid-19-induced proinflam-status was defined by +100% increase of dNLR between both timepoints. We built the FLARE score combining both Tumor and Infection-induced inflammation: T+/I+ (poor), if both proinflam-status; T+/I- (T-only), if inflammation only due to tumor; T-/I+ (I-only), if inflammation only due to Covid; T-/I- (favorable), if no proinflam-status. The IF of circulating neutrophils by flow cytometry was determined in a unicenter prospective cohort (PC) of pts with cancer during Covid-19 infection and in healthy volunteers (HV). Primary endpoint was 30-day mortality. Results: 524 pts were enrolled in the RC with a median follow-up of 84d (95%CI 78-90). Median age was 69 (range 35-98), 52% were male and 78% had baseline PS

Purpose: Total neoadjuvant treatment (TNT) is a valid strategy for patients with high-risk locally advanced rectal cancer (LARC). Biomarkers of response to TNT are an unmet clinical need. We aimed to determine the value of circulating tumor DNA (ctDNA) to predict tumor response, recurrence, and survival in patients with LARC treated with TNT. Experimental Design: The GEMCAD 1402 was a phase II randomized, multicentric clinical trial that randomized 180 patients with LARC to modified schedule of fluorouracil, leucovorin, and oxaliplatin (mFOLFOX6) +/− aflibercept, followed by chemoradiation and surgery. Plasma samples were collected at baseline and after TNT within 48 hours before surgery (presurgery). An ultrasensitive assay that integrates genomic and epigenomic cancer signatures was used to assess ctDNA status. ctDNA results were correlated with variables of local tumor response in the surgery sample, local/systemic recurrence, and survival. Results: A total of 144 paired plasma samples from 72 patients were included. ctDNA was detectable in 83% of patients at baseline and in 15% following TNT (presurgery). No association was found between ctDNA status and pathologic response. Detectable presurgery ctDNA was significantly associated with systemic recurrence, shorter disease-free survival (HR, 4; P = 0.033), and shorter overall survival (HR, 23; P < 0.0001). Conclusions: In patients with LARC treated with TNT, presurgery ctDNA detected minimal metastatic disease identifying patients at high risk of distant recurrence and death. This study sets the basis for prospective clinical trials that use liquid biopsy to personalize the therapeutic approach following TNT.

Introduction Hyperprogressive disease (HPD) as a consequence of immune checkpoint inhibitors in NSCLC has been reported in multiple studies. However, inconsistent results in incidence and survival outcomes within studies, together with different assessment methods, have led to increasing controversy regarding the concept of HPD. Methods Consecutive patients treated with nivolumab (N = 42) or docetaxel (N = 37) were evaluated. HPD was quantified by applying three different methods (tumor growth rate [TGR], tumor growth kinetics [TGK], and Response Evaluation Criteria in Solid Tumors version 1.1 [RECIST 1.1]). HPD rates were compared between and within both cohorts using the different methods. Results Using TGR, TGK, and RECIST 1.1, we identified seven (16.7%), seven (16.7%), and six (14.3%) patients with HPD in the nivolumab cohort and three (8.1%), four (10.8%), and five (13.6%) in the docetaxel cohort, respectively. We observed a higher concordance between TGR and TGK (90.1%) compared with RECIST 1.1 (31.3% and 37.5% with TGR and TGK, respectively). We found no significant differences in the overall survival between patients with progressive disease and HPD in either cohort. Conclusions TGR and TGK revealed high concordance rates for identifying patients with HPD in NSCLC. The incidence of HPD was numerically higher in patients treated with immune checkpoint inhibitors. Standardization of methods for measuring HPD and its exploration in larger studies are needed to establish its clinical meaning in NSCLC.

Breast cancer is the most common malignancy among women. Most of breast cancer patients are diagnosed in early stages and will be treated with curative intent. Despite this, some patients will relapse. The identification of patients at high risk remains an important challenge. CTCs can be useful to identify this patients, to assess tumor dynamics and to monitoring therapy. There is definitive evidence on the prognostic role of CTCs in early breast cancer (eBC) but its clinical utility in daily practice is still lacking. We have to take into consideration that the studies published to date mainly evaluated the presence of CTC based on the expression of epithelial surface markers. Future studies need to overcome this limitation and important advances in technical methods can assess CTCs and capture the heterogeneity of the tumor landscape. It is also tempting to speculate that CTCs may also provide complementary information on the interplay of tumor cells with the immune system. The combination of different methods to detect tumoral disease by liquid biopsy may provide new ways to personalize in an unprecedented manner the management of patients with eBC.

Breast cancer is the most common malignancy among women. Most of breast cancer patients are diagnosed in early stages and will be treated with curative intent. Despite this, some patients will relapse. The identification of patients at high risk remains an important challenge. CTCs can be useful to identify this patients, to assess tumor dynamics and to monitoring therapy. There is definitive evidence on the prognostic role of CTCs in early breast cancer (eBC) but its clinical utility in daily practice is still lacking. We have to take into consideration that the studies published to date mainly evaluated the presence of CTC based on the expression of epithelial surface markers. Future studies need to overcome this limitation and important advances in technical methods can assess CTCs and capture the heterogeneity of the tumor landscape. It is also tempting to speculate that CTCs may also provide complementary information on the interplay of tumor cells with the immune system. The combination of different methods to detect tumoral disease by liquid biopsy may provide new ways to personalize in an unprecedented manner the management of patients with eBC.

Introduction: Comprehensive genomic profiling (CGP) of tumors is available at many specialized cancer centers, potentially allowing oncologists to individualize patient therapy based on tumor molecular features. However, clinicians still lack a systematic approach to deal with the wealth of information provided by CGP. The proportion of plausible drug-matched alterations over the total number of alterations detected by CGP (Matching Score, MS) has been postulated to predict the likelihood of patient benefit. We performed a systematic analysis of MS and its association with the outcome of patients treated according to tumor DNA alterations within two recent precision oncology trials. Materials and Methods: We downloaded the supplementary data from WINTHER (NCT01856296) and I-PREDICT (NCT02534675) studies. We included patients who received treatment for one or more single-gene alterations, excluding eight patients that received immunotherapy due to high tumor mutational burden, PD-L1 positivity or microsatellite instability. We used MS cut-offs of 0.25 (as in WINTHER) and 0.5 (as in I-PREDICT) and also MS as a continuous variable to study its association with clinical variables such as age, sex, tumor type, and number of previous treatment lines (Kruskall-Wallis and Wilcoxon-rank tests), as well as disease control rate (DCR), progression-free survival (PFS) and overall survival (OS) (Cox proportional hazards models). Results: The pooled analysis included 144 patients. Median age was 60 years (range 21-86) and 64% had received ≥ 2 lines of therapy. Most patients had colorectal (37%), lung (14%), or breast (11%) tumors, and 58% were treated with 2 or more drugs. MS was not associated with any clinical variable nor with DCR. MS > 0.25 (n=98; 68%) was associated with longer PFS (median 3.5 vs. 1.9 months, HR [95%CI]: 0.60 [0.41-0.87]) but not with OS. In contrast, MS > 0.5 (n=41; 28%) was associated with longer OS (median 14.1 vs. 6.5 months, HR [95%CI]: 0.50 [0.29-0.54]) but not with PFS. As a continuous variable, increasing MS values were associated with better OS (HR [95% CI]: 0.43 [0.20-0.95]) and with a non-significant trend towards better PFS (HR [95% CI]: 0.54 [0.21-1.05]). In a multivariable model adjusted for Age and study, PFS/OS probabilities improved consistently with increasing MS values from 0 to 0.5. With MS values > 0.5, OS probabilities kept increasing while PFS probabilities plateaued. Conclusions: The significant and consistent relationship between MS and OS (in contrast with PFS) may be explained by patients with higher MS values having more targeted therapies available for successive treatment lines, although we cannot exclude the existence of other confounding prognostic variables. We note that, as better drugs are developed and new knowledge is incorporated into the clinic, the value of CGP will probably increase over time, improving the number of patients with actionable alterations and their outcome. Citation Format: David Casadevall, Michal Marczyk, Xavier Monzonis, Lajos Pusztai, Joan Albanell. Pooled analysis of matching score and patient outcome in I-PREDICT and WINTHER studies [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 423.

Background: Changes in the clinical subtype (CS) and intrinsic subtype (IS) between breast cancer (BC) metastases and corresponding primary tumours have been reported. However, their relationship with tumour genomic changes remains poorly characterised. Here, we analysed the association between genomic remodelling and subtype conversion in paired primary and metastatic BC samples. Methods: A total of 57 paired primary and metastatic tumours from GEICAM/2009-03 (ConvertHER, NCT01377363) study participants with centrally assessed CS (n = 57) and IS (n = 46) were analysed. Targeted capture and next-generation sequencing of 202 genes on formalin-fixed paraffin-embedded samples was performed. The cancer cell fraction (CCF) of mutations in primary and metastatic pairs was estimated as a surrogate of tumour clonal architecture. Changes in mutation CCF between matched primary and metastatic tumours were analysed in the presence or absence of subtype conversion. Findings: CS conversion occurred in 24.6% and IS conversion occurred in 36.9% of metastases. Primary tumours and metastases had a median of 11 (range, 3-29) and 9 (range, 1-38) mutations, respectively (P = 0.05). Overall, mutations in metastases showed a higher estimated CCF than in primary tumours (median CCF, 0.51 and 0.47, respectively; P = 0.042), consistent with increased clonal homogeneity. The increase in mutation CCF was significant in CS-converted (P = 0.04) but not in IS-converted (P = 0.48) metastases. Clonal remodelling was highest in metastases from hormone receptorepositive and human epidermal growth factor 2 (HER2)-positive tumours (P = 0.006). Conclusions(1): Mutations in BC metastases showed significantly higher estimated CCF than primary tumours. CCF changes were more prominent in metastases with CS conversion. Our findings suggest that changes in BC subtypes are linked to clonal remodelling during BC evolution. (C) 2019 Elsevier Ltd. All rights reserved.

OBJECTIVE: We aimed to assess MET intratumoral heterogeneity and its potential impact on biomarker-based patient selection as well as potential surrogate biomarkers of MET activation. METHODS: Our study included 120 patients with non-squamous Non-small-cell Lung Cancer (nsNSCLC), of which 47 were incorporated in tissue microarrays (TMA). Four morphologically distinct tumor areas were selected to assess MET heterogeneity. MET positivity by immunohistochemistry (IHC) was defined as an above-median H-score and by +2/+3 staining intensity in >50% of tumor cells (Metmab criteria). MET FISH positivity was defined by MET/CEP7 ratio ≥ 2.0 and/or MET ≥ 5.0. MET staining pattern (cytoplasmic vs. membranous) and mesenchymal markers were investigated as surrogates of MET activation. RESULTS: Median MET H-score was 140 (range 0-400) and 47.8% of patients were MET positive by Metmab criteria. Eight cases (6.8%) were MET FISH positive and showed higher H-scores (p = 0.021). MET positivity by IHC changed in up to 40% of cases among different tumor areas, and MET amplification in 25-50%. Cytoplasmic MET staining and positivity for vimentin predicted poor survival (p = 0.042 and 0.047, respectively). CONCLUSIONS: MET status is highly heterogeneous among different nsNSCLC tumor areas, hindering adequate patient selection for MET-targeted therapies. MET cytoplasmic staining and vimentin might represent surrogate markers for MET activation. This work was supported by RD12/0036/0051 /FEDER, PI13/00140/FEDER, a grant from Fundacio Marato de TV3. Ref.666/C/2013 and 2014 SGR 740. the “Xarxa de Bancs de tumors sponsored by Pla Director d’Oncologia de Catalunya (XBTC).” JA is recipient of intensification programme ISCIII.

Background Despite adequate treatment and follow-up, around one fifth of patients with localized bladder cancer will present with disease progression. Adequate prognostic biomarkers are lacking to define patients who are at risk. Mutations in chromatin remodeling genes are more frequently found in bladder cancer than in any other solid tumor. However, the prognostic relevance of epigenetic dysregulation has not been established and may offer an opportunity for biomarker discovery. Methods Looking for prognostic epigenetic factors, we performed a comprehensive PubMed search using keywords such as “bladder cancer”, “chromatin remodeling”, “gene methylation” and “epigenetics”. We only included studies reporting on the association of epigenetic markers with prognostic outcomes such as recurrence, progression or survival. Results Of 1113 results, 87 studies met the inclusion criteria, which represented a total of 85 epigenetic markers with potential prognostic relevance. No prospective studies were identified. Seventy-three percent (64/87) of the studies involved mixed cohorts of muscle invasive and non-muscle invasive bladder cancer. Promoter methylation of genes with putative prognostic value affected cellular processes such as cell cycle, apoptosis, cell-adhesion or migration, as well as critical pathways such as MAP-kinase or Wnt. Alteration of chromatin regulatory elements suggest a prognostic relevance alterations leading to a predominantly silenced chromatin state. Conclusions The prognostic impact of epigenetic alterations in bladder cancer is still unclear. Prospective evaluation of methylation marks and chromatin remodeling gene alterations using consistent methods and criteria is warranted.

Immune-checkpoint inhibitors against programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) have shown remarkable therapeutic activity in non-small-cell lung cancer (NSCLC). However, biomarker-based patient selection remains a challenge. Our aim was to assess the heterogeneity of various immune markers between different tumor areas of surgically resected NSCLC specimens.We included 94 adenocarcinoma (ADC) and 50 squamous cell carcinoma (SCC) specimens. Two distinct tumor areas of each tumor sample were selected and incorporated into tissue microarrays. PD-L1 expression in tumor cells (TCs) and immune cells (ICs) was assessed using clone SP142 (Ventana). PDL1 gene amplification was assessed using fluorescence in situ hybridization. CD3 and CD8 densities were quantified using digital image-based analysis. Heterogeneity was assessed using kappa agreement index (KI) and intraclass correlation coefficient.Prevalence of PD-L1 expression was 16.8% in TCs and 27.8% in ICs. Eleven tumors (7.6%) showed PDL1 amplification. In ADC, KI of PD-L1 TC and IC expression between cores was 0.465 and 0.260, compared with 0.274 and 0.124 in SCC, respectively. Higher concordance was observed for PDL1 amplification (KI, 0.647 in ADC and KI, 1 in SCC). Eleven (61.1%) of 18 amplified cores showed PD-L1 staining in 5% of TCs. Intraclass correlation coefficients for CD3 and CD8 were 0.293 and 0.186 in ADC and 0.489 and 0.610 in SCC samples, respectively.We found significant heterogeneity of PD-L1 expression in both ADC and SCC samples, especially in the IC compartment. Heterogeneous expression of PD-L1 could misclassify patients for PD-1/PD-L1-directed therapies.

Aims CD274 (PDL1) and JAK2 (9p24.1) gene amplifications have been recently described in pulmonary carcinomas in association with programmed death-ligand 1 (PD-L1) expression. Furthermore, PTEN loss has been explored preclinically in relation to PD-L1 expression. Our aim was to determine whether these genomic alterations affect PD-L1 expression levels in non-small-cell lung cancer. Methods and results PD-L1 and PTEN expression determined by immunohistochemistry (IHC), and CD274, JAK2 and PTEN copy number alterations (CNAs) determined by fluorescence in-situ hybridisation, were studied in 171 pulmonary carcinoma specimens. PD-L1 expression was positive in 40 cases (23.3%), and CD274 amplification was present in 14 tumours (8.8%). Concordance between both events was found in 12 of 14 amplified cases (P = 0.0001). We found nine JAK2-amplified cases (5.7%), seven with PD-L1 expression (P = 0.0006). Moreover, six of the seven cases had JAK2 and CD274 coamplification (9p24.1 genomic amplification). Remarkably, the average PD-L1 IHC score was higher in these amplified cases (230 versus 80; P = 0.001). Non-statistical associations were observed between PD-L1 expression and PTEN loss and PTEN deletions. Conclusions We describe a subset of patients (8.2%) who had 9p24.1 amplifications resulting in high expression of PD-L1. Our results provide evidence for genomic up-regulation of PD-L1 expression in non-small-cell lung cancer.

BACKGROUND: Approximately 50 % of malignant melanomas harbor activating point mutations in the BRAF gene. Typically, these mutations result in the substitution of the amino acid valine at codon 600 of the gene, and 90-95 % of mutations are either BRAF (V600E) or BRAF (V600K). Specific BRAF inhibitors such as dabrafenib and vemurafenib are the mainstays of treatment in patients with metastatic BRAF-mutant malignant melanomas. The third most common BRAF mutation is V600R, which also leads to increased BRAF signaling. Although evidence exists about the activity of dabrafenib and vemurafenib in patients with the BRAF (V600R) mutation, these patients have been systematically excluded from recent trials with targeted therapies. CASE PRESENTATION: Here, we report the positive results in terms of survival and quality of life obtained with dabrafenib in an 80-year-old Caucasian male patient with a Charlson Comorbidity Index of 8 diagnosed with metastatic malignant melanoma harboring the BRAF (V600R) mutation. Our patient was treated with dabrafenib for 7 months with minimal toxicity. We also report exploratory analyses of circulating tumor DNA during targeted treatment. Interestingly, the mutation was not detected after starting treatment and became detectable before radiological disease progression. CONCLUSIONS: Our report suggests that (1) a relevant benefit can be obtained with a BRAF inhibitor in real-world patients with a malignant melanoma harboring a BRAF (V600R) mutation, and that (2) circulating tumor DNA detection might be of help in assessing tumor burden in everyday clinical practice. The results reported here should encourage the inclusion of patients with BRAF (V600R)-mutated malignant melanomas in future prospective clinical trials with BRAF inhibitors.