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Depletion of CDA expression by short hairpin RNA in HeLa cells.

Figure S2 shows the ROC curve analysis of progression prediction according to the HPV ctDNA level at baseline. (AUC was 0.646 (95% CI 0.49 to 0.802) and 2940 copies/ml was determined as the ctDNA cut-off level for comparison. Sensitivity and specificity of the cut-off were 67% and 70% respectively).

Purpose: Triple-negative breast cancer (TNBC) patients with residual disease after neoadjuvant chemotherapy have a poor outcome. We developed patient-derived xenografts (PDX) from residual tumors to identify efficient chemotherapies and predictive biomarkers in a context of resistance to anthracyclines- and taxanes-based treatments.Experimental Design: PDX were established from residual tumors of primary breast cancer patients treated in neoadjuvant setting. TNBC PDX were treated by anthracyclines, taxanes, platins, and capecitabine. Predictive biomarkers were identified by transcriptomic and immunohistologic analysis. Downregulation of RB1 was performed by siRNA in a cell line established from a PDX.Results: Residual TNBC PDX were characterized by a high tumor take, a short latency, and a poor prognosis of the corresponding patients. With the exception of BRCA1/2-mutated models, residual PDX were resistant to anthracyclines, taxanes, and platins. Capecitabine, the oral prodrug of 5-FU, was highly efficient in 60% of PDX, with two models showing complete responses. Prior treatment of a responder PDX with 5-FU increased expression of thymidylate synthase and decreased efficacy of capecitabine. Transcriptomic and IHC analyses of 32 TNBC PDX, including both residual tumors and treatment-naïve derived tumors, identified RB1 and TYMP proteins as predictive biomarkers for capecitabine response. Finally, RB1 knockdown in a cell line established from a capecitabine-responder PDX decreased sensitivity to 5-FU treatment.Conclusions: We identified capecitabine as efficient chemotherapy in TNBC PDX models established from residual disease and resistant to anthracyclines, taxanes, and platins. RB1 positivity and high expression of TYMP were significantly associated with capecitabine response. Clin Cancer Res; 24(11); 2605–15. ©2018 AACR.

Boxplot of the expression of genes encoding proteins involved in capecitabine metabolism (TYMP, TYMS, TK1, DPYD) and cell-cycle control (RB1, CDKN2A, CCND1).

PDF file 97K, List of the 89 Wnt pathway selected genes

Purpose: Patients with luminal breast cancer (LBC) often become endocrine resistant over time. We investigated the molecular changes associated with acquired hormonoresistances in patient-derived xenografts of LBC.Experimental Design: Two LBC xenografts (HBCx22 and HBCx34) were treated with different endocrine treatments (ET) to obtain xenografts with acquired resistances to tamoxifen (TamR) and ovariectomy (OvaR). PI3K pathway activation was analyzed by Western blot analysis and IHC and responses to ET combined to everolimus were investigated in vivo. Gene expression analyses were performed by RT-PCR and Affymetrix arrays.Results: HBCx22 TamR xenograft was cross-resistant to several hormonotherapies, whereas HBCx22 OvaR and HBCx34 TamR exhibited a treatment-specific resistance profile. PI3K pathway was similarly activated in parental and resistant xenografts but the addition of everolimus did not restore the response to tamoxifen in TamR xenografts. In contrast, the combination of fulvestrant and everolimus induced tumor regression in vivo in HBCx34 TamR, where we found a cross-talk between the estrogen receptor (ER) and PI3K pathways. Expression of several ER-controlled genes and ER coregulators was significantly changed in both TamR and OvaR tumors, indicating impaired ER transcriptional activity. Expression changes associated with hormonoresistance were both tumor and treatment specific and were enriched for genes involved in cell growth, cell death, and cell survival.Conclusions: PDX models of LBC with acquired resistance to endocrine therapies show a great diversity of resistance phenotype, associated with specific deregulations of ER-mediated gene transcription. These models offer a tool for developing anticancer therapies and to investigate the dynamics of resistance emerging during pharmacologic interventions. Clin Cancer Res; 20(16); 4314–25. ©2014 AACR.

Supplementary Table 1 from Molecular Profiling of Inflammatory Breast Cancer

Supplementary Figure Legends 1-3 from Microarray-Based Identification of Tenascin C and Tenascin XB, Genes Possibly Involved in Tumorigenesis Associated with Neurofibromatosis Type 1

PDF file 161K, A simplified representation of the three different Wnt signalling pathways: the canonical pathway, the planar cell polarity pathway, and the Wnt/calcium pathway. In the absence of Wnt ligand, the 'destruction complex' composed of the core proteins Axin, adenomatous polyposis coli (APC), and glycogen synthase kinase-3 (GSK3) rapidly phosphorylates cytosolic beta-catenin, targeting it for subsequent proteasome-mediated destruction. Binding of Wnt to Frizzled (FZD) and low-density lipoprotein receptor-related protein 5/6 (LRP5/6) activates the cytosolic protein Dishevelled (DVL), leading to inhibition of the destruction complex. The resulting accumulated beta-catenin can then translocate to the nucleus to activate Wnt-responsive target genes regulated by lymphoid enhancer factor (LEF) and T cell factor (TCF) family transcription factors, leading to various cellular effects. The secreted inhibitor Dickkopf (DKK) can antagonize Wnt signalling by competitively binding to LRP5/6. Secreted FZD-related proteins (SFRPs) and Wnt inhibitory factor (WIF) are thought to antagonize Wnt signalling by sequestering Wnt ligand in the extracellular space. Binding of Wnt isoforms to FZD can trigger beta-catenin-independent downstream signalling events, other so-called non-canonical Wnt pathways that do not require the transcriptional activity of beta-catenin. One branch of non-canonical pathways involves the activation of RHO and RAC small G proteins to regulate the actin cytoskeleton. DVL-associated activator of morphogenesis 1 (DAAM1), when complexed with DVL and RHO, acts through the regulation of RHO-associated protein kinases (ROCK) and the DVL-RAC GTPase complex to affect actin remodelling. Another branch, when activated, is defined by a phospholipase C (PLC)-mediated increase in intracellular Ca2+ levels and Ca2+ fluxes that lead to the activation of Ca2+/calmodulin-dependent protein kinase (CaMK), protein kinase C (PKC), and nuclear factor of activated T cells (NFAT)

Purpose: The hallmark of neurofibromatosis type 1 (NF1) is the onset of dermal or plexiform neurofibromas, mainly composed of Schwann cells. Plexiform neurofibromas can transform into malignant peripheral nerve sheath tumors (MPNST) that are resistant to therapies.Experimental Design: The aim of this study was to identify an additional pathway in the NF1 tumorigenesis. We focused our work on Wnt signaling that is highly implicated in cancer, mainly in regulating the proliferation of cancer stem cells. We quantified mRNAs of 89 Wnt pathway genes in 57 NF1-associated tumors including dermal and plexiform neurofibromas and MPNSTs. Expression of two major stem cell marker genes and five major epithelial–mesenchymal transition marker genes was also assessed. The expression of significantly deregulated Wnt genes was then studied in normal human Schwann cells, fibroblasts, endothelial cells, and mast cells and in seven MPNST cell lines.Results: The expression of nine Wnt genes was significantly deregulated in plexiform neurofibromas in comparison with dermal neurofibromas. Twenty Wnt genes showed altered expression in MPNST biopsies and cell lines. Immunohistochemical studies confirmed the Wnt pathway activation in NF1-associated MPNSTs. We then confirmed that the knockdown of NF1 in Schwann cells but not in epithelial cells provoked the activation of Wnt pathway by functional transfection assays. Furthermore, we showed that the protein expression of active β-catenin was increased in NF1-silenced cell lines. Wnt pathway activation was strongly associated to both cancer stem cell reservoir and Schwann–mesenchymal transition.Conclusion: We highlighted the implication of Wnt pathway in NF1-associated tumorigenesis. Clin Cancer Res; 20(2); 358–71. ©2013 AACR.

Single-nucleotide polymorphisms identified by direct sequencing of the CDA gene in breast cell lines (S2A) and NCI60 cell lines (S2B).

Supplementary Table S7. Upstream regulators identified by the Upstream Regulator Analysis in HBCx22 TamR and HBCx34 TamR xenografts.

Supplementary Tables 1-4 from Microarray-Based Identification of Tenascin C and Tenascin XB, Genes Possibly Involved in Tumorigenesis Associated with Neurofibromatosis Type 1

Purpose: Inflammatory breast cancer (IBC) is a rare but particularly aggressive form of primary breast cancer. The molecular mechanisms responsible for IBC are largely unknown.Experimental Design: To obtain further insight into the molecular pathogenesis of IBC, we used real-time quantitative reverse transcription (RT)-PCR to quantify the mRNA expression of 538 selected genes in IBC relative to non-IBC.Results: Twenty-seven (5.0%) of the 538 genes were significantly up-regulated in IBC compared with non-IBC. None were down-regulated. The 27 up-regulated genes mainly encoded transcription factors (JUN, EGR1, JUNB, FOS, FOSB, MYCN, and SNAIL1), growth factors (VEGF, DTR/HB-EGF, IGFBP7, IL6, ANGPT2, EREG, CCL3/MIP1A, and CCL5/RANTES) and growth factor receptors (TBXA2R, TNFRSF10A/TRAILR1, and ROBO2). We also identified a gene expression profile, based on MYCN, EREG, and SHH, which discriminated subgroups of IBC patients with good, intermediate, and poor outcome.Conclusion: Our study has identified a limited number of signaling pathways that require inappropriate activation for IBC development. Some of the up-regulated genes identified here could offer useful diagnostic or prognostic markers and could form the basis of novel therapeutic strategies.

Purpose:Human papillomavirus (HPV) is found in 90% of squamous cell carcinomas of the anal canal (SCCA). We investigated the clinical validity of HPV circulating tumor DNA (ctDNA) detection in patients enrolled in the Epitopes-HPV02 trial that demonstrated the efficacy of docetaxel, cisplatin, and 5-FU as first-line chemotherapy in advanced SCCA.Experimental Design:According to the protocol, serum samples were collected before chemotherapy and on completion of chemotherapy. HPV16 ctDNA was quantified by droplet digital PCR (ddPCR) and correlated with prospectively registered patient characteristics and outcomes. A landmark was set at the time of chemotherapy completion for postchemotherapy progression-free survival (PFS) analyses.Results:Among 57 patients with HPV16-related advanced SCCA, HPV ctDNA was detected in 91.1% (95% confidence interval, 81.1–96.2) of baseline samples. Baseline HPV ctDNA levels were not associated with any patient characteristics; baseline ctDNA level below the cutoff obtained by AUC (area under the curve) was associated with a longer PFS (HR = 2.1; P = 0.04). Among the 36 patients who completed 5 months of chemotherapy, residual HPV ctDNA was detected after chemotherapy in 38.9% of patients. Residual HPV ctDNA detected at chemotherapy completion was associated with shorter postchemotherapy PFS (median PFS 3.4 months vs. not reached; HR = 5.5; P < 0.001) and a reduction of 1-year overall survival rate (OR = 7.0; P = 0.02).Conclusions:This prospective study in advanced SCCA demonstrated a significant prognostic impact of HPV ctDNA level before first-line chemotherapy and HPV ctDNA negativity after chemotherapy completion. With a limited cost and short turnaround, this assay is a promising tool to optimize the therapeutic management of SCCA.See related commentary by Morris, p. 2030

Cell lines (Supplementary Material 1), Primer sequences and Antibodies (Supplementary Material 2).

Pearson correlation between the levels of CDA transcripts and the cytotoxicity of a panel of drugs.

Purpose: Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder with a complex variety of clinical manifestations. The hallmark of NF1 is the onset of heterogeneous (dermal or plexiform) benign neurofibromas. Plexiform neurofibromas can give rise to malignant peripheral nerve sheath tumors, which are resistant to conventional therapies.Experimental Design: To identify new signaling pathways involved in the malignant transformation of plexiform neurofibromas, we applied a 22,000-oligonucleotide microarray approach to a series of plexiform neurofibromas and malignant peripheral nerve sheath tumors. Changes in the expression of selected genes were then confirmed by real-time quantitative reverse transcription-PCR.Results: We identified two tenascin gene family members that were significantly deregulated in both human NF1-associated tumors and NF1-deficient primary cells: Tenascin C (TNC) was up-regulated whereas tenascin XB (TNXB) was down-regulated during tumor progression. TNC activation is mainly due to the up-regulation of large TNC splice variants. Immunohistochemical studies showed that TNC transcripts are translated into TNC protein in TNC-overexpressing tumors. Aberrant transcriptional activation of TNC seems to be principally mediated by activator protein transcription factor complexes.Conclusion:TNXB and TNC may be involved in the malignant transformation of plexiform neurofibromas. Anti-TNC antibodies, already used successfully in clinical trials to treat malignant human gliomas, may be an appropriate new therapeutic strategy for NF1.

Supplementary materials and figures

Supplementary Figures 1-3 from Microarray-Based Identification of Tenascin C and Tenascin XB, Genes Possibly Involved in Tumorigenesis Associated with Neurofibromatosis Type 1

The Krüppel-like zinc finger protein ZNF217 is a candidate oncogene in breast cancer. In this study, we showed that high levels of expression of ZNF217 mRNA are associated with poor prognosis and the development of metastases in breast cancer. Overexpression of ZNF217 in breast cancer cells stimulated migration and invasion in vitro and promoted the development of spontaneous lung or node metastases in mice in vivo. ZNF217 also promoted epithelial–mesenchymal transition (EMT) in human mammary epithelial cells, and the TGF-β–activated Smad signaling pathway was identified as a major driver of ZNF217-induced EMT. In addition, a TGF-β autocrine loop sustained activation of the TGF-β pathway in ZNF217-overexpressing mammary epithelial cells, most likely because of ZNF217-mediated direct upregulation of TGFB2 or TGFB3. Inhibition of the TGF-β pathway led to the reversal of ZNF217-mediated EMT. Together, our findings indicate that ZNF217 mRNA expression may represent a novel prognostic biomarker in breast cancer. Therapeutic targeting of ZNF217 of the TGF-β signaling pathway may benefit the subset of patients whose tumors express high levels of ZNF217. Cancer Res; 72(14); 3593–606. ©2012 AACR.

PDF file - 3.2MB, complementary figures showing the impact of ZNF217 overexpression on MDA-MB-231 and HMLE cells phenotype

The lungs are a frequent target of metastatic breast cancer cells, but the underlying molecular mechanisms are unclear. All existing data were obtained either using statistical association between gene expression measurements found in primary tumors and clinical outcome, or using experimentally derived signatures from mouse tumor models. Here, we describe a distinct approach that consists of using tissue surgically resected from lung metastatic lesions and comparing their gene expression profiles with those from nonpulmonary sites, all coming from breast cancer patients. We show that the gene expression profiles of organ-specific metastatic lesions can be used to predict lung metastasis in breast cancer. We identified a set of 21 lung metastasis–associated genes. Using a cohort of 72 lymph node–negative breast cancer patients, we developed a 6-gene prognostic classifier that discriminated breast primary cancers with a significantly higher risk of lung metastasis. We then validated the predictive ability of the 6-gene signature in 3 independent cohorts of breast cancers consisting of a total of 721 patients. Finally, we show that the signature improves risk stratification independently of known standard clinical variables and a previously established lung metastasis signature based on an experimental breast cancer metastasis model. [Cancer Res 2008;68(15):6092–9]

PDF file - 107K, complementary information and methods on breast-tumor cohorts, HMLE cells and transcriptomic analysis

Supplementary Figure 1 from Characterization of a Germ-Line Deletion, Including the Entire INK4/ARF Locus, in a Melanoma-Neural System Tumor Family: Identification of ANRIL, an Antisense Noncoding RNA Whose Expression Coclusters with ARF

We have previously detected a large germ-line deletion, which included the entire p15/CDKN2B-p16/CDKN2A-p14/ARF gene cluster, in the largest melanoma-neural system tumor (NST) syndrome family known to date by means of heterozygosity mapping based on microsatellite markers. Here, we used gene dose mapping with sequence-tagged site real-time PCR to locate the deletion end points, which were then precisely characterized by means of long-range PCR and nucleotide sequencing. The deletion was exactly 403,231 bp long and included the entire p15/CDKN2B, p16/CDKN2A, and p14/ARF genes. We then developed a simple and rapid assay to detect the junction fragment and to serve as a direct predictive DNA test for this large French family. We identified a new large antisense noncoding RNA (named ANRIL) within the 403-kb germ-line deletion, with a first exon located in the promoter of the p14/ARF gene and overlapping the two exons of p15/CDKN2B. Expression of ANRIL mainly coclustered with p14/ARF both in physiologic (various normal human tissues) and in pathologic conditions (human breast tumors). This study points to the existence of a new gene within the p15/CDKN2B-p16/CDKN2A-p14/ARF locus putatively involved in melanoma-NST syndrome families and in melanoma-prone families with no identified p16/CDKN2A mutations as well as in somatic tumors. [Cancer Res 2007;67(8):3963–9]

Supplementary Figure 1 Legend from Characterization of a Germ-Line Deletion, Including the Entire INK4/ARF Locus, in a Melanoma-Neural System Tumor Family: Identification of ANRIL, an Antisense Noncoding RNA Whose Expression Coclusters with ARF

Supplementary Table 1 from A Six-Gene Signature Predicting Breast Cancer Lung Metastasis

Genomic alterations in KRAS/BRAF/NRA S wild-type xenografts, according to their response to cetuximab. Tumors were considered to be amplified if the gene copy number was >3. Gene overexpression is defined by an expression superior to average in all PDX panel + 1 SD.

PDF file - 104K, complementary tables on breast tumor clinical information, primers used for ChIP experiments and ZNF217-associated gene-expression deregulations

Development of targeted therapeutics required translationally relevant preclinical models with well-characterized cancer genome alterations. Here, by studying 52 colorectal patient-derived tumor xenografts (PDX), we examined key molecular alterations of the IGF2–PI3K and ERBB–RAS pathways and response to cetuximab. PDX molecular data were compared with that published for patient colorectal tumors in The Cancer Genome Atlas. We demonstrated a significant pattern of mutual exclusivity of genomic abnormalities in the IGF2–PI3K and ERBB–RAS pathways. The genomic anomaly frequencies observed in microsatellite stable PDX reproduce those detected in nonhypermutated patient tumors. We found frequent IGF2 upregulation (16%), which was mutually exclusive with IRS2, PIK3CA, PTEN, and INPP4B alterations, supporting IGF2 as a potential drug target. In addition to maintaining the genomic and histologic diversity, correct preclinical models need to reproduce drug response observed in patients. Responses of PDXs to cetuximab recapitulate also clinical data in patients, with partial or complete response in 15% (8 of 52) of PDXs and response strictly restricted to KRAS wild-type models. The response rate reaches 53% (8 of 15) when KRAS, BRAF, and NRAS mutations are concomitantly excluded, proving a functional cross-validation of predictive biomarkers obtained retrospectively in patients. Collectively, these results show that, because of their clinical relevance, colorectal PDXs are appropriate tools to identify both new targets, like IGF2, and predictive biomarkers of response/resistance to targeted therapies. Cancer Res; 75(8); 1560–6. ©2015 AACR.

Example of 2 PDXs treated with cetuximab (one red arrow for each injection).

Background: Up to 35% BC survivors who receive adjuvant treatment (tx) experience severe CRCI, which has a significant impact on quality of life, disrupting daily functioning as well as self-esteem, self-confidence, and work ability. However, limited tools exist to predict the risk of CRCI. We aimed to develop a comprehensive model of severe CRCI, including clinical and serum inflammatory protein data. Methods: We included 8875 patients (pts) with stage I-III BC from the multicenter, prospective CANTO cohort (NCT01993498). Longitudinal data were collected at diagnosis (dx), 1 (T1), 2 (T2) and 4 (T3) years post-dx. Our outcome of interest was severe cognitive impairment at T1, T2, and T3 (score < 75/100, EORTC QLQ-C30, Giesinger JM 2020). Multivariable logistic regression models retained associations between baseline clinical variables (sociodemographic, psychological, tumor, and tx-related) with severe CRCI by bootstrapped Augmented Backwards Elimination (ABE). Among a subset of patients with HR+/HER2- BC (N= 1151), blood samples were profiled at dx using a multi-biomarker inflammatory panel assessing IL6, TNFα, IL1RA, CRP, IL2, IL1β, IFNγ, IL10, IL1A, IL4, IL8, and monocyte chemoattractant protein-1 (MCP-1). All biomarkers were incorporated simultaneously into a model of severe CRCI and retained only if significantly associated with CRCI by ABE (p Table 1.Models of severe CRCI in the overall cohort: clinical predictors.T1 (N=7724)T2 (N=6825)T3 (N=4706)OR* (95% CI)OR* (95% CI)OR* (95% CI)Severe Pain**, vs no1.50 (1.09-2.07)1.93 (1.39-2.69)1.55 (1.03-2.34)Severe pre-tx CRCI**, vs no3.69 (2.70-5.05)2.53 (1.85-3.46)2.21 (1.47-3.32)Severe Fatigue**, vs no1.50 (1.06-2.11)1.61 (1.13-2.28)1.08 (0.69-1.70)Age (continous)0.98 (0.97-0.99)NR0.98 (0.96-0.99)Menopause, post- vs pre-NR0.73 (0.54-0.98)NRAnxiety, case vs normalNRNR1.82 (1.13-2.92)Anxiety, borderline vs normalNRNR1.84 (1.17-2.91)Hot flashes, vs no1.25 (0.92-1.69)1.20 (0.87-1.65)1.64 (1.10-2.43)Corrected AUC0.73 (0.70-0.76)0.69 (0.65-0.72)0.68 (0.63-0.72)OR= Odds Ratio, CI= Confidence Interval, NR= Not Retained; *Adjusted by BMI, alcohol, smoke, socioeconomic, psychological, tumor and tx; **QLQ-C30 Table 2.Models of severe CRCI in the overall cohort**: biological biomarkers.T1 (N=1094)T2 (N=1091)T3 (N=870)OR* (95% CI)OR* (95% CI)OR* (95% CI)IL6NR0.80 (0.46-1.40)1.01 (0.64-1.60)IL1RA0.66 (0.37-1.17)0.88 (0.50-1.55)NRCRP0.94 (0.60-1.48)1.44 (0.92-2.27)NRIL20.93 (0.55-1.57)1.10 (0.61-1.97)NRIL1βNR1.55 (0.71-3.40)NRIFNγ1.86 (0.69-5.01)0.75 (0.25-2.22)NRIL101.05 (0.34-3.27)1.27 (0.58-2.78)NRIL1A0.71 (0.15-3.33)0.80 (0.17-3.66)NRIL80.96 (0.58-1.58)NRNRTNFαNR1.35 (0.67-2.73)NRMCP-11.07 (0.64-1.78)0.83 (0.51-1.35)0.80 (0.48-1.31)Corrected AUC0.72 (0.67-0.77)0.70 (0.65-0.75)0.67 (0.62-0.72)OR= Odds Ratio, CI= Confidence Interval, NR= Not Retained; *Adjusted by BMI, alcohol, smoke, socioeconomic, psychological, tumor and tx; ** Clinical predictors from previous models were forced in the models at each time-point Citation Format: Daniele Presti, Florence Joly, Davide Soldato, Stergios Christodoulidis, Antonin Della Noce, Julie Havas, Florine Dubuisson, Barbara Pistilli, Valerie Camara-Clayette, Fabrice André, Anne-Laure Martin, Alexandra Jacquet, Sandrine Boyault, Ivan Bièche, Charles Coutant, Paul-Henry Cournede, Stefan Michiels, Caroline Pradon, Ines Vaz-Luis, Antonio Di Meglio. Cancer-related cognitive impairment (CRCI) in early breast cancer (BC) survivors [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 P4-11-09.

AimsUpper tract urothelial carcinoma (UTUC) is a rare malignancy with a poor prognosis which occurs sporadically or in few cases results from a genetic disorder called Lynch syndrome. Recently, examination of microsatellite instability (MSI) has gained importance as a biomarker: MSI tumours are associated with a better response to immunomodulative therapies. Limited data are known about the prevalence of MSI in UTUC. New detection methods using the fully automated Idylla MSI Assay facilitate analysis of increased patient numbers.MethodsWe investigated the frequency of MSI in a multi-institutional cohort of 243 consecutively collected UTUC samples using standard methodology (Bethesda panel), along with immunohistochemistry of mismatch repair (MMR) proteins. The same tumour cohort was retested using the Idylla MSI Assay by Biocartis.ResultsUsing standard methodology, 230/243 tumours were detected as microsatellite stable (MSS), 4/243 tumours as MSI and 9/243 samples as invalid. In comparison, the Idylla MSI Assay identified four additional tumours as MSS, equalling 234/243 tumours; 4/243 were classified as MSI and only 5/243 cases as invalid. At the immunohistochemical level, MSI results were supported in all available cases with a loss in MMR proteins. The overall concordance between the standard and the Idylla MSI Assay was 98.35%. Time to result differed between 3 hours for Idylla MSI Assay and 2 days with the standard methodology.ConclusionOur data indicate a low incidence rate of MSI tumours in patients with UTUC. Furthermore, our findings highlight that Idylla MSI Assay can be applied as an alternative method of MSI analysis for UTUC.

Background The phosphatidylinositol-3 kinase pathway is often altered in head and neck squamous cell carcinoma (HNSCC), and is involved in the resistance to EGFR inhibitors. Objective We investigated the dose-limiting toxicities (DLTs), maximum tolerated dose, pharmacokinetics, and preliminary efficacy of the combination of copanlisib, an intravenous, pan-class I PI3K inhibitor, with the anti-EGFR monoclonal antibody cetuximab in recurrent and/or metastatic HNSCC patients in a phase I dose-escalation trial. Patients and methods Copanlisib was given intravenously on days 1, 8, and 15 of 28-day cycles at the dose of 45 mg and 30 mg, in combination with standard doses of weekly cetuximab (400 mg/m2 loading dose followed by 250 mg/m2 on days 8, 15, and 22, and weekly thereafter). Results Three patients received copanlisib 45 mg, of whom two experienced grade 3 hyperglycemia during Cycle 1 that met the DLT criteria. Eight patients were then treated with copanlisib at the dose of 30 mg. Because of the occurrence of hyperglycemia, a premedication with metformine was introduced on the day of the injections. No DLTs were reported at this dose level. The trial was stopped early because of the unfavourable toxicity profile of the combination. Among eight evaluable patients for response, four patients (50%) had disease stabilization according to RECIST1.1 as best response. Conclusion Copanlisib combined with cetuximab demonstrated unfavorable toxicity and limited efficacy in heavily pretreated recurrent and/or metastatic HNSCC patients. Trial registration NCT02822482, Date of registration: June 2016.

Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder. The role of angiogenesis and VEGF pathway in the pathogenesis of neurofibromas and malignant peripheral nerve sheath tumors (MPNSTs) remains poorly understood. We assessed the expression of VEGF and VEGFR family members in cohorts of plexiform neurofibromas (pNF), MPNSTs and MPNST cell lines at transcript [pNF, n = 49; MPNST, n = 34] and protein levels [pNF, n = 21; MPNST, n = 9]. VEGF and VEGFR members were variably expressed in cell lines. VEGFA (p = 3.10

In advanced oestrogen receptor-positive, HER2-negative breast cancer, acquired resistance to aromatase inhibitors frequently stems from ESR1-mutated subclones, which might be sensitive to fulvestrant. The PADA-1 trial aimed to show the efficacy of an early change in therapy on the basis of a rising ESR1 mutation in blood (bESR1We did a randomised, open-label, phase 3 trial in 83 hospitals in France. Women aged at least 18 years with oestrogen receptor-positive, HER2-negative advanced breast cancer and an Eastern Cooperative Oncology Group performance status of 0-2 were recruited and monitored for rising bESR1From March 22, 2017, to Jan 31, 2019, 1017 patients were included, of whom 279 (27%) developed a rising bESR1PADA-1 is the first prospective randomised trial showing that the early therapeutic targeting of bESR1Pfizer.

Membrane Type 1 Matrix Metalloprotease (MT1-MMP) contributes to the invasive progression of breast cancers by degrading extracellular matrix tissues. Nucleoside diphosphate kinase, NME1/NM23-H1, has been identified as a metastasis suppressor; however, its contribution to local invasion in breast cancer is not known. Here, we report that NME1 is up-regulated in ductal carcinoma in situ (DCIS) as compared to normal breast epithelial tissues. NME1 levels drop in microinvasive and invasive components of breast tumor cells relative to synchronous DCIS foci. We find a strong anti-correlation between NME1 and plasma membrane MT1-MMP levels in the invasive components of breast tumors, particularly in aggressive histological grade III and triple-negative breast cancers. Knockout of NME1 accelerates the invasive transition of breast tumors in the intraductal xenograft model. At the mechanistic level, we find that MT1-MMP, NME1 and dynamin-2, a GTPase known to require GTP production by NME1 for its membrane fission activity in the endocytic pathway, interact in clathrin-coated vesicles at the plasma membrane. Loss of NME1 function increases MT1-MMP surface levels by inhibiting endocytic clearance. As a consequence, the ECM degradation and invasive potentials of breast cancer cells are enhanced. This study identifies the down-modulation of NME1 as a potent driver of the in situ-to invasive transition during breast cancer progression.

Significant rational is available for specific targeting of PI3K/AKT/mTOR pathway in the treatment of non-small cell lung cancer (NSCLC). However, almost all clinical trials that have evaluated Pi3K pathway-based monotherapies/combinations did not observe an improvement of patient's outcome. The aim of our study was therefore to define combination of treatment based on the determination of predictive markers of resistance to the mTORC1 inhibitor RAD001/Everolimus. An in vivo study showed high efficacy of RAD001 in NSCLC Patient-Derived Xenografts (PDXs). When looking at biomarkers of resistance by RT-PCR study, three genes were found to be highly expressed in resistant tumors, i.e., PLK1, CXCR4, and AXL. We have then focused our study on the combination of RAD001 + Volasertib, a PLK1 inhibitor, and observed a high antitumor activity of the combination in comparison to each monotherapy; similarly, a clear synergistic effect between the two compounds was found in an in vitro study. Pharmacodynamics study demonstrated that this synergy was due to (1) tumor vascularization decrease, increase of the HIF1 protein expression and decrease of the intracellular pH, and (2) decrease of the Carbonic Anhydrase 9 (CAIX) protein that could not correct intracellular acidosis. In conclusion, all these preclinical data strongly suggest that the inhibition of mTORC1 and PLK1 proteins may be a promising therapeutic approach for NSCLC patients.

Background Following the PALOMA-3 study results, the combination of palbociclib, a CDK4/6 inhibitor, with fulvestrant, a selective estrogen receptor degrader, has become a standard therapy in women with estrogen receptor-positive (ER+) HER2-negative (HER2−) metastatic breast cancer (MBC). Palbociclib has been shown to increase the progression-free survival (PFS) overall but no predictive biomarker of palbociclib efficacy has been validated so far. We thus evaluated whether early changes of circulating tumor DNA (ctDNA) levels are associated with palbociclib plus fulvestrant efficiency. Methods ER+ HER2− MBC patients were included in a prospective observational cohort before treatment initiation. Tumor response was assessed by radiological evaluation (RECIST v1.1) every 3 months. Plasma samples were collected before treatment (baseline), at day 15 (D15), at day 30 (D30), and at disease progression. We searched for somatic mutations from archived tumor tissues by targeted deep sequencing. For patients with somatic mutations identified, circulating tumor DNA (ctDNA) was tracked using digital droplet PCR. Ratios of ctDNA levels ([D15/baseline] and [D30/baseline]) were then correlated with prospectively registered patient characteristics and outcomes. Results Twenty-five of the 61 patients enrolled had a somatic mutation testable in plasma (NPIK3CA = 21, NTP53 = 2, NAKT1 = 2). At baseline, 84% of patients had detectable ctDNA levels but ctDNA levels had no prognostic impact on PFS (p = 0.10). Among those patients, ctDNA was still detected in 82% at D15 and 68% at D30. ctDNA clearance observed at day 30 was associated with longer PFS (HR = 7.2, 95% CI = 1.5–32.6, p = 0.004). On the contrary, a [D30/baseline] ctDNA ratio > 1 was associated with a shorter PFS (HR = 5.1, 95% CI = 1.4–18.3, p = 0.02) and all 5 patients with increased ctDNA levels at D30 showed disease progression after 3 months under palbociclib-fulvestrant. Finally, at the time of radiological tumor progression, ctDNA was detected in all patients tested. Conclusion Our study demonstrates that the efficiency of palbociclib and fulvestrant can be monitored by serial analyses of ctDNA before radiological evaluation and that early ctDNA variation is a prognostic factor of PFS.

Background Prognosis evaluation of advanced breast cancer and therapeutic strategy are mostly based on clinical features of advanced disease and molecular profiling of the primary tumor. Very few studies have evaluated the impact of metastatic subtyping during the initial metastatic event in a prospective study. The genomic landscape of metastatic breast cancer has mostly been described in very advanced, pretreated disease, limiting the findings transferability to clinical use. Methods We developed a multicenter, single-arm, prospective clinical trial in order to address these issues. Between November 2010 and September 2013, 123 eligible patients were included. Patients at the first, untreated metastatic event were eligible. All matched primary tumors and metastatic samples were centrally reviewed for pathological typing. Targeted and whole-exome sequencing was applied to matched pairs of frozen tissue. A multivariate overall survival analysis was performed (median follow-up 64 months). Results Per central review in 84 patients (out of 130), we show that luminal A breast tumors are more prone to subtype switching. By combining targeted sequencing of a 91 gene panel (n = 67) and whole-exome sequencing (n = 30), a slight excess of mutations is observed in the metastases. Luminal A breast cancer has the most heterogeneous mutational profile and the highest number of mutational signatures, when comparing primary tumor and the matched metastatic tissue. Tumors with a subtype change have more mutations that are private. The metastasis-specific mutation load is significantly higher in late than in de novo metastases. The most frequently mutated genes were TP53 and PIK3CA. The most frequent metastasis-specific druggable genes were PIK3CA, PTEN, KDR, ALK, CDKN2A, NOTCH4, POLE, SETD2, SF3B1, and TSC2. Long-term outcome is driven by a combination of tumor load and metastasis biology. Conclusions Profiling of the first, untreated, metastatic event of breast cancer reveals a profound heterogeneity mostly in luminal A tumors and in late metastases. Based on this profiling, we can derive information relevant to prognosis and therapeutic intervention, which support current guidelines recommending a biopsy at the first metastatic relapse. Trial registration The trial was registered at ClinicalTrials.gov (NCT01956552).

Introduction: Cell-free tumor DNA (ctDNA) is an emerging biomarker in head and neck squamous cell carcinoma (HNSCC) for disease staging, patients’ recurrence risk stratification and early detection of relapse. We aimed to compare variants identified in ctDNA versus surgical tumor specimen, and to study the evolution of the mutational landscape of ctDNA over time in HNSCC. Patients and Method: Forty-one HNSCC patients treated with curative-intent primary surgery from SCANDARE cohort (NCT03017573) were evaluated for longitudinal ctDNA-based NGS. Overall, 28 patients were treated with adjuvant (chemo)radiotherapy, and 31 experienced recurrence. Formalin-fixed paraffin-embedded tumor tissues at surgery were available for 41 patients. Serial contributive ctDNA were retrieved from all 41 patients at the date of surgery, 36 patients within 19 weeks after surgery, 20 patients at six months after surgery, and 22 patients at recurrence. Tissue DNA was personalized detected with a custom NGS panel of 571 genes (DRAGON) and ctDNA was sequenced using another personalized dedicated NGS panel including up to 15 genes (OncoFOLLOW). Results: Most frequently mutated genes in tissue included TP53 (15.9%), FAT1 (6.7%), NOTCH1 (5.5%) and PIK3CA (4.3%) with similar allelic ratio to ctDNA at baseline surgery. Higher prevalence of KRAS and TP53 mutations was found in ctDNA at recurrence in comparison with ctDNA and tissue, respectively, at baseline surgery (KRAS: 6.3% versus 1.6% and 0.6%; TP53: 31.2% versus 21.1% and 15.9%). Additional variants in NRAS, HRAS, TP53, JAK2 and SDHA were detected in 6 patients in ctDNA at surgery and were not found in tissue, suggesting spatial intratumor heterogeneity. Twenty-three/36 patients (64%) had detected ctDNA within 19 weeks after surgery among whom, 17/23 patients (74%) had disease recurrence. Eleven/20 patients (including 10 with adjuvant treatment) had detected ctDNA at six months after surgery among whom 6 patients (55%) had disease recurrence. Fifteen/22 patients (68%) had detected ctDNA at recurrence. Emerging pathogenic variants were found in patients with detected ctDNA after surgery (n=7/23; 30%), at six months after surgery (n=1/11; 9%) and at recurrence (n=4/15; 27%). Conclusion: Our study suggests spatial and longitudinal tumor heterogeneity and reports emerging mutations in ctDNA over time in HNSCC. Prognostic significance characterization of the ctDNA dominant clone allele frequency is ongoing. Citation Format: Grégoire Marret, Constance Lamy, Sophie Vacher, Mathieu Séné, Ladidi Ahmanache, Laura Courtois, Zakhia El Beiano, Jerzy Klijanienko, Charlotte Martinat, Nicolas Servant, Choumouss Kamoun, Linda Larbi Chérif, Thierry Bronzini, Cédric Balsat, Jean-François Laes, Aubray Prévot, Sébastien Sauvage, Maxime Lienard, Emmanuel Martin, Bérengère Genin, Nathalie Badois, Maria Lesnik, Antoine Dubray-Vautrin, Olivier Choussy, Wahib Ghanem, Rabah Taouachi, Julien Masliah Planchon, Ivan Bièche, Maud Kamal, Christophe Le Tourneau. Spatial and longitudinal tumor heterogeneity in head and neck squamous cell carcinoma patients treated with primary surgery [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 3363.

Background: The combination of a CDK4/6 inhibitor and an aromatase inhibitor (AI) is the gold standard for AI-sensitive first line treatment of ER+ HER2- advanced breast cancer. Nevertheless, some patients progress rapidly and may benefit from alternative strategies. Early ctDNA dynamics have been shown to predict disease course in several clinical situations. Here, we use samples from the PADA-1 trial to assess this strategy for patients receiving AI and palbociclib as first line treatment. PADA-1 was designed to assess the clinical utility of sequential analysis of ctDNA for emerging ESR1 mutations to trigger an early switch from AI plus palbociclib to fulvestrant plus palbociclib treatment. The study included 1,017 patients and was positive on its primary end-point. The objective of this translational study was to analyze the predictive value of 4-week molecular response (MR) for patient progression. Material & Method: First, a CLIA-validated targeted next-generation sequencing-based test (Guardant360 Response) was used to characterize changes in ctDNA level via detection of somatic single-nucleotide variants (SNVs), insertion/deletion mutations (indels), and gene fusions in 74 genes frequently mutated in cancer. A second analysis was restricted to cancer-associated mutations in 11 genes commonly mutated in breast cancer (PIK3CA, GATA3, TP53, AKT1, ERBB2, BRCA1, BRCA2, ATM, ESR1, PALB2 and RB1). The threshold for molecular response was defined as ≥ 50% decrease in ctDNA (MR score < 0.5). Subjects with ctDNA levels below the test’s limit of quantitation (ctDNA-low) were considered molecular responders. Results: 372 subjects with matched baseline and 4-weeks samples were available for analysis. Of these, 134 subjects (36%) were ctDNA-low, and 238 subjects (64%) quantifiable. Among the quantifiable subjects, 183 (77%) were molecular responders (MR+, MR < 0.5), and 55 (23%) were not (MR–, MR ≥ 0.5). PFS was moderately improved for both MR+ and ctDNA-low relative to MR– (HR=0.61 (95%CI 0.44-0.85), p< 0.01) over the full 29 months of follow up. Differential PFS event rate was observed only in the first 8 months following ctDNA assessment; during this time MR+ and ctDNA-low were associated with more significantly decreased risk of progression (HR 0.24, 95% CI 0.13 – 0.43, p=0.0001). Limiting ctDNA assessment to genes commonly mutated in breast cancer enhanced the predictive power of MR (HR=0.08, 95% CI 0.04 0.17, p< 0.001, for MR+ and ctDNA-low vs. MR– across 8 months post-assessment); however, fewer samples were quantifiable by this method (169 [45%] quantifiable; 203 [55%] ctDNA-low). Combining MR status with additional molecular features (e.g.tumor mutational burden and maximum mutation allele fraction) did not improve prediction of non-response. Conclusion: Changes in ctDNA fraction during the first weeks of treatment are predictive of long term clinical benefit on an individual patient basis, particularly during the first year of therapy. Adjusting the MR threshold and/or limiting to genes known to be relevant in the specific tumor can tailor the assessment of ctDNA change to specific clinical scenarios where greater sensitivity or specificity may be required. The identification of patients at high risk for early clinical failure at the onset of treatment may allow for therapy escalation and/or change to improve outcome in this population. Funding: Pfizer and Guardant Health Citation Format: Caroline Bailleux, Thomas Bachelot, Francois-Clement Bidard, Anne-Claire Hardy-Bessard, Ivan Bièche, Anne Pradines, Florian Clatot, thibault DE LA MOTTE ROUGE, Jean-Luc Canon, Barbara Pistilli, Kyle Chang, Katie J. Quinn, Heather L. Gustafson, Florence Dalenc, Cyril Foa, Hanifa Ammarguellat, Chantal Bernard-Marty, Brigitte Lucas, Sophie Barthier, Fabrice Lorchel, Olivier Gisserot, Laurent Arnould, Marjorie Mauduit, Jérôme Lemonnier, Frédérique Berger, Suzette Delaloge, Fabrice Andre. ctDNA Molecular Response based on breast cancer driver mutations predicts progression in aromatase inhibitor-sensitive first line treatment of oestrogen receptor-positive (ER+) HER2-negative (HER2-) advanced breast cancer. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD17-02.