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Evaluation of Clonal Origin of Malignant Mesothelioma


Evaluation of Clonal Origin of Malignant Mesothelioma

Sabahattin Comertpay et al. J Transl Med.


Background: 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.

Conclusions: 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.


Figure 1
Figure 1
Schematic diagram of the HUMARA assay. Maternal and paternal X chromosomes carry different numbers of CAG repeats at the Humara locus. HpaII methylation sensitive sites are located at the polyorphic CAG region. During embriogenesis, random X chromosome inactivation occurs in female individuals, resulting in methylation of either the paternal or maternal X chromosome in different cells. Therefore, a monoclonal cell population, derived from the division of a single ancestor cell, shares the same inactivated X chromosome, whereas a polyclonal population, derived from more than one ancestor cell, may contain cells with inactive maternal and paternal X chromosomes. HpaII digestion removes the unmethylated alleles, allowing amplification of the methylated HUMARA locus. Electrophoresis of the PCR products will indicate monoclonal or polyclonal cell populations, as a single band or two bands of different size, respectively. HpaII: denotes the methylation sensitive endonuclease sites; arrows: indicate primer annealing regions; cross bars: indicate the methylated chromosome.
Figure 2
Figure 2
HUMARA assay quality controls. HUMARA-PCR was performed on HpaII-digested (H+) and mock-digested DNA (H-) from a healthy male and analyzed by capillary gel electrophoresis, using the 3100 Genome Analyzer. Presence of a single PCR peak indicated complete DNA digestion by HpaII enzyme (A). Healthy female DNA sample (L-IV-II, upper panel) and DNA from a human monoclonal melanoma cell line (#1290, female, lower panel) were subjected to HUMARA assay. After HpaII digestion, gel (inserts) and capillary electrophoresis successfully detected a polyclonal pattern and a monoclonal pattern, respectively (B).
Figure 3
Figure 3
Sensitivity of the HUMARA assay by gel and capillary electrophoresis. To establish the sensitivity of the HUMARA assay for detection of minor alleles, different amounts of HpaII-digested (mono-allelic) and non-digested (representing bi-allelic DNA) #1290 DNA were mixed in different rations and subjected to PCR for detection of HUMARA locus. PCR products were resolved on a 3% agarose gel containing 0.5 ug/ml ethidium bromide and detected under UV light (A) or using the Applied Biosystems 3100 Genetic Analyzer (B). CTR denotes the no template control. 100, 75, 50, 25, 12.5, 6.25 and 0 indicate the percentage of bi-allelic DNA in the PCR reaction (A). Linear regression analysis calculated with Prism 6 software, shows comparison between input and detected allelic/biallelic ratios as calculated using Genescan software. (R2 > 0.98). The minor allele was reliably detectable when present at a fraction greater than or equal to 1/8 (12.5% of the input copies) (C).
Figure 4
Figure 4
X chromosome inactivation analysis by HUMARA assay shows a prevalent polyclonal pattern of Malignant Mesotheliomas. Gel electrophoresis (A). PCR products from mock digested (H-) and HpaII-digested (H+) DNA samples were separated on a 3% agarose gel and visualized under UV light, using ethidium bromide. Capillary-Electrophoresis (B, C). HUMARA PCR assay was performed using a 5FAM-labeled forward primer, and quantified by the Applied Biosystems 3100 Genetic Analyzer. Two major peaks denote the two allelic HUMARA loci PCR-amplified in HpaII-digested (H+) and mock-digested samples (H-). The allele intensities were measured as peak area of both alleles, which is proportional to the molar amount of DNA. Peak areas were calculated for each allele using Genescan software, as described in the Material and Methods. 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.
Figure 5
Figure 5
MMs originate as polyclonal tumors. Asbestos fibers travel through the airways to the lungs, and, from there, via the lymphatics, reach the pleura, exposing many mesothelial cells. The carcinogenic “field effect” of mineral fibers leads to several premalignant clones that give rise to these polyclonal malignancies.

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