Pathogenic alterations in PIK3CA and KMT2C are frequent and independent prognostic factors in anal squamous cell carcinoma treated with salvage abdominoperineal resection.

The management of anal squamous cell carcinoma (ASCC) has yet to experience the transformative impact of precision medicine. Conducting genomic analyses may uncover novel prognostic biomarkers and offer potential directions for the development of targeted therapies. To that end, we assessed the prognostic and theragnostic implications of pathogenic variants identified in 571 cancer-related genes from surgical samples collected from a homogeneous, multicentric French cohort of 158 ASCC patients who underwent abdominoperineal resection treatment. Alterations in PI3K/AKT/mTOR, chromatin remodeling, and Notch pathways were frequent in HPV-positive tumors, while HPV-negative tumors often harbored variants in cell cycle regulation and genome integrity maintenance genes (e.g., frequent TP53 and TERT promoter mutations). In patients with HPV-positive tumors, KMT2C and PIK3CA exon 9/20 pathogenic variants were associated with worse overall survival in multivariate analysis (Hazard ratio (HR)KMT2C = 2.54, 95%CI = [1.25,5.17], P value = .010; HRPIK3CA = 2.43, 95%CI = [1.3,4.56], P value = .006). Alterations with theragnostic value in another cancer type was detected in 43% of patients. These results suggest that PIK3CA and KMT2C pathogenic variants are independent prognostic factors in patients with ASCC with HPV-positive tumors treated by abdominoperineal resection. And, importantly, the high prevalence of alterations bearing potential theragnostic value strongly supports the use of genomic profiling to allow patient enrollment in precision medicine clinical trials.

Prognostic Value of Fusobacterium nucleatum after Abdominoperineal Resection for Anal Squamous Cell Carcinoma.

Main prognostic factors of anal squamous cell carcinoma (ASCC) are tumor size, differentiation, lymph node involvement, and male gender. However, they are insufficient to predict relapses after exclusive radiotherapy (RT) or chemoradiotherapy (CRT). Fusobacterium nucleatum has been associated with poor prognosis in several digestive cancers. In this study, we assessed the association between intratumoral F. nucleatum load and clinico-pathological features, relapse, and survival in patients with ASCC who underwent abdominoperineal resection (APR) after RT/CRT. We retrospectively analyzed surgical samples from a cohort of 166 patients with ASCC who underwent APR. F. nucleatum 16S rRNA gene sequences were quantified using real-time quantitative PCR. We associated F. nucleatum load with classical clinicopathological features, overall survival (OS), disease-free survival (DFS), and metastasis-free survival (MFS) using Cox regression univariate and multivariate analyses. Tumors harboring high loads of F. nucleatum (highest tercile) showed longer OS and DFS (median: not reached vs. 50.1 months, p = 0.01, and median: not reached vs. 18.3 months, p = 0.007, respectively). High F. nucleatum load was a predictor of longer OS (HR = 0.55, p = 0.04) and DFS (HR = 0.50, p = 0.02) in multivariate analysis. High F. nucleatum load is an independent favorable prognostic factor in patients with ASCC who underwent APR.

MISpheroID: a knowledgebase and transparency tool for minimum information in spheroid identity.

Spheroids are three-dimensional cellular models with widespread basic and translational application across academia and industry. However, methodological transparency and guidelines for spheroid research have not yet been established. The MISpheroID Consortium developed a crowdsourcing knowledgebase that assembles the experimental parameters of 3,058 published spheroid-related experiments. Interrogation of this knowledgebase identified heterogeneity in the methodological setup of spheroids. Empirical evaluation and interlaboratory validation of selected variations in spheroid methodology revealed diverse impacts on spheroid metrics. To facilitate interpretation, stimulate transparency and increase awareness, the Consortium defines the MISpheroID string, a minimum set of experimental parameters required to report spheroid research. Thus, MISpheroID combines a valuable resource and a tool for three-dimensional cellular models to mine experimental parameters and to improve reproducibility.

Longitudinal high-resolution imaging through a flexible intravital imaging window.

Intravital microscopy (IVM) is a powerful technique that enables imaging of internal tissues at (sub)cellular resolutions in living animals. Here, we present a silicone-based imaging window consisting of a fully flexible, sutureless design that is ideally suited for long-term, longitudinal IVM of growing tissues and tumors. Crucially, we show that this window, without any customization, is suitable for numerous anatomical locations in mice using a rapid and standardized implantation procedure. This low-cost device represents a substantial technological and performance advance that facilitates intravital imaging in diverse contexts in higher organisms, opening previously unattainable avenues for in vivo imaging of soft and fragile tissues.

Spontaneous mouse lymphoma in patient-derived tumor xenografts: The importance of systematic analysis of xenografted human tumor tissues in preclinical efficacy trials.

Patient-derived tumor xenograft (PDX) is now largely recognized as a key preclinical model for cancer research, mimicking patient tumor phenotype and genotype. Immunodeficient mice, well-known to develop spontaneous lymphoma, are required for PDX growth. As for all animal models used for further clinical translation, a robust experimental design is strongly required to lead to conclusive results. Here we briefly report unintentional co-engraftment of mouse lymphoma during expansion of well-established PDXs to illustrate the importance of systematic check of the PDX identity to avoid misinterpretation. Besides, this quality control based on complementary approaches deserves a more detailed description in materials and methods section to ensure experimental validity and reproducibility.

Interaction between IGF2-PI3K axis and cancer-associated-fibroblasts promotes anal squamous carcinogenesis.

Anal squamous cell carcinoma (ASCC) is a rare tumour, but its incidence is increasing. Standard chemoradiotherapy fails to cure 20% of patients and no targeted therapy is currently approved for recurrent ASCC. The PI3K/Akt/mTOR pathway is frequently altered in this poorly characterised carcinoma. IGF2 was identified here as a key factor in ASCC oncogenesis, as IGF2 was shown to play a crucial role in the PI3K pathway with frequent (~60%) and mutually exclusive genomic alterations in IGF2, IGF1R, PTEN and PIK3CA genes. We also demonstrated that IGF2 expression is mainly due to cancer-associated fibroblasts and that IGF2 secreted by cancer-associated fibroblasts from ASCC samples promotes proliferation of a human ASCC cell line via IGF2 paracrine signalling. Altogether, these results highlight the key role of the IGF2/PI3K axis, and the major role of cancer-associated fibroblasts in tumour growth via IGF2 secretion, suggesting a major role of IGF2/IGF1R inhibitors in ASCC therapies.

Array comparative genomic hybridization identifies high level of PI3K/Akt/mTOR pathway alterations in anal cancer recurrences.

Genomic alterations of anal squamous cell carcinoma (ASCC) remain poorly understood due to the rarity of this tumor. Array comparative genomic hybridization and targeted gene sequencing were performed in 49 cases of ASCC. The most frequently altered regions (with a frequency greater than 25%) were 10 deleted regions (2q35, 2q36.3, 3p21.2, 4p16.3, 4p31.21, 7q36.1, 8p23.3, 10q23.2, 11q22.3, and 13q14.11) and 8 gained regions (1p36.33, 1q21.1, 3q26.32, 5p15.33, 8q24.3, 9q34.3, 16p13.3, and 19p13.3). The most frequent minimal regions of deletion (55%) encompassed the 11q22.3 region containing ATM, while the most frequent minimal regions of gain (57%) encompassed the 3q26.32 region containing PIK3CA. Recurrent homozygous deletions were observed for 5 loci (ie, TGFR2 in 4 cases), and recurrent focal amplifications were observed for 8 loci (ie, DDR2 and CCND1 in 3 cases, respectively). Several of the focal amplified genes are targets for specific therapies. Integrated analysis showed that the PI3K/Akt/mTOR signaling pathway was the pathway most extensively affected, particularly in recurrences compared to treatment-naive tumors (64% vs 30%; P = .017). In patients with ASCC recurrences, poor overall survival (OS) was significantly correlated with a large number of altered regions (P = .024). These findings provide insight into the somatic genomic alterations in ASCC and highlight the key role of the druggable PI3K/Akt/mTOR signaling pathway.

Exome sequencing reveals aberrant signalling pathways as hallmark of treatment-naive anal squamous cell carcinoma.

Anal squamous cell carcinomas (ASCC) are rare tumours in humans. The etiological role of HPV infection is now well established but little is known about the molecular landscape and signalling pathways involved in the pathogenesis of this cancer. Here we report the results from a whole exome sequencing of a homogeneous group of 20 treatment-naive ASCC. A total of 2422 somatic single nucleotide variations (SNV) were found, with an overall moderate rate of somatic mutations per tumour (median: 105 relevant SNV per tumour) but a high mutational load in 3 tumours. The mutational signatures associated with age and APOBEC were observed in 100% and 60% of tumours respectively. The most frequently mutated genes were PIK3CA (25%) followed by FBXW7 (15%), FAT1 (15%), and TRIP12 (15%), the two last ones having never been described in ASCC. The main copy number alterations were gains of chromosome 3q (affecting PIK3CA) and losses of chromosome 11q (affecting ATM). The combined analysis of somatic mutations and copy number alterations show that recurrent alterations of the PI3K/AKT/mTOR pathway are frequent (60%) in these tumours, as well as potentially targetable alterations of other signalling pathways that have never been described in ASCC such as chromatin remodelling (45%) and ubiquitin mediated proteolysis (35%). These results highlight the possible implication of these aberrant signalling pathways in anal carcinogenesis and suggest promising new therapeutic approaches in ASCC. The high somatic mutation burden found in some tumours, suggesting an elevated neoantigen load could also predict sensitivity of ASCC to immunotherapy.

Interrogating open issues in cancer medicine with patient-derived xenografts.

This corrects the article DOI: 10.1038/nrc.2016.140.

The early pregnancy placenta foreshadows DNA methylation alterations of solid tumors.

The placenta relies on phenotypes that are characteristic of cancer to successfully implant the embryo in the uterus during early pregnancy. Notably, it has to invade its host tissues, promote angiogenesis-while surviving hypoxia-, and escape the immune system. Similarities in DNA methylation patterns between the placenta and cancers suggest that common epigenetic mechanisms may be involved in regulating these behaviors. We show here that megabase-scale patterns of hypomethylation distinguish first from third trimester chorionic villi in the placenta, and that these patterns mirror those that distinguish many tumors from corresponding normal tissues. We confirmed these findings in villous cytotrophoblasts isolated from the placenta and identified a time window at the end of the first trimester, when these cells come into contact with maternal blood, as the likely time period for the methylome alterations. Furthermore, the large genomic regions affected by these patterns of hypomethylation encompass genes involved in pathways related to epithelial-mesenchymal transition, immune response, and inflammation. Analyses of expression profiles corresponding to genes in these hypomethylated regions in colon adenocarcinoma tumors point to networks of differentially expressed genes previously implicated in carcinogenesis and placentogenesis, where nuclear factor kappa B is a key hub. Taken together, our results suggest the existence of epigenetic switches involving large-scale changes of methylation in the placenta during pregnancy and in tumors during neoplastic transformation. The characterization of such epigenetic switches might lead to the identification of biomarkers and drug targets in oncology as well as in obstetrics and gynecology.

Interrogating open issues in cancer precision medicine with patient-derived xenografts.

Patient-derived xenografts (PDXs) have emerged as an important platform to elucidate new treatments and biomarkers in oncology. PDX models are used to address clinically relevant questions, including the contribution of tumour heterogeneity to therapeutic responsiveness, the patterns of cancer evolutionary dynamics during tumour progression and under drug pressure, and the mechanisms of resistance to treatment. The ability of PDX models to predict clinical outcomes is being improved through mouse humanization strategies and the implementation of co-clinical trials, within which patients and PDXs reciprocally inform therapeutic decisions. This Opinion article discusses aspects of PDX modelling that are relevant to these questions and highlights the merits of shared PDX resources to advance cancer medicine from the perspective of EurOPDX, an international initiative devoted to PDX-based research.

Mutational analysis of anal cancers demonstrates frequent PIK3CA mutations associated with poor outcome after salvage abdominoperineal resection.

BACKGROUND: A better understanding of the molecular profile of anal squamous cell carcinomas (ASCCs) is necessary to consider new therapeutic approaches, and the identification of prognostic and predictive factors for response to treatment. METHODS: We retrospectively analysed tumours from ASCC patients for mutational analysis of KRAS, NRAS, HRAS, BRAF, PIK3CA, MET, TP53 and FBXW7 genes by HRM and Sanger sequencing analysis. RESULTS: Specimens from 148 patients were analysed: 96 treatment-naive tumours and 52 recurrences after initial radiotherapy (RT) or chemoradiotherapy (CRT). Mutations of KRAS, PIK3CA, FBXW7 and TP53 genes were present in 3 (2.0%), 30 (20.3%), 9 (6.1%) and 7 tumours (4.7%), respectively. The distribution of the mutations was similar between treatment-naive tumours and recurrences, except for TP53 mutations being more frequent in recurrences (P=0.0005). In patients treated with abdominoperineal resection (APR) after relapse (n=38, median follow-up of 18.2 years), overall survival (OS) was significantly correlated with HPV16 status (P=0.048), gender (P=0.045) and PIK3CA mutation (P=0.037). The PIK3CA status retained its prognostic significance in Cox multivariate regression analysis (P=0.025). CONCLUSIONS: Our study identified PIK3CA mutation as an independent prognostic factor in patients who underwent APR for ASCC recurrence, suggesting a potential benefit from adjuvant treatment and the evaluation of targeted therapies with PI3K/Akt/mTor inhibitors in PIK3CA-mutated patients.

Evaluating patient-derived colorectal cancer xenografts as preclinical models by comparison with patient clinical data.

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.

Spherical cancer models in tumor biology.

Three-dimensional (3D) in vitro models have been used in cancer research as an intermediate model between in vitro cancer cell line cultures and in vivo tumor. Spherical cancer models represent major 3D in vitro models that have been described over the past 4 decades. These models have gained popularity in cancer stem cell research using tumorospheres. Thus, it is crucial to define and clarify the different spherical cancer models thus far described. Here, we focus on in vitro multicellular spheres used in cancer research. All these spherelike structures are characterized by their well-rounded shape, the presence of cancer cells, and their capacity to be maintained as free-floating cultures. We propose a rational classification of the four most commonly used spherical cancer models in cancer research based on culture methods for obtaining them and on subsequent differences in sphere biology: the multicellular tumor spheroid model, first described in the early 70s and obtained by culture of cancer cell lines under nonadherent conditions; tumorospheres, a model of cancer stem cell expansion established in a serum-free medium supplemented with growth factors; tissue-derived tumor spheres and organotypic multicellular spheroids, obtained by tumor tissue mechanical dissociation and cutting. In addition, we describe their applications to and interest in cancer research; in particular, we describe their contribution to chemoresistance, radioresistance, tumorigenicity, and invasion and migration studies. Although these models share a common 3D conformation, each displays its own intrinsic properties. Therefore, the most relevant spherical cancer model must be carefully selected, as a function of the study aim and cancer type.

Vasculature analysis of patient derived tumor xenografts using species-specific PCR assays: evidence of tumor endothelial cells and atypical VEGFA-VEGFR1/2 signalings.

BACKGROUND: Tumor endothelial transdifferentiation and VEGFR1/2 expression by cancer cells have been reported in glioblastoma but remain poorly documented for many other cancer types. METHODS: To characterize vasculature of patient-derived tumor xenografts (PDXs), largely used in preclinical anti-angiogenic assays, we designed here species-specific real-time quantitative RT-PCR assays. Human and mouse PECAM1/CD31, ENG/CD105, FLT1/VEGFR1, KDR/VEGFR2 and VEGFA transcripts were analyzed in a large series of 150 PDXs established from 8 different tumor types (53 colorectal, 14 ovarian, 39 breast and 15 renal cell cancers, 6 small cell and 5 non small cell lung carcinomas, 13 cutaneous melanomas and 5 glioblastomas) and in two bevacizumab-treated non small cell lung carcinomas xenografts. RESULTS: As expected, mouse cell proportion in PDXs -evaluated by quantifying expression of the housekeeping gene TBP- correlated with all mouse endothelial markers and human VEGFA RNA levels. More interestingly, we observed human PECAM1/CD31 and ENG/CD105 expression in all tumor types, with higher rate in glioblastoma and renal cancer xenografts. Human VEGFR expression profile varied widely depending on tumor types with particularly high levels of human FLT1/VEGFR1 transcripts in colon cancers and non small cell lung carcinomas, and upper levels of human KDR/VEGFR2 transcripts in non small cell lung carcinomas. Bevacizumab treatment induced significant low expression of mouse Pecam1/Cd31, Eng/Cd105, Flt1/Vegfr1 and Kdr/Vefr2 while the human PECAM1/CD31 and VEGFA were upregulated. CONCLUSIONS: Taken together, our results strongly suggest existence of human tumor endothelial cells in all tumor types tested and of both stromal and tumoral autocrine VEGFA-VEGFR1/2 signalings. These findings should be considered when evaluating molecular mechanisms of preclinical response and resistance to tumor anti-angiogenic strategies.

Characterization of a large panel of patient-derived tumor xenografts representing the clinical heterogeneity of human colorectal cancer.

PURPOSE: Patient-derived xenograft models are considered to represent the heterogeneity of human cancers and advanced preclinical models. Our consortium joins efforts to extensively develop and characterize a new collection of patient-derived colorectal cancer (CRC) models. EXPERIMENTAL DESIGN: From the 85 unsupervised surgical colorectal samples collection, 54 tumors were successfully xenografted in immunodeficient mice and rats, representing 35 primary tumors, 5 peritoneal carcinoses and 14 metastases. Histologic and molecular characterization of patient tumors, first and late passages on mice includes the sequence of key genes involved in CRC (i.e., APC, KRAS, TP53), aCGH, and transcriptomic analysis. RESULTS: This comprehensive characterization shows that our collection recapitulates the clinical situation about the histopathology and molecular diversity of CRC. Moreover, patient tumors and corresponding models are clustering together allowing comparison studies between clinical and preclinical data. Hence, we conducted pharmacologic monotherapy studies with standard of care for CRC (5-fluorouracil, oxaliplatin, irinotecan, and cetuximab). Through this extensive in vivo analysis, we have shown the loss of human stroma cells after engraftment, observed a metastatic phenotype in some models, and finally compared the molecular profile with the drug sensitivity of each tumor model. Through an experimental cetuximab phase II trial, we confirmed the key role of KRAS mutation in cetuximab resistance. CONCLUSIONS: This new collection could bring benefit to evaluate novel targeted therapeutic strategies and to better understand the basis for sensitivity or resistance of tumors from individual patients.

The Wnt antagonist Dickkopf-1 increases endothelial progenitor cell angiogenic potential.

OBJECTIVE: To determine the role of Wnt antagonist Dickkopf (DKK) 1 in human endothelial colony-forming cells (ECFCs) in view of the emerging importance of Wnt pathways in vascular biology. METHODS AND RESULTS: Endothelial progenitor cells have been proposed to be crucial in tumor neovascularization. Recombinant DKK1 has been tested in ECFC angiogenic properties in vitro. DKK1 enhanced ECFC proliferation and the capacity of ECFCs to form pseudotubes in Matrigel. These effects have been attributed to enhancement of vascular endothelial growth factor receptor 2, SDF-1, and CXCR4. DKK1 gene silencing has been realized on ECFCs and mesenchymal stem cells, and we found that DKK1 silencing in the 2 cell types decreased their angiogenic potential. We then examined the possible role of DKK1 in tumor neovasculogenesis and found that blood vessels of breast cancer tissues expressed DKK1 far more strongly in human breast tumors than in normal breast tissues. By studying 62 human breast tumors, we found a significant positive correlation between DKK1 expression and von Willebrand factor. In vivo, DKK1 strongly enhanced the vascularization of Matrigel plugs and increased tumor size in a xenograft model of human breast carcinoma in nude mice. CONCLUSIONS: DKK1 enhances angiogenic properties of ECFCs in vitro and is required for ECFC and mesenchymal stem cell angiogenic phenotypes in vivo. DKK1 also increases tumoral angiogenesis. Thus, we demonstrated a major role of DKK1 in angiogenic processes.

In situ protein expression in tumour spheres: development of an immunostaining protocol for confocal microscopy.

BACKGROUND: Multicellular tumour sphere models have been shown to closely mimic phenotype characteristics of in vivo solid tumours, or to allow in vitro propagation of cancer stem cells (CSCs). CSCs are usually characterized by the expression of specific membrane markers using flow cytometry (FC) after enzymatic dissociation. Consequently, the spatial location of positive cells within spheres is not documented. Confocal microscopy is the best technique for the imaging of thick biological specimens after multi-labelling but suffers from poor antibody penetration. Thus, we describe here a new protocol for in situ confocal imaging of protein expression in intact spheroids. METHODS: Protein expression in whole spheroids (150 mum in diameter) from two human colon cancer cell lines, HT29 and CT320X6, has been investigated with confocal immunostaining, then compared with profiles obtained through paraffin immunohistochemistry (pIHC) and FC. Target antigens, relevant for colon cancer and with different expression patterns, have been studied. RESULTS: We first demonstrate that our procedure overcomes the well-known problem of antibody penetration in compact structures by performing immunostaining of EpCAM, a membrane protein expressed by all cells within our spheroids. EpCAM expression is detected in all cells, even the deepest ones. Likewise, antibody access is confirmed with CK20 and CD44 immunostaining. Confocal imaging shows that 100% of cells express beta-catenin, mainly present in the plasma membrane with also cytoplasmic and nuclear staining, in agreement with FC and pIHC data. pIHC and confocal imaging show similar CA 19-9 cytoplasmic and membranar expression profile in a cell subpopulation. CA 19-9+ cell count confirms confocal imaging as a highly sensitive method (75%, 62% and 51%, for FC, confocal imaging and pIHC, respectively). Finally, confocal imaging reveals that the weak expression of CD133, a putative colon CSC marker, is restricted to the luminal cell surface of colorectal cancer acini, with CD133+ cellular debris into glandular lumina. CONCLUSION: The present protocol enables in situ visualization of protein expression in compact three-dimensional models by whole mount confocal imaging, allowing the accurate localization and quantification of cells expressing specific markers. It should prove useful to study rare events like CSCs within tumour spheres.

Ralstonia pickettii-induced ataxia in immunodeficient mice.

We report here the characterization of an asymmetric ataxia syndrome (head tilt and circling, with death in the most severe cases) demonstrated by profoundly immunodeficient mice housed at the Institut Curie SPF facility. The immune system of the affected mice had been genetically modified so that they were deficient in both B and T cells. Extensive bacteriologic, parasitic, serologic, and histopathologic analysis of the affected animals and their healthy controls led us to identify Ralstonia pickettii as the causative agent of the ataxic syndrome. The outbreak was managed through a test-and-cull process. Even though they also carried Ralstonia pickettii, immunocompetent mice that were kept in the same facility, did not show any of the signs that were expressed by their immunodeficient counterparts. This case highlights the difficulty of maintaining immunocompetent and immunodeficient mice in the same microbiologic unit and the importance of enlarging the spectrum of health monitoring to opportunistic agents when investigating clinical cases in populations of immunocompromised rodents.