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Loss of imprinting (LOI) of the insulin-like growth factor II (IGFII) gene is a frequent phenomenon in colorectal tumor tissues. Previous reports indicated that subjects with colorectal neoplasias show LOI of IGFII in circulating lymphocytes. Furthermore, LOI of IGFII is strongly related to the methylation of a differentially methylated region (DMR) in intron 2 of IGFII, suggesting that the methylation status could serve as a biomarker for early detection. Thus, hypermethylation of this DMR, even at a systemic level, e.g., in lymphocyte DNA, could be used for screening for colon cancer. To validate this, we performed a case-control study of 97 colon cancer cases and 190 age-matched and gender-matched controls, nested within the prospective Northern Sweden Health and Disease Study cohort. Methylation levels of the IGFII-DMR in lymphocyte DNA were measured at two specific CpG sites of the IGFII-DMR using a mass-spectrometric method called short oligonucleotide mass analysis, the measurements of which showed high reproducibility between replicate measurements for the two CpG sites combined and showed almost perfect validity when performed on variable mixtures of methylated and unmethylated standards. Mean fractions of CpG methylation, for the two CpG sites combined, were identical for cases and controls (0.47 and 0.46, respectively; Pdifference = 0.75), and logistic regression analyses showed no relationship between colon cancer risk and quartile levels of CpG methylation. The results from this study population do not support the hypothesis that colon cancer can be predicted from the different degrees of methylation of DMR in the IGFII gene from lymphocyte DNA. [Cancer Res 2009;69(13):5400–5]

Aristolochic acid (AA) is a potent nephrotoxin and carcinogen (IARC Group 1) associated with urothelial, hepatobiliary, and renal carcinomas. Exposure to AA from dietary intake of traditional herbal medicines containing Aristolochia species poses a global health hazard, yet also an opportunity for disease prevention. Molecular epidemiology studies employing sensitive and specific biomarkers for screening of populations at risk are thus warranted. Genome-scale studies established a characteristic mutational signature of AA. Exploiting its unique features (predominant A>T transversions), alongside the knowledge of key affected cancer genes, we devised a cost-effective targeted resequencing (TRseq) method to detect AA etiology in upper tract urothelial carcinoma (UTUC) samples from South Korean patients. Aristolochia species (e.g., A. contorta, Bunge) are included in the Korean traditional pharmacopoeia although the local occurrence of AA-associated cancers remains unexplored. DNA from archived, paired kidney and UTUC samples of 16 Korean patients was used for adduct and TRseq analyses. Thirty-one cancer genes, previously found recurrently mutated in AA-induced UTUCs from Asia and Europe, were selectively captured using SeqCap EZ enrichment kit (Nimblegen Roche) and sequenced using Illumina MiSeq. Sets of UTUCs from Croatia (harboring AA signature) and from US patients (unlikely to be exposed) were included as respective positive and negative controls. Somatic variants were called against the patient-matched non-tumor DNA, using two distinct variant calling strategies. Three out of 16 kidney cortex samples tested positive for aristolactam-dA (AL-dA) adducts, indicative of past exposure to AA. One UTUC sample with A>T-mutated TP53 harbored 32 A>T mutations across the 31-gene panel, and another 8 cases, 3 of which were adduct-positive, harbored lower counts of A>T mutations (up to 3 per gene panel per sample). The allelic frequencies of the considered A>T mutations ranged between 10-83%. The positive control samples exhibited a strong A>T signal while the US samples were all A>T-negative, as expected. We propose that the TRseq approach can detect AA-driven mutagenesis in UTUC tumors, providing a sensitive complement to the AL-dA adduct analysis, at low per-sample cost. Importantly, the presence of adducts and AA signature mutations in South Korean UTUC cases highlights a previously uncharacterized population at risk. The future use of TRseq, either alone or in combination with adduct analysis, can address exposure to AA in extended UTUC case series, thereby assisting in the design of preventive measures against this global public health hazard. Funding: IARC; SW is Recipient of research support from the Drs. Martin & Dorothy Spatz Charitable Foundation; Henry and Marsha Laufer grant to KGD, VSS, APG. Citation Format: Shuhan Wang, Daeeun Choi, Jaesung Lim, Kathleen G. Dickman, Magali Olivier, Stephanie Villar, Viktoriya S. Sidorenko, Maude Ardin, Byeong H. Yun, Robert J. Turesky, Minho Shong, Jiri Zavadil, Arthur P. Grollman. New molecular evidence associating exposure to aristolochic acid with urothelial cancers in South Korean patients: Implications for global public health risk linked to carcinogenic herbal medicines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4252. doi:10.1158/1538-7445.AM2017-4252

Exposure to aristolochic acid (AA, IARC Group 1 carcinogen) present in some traditional herbal medicines leads to aristolochic acid nephropathy (AAN), often complicated by development of multiple urothelial carcinomas of sequential onset. We used genome-scale mutational signature analysis of multiple urinary tract tumors of AAN cases from a unique patient group to determine the relationships of the patients’ late-onset second cancers to the AA exposure as well as to the first cancers. Aristolactam-DNA adduct-positive AAN patients (n=4) who developed cancer within 8 years following the initial exposure to AA were chosen for analysis of their first cancers (upper tract urothelial carcinomas, UTUC) and second cancers of delayed onset (1-9 years after first-cancer diagnosis, involving the bladder or ureteral meatus). All patients had received a kidney transplant before developing the second cancers and had a functional renal graft prior to prophylactic nephroureterectomy. Genomic DNAs were isolated from FFPE sections of the renal cortex and from the upper and lower tract tumors of each patient using laser capture micro-dissection or macro-dissection of the tumor areas. Low-coverage (~15x) exome 100-bp paired-end sequencing was performed using Illumina HiSeq2500. Customized variant calling identified somatic variants absent in non-tumor tissues. Non-negative matrix factorization was applied to extract mutational signatures in the tumor tissues. In all cases, we established the mutational signature of AA (the COSMIC signature 22) in the first UTUC as well as second cancers involving the bladder or lower ureter (meatus). Additionally, the first and second cancers harbored considerable overlaps in exposure-specific (A>T) somatic mutations. This finding provides evidence that the delayed onset of bladder urothelial carcinomas in AAN patients is likely due to distal seeding of cancer cells originating from the primary UTUC tumors. Our first-of-its-kind study addresses the risk as well as mechanistic factors leading to the second, late-onset bladder urothelial carcinomas following kidney transplantation and primary UTUC development. Our results underline the importance of long-term bladder follow-up in high-risk populations with established or suspected AA exposure. Funding: IARC; NYU Genome Technology Center is partially supported by the NIH/NCI (P30CA016087) grant. Citation Format: Xavier Castells, Maude Ardin, Sandrine Rorive, Nilufer Broeders, Yan Song, Stephanie Villar, Christine Carreira, Pierre-Paul Bringuier, Adriana Heguy, Thierry Quackels, Thierry Roumeguere, Joelle Nortier, Jiri Zavadil. Tracking the genetic relationship between first and late-onset second urothelial cancers by mutational signature analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5738. doi:10.1158/1538-7445.AM2017-5738

Triple negative breast cancer (TNBC), defined by the lack of expression of the estrogen receptor (ER), progesterone receptor (PR) and human epidermal receptor 2 (HER2), is an aggressive form of breast cancer (BC) that is more prevalent in certain populations, in particular in low and middle-income regions. The detailed molecular features of TNBC in these regions remain unexplored as samples are mostly accessible as formalin-fixed paraffin embedded (FFPE) archived tissues, a challenging material for advanced genomic and transcriptomic studies. We performed an integrative genomic analysis on FFPE samples from TNBC patients to identify molecular programs associated with this disease in an understudied population. We implemented whole exome sequencing of 12 tumor tissues and blood pairs, complemented with miRNA and mRNA profiling of the tumor tissues, using FFPE archived pathological samples from Mexican women. Sequencing analyses found TP53 and RB1 genes as the most frequently mutated. Transcriptional programs were characterized by the overexpression of growth-promoting signals (EGFR, PDGFR, VEGF, PIK3CA, FOXM1), the repression of cell cycle control pathways (TP53, RB1), the deregulation of DNA-repair pathways, and alterations in epigenetic modifiers through miRNA:mRNA network de-regulation. The molecular programs identified were typical of those described in basal-like tumors in other populations. This work demonstrates the utility of archival clinical samples for advanced integrated genomics analyses. It thus opens up opportunities for investigating molecular features of tumors from regions where only FFPE tissues are available, allowing the development of treatment strategies and of retrospective studies on the exploration of the geographic diversity of BC. Citation Format: Felipe Vaca-Paniagua, Rosa María Alvarez-Gomez, Carlos Perez-Plasencia, Hector Aquiles Maldonado-Martínez, Veronica Fragoso-Ontiveros, Federico Lasa-Gonsebatt, Luis Alonso Herrera, David Cantú, Enrique Bargallo-Rocha, Alejandro Mohar, Geoffroy Durand, Nathalie Forey, Catherine Voegele, Maxime Vallee, Florence Le Calvez-Kelm, James McKay, Maude Ardin, Stephanie Villar, Jiri Zavadil, Magali Olivier. Revealing the molecular portrait of triple negative breast tumors from an understudied population through omics analysis of formalin-fixed and paraffin-embedded tissues. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4748. doi:10.1158/1538-7445.AM2015-4748