DNA aneuploidy as a topographic malignant transformation pattern in a pleomorphic adenoma of long-term evolution: a case report
© Gallego et al; licensee BioMed Central Ltd. 2011
Received: 31 August 2011
Accepted: 4 November 2011
Published: 4 November 2011
We present a case of long-term evolution of a submandibular pleomorphic adenoma. There is little information about topographic malignant transformation patterns of pleomorphic adenomas.
We extensively analyze a giant submandibular mixed tumor of 25-year evolution in a 57-year-old Caucasian woman. Deoxyribonucleic acid ploidy was evaluated in different superficial and deep areas using flow cytometry analysis and correlated with pathological and immunohistochemical characteristics. Superficial areas exhibited a typical histological pleomorphic adenoma pattern and were deoxyribonucleic acid diploid. Deep samples showed deoxyribonucleic acid aneuploidy, atypical histological benign features and expression of markers involved at an early-stage of malignant transformation, such as tumor protein 53 and antigen Ki67.
These findings revealed that deep tumor compartments may be involved in the initial stages of malignant transformation. Deoxyribonucleic acid ploidy analysis may provide an additional diagnosis tool and indicate 'uncertain' areas that require careful study to avoid diagnostic errors. Larger studies are needed to confirm our results and to evaluate the usefulness of the technique.
Pleomorphic adenomas (PAs) are the most common benign tumors arising in salivary glands and their malignant transformation to carcinoma ex pleomorphic adenomas (CXPAs) accounts for between 4.5% and 15% of all cancers of these glands . The diagnosis of CXPA is based on the coexistence of epithelial malignancy with histologically benign PA . These tumors are typically considered as high-grade carcinomas, with frequent metastases and disease-related deaths .
Long-term evolution of a PA might increase the risk of malignant transformation . Although rare, cases of giant PA have been reported, most of them involving the parotid gland [5–7]. Misdiagnosis is not rare in these cases, because the CXPA component may be small and therefore missed on histological analysis. There are few series published in the literature focusing on ploidy analysis for the prediction of tumor aggressiveness and for differentiating benign from malignant salivary gland tumors [8–10]. However, a lack of correlation between cytometric parameters and histological or immunohistochemical parameters has not been described.
The objective of this case report was to analyze a giant PA tumor of 25-year evolution. Deoxyribonucleic acid (DNA) ploidy was evaluated in different areas using flow cytometry analysis, and correlated with pathological and immunohistochemical characteristics in order to assess whether ploidy analysis may improve the diagnostic accuracy for predicting malignancy.
Half of the samples in each group were fixed in 4% buffered formalin, processed and embedded in paraffin according to routine procedures, for histological and immunohistochemical analysis. The remainder of the samples of fresh material for each group were immediately submitted for DNA flow cytometry. The rest of the surgical specimen was routinely studied in the Department of Pathology and diagnosed as a benign PA.
Samples of each group were minced with a scalpel in phosphate-buffered saline solution. Single nuclear suspensions were prepared by filtering through a 50-μm nylon mesh. The DNA contents were measured in a Cytomics FC500 (Beckman Coulter Inc., Fullerton, CA, USA) flow cytometer. DNA histograms of at least 10, 000 nuclei were plotted. The DNA-diploid cell population corresponding to surrounding normal tissue from the same location was used as an internal reference standard for the identification of DNA-aneuploid clones. The percentages of the cell cycle phases as well as the DNA indices of the aneuploid clones were calculated using the Modfit 5.2 software package. DNA histograms were classified as diploid if there was a single G0-G1 peak and aneuploid if additional G0-G1 peaks were present. The ratio of aneuploid G0-G1 peak values to diploid G0-G1 peak values was expressed as a DNA index. All specimens had a G0-G1 peak coefficient of variation of no more than 4%. The following were taken as cytometric variables: DNA ploidy, DNA index, and S-phase fraction. The cases with DNA indices between 0.9 and 1.10 were considered as DNA diploids, and those less than 0.9 or greater than 1.10 as DNA aneuploids.
Half of the paraffin-embedded samples of all three groups were routinely stained with hematoxylin and eosin. The rest of the paraffin samples were submitted to the labeled-polymer method of immunohistochemistry using antibodies against α-smooth-muscle actin (α-SMA), cytokeratin (CK) AE1/AE3, CK 8, protein 53 (p53), protein 63 (p63) and antigen Ki67.
PA is the most common benign salivary gland tumor [1, 2]. It is derived from the epithelium and typically presents as a cytologically benign circumscribed mass with variable encapsulation. This tumor is one of the few benign neoplasms than can undergo malignant transformation. The propensity for malignant transformation has been documented in the literature at 1.9% to 23.3% and CXPA represents approximately 12% of malignant neoplasms [2, 11, 12]. Otherwise, it has been estimated that approximately 25% of untreated mixed tumors would eventually develop into carcinomas . The likelihood of a malignant change in a PA increases with the duration of the tumor and with the age of the patient. The diagnosis of CXPA requires the presence of either a recognizable mixed tumor in association with a carcinoma or carcinoma developing as a recurrent neoplasm at the site of a previous mixed tumor. Criteria for malignancy include invasiveness, cellular anaplasia or pleomorphism, atypical mitosis and abnormal architectural patterns [1, 3].
It is currently postulated that malignant transformation is accompanied by genomic instability (cytogenetic and/or cytometric aneuploidy) . DNA ploidy analysis has proven to be a useful prognostic indicator in a variety of salivary gland neoplasms. Abnormal DNA content has been related to aggressive behavior in adenoid cystic carcinomas, acinic cell carcinomas, mucoepidermoid carcinomas and oncocytomas [8, 9]. Otherwise, most likely due to the low incidence of malignant salivary gland tumors, only a few attempts have been made to employ DNA ploidy analysis for diagnostic assessment [10, 15]. One purpose of the present study was to determine whether DNA ploidy analysis is a good diagnostic tool to distinguish malignant areas in PAs even before histopathological correlation.
Some authors reported that DNA diploidy may be seen in both benign and malignant lesions, but aneuploidy is mainly seen in malignant lesions. In the majority of previous studies, the PAs revealed a diploid pattern [8, 9, 15]. However, Martin et al. examined a series of 16 mixed tumors and found DNA aneuploidy in four cases, three of which were recurrent lesions . In our tumor, deep samples (Group 3) demonstrated aneuploidy correlated with atypical histological and immunohistochemical features. This central area exhibited a hypercellular pattern, with a predominantly epithelial pattern and a lack or poor myoepithelial cells and stroma. Immunohistochemistry for CKs AE1/AE3 and CK 8 confirmed the glandular (luminal) differentiation of the epithelial portion of this tumor. Otherwise, p63 antibody and α-SMA have been shown to be reliable myoepithelial and basal cell markers, and presented minor staining in the deep area. PA typical areas, the Group 1 and 2 samples, characteristically show a variable amount of myxochondroid stroma produced by myoepithelial cells. Chau and Radden studied 53 cases of intraoral PA, and reported that stroma-poor PAs were larger than stroma-rich ones, and suggested that cellular tumors may grow at a faster rate . Other studies described that, although the myoepithelial component predominates in most PAs, the genetic changes leading to malignant transformation occur more frequently in ductal luminal cells than in myoepithelial cells [18, 19].
In addition, p53 and Ki67 presented higher expression in deep compared to superficial areas. It has been suggested that the index of p53 and Ki67 accumulation could be a useful biomarker for detecting tumors at an early phase of malignant transformation and distinguish PA from CXPA areas . Therefore, Group 3 samples presented a hypercellular benign pattern, without dysplasia or obvious malignant change, but also showed DNA aneuploidy and expressed markers involved at an early stage of malignant transformation. It is tempting to postulate a future progression to CXPA in this area.
There is little information in the literature regarding topographic malignant transformation patterns of PA. This study revealed that deep tumor compartments may be involved in the initial stages of malignant transformation. The authors of the present study suggest that deep areas in long-term evolution PAs should be carefully assessed by serial sectioning to document evidence of malignancy. In conclusion, DNA ploidy analysis may provide an additional diagnosis tool for 'uncertain' areas. The prognosis and future therapy will depend on careful study of these lesions. Obviously, more cases are needed to evaluate these results and better understand this entity.
Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.
coefficient of variance
carcinoma ex pleomorphic adenoma
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