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Background: Mucosal-Associated Invariant T cells (MAITs) are an abundant subset of non-conventional T-cells with potent cytotoxic capacity (up to 20% of circulating T-cells) that are naturally resident in many tissues and solid tumors. T-cell redirection is a clinically validated approach to treating haematological cancers but has limited success in solid tumors. Classical T-cell engagers (TCE) bind the epsilon chain of the TCR leading to activation of all T-cells. MAIT cells utilize a semi-invariant TCR and recognize bacterial metabolites presented in the context of the MR1 protein. Biomunex has generated bispecific antibodies that bind the MAIT semi-invariant TCR (iTCR) and the HER2 receptor tyrosine kinase expressed on tumor cells. This enables the formation of an efficient immunological synapse and exclusive redirection of MAIT cells to directly kill cancer cells. Methods: Using the Biomunex proprietary BiXAb® platform, bispecific, tetravalent antibodies were generated that target the MAIT iTCR and HER2. Specific binding to the two targets was demonstrated by ELISA, DUAL ELISA and in FACS. In general, BiXAb®-mediated MAIT-cell activation, proliferation and degranulation were followed by gating on MAIT cells within a purified CD8 cell population. Tumor cell lines expressing varying amounts of HER2 were co-cultured with MAIT cells (peripheral and tumor-resident) and the BiXAb®s in several cytotoxic assays which were evaluated by measuring Chromium release, LDH release and FACS. Results: The iTCR x HER2 BiXAb® efficiently binds both target proteins with similar affinities to the parental Mabs and can bind both simultaneously, as judged by Dual ELISA. The iTCR x HER2 BiXAb® binds the MAIT-cell TCR and can bind cancer cells over a wide range of HER2 expression. BiXAb® engagement of MAIT cells and cancer cells leads to rapid activation, proliferation and degranulation of MAIT cells. In a population of PBMCs, only MAIT cells are activated by the BiXAb®. Even at low effector to target ratios (E:T = 2:1), MAIT cells efficiently kill engaged cancer cells in a HER2-dependent manner (over 50% cytotoxicity in 18 hrs). The MAIT-directed BiXAb® does not activate other T-cell subsets and hence has significantly reduced cytokine release when compared to a classical T-cell engager. Conclusions: BiXAb® mediated MAIT cell redirection leads to efficient killing of cancer cells and is a promising new approach for the treatment of solid tumors. The iTCR x HER2 BiXAb® has no impact on the general CD8 or CD4 T-cell population which may address some of the clinical limitations of classical TCEs. Citation Format: Simon Edward Plyte, Marie Fraudeau, Jonathan Grivel, Paloma Hougron, Katja Klausz, Dorothee Winterberg, Britta von Below, Alexandre Ivagnes, Claire Germain, Sebastian Amigorena, Eugene Zhukovsky, Matthias Peipp, Pierre-Emmanuel Gerard, Olivier Lantz, Julie Prigent. MAIT engagers: An efficacious novel modality in the field of T-cell engagers for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2954.

Background: Human conventional dendritic cells (cDC) are essential for the anti-tumor immune response. Their immunogenic and tolerogenic states in cancer remain controversial. Our objective was to define the range of DC activation states in vitro and to determine their relevance ex vivo in cancer. Methods: First, in vitro, we activated healthy donor cDC with 16 different stimuli and measured cDC and T cells cytokine and chemokine secretion upon co-culture (n = 130 individual experiments) to define new maturation archetypes. Second, we established the relevance of these archetypes in cancer, ex vivo in human. We performed in depth analysis of cDC infiltrating head and neck cancer (HNSCC) by flow cytometry (n = 22 patients), transcriptomic analysis (n = 6 patients), and cDC-enriched single-cell transcriptomics (ScRNAseq) (n = 10503 cells from 2 patients). Then we performed a merged analysis of cDC from in house and public ScRNAseq datasets (n = 36 tumor samples from HNSCC, Lung, and Breast cancer, and, as comparator, n = 33 blood and juxtatumor samples, n = 19 healthy donor samples, and n = 23 samples from inflammatory diseases). Results: In vitro, we identified two archetypes of cDC activation: (i) PD-L1highICOSLlow cDC that produce large amounts of inflammatory cytokines and chemokines, labeled Secretory cDC; (ii)PD-L1lowICOSLhigh cDC that induce a broad range of Th cytokines, labeled Helper cDC. This functional dichotomy of cDC was mutually exclusive and not receptor specific. In cancer, we identified Secretory cDC, and these cells aligned with mature LAMP3+cDC. Helper cDC were not found ex vivo. Secretory cDC simultaneously expressed stimulatory (CD40, TNFRSF9 (4-1BB)), inhibitory (CD274, CD200, IDO1), and mixed (PVR) checkpoints. Secretory cDC were associated with T cell inflammation in HNSCC, triple-negative breast cancer (TNBC), and melanoma (all p < 1.10-15), with improved overall survival in HNSCC and TNBC (all p < 0.01), and with response to checkpoint blockade in 2 melanoma cohorts (all p < 0.01). Conclusion: We identify and characterize Secretory cDC in several solid cancers. This novel phenotypic and functional dichotomy of human cDC activation states has broad implications for all types of immunotherapies and provides rational for drug combinations. Citation Format: Caroline Hoffmann, Floriane Noël, Maximilien Grandclaudon, Lucile Massenet-Regad, Paula Michea, Philemon Sirven, Lilith Faucheux, Aurore Surun, Olivier Lantz, Mylene Bohec, Jian Ye, Weihua Guo, Juliette Rochefort, Jerzy Klijanienko, Sylvain Baulande, Charlotte Lecerf, Maud Kamal, Christophe Le Tourneau, Maude Guillot-Delost, Vassili Soumelis. PD-L1 high ICOSL low Secretory dendritic cells infiltrate human solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2105.

Background: Cancer mutations acquired during carcinogenesis are not subject to central tolerance and thus, are cornerstone in cancer immunosurveillance. The use of mutation-derived neoepitopes to design cancer vaccines is an unprecedented opportunity to target efficient antigens, but the stochastic occurrence of mutations implies that a tailored vaccine needs to be designed and manufactured for each patient. We used next generation sequencing, machine learning based genomic data analysis and advances in genome editing to systematically identify private neoepitopes in high-risk HPV negative HNSCC and relapsing OvC patients. Methods: Mutations were identified by DNA and RNA sequencing and putative epitopes were selected for vaccine design on their likelihood to elicit a class I or II response based on HLA binding, allelic frequency, prediction of processing and expression at RNA level. The vaccine was a recombinant virus of the modified Vaccinia Ankara (MVA) strain encoding for up to 30 sequences of 29 amino acids centred on the predicted epitope and flanked with the normal germline sequence in case of missense mutations, and of the mutated protein sequence in case of frameshift mutations. The viral vaccine was amplified under GMP conditions and administered to patients after curative intent treatment in two phase I trials. Treatment was started for OvC patients at relapse defined as elevation of CA-125 or onset of suspicious radiological findings and, after first line therapy for HNSCC patients with locally advanced disease with either extracapsular extension or positive resection margins following upfront surgery. The vaccine was administered for 6 weeks and a booster dose every three weeks over a year, in absence of other cancer therapy. Results: Six patients (4 OvC and 2 HNSCC) were treated and experienced only mild related adverse events. After 6 doses of vaccine, we measured immunoreactive T cells in PBMCs against each of the targeted epitopes using an ex vivo IFNγ ELISPOT showed robust responses in all evaluable patients. Positive responses to vaccination were observed for a total of 46 epitopes over 115 tested with a median of 11 epitopes (6 to 18) per patient. Responses were directed against both class I and II epitopes and were either de novo responses (64%) or amplification of pre-existing responses (36%). In parallel, phenotyping of patient PBMCs showed, in all treated patients, a decline in naïve CD4 and CD8 cells and the onset of effector CD27- CD4 and CD27-/CD57+ CD8; similarly, CD56dim NK cells increased suggesting a stimulation of antitumor innate immunity. In one ovarian cancer patient, these immune changes correlated with a normalization of CA-125 that lasted 7 months. Conclusions: These data show that the personalized approach with TG4050 is feasible, safe and immunogenic. The two phase I trials are ongoing. Citation Format: Matthew S. Block, Jean-Pierre Delord, Christian H. Ottensmeier, Christophe Le Tourneau, Ana Lalanne, Olivier Lantz, Keith L. Knutson, Gisèle Lacoste, Annette Tavernaro, Maud Brandely, Nathalie Silvestre, Benoit Grellier, Yoshiko Yamashita, Onoue Kousuke, Naoko Yamagata, Eric Quemeneur, Kaidre Bendjama. Phase I trials of personalized cancer vaccine TG4050 in surgically treated high-risk head and neck squamous cell carcinoma (HNSCC) and relapsing ovarian cancer (OvC) patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT182.

Background Preliminary reports suggested that circulating tumor DNA (ctDNA) can be used as a prognostic marker in a way akin to circulating tumor cells (CTC) in metastatic breast cancer patients. However ctDNA detection is often performed on multiple mutations, combining heterogeneous techniques. Here we used the high prevalence of TP53 mutations in triple-negative metastatic breast cancer (TNMBC) to compare CTC and ctDNA detection rates and prognostic value. Methods A cohort of 40 patients treated at the Institut Curie (Paris, France) was enrolled before starting a new line of treatment for TNMBC. CTC were detected by the CellSearch system (in 7.5 mL of blood). Using massively parallel sequencing (NGS), TP53 mutations were first characterized in tumor tissue, then in plasma DNA extracted from fresh frozen plasma samples (from 15-20 mL of blood). ctDNA detection was performed using high depth targeted sequencing using two platforms in parallel (Illumina HiSeq 2500 and Roche 454). Libraries for Illumina were prepared following the TAm-Seq procedure (Forshew et al, Sci Transl Med 2012), with preamplification of all coding TP53 exons and flanking untranslated regions followed by both paired-end 150bp Illumina and 454/Roche sequencing. CTC, ctDNA and usual patient characteristics were correlated with time to progression (TTP) and overall survival (OS). Results Archived tumor (FFPE or frozen) tissue was available for 36 patients, and 31 were successfully sequenced: TP53 mutations were found in 27 patients. As measured on the Illumina platform, ctDNA was detected in 21/27 patients (81%), ranging from 48 to 648,000 copies/mL of plasma (median 1620). Mutant allele fraction in circulating cell-free DNA ranged from 2 to 70% (median 5%). Comparison between ctDNA levels measured by Illumina and 454/Roche platforms in plasma displayed a good correlation (R2 = 0.903), with a single discordance. ≥1 CTC were detected in 19 of these 27 patients (70%). Strikingly, high ctDNA levels had prognostic impact neither on OS, nor on TTP, whatever the dataset used (Illumina or 454) whereas CTC≥5/7.5 mL were correlated with OS (p=0.04) and marginally with TTP (p=0.06). Other known usual factors, such as poor performance status, elevated LDH and number of previous treatment lines had also significant prognostic factors in this cohort. CTC and ctDNA early changes during treatment were available for 12 patients and changes (increase/decrease) of the two biomarkers were globally similar. Conclusion Demonstrating a good sensitivity (81%), ctDNA by the TAm-Seq is more frequently detected than CTCs in the 27 TNMBC with TP53 mutations. The observed correlation between the 2 massively parallel sequencing approaches suggested that ctDNA levels data were quantitative. In contrast to other usual prognostic factors, baseline ctDNA level did not demonstrate a prognostic impact,in this proof-of-principle study, suggesting that mechanisms of ctDNA release in TNMBC rely on biological features that do not dramatically impact patient’s outcome. Citation Format: Francois-Clement Bidard, Jordan Madic, Anna Kiialainen, Fabian Birzele, Guillemette Ramey, Quentin Leroy, Thomas Rio Frio, Virginie Raynal, Virginie Bernard, Alban Lermine, Inga Clausen, Nicolas Giroud, Roland Schmucki, Carsten Horn, Olivia Spleiss, Olivier Lantz, Marc-Henri Stern, Martin Weisser, Ronald Lebofsky, Jean-Yves Pierga. Circulating tumor DNA and circulating tumor cells in metastatic triple negative breast cancer patients [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr PD3-8.

Inhibitory CD94-NKG2A (Natural Killer Group 2A) receptors are expressed on subsets of natural killer (NK) cells, γδ and CD8+ T cells. HLA-E (Human Leukocytes Antigen-E), the ligand for CD94-NKG2A, is upregulated on cancer cells of several solid tumors, providing a negative regulatory signal to tumor-infiltrating lymphocytes (TILs). Blockade of this immune checkpoint pathway with anti-NKG2A monoclonal antibodies (Ab) enhances NK cell responses to tumor cells in vitro and in humanized mice. Here, we describe NK and CD8+ T cell infiltrates in several human solid tumors by immunohistochemistry (IHC) and multicolor flow cytometry. We then studied the effects of in vitro targeting both pathways on primary human NK and CD8+ T cells. By using IHC on formalin-fixed paraffin-embedded samples on cohorts of solid tumors i.e. non-small cell lung cancer (NSCLC, n=45), stomach tumor (n=76), colorectal cancer (CRC, n=103), head and neck tumor (HNSCC, n=68), pancreatic tumors (n=77) and renal cell carcinoma (RCC, n=75), we observed NKp46+ NK cells in the large majority of cancer types (especially in RCC, HNSCC and stomach cancers). CD8+ T cells were found at significantly higher densities in all tumor types. In another cohort of HNSCC samples (n=60), higher proportions of CD94+ lymphocytes were found within the tumor islets both in the primary tumors and in the metastatic lymph nodes. In addition, in CRC patients with liver metastasis (n=101), high CD94+ cell densities were associated with poor overall survival. These data suggest that NKG2A blockade might unleash NK and T cells that are present in close contact to tumor cells. As previously shown, we confirmed that CD94-NKG2A ligand, HLA-E, was expressed by tumor cells in the large majority of solid tumor. In addition, high PD-L1 expressions were observed in NSCLC, stomach tumors, CRC and HNSCC. Flow cytometry analyses revealed that NK and CD8+ T cells co-expressing NKG2A and PD-1 were more numerous in tumor compared to matched peripheral blood and adjacent tissues of NSCLC and HNSCC patients. To mimic the “NKG2A+PD-1+” TILs phenotype, chronic stimulation of peripheral blood cells from healthy volunteers was performed using IL-15. The conditions led to the induction of NKG2A and PD-1 on NK and antigen-specific CD8+ T cell subsets. These effector cells were then co-cultured with tumor cell lines expressing HLA-E +/- PD-L1. We showed that monalizumab, a first-in class anti-NKG2A Ab, combined with durvalumab resulted in higher responses (CD107 mobilization and IFN-γ production) compared to each Ab alone. These data were confirmed in a syngeneic mouse model where both NK and CD8+ T cells were found to be involved in tumor rejection. Together, these data confirm that blocking NKG2A can potentiate the anti-tumor efficacy of PD-1 inhibitors and support the rationale for ongoing clinical trials investigating the monalizumab/durvalumab combination (NCT02671435 and NCT03088059). Citation Format: Caroline Soulas, Romain Remark, Vedran Brezar, Julie Lopez, Elodie Bonnet, Flavien Caraguel, Ana Lalanne, Caroline Hoffmann, Caroline Denis, Thomas Arnoux, Clarisse Caillet, Arnaud Dujardin, Guillaume Habif, Olivier Lantz, Cécile Bonnafous, Eric Vivier, Pascale Andre. Combination of monalizumab and durvalumab as a potent immunotherapy treatment for solid human cancers [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 2714.

Monalizumab (IPH2201) is a first-in-class humanized IgG4 targeting NKG2A (Natural Killer Group 2A), which is expressed as a heterodimer with CD94 at the surface of subsets of NK (Natural Killer) cells, γδ T cells and tumor infiltrating CD8+ T cells. This inhibitory receptor binds to HLA-E (Human Leukocytes Antigen-E) molecules that are frequently up-regulated on human cancer cells, preventing from killing by NKG2A+ immune cells. HLA-E is expressed on most of the patients with Head and Neck Squamous Cell Cancer (HNSCC). Monalizumab blocks the binding of CD94/NKG2A to HLA-E, reducing inhibitory signaling thereby unleashing NK and T cell responses. High expression of EGFR occurs in most epithelial malignancies and particularly in HNSCC and is associated with poor prognosis. The anti-EGFR monoclonal antibody cetuximab (Ctx) is thought to act through blocking oncogenic signaling and by inducing Fcγ receptor-mediated antibody dependent cell cytotoxicity (ADCC) which involves human NK cells. We investigated ex vivo and in vitro the rationale of combining monalizumab with Ctx in the treatment of oral cancers. We first analyzed formalin-fixed paraffin-embedded samples of HNSCC patients by immunohistochemistry (n=65). We confirmed that HLA-E was expressed on carcinoma cells in all patients. We also observed that most of the tumors were highly infiltrated by CD8+ T cells both in the tumor beds and in the stroma and that the majority of tumors were also infiltrated by NKp46+ NK cells in higher numbers in the non-metastatic tumors. Interestingly, HNSCC was found as being one of the tumor types with the highest NKp46+ cell density as compared with renal cell carcinoma, non-small cell lung cancer, colorectal cancer or pancreatic tumors. Moreover CD94+ lymphocytes were detected in the stroma and tumor beds in close contact to tumor cells in about half of the patients. Using multicolor flow cytometry, NK cells and CD8+ T cells from tumor and non-involved adjacent tissues, metastatic lymph nodes and peripheral blood from patients relapsing post-chemotherapy were characterized for expression of activating and inhibitory receptors. Interestingly, NKG2A expression was higher on tumor infiltrating NK cells (CD56dim or CD56bright) and CD8+ T cell compared to the one observed in non-involved adjacent tissue, metastatic lymph node or blood. In vitro, monalizumab increased CD107 mobilization and CD137 upregulation on NKG2A+ NK cells in response to HNSCC cell lines with endogenous HLA-E expression and enhanced Ctx-mediated ADCC in a dose dependent manner. Altogether, these data support the rationale for investigating monalizumab in HNSCC patients and in combination with cetuximab in clinical trials (NCT02643550). Citation Format: Caroline Soulas, Ana Lalanne, Cécile Bonnafous, Caroline Hoffman, Elodie Bonnet, Arnaud Dujardin, Violette Breso, Mathieu Bléry, Olivier Lantz, Romain Remark, Eric Vivier, Pascale Andre. NKG2A immune checkpoint blockade potentiates cetuximab induced ADCC in head and neck cancer preclinical model [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 1690.

Exosomes are nanovesicles originating from late endosomal compartments and secreted by most living cells in ex vivo cell culture conditions. The interest in exosomes was rekindled when B-cell and dendritic cell-derived exosomes were shown to mediate MHC-dependent immune responses. Despite limited understanding of exosome biogenesis and physiological relevance, accumulating evidence points to their bioactivity culminating in clinical applications in cancer. This review focuses on the preclinical studies exploiting the immunogenicity of dendritic cell-derived exosomes (Dex) and will elaborate on the past and future vaccination trials conducted using Dex strategy in melanoma and non-small cell lung cancer patients. Cancer Res; 70(4); 1281–5

T cells need to migrate to and accumulate inside tumors before mediating rejection of the tumor. The number of specific T cells inside tumors may depend on the efficiency of priming in the draining lymph node (DLN), intratumor deletion, suppressive phenomena, or both. We used monoclonal anti-male antigen CD4 (Marilyn) T cells and tumor cell lines expressing or not the corresponding antigen (Dby) to analyze CD4 T-cell accumulation in tumors. Priming by MHC II+ or MHC II− male splenocytes or Dby+ tumor cells induced similar Marilyn T-cell expansion in the DLN and recirculation in other lymph nodes and capacity to produce IFN-γ. However, intratumor accumulation was different for each priming condition. In mice with Dby− tumors, MHC II+ male splenocyte priming induced greater, although not statistically significant, Marilyn T-cell accumulation in the tumors than MHC II− male splenocyte priming. In mice with Dby+ tumors, priming in the tumor DLN induced less Marilyn T-cell intratumor accumulation than priming by MHC II+ male splenocytes. We saw comparable differences for Marilyn T-cell accumulation in gut lamina propria, suggesting that priming affects effector T-cell accumulation in inflamed tissues. Mature dendritic cells were loaded with graded doses of Dby peptide to control for antigen-presenting cell characteristics during priming. We observed similar proliferation, with higher concentrations inducing higher intratumor accumulation. Thus, intratumor accumulation requires stronger stimulation than for proliferation or the capacity to secrete lymphokines. In this system, priming intensity alone can explain the number of intratumor T cells without having to call for intratumor deletion or suppression phenomena. (Cancer Res 2006; 66(10): 5443-51)

CD4+ T cells influence tumor immunity in complex ways that are not fully understood. In this study, we characterized a population of human differentiated effector CD4+ T cells that is defined by low levels of the interleukin (IL)-2 and IL-7 receptors (CD25−CD127−). We found that this cell population expands in patients with various types of cancer, including breast cancer, to represent 2% to 20% of total CD4+ blood T lymphocytes as compared with only 0.2% to 2% in healthy individuals. Notably, these CD25−CD127−CD4 T cells expressed effector markers such as CD244 and CD11b with low levels of CD27, contrasting with the memory phenotype dominating this population in healthy individuals. These cells did not cycle in patients, nor did they secrete IL-10 or IL-17, but instead displayed cytotoxic features. Moreover, they encompassed oligoclonal expansions paralleling an expansion of effector CD8+ T cells that included tumor antigen–specific T cells. During neoadjuvant chemotherapy in patients with breast cancer, we found that the increase in CD25−CD127− CD4+ T cells correlated with tumor regression. This observation suggested that CD4+ T cells included tumor antigen–specific cells, which may be generated by or participate in tumor regressions during chemotherapy. In summary, our results lend support to the hypothesis that CD4+ T cells are involved in human antitumor responses. Cancer Res; 74(8); 2204–16. ©2014 AACR.

Background: Recent progress in the ‘liquid biopsy’ field in combination with the development of the droplet digital PCR (ddPCR) technology now enables non-invasive monitoring of cancer-related genomic alterations with high detection accuracy. Current ddPCR techniques represent the gold standard for the detection of point mutations that have been previously characterized on tumor tissues. However, ddPCR screen for only one mutation (or a few mutations, with multicolor ddPCR) per reaction. This is a clear limitation to use it as a discovery tool, to detect resistance-associated mutations that may appear during therapy (e.g. ESR1 activating mutations in breast cancer) or when tumor tissue is not available (e.g. EGFR activating mutations in lung cancers). Methods: We developed the Multiple Hotspot mutations detection by Single droplet digital PCR (MHS-ddPCR) method, a variant of the conventional ddPCR technique, which detects all genomic alterations within a hotspot region, using a unique couple of Taqman oligo-probes. We first established two specific assays covering KRAS and EGFR mutation hotspot regions, which are of clinical importance in the context of colorectal cancer. The assay for KRAS scans for the 7 most common mutations in codons 12 and 13 of the gene as well as all other mutations with lower frequency ( Results: Sensitivity and specificity of MHS-ddPCR were assessed on decreasing fractions of tumor DNA mutated for KRAS. The KRAS assay reaches a sensitivity of 0.02% and was validated on plasma and tumor samples harboring a panel of different KRAS mutations. Similarly, the EGFR assay can detect numerous exon 19 deletions from patient samples and with limited amounts of tumor DNA. Conclusions: The MHS-ddPCR is extremely sensitive, works for numerous hotspot regions with different types of alterations (SNV, deletion) and is cost-effective as it combines multiple assays in one reaction. Citation Format: Charles Decraene, Francois-Clement Bidard, Samia Belaabi, Etienne Rouleau, Adrien Saliou, Alexandre Houy, Maud Milder, Olivier Lantz, Marc Ychou, Jean-Yves Pierga, Marc-Henri Stern, Charlotte Proudhon. Exhaustive scanning of mutation hotspot regions using a new tool: The MHS-ddPCR. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 410.

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Rationale: The noninvasive biomarker circulating cell-free tumor DNA (ctDNA) has the potential to enable noninvasive diagnostic tests for personalized medicine in providing similar molecular information as that derived from invasive tumor biopsies. The ongoing large multicentric randomized histology-independent phase II trial (SHIVA, [NCT01771458][1]) compares molecularly targeted therapy based on tumor molecular profiling versus conventional therapy in patients with any type of refractory cancer. The screening phase consists of the invasive biopsy of metastatic tumor tissue and downstream analysis using Ion Torrent's Ampliseq hotspot cancer panel. We evaluated whether ctDNA analysis would identify the same mutations as those obtained through invasive biopsy. Patients and methods: de novo detection of somatic mutations using ctDNA in 34 patients covering 18 different tumor types, scanning ∼50 genes and more than 2500 mutations with a multiplexed next generation sequencing panel. Results: In 27 patients, 27 of 28 actionable mutations identified in metastasis biopsies (96%) were detected in matched ctDNA. Among these 27 patients, two additional mutations were found in ctDNA only. In the seven other patients, mutation detection from metastasis biopsy failed due to inadequate biopsy material, but was successful in all plasma DNA samples providing three more potential actionable mutations. Conclusion; These results suggest that ctDNA analysis is a cheaper, safer and quicker alternative to invasive biopsy of metastasis, irrespective of cancer type and metastatic site, for multiplexed mutation detection in selecting personalized therapies based on the patient's tumor genetic content. Citation Format: Jean-Yves Pierga, Charles Decraene, Virginie Bernard, Maud Kamal, Anthony Blin, Quentin Leroy, Thomas Rio Frio, Gaelle Pierron, Celine Callens, Ivan Bieche, Adrien Saliou, Jordan Madic, Etienne Rouleau, Francois-Clement Bidard, Olivier Lantz, Marc-Henri Stern, Ronald Lebofsky, Christophe Le Tourneau. Circulating tumor DNA as a non-invasive substitute to metastasis biopsy for targeted sequencing in a prospective randomized trial for personalized treatment in all tumor type: The SHIVA study. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2400. doi:10.1158/1538-7445.AM2015-2400 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01771458&atom=%2Fcanres%2F75%2F15_Supplement%2F2400.atom

Circulating tumor DNA (ctDNA) analysis requires absolute single molecule sensitivity, since only a few copies of the mutation may be present per blood draw. Recent developments in next-generation sequencing (NGS) sample preparation have improved the relative sensitivity to detect Citation Format: Ronald Lebofsky, Jordan Madic, Adrien Saliou, Francois-Clement Bidard, Olivier Lantz, Marc-Henri Stern, Jean-Yves Pierga. Absolute single-molecule sensitive NGS by SPLITSEQ: a new ctDNA analysis tool. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-76. doi:10.1158/1538-7445.AM2014-LB-76

The incidence of skin cancer is increased in transplant recipients. UV radiation, papillomaviruses, and immunosuppression participate in the pathogenesis of these tumors. In addition, donor cells may leave the grafted organ, reach peripheral tissues and either induce immune phenomena or possibly take part in tissue remodeling. Herein, we investigated the possible involvement of donor cells in the development of skin tumors in kidney allograft recipients. We analyzed a series of 48 malignant and benign cutaneous tumors developing in 14 females who had been grafted with a male kidney. The number of male cells was measured on microdissected material by quantitative PCR for Y chromosome. In the samples with high levels of male cells, fluorescent in situ hybridization (FISH) with X and Y probes and/or immuno-FISH with anticytokeratin antibodies were carried out. Male cells were detected in 5/15 squamous cell carcinomas and Bowen disease (range 4-180 copies), 3/5 basal cell carcinomas (91-645), 6/11 actinic keratosis (7-102), 2/4 keratoacanthoma (22-41), and 2/5 benign cutaneous lesions (14-55). In a basal cell carcinoma specimen with a high number of male cells, FISH showed that most cells within the tumoral buds were XY. In this lesion, immuno-FISH showed the presence of XY cytokeratin-positive cells indicating that the tumor nests contained male keratinocytes. In contrast, in other female transplants, male cells present in the tumors were not epithelial. In conclusion, stem cells originating from a grafted kidney may migrate to the skin, differentiate, or fuse as keratinocytes that could, rarely, undergo cancer transformation.