We have found that cellular patterns of histone modifications provide significant and independent prognostic information in two large cohorts of patients with pancreatic adenocarcinoma. Similar to published findings with H3K27me3,16
we found a significant association between reduced cellular levels of H3K4me2, H3K9me2, or H3K18ac and worse prognosis in pancreatic adenocarcinoma. Our work here and other studies12,13
highlight the potential widespread applicability of cellular histone modification levels as cancer prognostic markers that could be incorporated into a standardized immunohistochemical assay. Such an assay will need to be additionally developed with carefully calibrated internal tissue and total histone antibody controls, as well as with absolute percent cell cutoffs for each tumor type, to be used reliably in the clinical laboratory setting.
Survival varies widely for patients with pancreatic adenocarcinoma, even within subsets of patients stratified by clinicopathologic criteria, such as tumor grade, stage, or lymph node status. In both of our TMA data sets, histone levels were prognostic for the subset of patients with node-negative pancreatic cancer. This is consistent with previous reports, in which the prognostic value of histone modifications were largely confined to less-aggressive or early-stage cancers, including lower Gleason score prostate cancer,13
lower-stage lung cancer,12
and localized kidney cancer.12
Thus, cellular histone levels appear best suited as biomarkers when used in conjunction with routine clinicopathologic staging information and, perhaps specifically, in the context of early stage pancreatic cancer.
Genome-wide profiling studies that compare normal versus cancer cells indicate a dynamic interplay between active or repressive histone marks and altered gene expression.20
Although changes in a histone modification at a particular genetic locus may predictably alter gene expression, the consequences of global changes in the levels of multiple histone modifications is more difficult to forecast, given their potential opposing functional effects on transcriptional activity and our incomplete understanding of their distribution across the cancer genome. Although decreased levels of nearly all histone modifications studied thus far have been linked to worse prognosis, these same histone modifications are variably associated with transcriptional activation (eg, H3K4me2 and H3K18ac) or transcriptional silencing (eg, H3K27me3 or H3K9me2).5
One possible explanation is that, similar to global DNA hypomethylation in cancer,21
bulk reductions in histone modifications may lead to genomic instability. In support of this hypothesis, prostate cancer cell lines with large differences in H3K9me2 levels have been shown to alter the distribution H3K9me2 almost exclusively at repetitive DNA elements and not at gene promoters.12
Likewise, global losses of H4K16ac and H4K20me3 in cancer cells have been shown to occur primarily at repetitive elements in combination with DNA hypomethylation.22
Experimentally, reduction in H3K9me2 levels by knockdown of the histone methyltransferase G9a has been shown to induce chromosomal instability, whereas it has little impact on gene expression in cancer cell lines.23
Additional studies are needed to determine the global distribution of histone modifications and the underlying reasons for their reduced levels in subsets of more clinically aggressive pancreatic cancer; studies that establish the direct effects of altered histone modification levels on genomic instability and gene transcription are needed as well.
The present approach for adjuvant chemotherapy in resected pancreatic cancer primarily involves the choice between gemcitabine versus fluorouracil, and the evolving consensus is that gemcitabine provides improved survival benefit.24
However, RTOG 9704 concluded that adjuvant gemcitabine provided only a nonstatistically significant survival benefit compared with adjuvant fluorouracil in the setting of fluorouracil-based chemoradiotherapy.18
The ESPAC-3 multicenter, randomized, controlled phase III trial of resected pancreatic ductal adenocarcinoma also recently reported no survival difference between adjuvant fluorouracil/folinic acid versus gemcitabine.25
These studies highlight the need for predictive biomarkers better able to inform treatment decisions. Toward this end, accumulating data suggest the levels of one or more mediators of drug transport or metabolism may be useful in predicting response to gemcitabine26–29
chemotherapy in cancer. Our data here indicate cellular histone modification levels are an additional class of biomarkers that could help to predict response to fluorouracil. In keeping with our observation that low H3K18ac is associated with worse response to fluorouracil, certain histone deacetylase inhibitors (which will act to increase global levels of H3K18ac) have been shown to act in synergy with fluorouracil to increase its cytotoxic and growth inhibitory effects in cancer cell lines.31,32
This appears to be caused at least in part by reduction in the levels of thymidylate synthase,31,33
which has been associated with resistance to fluorouracil chemotherapy. By extension, cellular levels of H3K18ac may help identify patients who are more likely to benefit from the addition of an histone deacetylase inhibitor to fluorouracil chemotherapy.
We have shown that histone modification levels indicate patients in RTOG 9704 who were more or less likely to derive survival benefit from adjuvant fluorouracil relative to gemcitabine. Although differences were modest and require validation, they raise the possibility that histone modification levels could serve as predictive biomarkers for adjuvant fluorouracil in pancreatic cancer, perhaps in combination with other fluorouracil predictive markers, such as thymidylate synthase or dihydropyrimidine dehydrogenase expression.30
These results also merit the investigation of histone modification levels as predictive biomarkers in other cancers (ie, colorectal or breast) treated with fluorouracil. In conclusion, cellular histone modification levels define previously unrecognized subsets of patients with pancreatic adenocarcinoma with distinct epigenetic phenotypes and clinical outcomes, with potential clinical value for prognosis or in predicting response to fluorouracil or histone-modifying agents.