October 2008

Editor:
Michael Cibull, MD
Melissa Kesler, MD

Using biomarkers to distinguish high-grade SIL from its mimics
Nm23 as a prognostic biomarker in ovarian serous carcinoma
Evidence-based pathology and the pathologic evaluation of thymomas
Genetic abnormalities in the normal-appearing skin of women with breast cancer
Link between expression of Smad2 and Smad4 and cervical cancer
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Using biomarkers to distinguish high-grade SIL from its mimics

Topoisomerase II-α and minichromosome maintenance protein 2 are associated with aberrant S-phase induction. The authors evaluated the performance of these biomarkers (ProEx C, TriPath Oncology) against p16INK4A and MiB-1 for distinguishing high-grade squamous intraepithelial lesions (HSILs) from HSIL mimics. They collected archival cervical biopsy, cone, and curettage specimens from 96 cases in which the differential diagnosis of HSIL versus reactive epithelial changes was considered. Three pathologists independently reviewed hematoxylin-and-eosin-stained slides and scored them for the presence or absence of SIL. Immunostains for ProEx C, p16, and MiB-1 were available for 95, 96, and 59 samples, respectively, and classified blinded to histological interpretation. Strong nuclear and cytoplasmic staining for p16 and staining for MiB-1 and ProEx C that extended beyond the lower one-third of the epithelium were scored as positive. Receiver operating characteristic analysis and Χ² tests were conducted to statistically compare biomarker immunostaining performance against majority histological interpretation of SIL. Agreement between pathologists was also assessed by the κ-statistic. Inter-observer agreement ranged from fair to moderate (κ=0.37 to 0.57). All three biomarkers correlated strongly with the majority diagnosis of SIL (P<.001). Positive staining for ProEx C, p16, and MiB-1 was observed in 87 percent (n=52 of 60), 84 percent (n=51 of 61), and 94 percent (n=34 of 36) of SIL, respectively. Staining was negative in 71 percent (n=25 of 35), 63 percent (n=22 of 35), and 52 percent (n=12 of 23) of majority diagnoses of NoSIL, respectively. The combination of p16/ProEx C predicted more SIL (92 percent; n=33 of 36) and NoSIL (61 percent; n=14 of 23) than p16 plus MiB-1 (94 percent, n=34 of 36 and 43 percent, n=10 of 23), although this difference was not statistically significant. The authors concluded that ProEx C appears to provide an equivalent level of sensitivity and a higher level of specificity for HSIL alone or in conjunction with p16. Its principal value may be in providing a lower false-positive rate for NoSIL relative to MiB-1.

Pinto AP, Schlecht NF, Woo TY, et al. Biomarker (ProEx C, p16INK4A, and MiB-1) distinction of high-grade squamous intraepithelial lesion from its mimics. Mod Pathol. 2008;21:1067–1074.

Correspondence: Dr. E. S. Cibas at ecibas@partners.org
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Nm23 as a prognostic biomarker in ovarian serous carcinoma

Studies have indicated that expression of the nm23 gene, a reported metastasis suppressor gene, has tissue specificity. However, the role of nm23 in ovarian cancer is still controversial. The authors examined the prognostic significance of nm23 expression in patients with serous ovarian carcinoma. Following comparative proteomics in 13 fresh-frozen ovarian serous cancer tissues with other histological types of ovarian cancers, validation was performed using immunohistochemistry on 73 clinically well-designed ovarian serous carcinoma microarray samples taken from ovarian cancer patients between 1990 and 2003. Statistical analysis of the results was performed using the Χ² test, Cox proportional regression, Kaplan-Meier method, and log-rank test. The authors found that the expression of nm23 inversely correlated with peritoneal seeding (P=.009). However, strong nm23 expression was associated with mortality in patients with ovarian carcinoma in univariate analysis (P=.04). Poor prognostic factors for disease-free survival included tumor residue of more than 2 cm (P=.02), bilaterality (P=.01), and peritoneal seeding (P<.01), whereas poor prognostic factors affecting overall survival included peritoneal seeding (P=.05). In Kaplan-Meier analysis, strong nm23 immunoreactivity correlated with poor overall survival (P=.04) but not with poor disease-free survival. The authors concluded that overexpression of nm23 is independently associated with decreased overall survival in patients with ovarian carcinoma and correlates significantly with mortality. Nm23 may have a biological function that leads to poor clinical outcomes in ovarian carcinoma.

Youn BS, Kim DS, Kim JW, et al. Nm23 as a prognostic biomarker in ovarian serous carcinoma. Mod Pathol. 2008;21:885–892.

Correspondence: N. H. Cho at cho1988@yumc.yonsei.ac.kr
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Evidence-based pathology and the pathologic evaluation of thymomas

The clinical validity and applicability of the World Health Organization’s histopathologic classification of thymomas has been questioned. Evidence-based pathology promotes the use of systematic reviews and the study of data using meta-analysis rather than subjective reviews of the literature. The authors analyzed the English literature from 1999 to the present to identify “best evidence” regarding the use of the WHO classification. The data were scrutinized using meta-analysis software. To the authors’ knowledge, only level three or four evidence published in retrospective case series is available for the WHO classification. Meta-analysis demonstrated that only three WHO categories of thymomas are associated with significant survival differences: A/AB/B1, B2, and B3. It also indicated significant heterogeneity with regard to results published in other studies. To the authors’ knowledge, there is no current evidence to determine whether thymoma types are significant prognostic features for patients previously stratified by stage. The authors concluded that there is a lack of randomized clinical trials evaluating the prognosis for patients with thymomas and the effects of various treatment modalities. The WHO classification of thymomas needs to be revised and could most likely be simplified into fewer classes with significant prognostic value. Additional studies are needed to evaluate the prognostic or predictive value, or both, for thymoma patients previously stratified by stage. The latter information is important to help select patients who may benefit from neoadjuvant chemotherapy or postoperative radiotherapy and other modalities.

Marchevsky AM, Gupta R, McKenna RJ, et al. Evidence-based pathology and the pathologic evaluation of thymomas: the World Health Organization classification can be simplified into only 3 categories other than thymic carcinoma. Cancer. 2008;112:2780–2788.

Correspondence: Dr. Alberto M. Marchevsky at alberto.marchevsky@cshs.org
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Genetic abnormalities in the normal-appearing skin of women with breast cancer

Genetic abnormalities in microenvironmental tissues with subsequent alterations of reciprocal interactions between epithelial and mesenchymal cells play a role in breast carcinogenesis. Although a few reports have demonstrated abnormal fibroblastic functions in normal-appearing fibroblasts taken from the skin of breast cancer patients, the genetic basis of this phenomenon and its implication for carcinogenesis are unexplored. The authors analyzed 12 mastectomy specimens showing invasive ductal carcinomas. In each case, morphologically normal epidermis and dermis, carcinoma, normal stroma close to carcinoma, and stroma at a distance from carcinoma were microdissected. Metastatic-free lymphatic tissues from lymph nodes served as a control. Using polymerase chain reaction, DNA extracts were examined with 11 microsatellite markers known for a high frequency of allelic imbalances in breast cancer. Losses of heterozygosity or microsatellite instability, or both, were detected in 83 percent of the skin samples occurring concurrently with or independently from the cancerous tissues. In 80 percent of these cases, at least one microsatellite marker displayed loss of heterozygosity or microsatellite instability in the skin, which was absent in carcinoma. Forty-one percent of samples showed alterations of certain loci observed exclusively in the carcinoma but not in the skin compartments. This study suggests that breast cancer is not just a localized genetic disorder, but rather part of a larger field of genetic alterations or instabilities affecting multiple cell populations in the organ with various cellular elements, ultimately contributing to the manifestation of more localized carcinoma. The data indicate that additional global assessment of tumor microenvironment and macroenvironment is crucial to furthering understanding of breast carcinogenesis.

Moinfar F, Beham A, Friedrich G, et al. Macro-environment of breast carcinoma: frequent genetic alterations in the normal appearing skins of patients with breast cancer. Mod Pathol. 2008;21:639–646.

Correspondence: Dr. F. Moinfar at farid.moinfar@meduni-graz.at
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Link between expression of Smad2 and Smad4 and cervical cancer

Alterations in transforming growth factor-β signaling, due to a decrease in Smad2 and Smad4 expression, primarily have been reported in pancreatic and colorectal cancers, although loss of the chromosomal region 18q21.1, containing the loci of Smad2 and Smad4, is among the most frequent molecular alterations in cervical cancer. The authors conducted a study to investigate whether decreased Smad2 and Smad4 protein expression in primary cervical cancers is associated with molecular alterations at 18q21.1, mutations in the functional domains of Smad2 and Smad4, or hypermethylation, and to assess the biological relevance of decreased Smad2 and Smad4 expression. Subsequently, Smad2, Smad4, and p21 protein expression were determined by immunohistochemistry in 117 primary cervical carcinomas assembled in a tissue array. Smad signaling was shown to be associated with p21 mRNA expression. All of the tumors studied expressed Smad2 or Smad4. Weak cytoplasmic Smad2 or weak cytoplasmic Smad4 expression could not be attributed to loss of heterozygosity at 18q21.1. Despite weak/moderate Smad2 expression and absent nuclear Smad4 expression, the coding regions of the functional MH1 and MH2 domains of Smad2 and Smad4 were unchanged, as assessed by sequence analysis. The Smad4 promoter region was unmethylated in tumor samples with weak/moderate cytoplasmic Smad4 expression. Both weak cytoplasmic Smad4 expression and absence of nuclear Smad4 expression significantly correlated with poor disease-free (P=.003 and P=.003, respectively) and overall five-year (P=.003 and P=.010, respectively) survival. These findings support the hypothesis that Smad4 is a target molecule for functional inactivation in cervical cancer.

Kloth JN, Kenter GG, Spijker HS, et al. Expression of Smad2 and Smad4 in cervical cancer: absent nuclear Smad4 expression correlates with poor survival. Mod Pathol. 2008;21:866–875.

Correspondence: Dr. A. Gorter at a.gorter@lumc.nl
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Disease-free survival rates in locally advanced rectal cancer patients after preoperative chemoradiation

Preoperative chemoradiation is the standard treatment for locally advanced rectal cancer. However, it is uncertain whether pretreatment clinical stage, degree of response to neoadjuvant treatment, or pathologic stage is the most reliable predictor of outcome. The authors conducted a study in which they compared various staging elements and treatment-related variables to identify which factors or combination of factors prognosticates disease-free survival in rectal cancer patients receiving neoadjuvant combined modality therapy. The authors identified, from a prospectively maintained single-institution database, 342 consecutive patients with locally advanced rectal cancer staged by endorectal ultrasound. Patients underwent rectal resection four to eight weeks after a five-and-a-half-week course of pelvic radiotherapy/concurrent chemotherapy. The degree of tumor regression was graded histologically on each resection specimen using a previously reported response scale of zero to 100 percent. Predictive models of disease-free survival were created using available pretherapy and postoperative staging elements in addition to the degree of tumor regression noted histologically. Model accuracy was measured and compared by concordance index, with a 95 percent confidence interval (CI). Stratifying patients by degree of tumor regression predicted outcome with a concordance index of 0.65 (95 percent CI, 0.59–0.71), which was significantly better than models using preoperative staging elements (concordance index, 0.54; 95 percent CI, 0.50–0.58). However, the model found to be most predictive of disease-free survival stratified patients by final pathologic T classification and N classification elements, with a concordance index of 0.75 (95 percent CI, 0.70–0.80). The authors concluded that tumor response to preoperative therapy is a strong predictor of disease-free survival. However, outcome is most accurately estimated by final pathologic stage, which is influenced by preoperative stage and response to therapy.

Quah HM, Chou JF, Gonen M, et al. Pathologic stage is most prognostic of disease-free survival in locally advanced rectal cancer patients after preoperative chemoradiation. Cancer. 2008;113:57–64.

Correspondence: Dr. Martin R. Weiser at weiser1@ mskcc.org
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