6q12-22 is the second most commonly deleted genomic region in prostate cancer. Mapping studies have described a minimally deleted area at 6q15, containing MAP3K7/TAK1, which was recently shown to have tumor suppressive properties. To determine prevalence and clinical significance of MAP3K7 alterations in prostate cancer, a tissue microarray containing 4699 prostate cancer samples was analyzed by fluorescence in situ hybridization. Heterozygous MAP3K7 deletions were found in 18.48% of 2289 interpretable prostate cancers. MAP3K7 deletions were significantly associated with advanced tumor stage (Po0.0001), high Gleason grade (Po0.0001), lymph node metastasis (Po0.0108) and early biochemical recurrence (Po0.0001). MAP3K7 alterations were typically limited to the loss of one allele as homozygous deletions were virtually absent and sequencing analyses revealed no evidence for MAP3K7 mutations in 15 deleted and in 14 non-deleted cancers. There was a striking inverse association of MAP3K7 deletions and TMPRSS2:ERG fusion status with 26.7% 6q deletions in 1125 ERG-negative and 11.1% 6q deletions in 1198 ERG-positive cancers (Po0.0001). However, the strong prognostic role of 6q deletions was retained in both ERG-positive and ERG-negative cancers (Po0.0001 each). In summary, our study identifies MAP3K7 deletion as a prominent feature in ERG-negative prostate cancer with strong association to tumor aggressiveness. MAP3K7 alterations are typically limited to one allele of the gene. Together with the demonstrated tumor suppressive function in cell line experiments and lacking evidence for inactivation through hypermethylation, these results indicate MAP3K7 as a gene for which haploinsufficency is substantially tumorigenic. Modern Pathology (2013) 26, 975-983; doi:10.1038/modpathol.2012 published online 1 February 2013 Keywords: ERG; MAP3K7; prostate cancerIn prostate cancer a variety of chromosomal deletions occur frequently, whereas gains of chromosomal material and high-level amplification occur rarely in this tumor. 1,2 Deletions of 6q12-22 have been described to occur in 22-62% of prostate cancers. 1-5 6q12-22 deletions rank second in the

Prostate cancer is characterized by recurrent deletions that can considerably vary in size. We hypothesized that large deletions develop from small deletions and that this “deletion lengthening” might have a “per se” carcinogenic role through a combinatorial effect of multiple down regulated genes. In vitro knockdown of 37 genes located inside the 6q12-q22 deletion region identified 4 genes with additive tumor suppressive effects, further supporting a role of the deletion size for cancer aggressiveness. Employing fluorescence in-situ hybridization analysis on prostate cancer tissue microarrays, we determined the deletion size at 6q and 16q in more than 3,000 tumors. 16q and 6q deletion length was strongly linked to poor clinical outcome and this effect was even stronger if the length of both deletions was combined. To study deletion lengthening in cancer progression we eventually analyzed the entire cancers from 317 patients for 6q and 16q deletion length heterogeneity and found that the deletion expanded within 50-60% of 6q and 16q deleted cancers. Taken together, these data suggest continuous “deletion lengthening” as a key mechanism for prostate cancer progression leading to parallel down regulation of genes with tumor suppressive properties, some of which act cooperatively.

Deletions of chromosome 6q12-22 represent the second common deletion in prostate cancer. Although most of these deletions are large and span > 50 megabases, a minimal deleted region containing 3-4 megabases was described at 6q15. MAP3K7 (at 6q15) was suggested as a potential target gene in this area. Based on the hypothesis, that large 6q deletions compromising a multitude of genes may have a different impact on the biology of prostate cancer cells than small circumscribed 6q15 deletions, a tissue microarray (TMA) containing over 7000 prostate cancers was analyzed by fluorescence in situ hybridization (FISH). All cancers were analyzed with probes for 6q12, 6q14, 6q15 (MAP3K7), 6q16, and 6q22. 3888 tumors were interpretable for all 5 probes including 463 (10.6%) showing a 6q deletion. 6q deletions were tightly linked to a negative ERG status with 15.6% deletions in ERG negative and 5.8% deletions in ERG positive cancers (p<0.0001). The frequency of 6q deletions increased with Gleason grade and pT stage. 6q deletions were found in 5.4% of ≤3+3, 10.6% of 3+4, 19.6% of 4+3, and 18.8% of ≥4+4 cancers (p<0.0001). 6q deletions occurred in 9.6% of pT2, 11.5% of pT3a, and 14.8% of pT3b carcinomas (p=0.0160). 6q deletions were also linked to early PSA recurrence (p<0.0001). Remarkably, the clinical outcome varied markedly with deletion size. PSA recurrence was more likely in cancers with 6q12-22 deletions as compared to tumors with 6q14-16 deletions (p<0.0001). Twelve genes located within 6q14-16 were functionally evaluated applying colony formation assays to prostate cell lines BPH1, DU-145, and PC-3. These analyses failed to identify a cell behavior consistent with a role as a tumor suppressor gene for MAP3K7, as well as RRAGD, ANKRD6, LYRM2, MDN1, CASP8AP2, BACH2, COQ3, MANEA, RNGTT, SLC35A1, and MMS22L. In conclusion, the strong impact of the deletion size suggests, that multiple genes on chromosome 6q12-22 might have a tumor suppressive role in prostate cancer and that alterations of more than one of these has an additive adverse effect to cells. Our data do not confirm MAP3K7 as the main target of 6q deletions in prostate cancer Citation Format: Martina Kluth, Nina Amschler, Anna Heinl, Simon Jung, Ronald Simon, Thorsten Schlomm, Guido Sauter, Sarah Jane Pauline Minner. Clinical impact of 6q deletion patterns in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1972. doi:10.1158/1538-7445.AM2013-1972