Elsevier

The Lancet Oncology

Volume 17, Issue 10, October 2016, Pages 1386-1395
The Lancet Oncology

Articles
Epigenetic profiling to classify cancer of unknown primary: a multicentre, retrospective analysis

https://doi.org/10.1016/S1470-2045(16)30297-2Get rights and content

Summary

Background

Cancer of unknown primary ranks in the top ten cancer presentations and has an extremely poor prognosis. Identification of the primary tumour and development of a tailored site-specific therapy could improve the survival of these patients. We examined the feasability of using DNA methylation profiles to determine the occult original cancer in cases of cancer of unknown primary.

Methods

We established a classifier of cancer type based on the microarray DNA methylation signatures (EPICUP) in a training set of 2790 tumour samples of known origin representing 38 tumour types and including 85 metastases. To validate the classifier, we used an independent set of 7691 known tumour samples from the same tumour types that included 534 metastases. We applied the developed diagnostic test to predict the tumour type of 216 well-characterised cases of cancer of unknown primary. We validated the accuracy of the predictions from the EPICUP assay using autopsy examination, follow-up for subsequent clinical detection of the primary sites months after the initial presentation, light microscopy, and comprehensive immunohistochemistry profiling.

Findings

The tumour type classifier based on the DNA methylation profiles showed a 99·6% specificity (95% CI 99·5–99·7), 97·7% sensitivity (96·1–99·2), 88·6% positive predictive value (85·8–91·3), and 99·9% negative predictive value (99·9–100·0) in the validation set of 7691 tumours. DNA methylation profiling predicted a primary cancer of origin in 188 (87%) of 216 patients with cancer with unknown primary. Patients with EPICUP diagnoses who received a tumour type-specific therapy showed improved overall survival compared with that in patients who received empiric therapy (hazard ratio [HR] 3·24, p=0·0051 [95% CI 1·42–7·38]; log-rank p=0·0029).

Interpretation

We show that the development of a DNA methylation based assay can significantly improve diagnoses of cancer of unknown primary and guide more precise therapies associated with better outcomes. Epigenetic profiling could be a useful approach to unmask the original primary tumour site of cancer of unknown primary cases and a step towards the improvement of the clinical management of these patients.

Funding

European Research Council (ERC), Cellex Foundation, the Institute of Health Carlos III (ISCIII), Cancer Australia, Victorian Cancer Agency, Samuel Waxman Cancer Research Foundation, the Health and Science Departments of the Generalitat de Catalunya, and Ferrer.

Introduction

Cancer of unknown primary accounts for about 3–9% of all cancer diagnoses,1 and in the USA alone, more than 80 000 patients receive a diagnosis of cancer of unknown primary every year.2 With a median age at presentation of 60 years, cancers of unknown primary are the fourth most common cause of cancer-related deaths worldwide.3 Cancers of unknown primary are a molecularly heterogeneous group of cancers, in which there has not yet been elucidation of the biological mechanisms that allow the primary site to remain obscure after metastasis. No common molecular signature has been identified that produces this particular clinical phenotype, and cancers of unknown primary present a wide variety of mutations and genomic alterations.4 From a clinical standpoint, the prognosis for patients with cancer of unknown primary is poor: patients attain a median survival of 9 months (95% CI 8·3–10·0)5 after diagnosis and only 25% survive for 1 year or more.6 For most patients with cancer of unknown primary, recommended treatments involve empirical chemotherapy—defined as the chemotherapy that the oncologist think will work best based on their experience treating other people with similar characteristics—usually with a taxane plus platinum, or gemcitabine plus a platinum regimen,7, 8, 9 which produce the described modest clinical benefit. However, accurate identification of the primary tumour type and subsequent treatment with site-specific therapy could result in improved survival.10, 11, 12, 13

If the initial assessment of a patient with a cancer of unknown primary, which usually involves CT scanning and specific signs or symptoms, is uninformative, the first attempt to identify a tissue of origin relies on pathological assessment, including an immunohistochemical examination. Several immunohistochemical panels have been developed for the diagnoses of cancer of unknown primary, but even after the full diagnostic work-up, the primary site of a cancer of unknown primary remains unknown in about 75% of patients.1 A post-mortem examination is done in only a few patients with cancer of unknown primary, in which a complete autopsy only reveals 55–85% of the primaries.6 Our increasing understanding of cancer biology has prompted the search for molecular markers that, being present in the cancer of unknown primary, might retain the signature of the putative primary origin. In this regard, the use of expression microarray-based classifiers has achieved a prediction accuracy of the primary site in about 75% of patients.1, 14, 15 However, the limitations in the numbers, types, and subtypes of tumours included in these assays, in addition to the required amount and state of preservation of the studied biological material, and the cost of the procedure, warrant further development of complementary diagnostic instruments for cancer of unknown primary. In this regard, we examined DNA methylation,16, 17, 18 a stable marker of DNA that has already been clinically successful in the pharmacogenetic management of gliomas19, 20 and has different profiles among distinct tumour types.21 Herein, we attempt to diagnose the primary tumour site for all cancers of unknown primary, and we have devised a new strategy based on the DNA methylation profiles of the metastasis sample.

Research in context

Evidence before this study

This study was initiated based on our preclinical data showing that DNA methylation patterns are tumour type specific, a finding that could be helpful in the identification of the site of origin of cancer of unknown primary. Additionaly, we searched PubMed on April 8, 2016, unrestricted by language or date limits, to identify scientific literature focused on the diagnosis and therapies of cancer of unknown primary using the search term “cancer of unknown primary”. We also searched abstracts from the American Society of Clinical Oncology and the European Society for Medical Oncology. We found no studies that examined the use of epigenetic profiling to improve the clinical management of cancer of unknown primary.

Added value of this study

Our findings show that a classifier of cancer type based on microarray DNA methylation signatures shows a high specificity, sensitivity, positive predictive value, and negative predictive value for the prediction of the original primary tumour site. We validated the accuracy of the test using autopsy examination, subsequent clinical detection of the primary site, light microscopy, and comprehensive immunohistochemistry profiling. Additionally, our results suggest that patients with cancer of unknown primary who received a tumour type-specific therapy showed improved overall survival compared with that in patients who received empiric therapy. The test also suggested the presence of actionable targets such as HER2 and C-MET amplification and EGFR mutation.

Implications of all the available evidence

The results of this study could change the diagnosis of patients with cancer of unknown primary where the approaches routinely used to determine the tissue of origin provide conclusive results in only 25% of cases. Our data support the use of epigenetic profiling to significantly improve cancer of unknown primary diagnoses and guide more precise therapies associated with better outcomes.

Section snippets

Patients and samples

Between March 2, 2011, and Dec 2, 2015, samples for the training (n=692) and validation (n=1948) sets were obtained from the Cancer Epigenetics and Biology Program (PEBC) of the Bellvitge Biomedical Research Institute (IDIBELL; Barcelona, Catalonia, Spain). The histopathology findings and the clinical data from the PEBC samples were obtained from the authors' institutions, according to the protocol approved by the Bellvitge University Hospital Clinical Investigation Ethics Committee (PR133/14).

Results

To obtain the basal DNA methylation landscapes associated with 38 tumour types, we analysed 10 481 tumour samples of known origin: 2790 in the training set and 7691 in the validation set (table 1). Each tumour type had at least 25 cases adding training plus validation set. We studied 692 tumour samples of known origin from the PEBC cohort to establish a reference dataset of DNA methylation profiles associated with 38 tumour types that included those typically associated with cancer of unknown

Discussion

Our study shows that the use of DNA methylation profiling provides a consistent diagnosis of the primary tumour site in cases of cancer of unknown primary. Furthermore, our results support that the determination of the original tumour type followed by site-specific therapies improves the outcome of these patients compared with those treated empirically, and, in this regard, addresses an unmet need in this area, because only 25% of cases of unknown primary cancer receive a single putative

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