Showing papers by "Marc-Henri Stern published in 2022"

MBD4 deficiency is predictive of response to immune checkpoint inhibitors in metastatic uveal melanoma patients.

Abstract: BACKGROUND MBD4 mutations have been reported in uveal melanomas, acute myeloid leukemias, colorectal adenocarcinomas, gliomas, and spiradenocarcinomas and cause a hypermutated phenotype. Although metastatic uveal melanomas (mUM) are usually resistant to immune checkpoint inhibitors (ICI), the first reported MBD4-mutated (MBD4m) patient responded to ICI, suggesting that MBD4 mutation may predict response to ICI. METHODS Retrospective cohort of mUM patients treated with ICI. MBD4 was sequenced in a subset of these patients. RESULTS Three hundred mUM patients were included. Median follow-up was 17.3 months. Ten patients with an objective response and 20 cases with stable disease for >12 months were observed, corresponding to an objective response rate of 3.3% and a clinical benefit (i.e., responder patients and stable disease) rate of 10%. Of the 131 tumors sequenced for MBD4, five (3.8%) were mutated. MBD4 mutation was associated with a better objective response rate as three out of five MBD4m versus 4% of MBD4 wild-type patients responded (p < 0.001). Of these five responders, three presented progressive disease at 2.8, 13.9, and 22.3 months. Median PFS was 4.0 months in MBD4 wild-type and 22.3 months in MBD4m patients (HR = 0.22; p = 0.01). Median OS in MBD4def patients was unreached as compared to 16.6 months in MBD4pro (HR = 0.11; 95% CI: 0.02-0.86; log-rank p-test = 0.04; Fig. 2e). CONCLUSIONS In mUM patients, MBD4 mutation is highly predictive for the response, PFS, and overall survival benefit to ICI. MBD4 could be a tissue-agnostic biomarker and should be sequenced in mUM, and other tumor types where MBD4 mutations are reported.

Real-Time Detection of ESR1 Mutation in Blood by Droplet Digital PCR in the PADA-1 Trial: Feasibility and Cross-Validation with NGS.

Abstract: The clinical actionability of circulating tumor DNA requires sensitive detection methods with a short turnaround time. In the PADA-1 phase 3 trial (NCT03079011), metastatic breast cancer patients treated with an aromatase inhibitor and palbociclib were screened every 2 months for activating ESR1 mutations in blood (bESR1mut). We report the feasibility of the droplet digital polymerase chain reaction (ddPCR) and cross-validation with next-generation sequencing (NGS). bESR1mut testing was centralized in two platforms using the same ddPCR assay. Results were reported as copies/mL of plasma and mutant allele frequency (MAF). We analyzed 200 positive ddPCR samples with an NGS assay (0.5-1% sensitivity). Overall, 12,552 blood samples were collected from 1017 patients from 83 centers. Among the 12,525 available samples with ddPCR results, 11,533 (92%) were bESR1mut-negative. A total of 267 patients newly displayed bESR1mut (26% patients/2% samples) with a median copy number of 14/mL (range: 4-1225) and a median MAF of 0.83% (0.11-35), 648 samples (20% patients/5% samples) displayed persistent bESR1mut, and 77 (<1%) samples encountered a technical failure. The median turnaround time from blood drawing to result notification was 13 days (Q1:9; Q3:21 days). Among 200 ddPCR-positive samples tested, NGS detected bESR1mut in 168 (84%); 25 of the 32 cases missed by NGS had low MAF and/or low coverage. In these 200 samples, bESR1mut MAF by both techniques had an excellent intraclass correlation coefficient (ICC = 0.93; 95% CI [0.85; 0.97]). These results from a large-scale trial support the feasibility and accuracy of real-time bESR1mut tracking by ddPCR, opening new opportunities for therapeutic interventions.

Concurrent Olaparib and Radiotherapy in Patients With Triple-Negative Breast Cancer: The Phase 1 Olaparib and Radiation Therapy for Triple-Negative Breast Cancer Trial.

Abstract: Importance Triple-negative breast cancer (TNBC) cells are sensitive to poly(adenosine diphosphate ribose) polymerase (PARP) inhibitors used as radiosensitizers. Whether combining PARP inhibitors with radiotherapy in patients with TNBC would enhance the biological effectiveness of the irradiation and improve locoregional control is unclear. Objective To assess the safety and tolerability of PARP inhibition with olaparib used concurrently with radiotherapy in patients with TNBC with residual disease after neoadjuvant chemotherapy. Design, Setting, and Participants This phase 1 prospective dose-escalation trial (Olaparib and Radiation Therapy for TNBC [RadioPARP] trial) using a time-to-event continual reassessment method was performed from September 2017 to November 2019, with follow-up until November 2021. Participants had an incomplete pathologic response after neoadjuvant chemotherapy or unresectable TNBC despite previous neoadjuvant chemotherapy, an Eastern Cooperative Oncology Group Performance Status score of 0 or 1, and adequate organ functions. Interventions Olaparib was administered orally in the form of tablets and given at increasing doses (50 mg, 100 mg, 150 mg, or 200 mg twice daily). Olaparib therapy was started 1 week before radiotherapy and was continued concomitantly with radiotherapy. After breast-conserving surgery, a total dose of 50.4 Gy was delivered to the whole breast, with a 63-Gy simultaneously integrated boost to the tumor bed for patients younger than 60 years. After radical mastectomy or for unresectable tumors despite neoadjuvant chemotherapy, a total dose of 50.0 Gy was delivered to the chest wall (after mastectomy) or to the whole breast (for unresectable tumors). Regional lymph node stations could be treated with a total dose of 50.0 Gy to 50.4 Gy in cases of node-positive disease. Main Outcomes and Measures Main outcomes were the safety and tolerability of PARP inhibition with radiotherapy for early-stage, high-risk TNBC. Secondary outcomes included overall survival (OS) and event-free survival (EFS). Results Among the 24 patients included in the trial (100% female; median age, 46 years [range, 25-74 years]), no dose-limiting toxic effects were observed, and olaparib was escalated to 200 mg twice daily without reaching the maximum tolerated dose. No late treatment-related grade 3 or greater toxic effect was observed, and the maximum observed treatment-related toxic effects at the 2-year follow-up were grade 2 breast pain, fibrosis, and deformity in 1 patient (4.2%). Three-year OS and EFS were 83% (95% CI, 70%-100%) and 65% (95% CI, 48%-88%), respectively. Homologous recombination status was not associated with OS or EFS. Conclusions and Relevance The findings of this phase 1 dose-escalation trial suggest that PARP inhibition with olaparib concurrently with radiotherapy for early-stage, high-risk TNBC is well tolerated and should continue to be evaluated in further clinical trials. Trial Registration ClinicalTrials.gov Identifier: NCT03109080.

A catalog of numerical centrosome defects in epithelial ovarian cancers

Abstract: Centrosome amplification, the presence of more than two centrosomes in a cell is a common feature of most human cancer cell lines. However, little is known about centrosome numbers in human cancers and whether amplification or other numerical aberrations are frequently present. To address this question, we have analyzed a large cohort of primary human epithelial ovarian cancers (EOCs) from 100 patients. We found that rigorous quantitation of centrosome number in tumor samples was extremely challenging due to tumor heterogeneity and extensive tissue disorganization. Interestingly, even if centrosome clusters could be identified, the incidence of centrosome amplification was not comparable to what has been described in cultured cancer cells. Surprisingly, centrosome loss events where a few or many nuclei were not associated with centrosomes were clearly noticed and overall more frequent than centrosome amplification. Our findings highlight the difficulty of characterizing centrosome numbers in human tumors, while revealing a novel paradigm of centrosome number defects in EOCs.

Rare germline heterozygous missense variants in BRCA1-associated protein 1, BAP1, cause a syndromic neurodevelopmental disorder.

Abstract: Nuclear deubiquitinase BAP1 (BRCA1-associated protein 1) is a core component of multiprotein complexes that promote transcription by reversing the ubiquitination of histone 2A (H2A). BAP1 is a tumor suppressor whose germline loss-of-function variants predispose to cancer. To our knowledge, there are very rare examples of different germline variants in the same gene causing either a neurodevelopmental disorder (NDD) or a tumor predisposition syndrome. Here, we report a series of 11 de novo germline heterozygous missense BAP1 variants associated with a rare syndromic NDD. Functional analysis showed that most of the variants cannot rescue the consequences of BAP1 inactivation, suggesting a loss-of-function mechanism. In T cells isolated from two affected children, H2A deubiquitination was impaired. In matching peripheral blood mononuclear cells, histone H3 K27 acetylation ChIP-seq indicated that these BAP1 variants induced genome-wide chromatin state alterations, with enrichment for regulatory regions surrounding genes of the ubiquitin-proteasome system (UPS). Altogether, these results define a clinical syndrome caused by rare germline missense BAP1 variants that alter chromatin remodeling through abnormal histone ubiquitination and lead to transcriptional dysregulation of developmental genes. Nuclear deubiquitinase BAP1 (BRCA1-associated protein 1) is a core component of multiprotein complexes that promote transcription by reversing the ubiquitination of histone 2A (H2A). BAP1 is a tumor suppressor whose germline loss-of-function variants predispose to cancer. To our knowledge, there are very rare examples of different germline variants in the same gene causing either a neurodevelopmental disorder (NDD) or a tumor predisposition syndrome. Here, we report a series of 11 de novo germline heterozygous missense BAP1 variants associated with a rare syndromic NDD. Functional analysis showed that most of the variants cannot rescue the consequences of BAP1 inactivation, suggesting a loss-of-function mechanism. In T cells isolated from two affected children, H2A deubiquitination was impaired. In matching peripheral blood mononuclear cells, histone H3 K27 acetylation ChIP-seq indicated that these BAP1 variants induced genome-wide chromatin state alterations, with enrichment for regulatory regions surrounding genes of the ubiquitin-proteasome system (UPS). Altogether, these results define a clinical syndrome caused by rare germline missense BAP1 variants that alter chromatin remodeling through abnormal histone ubiquitination and lead to transcriptional dysregulation of developmental genes. Main textProtein degradation by the ubiquitin-proteasome system (UPS) is essential for the maintenance of proteostasis in eukaryotic cells.1Chiti F. Dobson C.M. Protein Misfolding, Amyloid Formation, and Human Disease: A Summary of Progress Over the Last Decade.Annu. Rev. Biochem. 2017; 86: 27-68Crossref PubMed Scopus (1293) Google Scholar It prevents the accumulation of potentially cytotoxic misfolded or short-lived proteins whose functional conformation can no longer be restored by chaperones.2Franić D. Zubčić K. Boban M. Nuclear Ubiquitin-Proteasome Pathways in Proteostasis Maintenance.Biomolecules. 2021; 11: 54Crossref PubMed Scopus (12) Google Scholar,3Samant R.S. Livingston C.M. Sontag E.M. Frydman J. Distinct proteostasis circuits cooperate in nuclear and cytoplasmic protein quality control.Nature. 2018; 563: 407-411Crossref PubMed Scopus (91) Google Scholar Before being transported to the proteasome for hydrolysis, proteins destined to be degraded are specifically tagged by the addition of ubiquitin molecules through a cascade of reactions involving activating-, conjugating-, and ligating-enzymes.4Pickart C.M. Mechanisms underlying ubiquitination.Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2887) Google Scholar,5Pickart C.M. Eddins M.J. Ubiquitin: structures, functions, mechanisms.Biochim. Biophys. Acta. 2004; 1695: 55-72Crossref PubMed Scopus (1003) Google Scholar However, the ubiquitination process can be reversed by deubiquitinases (DUBs), which are able to cleave and disassemble the polyubiquitin chains of tagged substrates, thus avoiding their degradation by the proteasome.6Meyer-Schwesinger C. The ubiquitin-proteasome system in kidney physiology and disease.Nat. Rev. Nephrol. 2019; 15: 393-411Crossref PubMed Scopus (57) Google Scholar This action of DUBs is important for recycling the ubiquitin, avoiding proteasome overload and regulating protein turnover. Approximately a hundred DUBs are divided into four main families, including the ubiquitin C-terminal hydrolases (UCH) to which BRCA1-associated protein 1 (BAP1) belongs.7Szczepanski A.P. Wang L. Emerging multifaceted roles of BAP1 complexes in biological processes.Cell Death Discov. 2021; 7: 20Crossref PubMed Scopus (10) Google Scholar,8Masclef L. Ahmed O. Estavoyer B. Larrivée B. Labrecque N. Nijnik A. Affar E.B. Roles and mechanisms of BAP1 deubiquitinase in tumor suppression.Cell Death Differ. 2021; 28: 606-625Crossref PubMed Scopus (36) Google ScholarBAP1 is a nuclear DUB recognized for its tumor-suppressor properties that was proposed to depend on its ability to bind to the RING finger domain of BRCA1.9Jensen D.E. Rauscher 3rd, F.J. Defining biochemical functions for the BRCA1 tumor suppressor protein: analysis of the BRCA1 binding protein BAP1.Cancer Lett. 1999; 143: S13-S17Crossref PubMed Scopus (32) Google Scholar Nonetheless, later studies have shown that BAP1 acts as a tumor-suppressor gene independently of BRCA1 (MIM: 113705). BAP1 (MIM: 603089) is frequently inactivated in tumors by somatic loss-of-function (LoF) variants and its germline variants predispose to a tumor syndrome (BAP1-TPDS [MIM: 614327]) that encompasses various cancers, notably uveal melanoma, malignant pleural mesothelioma, and cutaneous melanoma.10Louie B.H. Kurzrock R. BAP1: Not just a BRCA1-associated protein.Cancer Treat. Rev. 2020; 90: 102091Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar,11Popova T. Hebert L. Jacquemin V. Gad S. Caux-Moncoutier V. Dubois-d’Enghien C. Richaudeau B. Renaudin X. Sellers J. Nicolas A. et al.Germline BAP1 mutations predispose to renal cell carcinomas.Am. J. Hum. Genet. 2013; 92: 974-980Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar In the nucleus, BAP1 acts as a chromatin-associated protein exerting its deubiquitinating function through the multiprotein complexes formed with transcription factors and co-factors. A prominent role of BAP1 is the modulation of chromatin through the complexes formed with the additional sex comb-like proteins ASXL1, ASXL2, and ASXL3 (ASXL1/2/3).12Srivastava A. Ritesh K.C. Tsan Y.C. Liao R. Su F. Cao X. Hannibal M.C. Keegan C.E. Chinnaiyan A.M. Martin D.M. Bielas S.L. De novo dominant ASXL3 mutations alter H2A deubiquitination and transcription in Bainbridge-Ropers syndrome.Hum. Mol. Genet. 2016; 25: 597-608Crossref PubMed Scopus (45) Google Scholar BAP1 complexes remove mono-ubiquitin from lysine 119 of histone H2A (Ub-H2A) previously added by Polycomb-repressive complex 1 (PRC1), thus antagonizing gene silencing mediated by PRC1 and activating expression of genes that contribute in particular to embryonic development or differentiation.10Louie B.H. Kurzrock R. BAP1: Not just a BRCA1-associated protein.Cancer Treat. Rev. 2020; 90: 102091Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar,13Campagne A. Lee M.K. Zielinski D. Michaud A. Le Corre S. Dingli F. Chen H. Shahidian L.Z. Vassilev I. Servant N. et al.BAP1 complex promotes transcription by opposing PRC1-mediated H2A ubiquitylation.Nat. Commun. 2019; 10: 348Crossref PubMed Scopus (63) Google Scholar,14Scheuermann J.C. de Ayala Alonso A.G. Oktaba K. Ly-Hartig N. McGinty R.K. Fraterman S. Wilm M. Muir T.W. Müller J. Histone H2A deubiquitinase activity of the Polycomb repressive complex PR-DUB.Nature. 2010; 465: 243-247Crossref PubMed Scopus (551) Google Scholar In addition, still in association with ASLX proteins that stabilize it,13Campagne A. Lee M.K. Zielinski D. Michaud A. Le Corre S. Dingli F. Chen H. Shahidian L.Z. Vassilev I. Servant N. et al.BAP1 complex promotes transcription by opposing PRC1-mediated H2A ubiquitylation.Nat. Commun. 2019; 10: 348Crossref PubMed Scopus (63) Google Scholar BAP1 has been shown to regulate a wide range of other cellular processes via its interactions with partner proteins involved in DNA damage response (BRCA1, BARD1), cell cycle control and proliferation (HCF1, YY1, FoxK1/K2), ferroptosis (SLC7A11), apoptosis (IP3R3), or even the immune response.10Louie B.H. Kurzrock R. BAP1: Not just a BRCA1-associated protein.Cancer Treat. Rev. 2020; 90: 102091Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar,15Bononi A. Giorgi C. Patergnani S. Larson D. Verbruggen K. Tanji M. Pellegrini L. Signorato V. Olivetto F. Pastorino S. et al.BAP1 regulates IP3R3-mediated Ca2+ flux to mitochondria suppressing cell transformation.Nature. 2017; 546: 549-553Crossref PubMed Scopus (232) Google Scholar, 16Zhao W. Steinfeld J.B. Liang F. Chen X. Maranon D.G. Jian Ma C. Kwon Y. Rao T. Wang W. Sheng C. et al.BRCA1-BARD1 promotes RAD51-mediated homologous DNA pairing.Nature. 2017; 550: 360-365Crossref PubMed Scopus (176) Google Scholar, 17Ladanyi M. Zauderer M.G. Krug L.M. Ito T. McMillan R. Bott M. Giancotti F. New strategies in pleural mesothelioma: BAP1 and NF2 as novel targets for therapeutic development and risk assessment.Clin. Cancer Res. 2012; 18: 4485-4490Crossref PubMed Scopus (63) Google Scholar, 18Liao L. Testa J.R. Yang H. The roles of chromatin-remodelers and epigenetic modifiers in kidney cancer.Cancer Genet. 2015; 208: 206-214Abstract Full Text Full Text PDF PubMed Scopus (44) Google ScholarWe show herein that the consequences of BAP1 germline variants are not limited to cancer predisposition but also extend to developmental disabilities. In the frame of an international collaborative effort initiated by the Western France consortium HUGODIMS, we compiled the clinical findings for a series of 11 unrelated individuals exhibiting a syndromic form of intellectual disability (ID) and/or developmental delay (DD) due to de novo heterozygous missense single-nucleotide variants (SNVs) in BAP1. The identification of the cases was partly facilitated by the web-based tool GeneMatcher.19Sobreira N. Schiettecatte F. Valle D. Hamosh A. GeneMatcher: a matching tool for connecting investigators with an interest in the same gene.Hum. Mutat. 2015; 36: 928-930Crossref PubMed Scopus (775) Google Scholar The variants were identified by subject-parents trio-based exome or genome sequencing (ES/GS) in diagnostic or research settings. In this study, which was approved by the local ethics committee of the University Hospital Center (CHU) of Nantes (number CCTIRS: 14.556), all participants were clinically assessed by at least one expert clinical geneticist from each of the centers involved. Written informed consent was obtained from the parents or legal guardians of all study participants and written authorization for the publication of photographs shown in Figure 1B.All affected individuals harboring a de novo BAP1 variant had DD or ID (11/11) characterized notably by speech (11/11) and motor delay (6/11) (Table 1; Figure 1A). Most of them had hypotonia (7/11), seizures (6/11), and abnormal behavior (8/10), including autism spectrum disorder, attention deficit hyperactivity disorder, and hypersensitivity. Almost all individuals showed dysmorphic facial features (10/11), and more than half (6/11) had skeletal malformations (involving the hands [4/11], feet [3/11], or spine [2/11],). Most of the individuals had growth failure (9/11), including four individuals with a very short stature (ranging from −3.18 to −6 SD). Organ abnormalities were inconsistent and heterogeneous and involved the eye (5/10), heart (3/10), and kidney or urogenital system (2/10). Other findings included sleep disorders, reported in 3/5 individuals, frequent episodes of otitis media in 4/11 (followed by hearing loss in two individuals), hypertrichosis in 3/11 and alopecia in 1/11, and feeding difficulties in 4/8. It is noteworthy that we could clinically distinguish two subgroups of affected individuals: one is represented by individuals 5 to 9 with a very syndromic phenotype who exhibit the most severe symptoms (severe ID, very short stature, facial dysmorphism, and congenital malformations), whereas the second subgroup with the six remaining individuals has a less syndromic phenotype with generally milder symptoms. Of note, the initial diagnostic impression was Cornelia de Lange syndrome 1 (MIM: 122470) for individuals 5 and 6 and Smith-Magenis syndrome (MIM: 182290) for individual 3.20Berger S.I. Ciccone C. Simon K.L. Malicdan M.C. Vilboux T. Billington C. Fischer R. Introne W.J. Gropman A. Blancato J.K. et al.Exome analysis of Smith-Magenis-like syndrome cohort identifies de novo likely pathogenic variants.Hum. Genet. 2017; 136: 409-420Crossref PubMed Scopus (20) Google ScholarTable 1Phenotype of affected individuals with BAP1 de novo variantsIndividual (family)1 (F1)2 (F2)3 (F3)aIndividual 3 was first reported by Berger et al., 201720 in a Smith-Magenis syndrome (SMS)-like cohort.4 (F4)5 (F5)6 (F6)7 (F7)8 (F8)9 (F9)10 (F10)11 (F11)TotalVariant nomenclature Chr3: (GRCh37; GenBank: NM_004656.3)g.52443861G>Tg.52443861G>Cg.52443601C>Tg.52442599A>Gg.52442077C>Gg.52442078A>Cg.52442078A>Gg.52442078A>Gg.52441264T>Cg.52441264T>Cg.52436341C>T11 variants de novoc.34C>Ac.34C>Gc.91G>Ac.146T>Cc.272G>Cc.271T>Gc.271T>Cc.271T>Cc.506A>Gc.506A>Gc.2153G>Ap.Pro12Thrp.Pro12Alap.Glu31Lysp.Leu49Prop.Cys91Serp.Cys91Glyp.Cys91Argp.Cys91Argp.His169Argp.His169Argp.Arg718GlnClinVar accession numberSCV001572228SCV001738368SCV001738369SCV001738370SCV001738372SCV001738373SCV001738374SCV001738374SCV001738375SCV001738375SCV001738376Variant annotationMobiDetailsbThe detailed predictions for variants can be accessed via MobiDetails21 (see web resources for the URL for variant 22880; to access other variants’ information, replace “22880” with the relevant variant number in the URL).2288022881228822288322865228842288522885228862288622888CADD (GRCh37-v1.6)26.525.729.429.726.128.529.129.125.725.724.4gnomAD v2.1.1absentabsentabsentabsentabsentabsentabsentabsentabsentabsentpresent (x1):4.7E−6Metadome (tolerance score)HI (0.13)HI (0.13)I (0.26)HI (0.11)I (0.35)I (0.35)I (0.35)I (0.35)I (0.34)I (0.34)SI (0.94)Method of variant detectionESESESESESESESESESESES and GSSexfemalemalefemalemalefemalefemalemalefemalemalefemalefemale7 F/4 MAge at last investigation10 years3 years14 years6 years11 years 5 months15 years1 years 11 months4 years16 years8 years 4 months12 years 1 month1 year 11 month to 15 yearsGrowth failure−−+++++++++9/11Weight at age last investigation (kg/SD)36.2/+1.512.3/−1.548.6/+0.520/−0.9926.5/−230/−4.3512.17/+0.0313.7/−1.2850/−1.1730/+126/−2.5−4.35 to +1Height at age last investigation (cm/SD)128.5/−1.588.9/−1.5149.4/−2.5109.7/−2.44116/−4.5125.7/−678/−3.1890/−2.71142/−3.69117.4/−2135.3/−2−6 to −1.5Head circumference at age last investigation (cm/SD)51/−147.9/−256/+154.7/+1.6752/−150/−2.8649/+0.5650/−0.0153/−1.4148/+0 at 27 months54.5/+1−2.86 to +1.67Developmental delay or ID+++; mild++; severe+; severe+++++11/11Age of walking18 months19 months18 months2 yearsnot walkingnot achieved17 months2.5 years2 years23 months15 monthsnot applicableSpeech delay+++++++++++11/11Hypotonia−+++++−+−+−7/11Seizures+−−−+−−++++6/11Behavioral anomalies+; ADHD+; ASD+; ADHD+; ASDN/A+; ASD−++++; ADHD9/10Cardiac anomalies−++−−−N/A−−+; VSD−3/10Eye anomalies−++−−+N/A+−+−5/10Urogenital/kidney anomalies−−−−+−N/A−−+−2/10Hands−+−+−+−−+−−4/11Feet−−−−++−−+−−3/11Feeding difficulties−−+−N/A+N/A+−+N/A4/8Facial dysmorphism++++++++++−10/11Other recurrent signs−hearing lossfrequent otitishypertrichosishypertrichosishypertrichosisrecurrent otitis−hearing loss−−not applicableHI, highly intolerant; I, intolerant; SI, slightly tolerant; ES, exome sequencing; GS, genome sequencing; N/A, not analyzed; VSD, ventricular septal defect; ASD, autism spectrum disorder; ADHD, attention deficit hyperactivity disorder; VUR, vesicoureteral reflux.a Individual 3 was first reported by Berger et al., 201720Berger S.I. Ciccone C. Simon K.L. Malicdan M.C. Vilboux T. Billington C. Fischer R. Introne W.J. Gropman A. Blancato J.K. et al.Exome analysis of Smith-Magenis-like syndrome cohort identifies de novo likely pathogenic variants.Hum. Genet. 2017; 136: 409-420Crossref PubMed Scopus (20) Google Scholar in a Smith-Magenis syndrome (SMS)-like cohort.b The detailed predictions for variants can be accessed via MobiDetails21Baux D. Van Goethem C. Ardouin O. Guignard T. Bergougnoux A. Koenig M. Roux A.F. MobiDetails: online DNA variants interpretation.Eur. J. Hum. Genet. 2021; 29: 356-360Crossref PubMed Scopus (12) Google Scholar (see web resources for the URL for variant 22880; to access other variants’ information, replace “22880” with the relevant variant number in the URL). Open table in a new tab In total, we found 11 missense SNVs, two of them recurrent (c.271T>C [p.Cys91Arg] and c.506A>G [p.His169Arg]) and eight restricted to three codons: 12 (2/11), 91 (4/11), and 169 (2/11) (Table 1; Figure 1H). Almost all variants were absent in public variant databases, and bioinformatics predictions were in favor of their pathogenicity; only c.2153G>A (p.Arg718Gln) was present in one heterozygote in gnomAD. According to gnomAD,22Karczewski K.J. Francioli L.C. Tiao G. Cummings B.B. Alföldi J. Wang Q. Collins R.L. Laricchia K.M. Ganna A. Birnbaum D.P. et al.The mutational constraint spectrum quantified from variation in 141,456 humans.Nature. 2020; 581: 434-443Crossref PubMed Scopus (2753) Google Scholar BAP1 is intolerant of LoF variants (probability of being loss-of-function intolerant [pLI] = 0.99; observed/expected variants (o/e) = 0.12 [0.06–0.28]) and moderately intolerant to missense variants across the entire gene (Z score = 2.64; o/e = 0.64 [0.58–0.71]). Yet, the analysis by missense tolerant ratio (MTR) Gene Viewer23Traynelis J. Silk M. Wang Q. Berkovic S.F. Liu L. Ascher D.B. Balding D.J. Petrovski S. Optimizing genomic medicine in epilepsy through a gene-customized approach to missense variant interpretation.Genome Res. 2017; 27: 1715-1729Crossref PubMed Scopus (93) Google Scholar shows that the region encoding the UCH domain, where 10/11 variants in the study reside (Figure 1H), is much less tolerant to missense variants than the rest of the protein (Figure S1). All amino acid residues affected by the variants are highly conserved across the species (to Caenorhabditis elegans for the variants harbored by individuals 1–10 and to Drosophila melanogaster for c.2153G>A [p.Arg718Gln]). Most variants are localized to a small region of the protein within the catalytic domain. Several alter critically important functional residues, including Cys91, which is the critical active site residue of the enzyme and whose substitutions disrupt BAP1 activity, and His169, which is also part of the enzyme active site and interacts directly with Cys9124Jensen D.E. Proctor M. Marquis S.T. Gardner H.P. Ha S.I. Chodosh L.A. Ishov A.M. Tommerup N. Vissing H. Sekido Y. et al.BAP1: a novel ubiquitin hydrolase which binds to the BRCA1 RING finger and enhances BRCA1-mediated cell growth suppression.Oncogene. 1998; 16: 1097-1112Crossref PubMed Scopus (575) Google Scholar (supplemental information). The only variant not located in the catalytic domain,c.2153G>A (p.Arg718Gln) (GenBank: NM_004656.3), affects an amino acid within the nuclear-localization signal (NLS) domain. However, no clear genotype-phenotype correlation could be established that supports the observation of two distinct phenotypes made during clinical assessment. Secondary molecular findings were made in exome data for some of the affected individuals (Table S1), but without any evidence of their pathogenicity.In order to evaluate directly whether the mutations described above could affect the activity of BAP1, we used a previously characterized model cell line (HAP1) in which we knocked out BAP1 by genome editing and then rescued its expression through expression of a retrovirus carrying BAP1 cDNA.13Campagne A. Lee M.K. Zielinski D. Michaud A. Le Corre S. Dingli F. Chen H. Shahidian L.Z. Vassilev I. Servant N. et al.BAP1 complex promotes transcription by opposing PRC1-mediated H2A ubiquitylation.Nat. Commun. 2019; 10: 348Crossref PubMed Scopus (63) Google Scholar We generated rescued cell lines for six variants and first checked by immunoblot that the mutant proteins are expressed and stable (Figure 2A). H2AK119ub is a major substrate for BAP1 enzymatic activity,14Scheuermann J.C. de Ayala Alonso A.G. Oktaba K. Ly-Hartig N. McGinty R.K. Fraterman S. Wilm M. Muir T.W. Müller J. Histone H2A deubiquitinase activity of the Polycomb repressive complex PR-DUB.Nature. 2010; 465: 243-247Crossref PubMed Scopus (551) Google Scholar so we therefore used its quantification as a proxy for BAP1 activity. As expected, H2AK119ub level increases by about 2-fold in the BAP1 knockout cells and reaches the original level in the BAP1-WT rescue line (Figure 2B). In contrast, four out of the six variants, namely p.Pro12Thr, p.Cys91Arg, p.Cys91Ser, and p.His169Arg are unable to rescue H2AK119ub levels. The level of H2AK119ub with the variant p.Pro12Ala rescue is near that of wild type (WT), suggesting that this variant is either partially or fully enzymatically active, at least on this substrate. The p.Arg718Gln variant displays a normal deubiquitinase activity toward H2AK119ub. We also analyzed the subcellular localization of BAP1 in the cell lines rescued with the six different variants. All showed prominent nuclear staining (Figure 2C), even the variant located in the NLS domain, p.Arg718Gln. This finding suggests that this domain is not strictly required for the nuclear localization and rules out the possibility that p.Arg718Gln could compromise the function of BAP1 by causing its cytosolic accumulation.7Szczepanski A.P. Wang L. Emerging multifaceted roles of BAP1 complexes in biological processes.Cell Death Discov. 2021; 7: 20Crossref PubMed Scopus (10) Google Scholar Finally, we investigated whether the regulation of H2AK119ub correlates with BAP1-mediated transcriptional regulation. We had shown that TMSB4X (MIM: 300159) and S100A11 (MIM: 603114) expression are regulated by BAP1 in HAP1 cells.13Campagne A. Lee M.K. Zielinski D. Michaud A. Le Corre S. Dingli F. Chen H. Shahidian L.Z. Vassilev I. Servant N. et al.BAP1 complex promotes transcription by opposing PRC1-mediated H2A ubiquitylation.Nat. Commun. 2019; 10: 348Crossref PubMed Scopus (63) Google Scholar Indeed, RT-qPCR experiments confirmed that their expression decreases in the absence of BAP1 and is restored in the WT rescue line. However, five out of six variants did not rescue the expression of those genes, including the p.Pro12Ala variant, although it is enzymatically active on H2AK119ub (Figure 2D). Only the p.Arg718Gln variant behaves similarly to the WT, suggesting that this might not be an LoF variant; further investigation will be necessary to determine whether it is pathogenic. In the meantime, we consider it as a variant of uncertain significance (VUS).Figure 2Assessment of BAP1 variants effect in model cell linesShow full caption(A) Nuclear extract of HAP1 cells WT, BAP1 knockout or BAP1-knockout-rescued with the different variants indicated on top were probed by immunoblot with anti-BAP1 antibody (top) or anti-HDAC1 (bottom, loading control). Representative result.(B) Same as in (A), but this time probed with anti-H2AK119ub antibody (top) or anti-Histone H3 (bottom, loading control). Immunoblots were quantified, the ratio between H2AK119ub and H3 is indicated below after normalization to 1 for HAP1 WT cells. Representative result.(C) Detection of BAP1 by immunofluorescence microscopy (top), nuclear staining with DAPI (bottom), representative experiment.(D) Gene expression was analyzed by RT-qPCR in the different cell lines described in (A). TMSB4X (MIM: 300159) and S100A11 (MIM: 603114) levels normalized to TBP (MIM: 600075) expression are shown (n = 3, biological replicates).View Large Image Figure ViewerDownload Hi-res image Download (PPT)We assessed T cells isolated from affected children (individuals 1 and 5) carrying BAP1 variants c.34C>A (p.Pro12Thr) and c.272G>C (p.Cys91Ser) for their contents of ubiquitinated H2A (Ub-H2A) by immunoblotting. As shown in Figure 3A, although the amounts of H2A were similar between control and affected individuals, the steady-state level of Ub-H2A was substantially increased in cells of the affected children when compared to those of their respective related controls (i.e., father and/or mother of the probands). Specifically, densitometry analysis of the band intensities revealed that Ub-H2A was 1.5-fold and 2.0-fold enriched in the subjects bearing the p.Cys91Ser and p.Pro12Thr BAP1 variants, respectively (Figure 3A, bottom). These data are fully in line with our previous observation that both of these variants were unable to rescue H2A deubiquitination in BAP1-knockout cells (Figure 2) and confirm that the missense variants p.Pro12Thr and p.Cys91Ser cause LoF.Figure 3Subjects with BAP1 variants exhibit increased amounts of Ub-H2A and pro-β5 proteasome subunitShow full caption(A) Top: whole-cell lysates from T cells of affected individuals carrying the p.Pro12Thr or p.Cys91Ser BAP1 variants (labeled probands 1 and 5) and control T cells (subjects’ father and/or mother) were subjected to protein extraction and SDS-PAGE/immunoblotting with antibodies directed against Ub-H2A, H2A as well as α-tubulin and GAPDH (loading control), as indicated. Bottom: densitometry analysis of the shown immunoblots (top) depicting the Ub-H2A contents detected in control (black) and BAP1 subjects (red). The y axis represents the fold changes of normalized Ub-H2A (Ub-H2A/α-tubulin) in densitometry measurements, which were set as 1 for subjects’ father or mother, as indicated.(B) Native-PAGE analysis from control and affected individuals’ T cells with proteasome bands being visualized by exposing the gel to 0.1 mM of the LLVY fluorescent peptide (left) and staining the gels with a monoclonal antibody specific for the α6 subunit (right), as indicated.(C) Top: whole-cell lysates from control and affected individuals’ T cells were separated by SDS-PAGE and analyzed by immunoblotting with antibodies directed against, RPT1, RPT2, RPT3, RPT4, RPT5, RPT5, β1, β2, β5, and β5i, as indicated. Equal loading was ensured by probing the membranes with an anti-GAPDH antibody, as indicated. Bottom: densitometry analysis of the shown immunoblots (top) depicting the pro-β5 contents detected in control (black) and affected individuals (red). The y axis represents the fold changes of normalized pro-β5 (pro-β5/GAPDH) in densitometry measurements, which were set as 1 for subjects’ father or mother, as indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Because several recent studies have suggested the role of deubiquitination in the regulation of the UPS,25Santiago-Sim T. Burrage L.C. Ebstein F. Tokita M.J. Miller M. Bi W. Braxton A.A. Rosenfeld J.A. Shahrour M. Lehmann A. et al.Biallelic Variants in OTUD6B Cause an Intellectual Disability Syndrome Associated with Seizures and Dysmorphic Features.Am. J. Hum. Genet. 2017; 100: 676-688Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar,26Garret P. Ebstein F. Delplancq G. Dozieres-Puyravel B. Boughalem A. Auvin S. Duffourd Y. Klafack S. Zieba B.A. Mahmoudi S. et al.Report of the first patient with a homozygous OTUD7A variant responsible for epileptic encephalopathy and related proteasome dysfunction.Clin. Genet. 2020; 97: 567-575Crossref PubMed Scopus (14) Google Scholar we next sought to determine whether these BAP1 variants were associated with impaired proteasome expression and/or function. To this end, T cells from affected children carrying the p.Pro12Thr and p.Cys91Ser BAP1 variants were subjected to a non-denaturing cell lysis prior to analysis of their proteasome complexes by native-PAGE and immunoblotting. As illustrated in Figure 3B, the chymotrypsin-like activity and amounts of the 20S and 26S proteasomes did not substantially vary between control and affected children’s cells, as determined by in-gel overlay assay and α6 staining. To further ascertain the impact of BAP1 variants on proteasomes, we next compared the proteasome subunit composition between control and affected children’s T cells by SDS-PAGE and immunoblotting. Our data show that the proteasome expression profile in T cells of affected individuals was similar to that detected in control cells with no major changes in the abundance of the AAA+-ATPase subunits and most of the catalytic β-subunits (Figure 3C). Strikingly, the β5 subunit precursor, however, consistently accumulated in probands’ T cells, as determined by immunoblotting (Figure 3C). Importantly, it is highly unlikely that such pro-β5 upregulation reflects greater amounts of proteasomes in affected individuals’ T cells, as our analysis of the 20S/26S native complexes failed to show any quantitative differences between control and affected children (Figure 3B). The enrichment of the β5 precursor protein (pro-β5), which was confirmed by densitometry analysis in both probands (Figure 3C, bottom), strongly suggests that BAP1 LoF is asso

Abstract P1-07-01: Real time detection of ESR1 mutation in blood by droplet digital PCR in the PADA-1 trial: Feasibility and cross-validation with NGS

Abstract: Background: Mutations of ESR1 (ESR1mut), the gene encoding for Estrogen Receptor (ER) alpha, have been functionally characterized as driving resistance to aromatase inhibitors (AI) given to in ER+ HER2- metastatic breast cancer (mBC). In the randomized multicenter phase 3 PADA-1 trial (NCT03079011), ER+ HER2- mBC patients treated with first line AI+Palbociclib were screened every two months for ESR1mut in blood (bESR1mut), by subjecting cell-free circulating DNA (cfcDNA) to a droplet digital PCR (ddPCR) assay. Upon the onset of a rising (i.e., increasing or appearing) bESR1mut, patients with no concomitant disease progression were randomized between keeping the same regimen (AI-Palbociclib) or switching to Fulvestrant-Palbociclib. We report herein the sample flow of this first-in-its-class trial, from April 2017 to March 2021, and the cross-validation of ddPCR results with NGS. Methods: At each time point, 20 mL of blood were drawn into BCT Streck® tubes. cfcDNA was extracted from 4 mL of plasma. bESR1mut testing was centralized in 2 platforms using the same ddPCR assay, which combines a drop-off ddPCR, targeting the clustered hotspot L536, Y537 and D538 mutations found in exon 8, with an unconventional ddPCR interrogating specifically the E380Q mutation, located in exon 5 (Jeannot et al, Oncogene 2020). Results were expressed as the number of ESR1mut copies detected per mL of plasma together with the Mutant Allele Frequency (MAF). As a control, we submitted 200 ESR1mut+ (by ddPCR) left-over samples to an amplicon-based Next Generation Sequencing (NGS) assay covering all ESR1 exons - with a nominal sensitivity of >0.5% MAF. Results: From 03/2017 to 03/2021, 1,017 MBC patients have been included in 83 centers and 12,552 blood samples have been collected. The median time of delivery to central platforms was 1 day (range: [0-11]). A median volume of 9.5 mL [2-20] of plasma was obtained after 2 centrifugations. bESR1mut results were notified to investigators with a median turnaround time of 13 days [1-813] (32 days in 2017, 9 days in 2019, 8 days in 2021). Among the 12,525 available ddPCR results: N=77 (<1%) were considered as non-contributive (failed); N=11,533 (92%) samples were bESR1mut-; 267 (2%) samples allowed to find a rising bESR1mut (first occurrence of bESR1mut in a given patient under AI-Pal, triggering randomization in the absence of concomitant progression), while 648 (6%) other bESR1mut+ samples were not labeled as rising (e.g. repeated samplings in randomized patients, etc.). Samples with rising bESR1mut had a median MAF of 0.83% [0.11-35] and a median number of 14.5 ESR1mut copies/mL [4-1225]. The 200 bESR1mut+ samples by ddPCR submitted to NGS had a median MAF of 1.39% [0.1-46.6] and a median number of 24 ESR1mut copies/mL [4-6,493]. 168 samples (84%) were confirmed by NGS as displaying an ESR1mut in exon 5 and/or 8, of which 50 samples harbored polyclonal mutations. MAFs retrieved by NGS and ddPCR showed an excellent intraclass correlation coefficient (ICC=0.93; 95%CI[0.85;0.97]). Lack of ESR1mut detection by NGS in 32 samples could be primarily attributed to low MAF (MAF<0.5% by ddPCR, N=14/32; MAF<1%, N=29/32) and/or low coverage (<1,000x) (N=11/32). Conclusion: PADA-1 demonstrated that large scale, real time ESR1mut screening is feasible. The ddPCR assay yielded to valid results in a short turnaround time and at a limited cost. The primary objective of PADA-1, which tested the actionability of bESR1mut, will be reported in another presentation. Funding: Pfizer, INCA PRT-K (Grant nb PRT-K19-110) Citation Format: Céline Callens, François-Clément Bidard, Anais Curto-Taribo, Olfa Trabelsi-Grati, Samia Melaabi, Suzette Delaloge, Anne-Claire Hardy-Bessard, Thomas Bachelot, Florian Clatot, Thibault De La Motte Rouge, Jean-Luc Canon, Laurent Arnould, Fabrice André, Sandrine Marques, Marc-Henri Stern, Jean-Yves Pierga, Anne-Vincent Salomon, Emmanuelle Jeannot, Frederique Berger, Ivan Bieche, Anne Pradines. Real time detection of ESR1 mutation in blood by droplet digital PCR in the PADA-1 trial: Feasibility and cross-validation with NGS [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-07-01.

Functional characterization of 5p15.33 risk locus in uveal melanoma reveals rs452384 as a functional variant and NKX2.4 as an allele-specific interactor

Abstract: The TERT/CLPTM1L risk locus on chromosome 5p15.33 is a pleiotropic cancer risk locus in which multiple independent risk alleles have been identified, across well over ten cancer types. We previously conducted a genome-wide association study in uveal melanoma (UM), which uncovered a role for the TERT/CLPTM1L risk locus in this intraocular tumor and identified multiple highly correlated risk alleles. Aiming to unravel the biological mechanisms in UM of this locus, which contains a domain enriched in active chromatin marks and enhancer elements, we demonstrated the allele-specific enhancer activity of this risk region using reporter assays. In UM, we identified the functional variant rs452384, of which the C risk allele is associated with higher gene expression, increased CLPTM1L expression in UM tumors, and a longer telomere length in peripheral blood mononuclear cells. Electrophoretic mobility shift assays and quantitative mass spectrometry identified NKX2.4 as an rs452384-T-specific binding protein, whereas GATA4 preferentially interacted with rs452384-C. Knockdown of NKX2.4 but not GATA4 resulted in increased TERT and CLPTM1L expression. In summary, the UM risk conferred by the 5p locus is at least partly due to rs452384, for which NKX2.4 presents strong differential binding activity and regulates CLPTM1L and TERT expression. Altogether, our work unraveled some of the complex regulatory mechanisms at the 5p15.33 susceptibility region in UM, and this might also shed light on shared mechanisms with other tumor types affected by this susceptibility region.

Oral Etoposide and Trastuzumab Use for HER2-Positive Metastatic Breast Cancer: A Retrospective Study from the Institut Curie Hospitals

Abstract: Simple Summary Oral etoposide (VP16), an inhibitor of topoisomerase-II, has demonstrated clinical activity in metastatic breast cancer (MBC). To our knowledge, oral VP16 combined with trastuzumab (VP16-T) in HER2+ MBC has not been evaluated before. This combination is biologically relevant, as TOP2A, the gene encoding topoisomerase II, is often co-amplified with ERBB2. We report a retrospective analysis of the impact of oral VP16-trastuzumab on HER2+ MBC patients, together with TOP2A/ERBB2 co-amplification status, assessed through shallow whole genome sequencing. In addition to its low cost and convenience, the oral VP16-trastuzumab regimen has shown a satisfactory activity and excellent tolerability. Abstract Background: The TOP2A and ERBB2 genes are co-amplified in about 40% of HER2 positive (HER2+) breast cancers. Oral etoposide (VP16), an inhibitor of topoisomerase-II (encoded by TOP2A), has demonstrated clinical activity in metastatic breast cancer (MBC). The benefit of oral VP16 combined with trastuzumab (VP16-T) in HER2+ MBC has not yet been evaluated. Methods: Patients treated at the Institut Curie Hospitals with VP16-T for HER2+ MBC were retrieved by an in silico search. Progression-free survival (PFS), overall survival (OS), response rate, prolonged PFS (defined as at least 6 months), clinical benefit, and toxicity were assessed. The co-amplification of ERBB2 and TOP2A was assessed by shallow whole genome sequencing on tumor tissue whenever available. Results: Forty-three patients received VP16-T after a median number of six prior treatment lines for HER2+ MBC. Median PFS and OS were 2.9 months (95% CI [2.4–4.7]) and 11.3 months (95% CI [8.3–25.0]), respectively. Three patients had a complete response, while 12/40 (30%) experienced clinical benefit. Only three patients stopped treatment for toxicity. Seven (35%) patients displayed a TOP2A/ERBB2 co-amplification. No statistically significant correlation was found between outcome and TOP2A/ERBB2 co-amplification. Conclusion: Our analysis suggests a favorable efficacy and toxicity profile for VP16-T in patients with heavily pretreated HER2+ MBC.

Concurrent Olaparib with Radiotherapy in Patients with Triple Negative Breast Cancer: Final Results of the RADIOPARP Phase 1 Trial

Abstract:

Purpose/Objective(s)

Preclinical studies have demonstrated that triple-negative breast cancer (TNBC) cells are sensitive to PARP inhibitors when used as radiosensitizers. Combining PARP-inhibitors with radiotherapy in TNBC patients may consequently enhance the biological effectiveness of the irradiation ultimately leading to improved locoregional control. We aimed to establish the appropriate dosing and safety profile of Olaparib used as a radiosensitizer in combination with radiotherapy in TNBC patients with residual disease after neoadjuvant chemotherapy.

Materials/Methods

RADIOPARP (NCT03109080) was a prospective phase I dose-escalation trial establishing the tolerance profile of Olaparib combined with breast radiotherapy in high-risk early TNBC patients. Inclusion criteria were resected TNBC with non-complete pathological response after neoadjuvant chemotherapy, or unresectable TNBC despite prior neoadjuvant chemotherapy. Olaparib was started seven days before irradiation and continued during radiotherapy. A time-to-event continual reassessment method was used to increase Olaparib through four increasing dose levels (50mg, 100mg, 150mg or 200mg twice a day) with a 25% maximum probability rate of dose-limiting toxicities (DLT). Radiotherapy delivered 50 Gy to the breast or the chest wall, with or without lymph node irradiation. Toxicities were graded according to CTCAE (version 4.03). Homologous recombination (HR) proficiency status was genetically determined based on shallow whole genome sequencing.

Results

Twenty-four TNBC patients were enrolled between 09/2017 and 11/2019. Olaparib was escalated to 200 mg twice a day without DLT and the MTD was not reached. With a median follow-up of 34 months, no late treatment-related grade ≥ 3 toxicity was observed, and the maximum observed treatment-related toxicities were limited to grade 2 breast pain (n=2), fibrosis (n=2), deformity (n=1) and telangiectasia (n=1). Three-year OS and EFS were 83% [95% CI: 70%-100%] and 68% [51%-91%], respectively. HR proficiency status was not associated with OS and EFS.

Conclusion

Olaparib used as a radiosensitizer in combination with breast radiotherapy in TNBC patients was well-tolerated. The MTD was not reached, and no significant late toxicity was reported. For future trials evaluating the anti-tumor efficacy of this combination, an Olaparib dose of 200 mg twice a day should be considered.

Multi-omics comparison of malignant and normal uveal melanocytes reveals novel molecular features of uveal melanoma

Abstract: Uveal Melanoma (UM) is a rare cancer resulting from transformation of melanocytes residing in the uveal tract. Integrative analysis has identified four molecular and clinical subsets in UM. To improve our molecular understanding of UM we performed extensive multi-omics characterization comparing two aggressive UM patient-derived xenograft models with normal choroidal melanocytes, including DNA optical mapping, specific histone modifications, and DNA topology analysis by Hi-C. Our gene expression and cytogenetic analyses suggest that genomic instability is a hallmark of UM. We also identified a recurrent deletion in the BAP1 promoter resulting in loss of expression and associated with high risk of metastases in UM patients. Hi-C revealed chromatin topology changes associated with up-regulation of PRAME, an independent prognostic biomarker in UM and potential therapeutic target. Our findings illustrate how multi-omics approaches can improve the understanding of tumorigenesis and reveals two novel mechanisms of gene expression dysregulation in UM.

Glycolysis Dependency as a Hallmark of SF3B1-Mutated Cells

Abstract: Simple Summary Cancer-associated SF3B1 mutations result in aberrant transcripts whose fate remains unknown. We aimed to investigate the functional consequences of these splice aberrations. Our results show that SF3B1 mutation alters the translation of transcripts encoding proteins involved in metabolism, which triggers a metabolic switch toward an increased glucose uptake. Consequently, SF3B1-mutated cells are more sensitive to glycolysis inhibition than SF3B1 wild-type cells. Abstract SF3B1 mutations are recurrent in cancer and result in aberrant splicing of a previously defined set of genes. Here, we investigated the fate of aberrant transcripts induced by mutant SF3B1 and the related functional consequences. We first demonstrate that mutant SF3B1 does not alter global nascent protein synthesis, suggesting target-dependent consequences. Polysome profiling revealed that 35% of aberrantly spliced transcripts are more translated than their corresponding canonically spliced transcripts. This mostly occurs in genes with enriched metabolic functions. Furthermore, LC-MS/MS analysis showed that mutant SF3B1 impacts the abundance of proteins involved in metabolism. Functional metabolic characterization revealed that mutant SF3B1 decreases mitochondrial respiration and promotes glycolysis to compensate for defective mitochondrial metabolism. Hence, mutant SF3B1 induces glycolysis dependency, which sensitizes cells to glycolysis inhibition. Overall, we provide evidence of the oncogenic involvement of mutant SF3B1 in uveal melanoma through a metabolic switch to glycolysis, revealing vulnerability to glycolysis inhibitors as a promising therapeutic strategy.

Safety and tolerability of olaparib combined with breast radiotherapy in patients with triple-negative breast cancer: Final results of the RADIOPARP phase 1 trial.

Abstract: 534 Background: Preclinical studies have demonstrated that triple-negative breast cancer (TNBC) cells were sensitive to PARP inhibitors when used as radiosensitizers. Combining PARP-inhibitors with radiotherapy may consequently enhance the biological effectiveness of the irradiation, leading to improved locoregional control in TNBC patients. We aimed to establish the appropriate dosing and the safety profile of Olaparib used as a radiosensitizer in combination with radiotherapy in TNBC patients with residual disease after neoadjuvant chemotherapy. Methods: RADIOPARP (NCT03109080) was a prospective phase I dose-escalation trial establishing the tolerance profile of Olaparib combined with breast radiotherapy in high-risk early TNBC patients. Inclusion criteria were resected TNBC with non-complete pathological response after neoadjuvant chemotherapy, or unresectable TNBC despite prior neoadjuvant chemotherapy. Olaparib was started seven days before irradiation and continued during radiotherapy. A time-to-event continual reassessment method was used to increase Olaparib through four increasing dose levels (50mg, 100mg, 150mg or 200mg twice a day) with a 25% maximum probability rate of dose-limiting toxicities (DLT). Radiotherapy delivered 50 Gy to the breast or to the chest wall, with or without lymph node irradiation. Toxicities were graded according to CTCAE (version 4.03). Homologous recombination (HR) proficiency status was genetically determined based on shallow whole genome sequencing. Results: Twenty-four TNBC patients were enrolled between 09/2017 and 11/2019. Olaparib was escalated to 200 mg twice a day without DLT and the MTD was not reached. With a median follow-up of 34 months, no late treatment-related grade ≥ 3 toxicity was observed, and the maximum observed toxicities were limited to grade 2 breast pain (n=2), fibrosis (n=2), deformity (n=1) and telangiectasia (n=1). Three-year OS and EFS were 83% [95% CI: 70%-100%] and 65% [51%-91%], respectively. HR proficiency status was not associated with OS and EFS. Conclusions: Olaparib used as a radiosensitizer in combination with radiotherapy in TNBC patients was well-tolerated. The MTD was not reached, and no significant late toxicity was reported. For future trials evaluating the anti-tumor efficacy of this combination, an Olaparib dose of 200 mg twice a day should be considered. Clinical trial information: NCT03109080.

Oral etoposide and trastuzumab in HER2-positive metastatic breast cancer: a retrospective study at Institut Curie Hospitals.

Abstract: Background: The TOP2A and ERBB2 genes are co-amplified in about 40% of HER2 positive (HER2+) breast cancers. Oral etoposide (VP16), an inhibitor of topoisomerase-II (encoded by TOP2A), has demonstrated clinical activity in metastatic breast cancer (MBC). However, the clinical benefit of oral VP16 combined with trastuzumab (VP16-T) in HER2+ MBC has not been evaluated.Methods: Patients treated at Institut Curie Hospitals with VP16-T for HER2+ MBC were retrieved by an in-silico search. Trained medical oncologists retrospectively assessed progression-free survival (PFS), overall survival (OS), response rate, prolonged PFS (defined as a duration of at least 6 months), 6 months clinical benefit rate and toxicity. Co-amplification of ERBB2 and TOP2A was assessed by shallow whole genome sequencing on tumor tissue whenever available.Results: Forty-three patients received VP16-T after a median number of six prior treatment lines for HER2+ MBC. Median PFS and OS were 2.9 months (95% CI [2.4-4.7]) and 11.3 months (95% CI [8.3-25.0]), respectively. Three patients had a complete response while 12/40 (30%) had a clinical benefit. Only 3 patients stopped treatment for toxicity. Median PFS in the population with and without TOP2A/ERBB2 co-amplification was respectively 4.7 months (95% CI [2.3-NA]) and 2.9 months (95% CI [1.2-NA]; p=0.36).Conclusion: Our analysis suggests a favorable efficacy and toxicity profile for VP16-T in patients with heavily pretreated HER2+ MBC.

Optical Genome Mapping for detecting Homologous Recombination Deficiency (HRD) in human breast cancers

Abstract: Homologous recombination deficiency (HRD) leads to genomic instability that marks HRD tumor genome with a specific genomic scar. Present in many cancers, HRD is important to be detected as it is associated with a hyper-sensitivity to some classes of drugs, in particular the PARP inhibitors. Here, we investigate the use of structural variants detected by the Optical Genome Mapping (OGM) technology to identify HRD tumors. We first compared the performance of OGM and whole genome sequencing (WGS) in an HRD triple negative breast carcinoma (TNBC) carrying a germline BRCA2 deleterious mutation. We showed the excellent performance of OGM and its ability to recognize subclonal events not detected by WGS. We then analyzed by RVA OGM data from fifteen TNBC samples from the clinical trial RadioPARP. We defined two features characteristic of HRD. Tandem duplication (TD) and insertion events were found increased in HRD tumors. We showed that insertion calls were probably mostly TD too small to be called as TD by RVA. The insertion/TD feature fully discriminated HRD from all homologous recombination proficient (HRP) TNBC but one. This outlier carried a CCNE1 amplicon probably explaining the excess of insertion/TD. Total numbers of translocations were similar in HRP and HRD TNBC. We suggested a novel feature, translocations and intra-fusions isolated from another event by 3 megabases. Isolated translocations and intra-fusions perfectly discriminated HRD from HRP TNBC. Our results demonstrate that the OGM technology is an affordable way of getting an insight of the structural variants present in solid tumors, even with low tumoral cellularity. It represents an alternative technology for HRD diagnosis, which should now be evaluated in independent series of tumors of different tissue origins.

Cytidine analogs are synthetic lethal with base excision repair default due to MBD4 deficiency

Abstract: Abstract Inactivating mutations of MBD4 have been reported in subsets of various tumors. A deficiency of this DNA glycosylase, recognizing specifically T:G mismatch resulting from the deamination of methyl-cytosine, results in a hypermutated phenotype due to the accumulation of CpG>TpG transitions. Here, we hypothesize that the difference in DNA metabolism consecutive to MBD4 deficiency may result in specific cytotoxicities in MBD4-deficient tumor cells in a synthetic lethality fashion. After a large-scale drug repurposing screen, we show in two isogenic MBD4 knock-out cell models that the inactivation of MBD4 sensitizes cancer cells to cytidine analogs. We further confirm the exquisite activity of gemcitabine in an MBD4 -deficient co-clinical model as (i) it completely prevented the development of an MBD4 -deficient uveal melanoma patient-derived xenograft and (ii) treatment in the corresponding patient resulted in an exceptional tumor response. These data suggest that patients harboring MBD4-deficient tumors may be treated efficiently by cytidine analogs.