Evaluation of clonal origin of malignant mesothelioma
- Sabahattin Comertpay1,
- Sandra Pastorino1,
- Mika Tanji1,
- Rosanna Mezzapelle2,
- Oriana Strianese1,
- Andrea Napolitano1, 3,
- Francine Baumann1,
- Tracey Weigel4,
- Joseph Friedberg5,
- Paul Sugarbaker6,
- Thomas Krausz7,
- Ena Wang8,
- Amy Powers1,
- Giovanni Gaudino1,
- Shreya Kanodia9,
- Harvey I Pass10,
- Barbara L Parsons11,
- Haining Yang1 and
- Michele Carbone1, 12Email author
© Comertpay et al.; licensee BioMed Central Ltd. 2014
Received: 11 September 2014
Accepted: 16 October 2014
Published: 4 December 2014
The hypothesis that most cancers are of monoclonal origin is often accepted as a fact in the scientific community. This dogma arose decades ago, primarily from the study of hematopoietic malignancies and sarcomas, which originate as monoclonal tumors. The possible clonal origin of malignant mesothelioma (MM) has not been investigated. Asbestos inhalation induces a chronic inflammatory response at sites of fiber deposition that may lead to malignant transformation after 30-50 years latency. As many mesothelial cells are simultaneously exposed to asbestos fibers and to asbestos-induced inflammation, it may be possible that more than one cell undergoes malignant transformation during the process that gives rise to MM, and result in a polyclonal malignancy.
Methods and results
To investigate the clonality patterns of MM, we used the HUMARA (Human Androgen Receptor) assay to examine 16 biopsies from 14 women MM patients. Out of 16 samples, one was non-informative due to skewed Lyonization in its normal adjacent tissue. Fourteen out of the 15 informative samples revealed two electrophoretically distinct methylated HUMARA alleles, the Corrected Allele Ratio (CR) calculated on the allele peak areas indicating polyclonal origin MM.
Our results show that MM originate as polyclonal tumors and suggest that the carcinogenic “field effect” of mineral fibers leads to several premalignant clones that give rise to these polyclonal malignancies.
KeywordsMalignant mesothelioma Clonal origin HUMARA assay Carcinogenesis Polyclonal tumors
Malignant mesothelioma (MM) is an aggressive cancer arising from the transformation of the mesothelial lining of the pleura, peritoneum and pericardium. It is a lethal cancer affecting approximately 3,200 individuals each year in the US, most of them die within 1 year from diagnosis . MM incidence has remained stable in the US since 2003, but it continues to increase worldwide, due to exposure to asbestos fibers, which are widely used for industrial purposes . Occupational exposure accounts for a male to female MM incidence ratio of 6-8 to 1, as males are much more often involved in the asbestos industrial trades . Asbestos, erionite and other mineral fibers are also naturally present in developing rural areas where they pose a major risk factor for MM ,. Asbestos inhalation induces a chronic inflammatory response at sites of fibers deposition that may lead to malignant mesothelial cell transformation after a latency of 30- to 50-years . In addition to the environmental and inflammatory components of MM etiology, we have recently discovered that germline mutations of the BAP1 gene cause a novel cancer syndrome characterized by a very high incidence of MM, and other malignancies .
MMs show different histology. There are 3 main subtypes: about 50% of MM show an epithelioid morphology and they look like carcinomas, 10% have a spindle cell morphology similar to sarcomas, and about 35% are biphasic, composed of both epithelioid and spindle cells in different proportions. In addition there are less common histological variants . Diagnosis is further complicated by the presence of intra-tumoral pleomorphisms and phenotypic heterogeneity, raising the question of whether MM results from genetic and epigenetic alterations, which drive clonal tumor evolution into these different morphologies, or it arises from different subsets of mesothelial cells that interact or cooperate to drive malignant progression.
Multistep carcinogenesis is the currently accepted hypothesis to explain genetic diversity in tumors -. This hypothesis is based on the notion that somatic mutations are rare events that are unlikely to co-occur in different single normal cells, and that the mutations leading to the genetic diversification of the tumor architecture occur during the process of clonal expansion and selection. A cancer is considered monoclonal when all cells within the tumor can be traced back to a single progenitor/initiator cell. Instead, a polyclonal malignancy derives from the concomitant transformation of two or more different ancestor cells. Determining the clonal status of a cancer can be quite challenging, because of the inherent plasticity of the cancer genome that can acquire many somatic mutations during malignant cell growth. Analysis of the X chromosome inactivation pattern in female cancer biopsies, by measuring the methylation status of the polymorphic human androgen receptor (HUMARA) locus, is considered the most accurate method to assess clonality . During early female embryogenesis, one of the two X chromosomes is randomly inactivated and the pattern of X-chromosome inactivation is stably transmitted from parent cell to the progeny (Lyonization) . Therefore, the presence of the same inactivated X chromosome in all cancer cells has been interpreted as an indication of monoclonality. Although the current dogma is that cancers originate as monoclonal growths, some studies suggest that some cancers may arise as polyclonal ,.
To the best of our knowledge, the clonal origin of MM has never been investigated. However MMs are “assumed” to be monoclonal as most tumors are usually assumed to be. Here we tested the hypothesis that MMs are monoclonal using the HUMARA assay on 16 MM biopsies from 14 female patients. We found that human MMs are polyclonal in origin.
Clinical specimens and study approval
Human MM biopsies were collected at the following institutions: Department of Cardiothoracic Surgery, New York University, New York, NY; MedStar Washington Hospital Center, Washington, DC; University of Wisconsin School of Medicine and Public Health Department of Surgery, Madison, WI, and at the Department of Surgery, Penn Presbyterian Medical Center, Philadelphia, PA.
Clinical features and clonality pattern in 16 biopsies from 14 female MM patients
Laser tissue microdissection and DNA extraction
HUMARA clonality assessment ,, was conducted on stage I to III MM biopsies (Table 1). A limitation of this test is that, it can be used only on female specimens. Although MM is rare in women, we were able to collect and study clonality in 16 MM biopsies from 14 women that were treated by some of the co-authors. In 2 out of 14 cases, two distinct nodules from the same pleura were available for comparison. Tumors and adjacent normal tissues were dissected by Laser Capture Microdissection, using MMI CellCut Plus (Molecular Machines & Industries, MI, USA). Hematoxylin and Eosin (H&E) staining was used to help identify tissue purity. Microdissection was performed on serial sections stained with Hematoxilin (i.e., cut next to the one stained with H/E) on tumor areas containing less than 5% of infiltrating inflammatory cells and in areas containing only normal tissue (control) (Additional file 1: Figure S1).
HUMARA assay was performed on DNA extracted from microdissected tissues. DNA was extracted from microdissected tissue using the QiAamp DNA Micro Kit DNA Extraction Protocol (Qiagen). DNAs were then digested with HpaII enzyme as previously described : briefly, 100 ng of either tumor or normal DNA were digested with 10 U Hpa II restriction enzyme (New England Biolabs, Ipswich, MA, USA) at 37°C overnight in a 20 μl reaction volume. Separate aliquots of DNA were subjected to mock digestion without the enzyme. After incubation, the restriction enzyme was inactivated at 80°C for 20 min. HpaII-digested or mock-digested DNA was then subjected to PCR reaction, using the following primers: 5 FAM-labeled forward primer, 5’ACC GAG GAG CTT TCC AGA AT3’; reverse primer, 5’TGG GGA GAA CCA TCC TCA C3’. Thermal cycling conditions included the following steps: denaturation at 95°C for 10 minutes; 30 cycles at 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds; and a final extension at 72°C for 10 minutes. Products of PCR amplification were analyzed by gel and capillary electrophoresis. Gel electrophoresis was performed on 3% agarose gel containing ethidium bromide (10ug/ml), and resolved DNA bands were visualized on a UV transilluminator (Biorad). For capillary electrophoresis, PCR products were mixed with 95% formamide and loading buffer (5% blue dextran, 25 mM EDTA) containing Rox-500. The mixture was then loaded on a 5% Long Ranger–6 M urea gel in TBE buffer. Electrophoresis was performed at 200 W for 2.25 hours, and the data were analyzed by an on a ABI 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA) and quantified by Genescan 3.1 software (Applied Biosystems). Mock-digested samples were used to monitor possible false positive results and to correct allele ratios. All samples were analyzed in triplicate. The ratio SD/mean CR was <2% for all replicates indicating that results were 100% reproducible and reliable.
For each sample, the allele intensities were measured as the peak areas of both alleles, which are proportional to the molar amount of DNA. The allele ratios (AR) were first calculated by dividing the ratio (RU = A1U/A2U) of the non-HpaII digested sample by the ratio (RD = A1D/A2D). The AR calculation (AR = RU/RD) corrects for any preferential amplification of one allele that might occur if the alleles are different in length. The clonality ratio (CR) is then calculated by dividing the AR of the tumor DNA by the AR calculated for the normal tissue (CR = ART÷ARN). This final calculation corrects for a potential skewed Lyonization ,. A CR ≥3.0 or ≤0.33, representing a preferential loss of intensity in the digested sample of one of the two alleles present in the tumor sample, was scored as a monoclonal pattern ,.
The Wald method was used to calculate the confidence interval for the observed proportion of polyclonality in our sample population.
Results and discussion
Our data indicate that MM arise as polyclonal tumors, a finding that has both pathogenesis and clinical implications. For example, MM patients whose tumors are removed at Stage Ia, most often experience recurrence after surgery in spite of apparent successful MM eradication .
Our data suggest that, in contrast to current dogma, recurrence may represent novel malignancies, occurring because of the carcinogenic “field effect” of asbestos, its related chronic inflammation, and/or because of ubiquitous genetic predisposition in patients carrying germline BAP1 mutations. Therefore, MM may arise from a large pool of independent and mostly covert cancers, as observed in some other malignancies . Accordingly, the multiple minuscule pleural nodules that are characteristically found on the pleura of early-stage MM patients are likely pre-malignant lesions or early tumors rather than early local metastases. Our findings underscore the need to attack simultaneously several different molecular targets to try to eliminate the different MM cell clones, as each clone may carry its own distinct set of molecular alterations.
- HUMARA :
Human androgen receptor
Hematoxylin and Eosin
We thank Dr. Patricia Ward for advice, Ms. Kimberly Theos and Dr. Masaki Nasu for technical support. This work was supported by National Institute of Health [grant numbers R01CA106567, P01CA114047, P30CA071789 to MC and R01CA160715-0A to HY]; the DoD CDMRP PRCRP Career Development Award to HY, and the V Foundation to MC and HY, the P30 CA071789 (UHCC Pathology Shared Resource); the Mesothelioma Applied Research Foundation to HY, the United-4 A Cure, the Hawai’i Community Foundation to HY, and the University of Hawai’i Foundation, which received donations to support mesothelioma research from Honeywell International Inc., to MC. The contents of this manuscript do not necessarily reflect the views or polices of the U.S. FDA, nor does the mention of trade names or commercial products constitute endorsement or recommendation for use.
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