Final validation of the ProMisE molecular classifier for endometrial carcinoma in a large population-based case series.

Background: We have previously developed and confirmed a pragmatic molecular classifier for endometrial cancers; ProMisE (Proactive Molecular Risk Classifier for Endometrial Cancer). Inspired by the Cancer Genome Atlas, ProMisE identifies four prognostically distinct molecular subtypes and can be applied to diagnostic specimens (biopsy/curettings) enabling earlier informed decision-making. We have strictly adhered to the Institute of Medicine (IOM) guidelines for the development of genomic biomarkers, and herein present the final validation step of a locked-down classifier before clinical application. Patients and methods: We assessed a retrospective cohort of women from the Tübingen University Women's Hospital treated for endometrial carcinoma between 2003 and 2013. Primary outcomes of overall, disease-specific, and progression-free survival were evaluated for clinical, pathological, and molecular features. Results: Complete clinical and molecular data were evaluable from 452 women. Patient age ranged from 29 to 93 (median 65) years, and 87.8% cases were endometrioid histotype. Grade distribution included 282 (62.4%) G1, 75 (16.6%) G2, and 95 (21.0%) G3 tumors. 276 (61.1%) patients had stage IA disease, with the remaining stage IB [89 (19.7%)], stage II [26 (5.8%)], and stage III/IV [61 (13.5%)]. ProMisE molecular classification yielded 127 (28.1%) MMR-D, 42 (9.3%) POLE, 55 (12.2%) p53abn, and 228 (50.4%) p53wt. ProMisE was a prognostic marker for progression-free (P = 0.001) and disease-specific (P = 0.03) survival even after adjusting for known risk factors. Concordance between diagnostic and surgical specimens was highly favorable; accuracy 0.91, κ 0.88. Discussion: We have developed, confirmed, and now validated a pragmatic molecular classification tool (ProMisE) that provides consistent categorization of tumors and identifies four distinct prognostic molecular subtypes. ProMisE can be applied to diagnostic samples and thus could be used to inform surgical procedure(s) and/or need for adjuvant therapy. Based on the IOM guidelines this classifier is now ready for clinical evaluation through prospective clinical trials.

Assessment of DNA Ploidy in the ProMisE molecular subgroups of endometrial cancer.

OBJECTIVE: We sought to determine whether DNA ploidy correlates with the four molecular subgroups of endometrial carcinoma (EC) as determined using ProMisE (Proactive Molecular Risk Classifier for Endometrial Cancer). METHODS: 90 cases of EC previously characterized by clinicopathological parameters, outcomes, and ProMisE molecular subgroup (POLE EDM, MMR-D, p53 wt or p53 abn) were assessed for DNA ploidy using image cytometry. Associations of ploidy with traditional clinicopathological parameters were also tested. RESULTS: Abnormal DNA ploidy status differed amongst the ProMisE groups (p<0.001) and was found in 80.9% (17/21) of p53 abn, 37.0% (10/27) of p53 wt, 28.6% (4/14) of POLE EDM and 14.3% (4/28) of MMR-D. Abnormal DNA content was significantly associated with lower BMI (p=0.034) and grade 3 tumors (p=0.001). In the entire cohort, abnormal DNA content was significantly associated with worse progression free survival (p=0.0094) but not disease specific survival (p=0.249) or overall survival (p=0.187). When examining ploidy within each of the ProMisE groups, abnormal DNA content correlated with worse overall survival (p=0.041) and progression free survival (p=0.011) in the MMR-D group. No statistically significant relationship was seen in the remaining 3 groups. CONCLUSION: Abnormal DNA ploidy status did correlate with the molecular subgroups of EC; abnormal DNA content was seen in the large majority of p53 abn cases. Abnormal ploidy however was also seen in smaller numbers in the p53 wt, POLE EDM and MMR-D groups; therefore abnormal DNA content was not a specific marker for any one molecular group. The addition of ploidy to the ProMisE molecular categories conferred additional prognostic value within the MMR-D group, which merits further study.

Elevated CDCP1 predicts poor patient outcome and mediates ovarian clear cell carcinoma by promoting tumor spheroid formation, cell migration and chemoresistance.

Hematogenous metastases are rarely present at diagnosis of ovarian clear cell carcinoma (OCC). Instead dissemination of these tumors is characteristically via direct extension of the primary tumor into nearby organs and the spread of exfoliated tumor cells throughout the peritoneum, initially via the peritoneal fluid, and later via ascites that accumulates as a result of disruption of the lymphatic system. The molecular mechanisms orchestrating these processes are uncertain. In particular, the signaling pathways used by malignant cells to survive the stresses of anchorage-free growth in peritoneal fluid and ascites, and to colonize remote sites, are poorly defined. We demonstrate that the transmembrane glycoprotein CUB-domain-containing protein 1 (CDCP1) has important and inhibitable roles in these processes. In vitro assays indicate that CDCP1 mediates formation and survival of OCC spheroids, as well as cell migration and chemoresistance. Disruption of CDCP1 via silencing and antibody-mediated inhibition markedly reduce the ability of TOV21G OCC cells to form intraperitoneal tumors and induce accumulation of ascites in mice. Mechanistically our data suggest that CDCP1 effects are mediated via a novel mechanism of protein kinase B (Akt) activation. Immunohistochemical analysis also suggested that CDCP1 is functionally important in OCC, with its expression elevated in 90% of 198 OCC tumors and increased CDCP1 expression correlating with poor patient disease-free and overall survival. This analysis also showed that CDCP1 is largely restricted to the surface of malignant cells where it is accessible to therapeutic antibodies. Importantly, antibody-mediated blockade of CDCP1 in vivo significantly increased the anti-tumor efficacy of carboplatin, the chemotherapy most commonly used to treat OCC. In summary, our data indicate that CDCP1 is important in the progression of OCC and that targeting pathways mediated by this protein may be useful for the management of OCC, potentially in combination with chemotherapies and agents targeting the Akt pathway.

A clinically applicable molecular-based classification for endometrial cancers.

BACKGROUND: Classification of endometrial carcinomas (ECs) by morphologic features is inconsistent, and yields limited prognostic and predictive information. A new system for classification based on the molecular categories identified in The Cancer Genome Atlas is proposed. METHODS: Genomic data from the Cancer Genome Atlas (TCGA) support classification of endometrial carcinomas into four prognostically significant subgroups; we used the TCGA data set to develop surrogate assays that could replicate the TCGA classification, but without the need for the labor-intensive and cost-prohibitive genomic methodology. Combinations of the most relevant assays were carried forward and tested on a new independent cohort of 152 endometrial carcinoma cases, and molecular vs clinical risk group stratification was compared. RESULTS: Replication of TCGA survival curves was achieved with statistical significance using multiple different molecular classification models (16 total tested). Internal validation supported carrying forward a classifier based on the following components: mismatch repair protein immunohistochemistry, POLE mutational analysis and p53 immunohistochemistry as a surrogate for 'copy-number' status. The proposed molecular classifier was associated with clinical outcomes, as was stage, grade, lymph-vascular space invasion, nodal involvement and adjuvant treatment. In multivariable analysis both molecular classification and clinical risk groups were associated with outcomes, but differed greatly in composition of cases within each category, with half of POLE and mismatch repair loss subgroups residing within the clinically defined 'high-risk' group. Combining the molecular classifier with clinicopathologic features or risk groups provided the highest C-index for discrimination of outcome survival curves. CONCLUSIONS: Molecular classification of ECs can be achieved using clinically applicable methods on formalin-fixed paraffin-embedded samples, and provides independent prognostic information beyond established risk factors. This pragmatic molecular classification tool has potential to be used routinely in guiding treatment for individuals with endometrial carcinoma and in stratifying cases in future clinical trials.

Detection of DNA mismatch repair (MMR) deficiencies by immunohistochemistry can effectively diagnose the microsatellite instability (MSI) phenotype in endometrial carcinomas.

BACKGROUND: A proportion of endometrial carcinomas (ECs) are associated with deficient DNA mismatch repair (MMR). These tumors are characterized by high levels of microsatellite instability (MSI). Identification of MSI is important in identifying women who should be tested for Lynch syndrome and identifying a phenotype that may have specific prognostic and predictive implications. Genomic characterization of ECs has shown that MSI tumors form a distinct subgroup. The two most common methodologies for MSI assessment have not been compared in EC. METHODS: Pentaplex mono and di-nucleotide PCR for MSI testing was compared to MMR IHC (presence/absence of MLH1, MSH2, MSH6, PMS2) in a cohort of patients with EC. Concordance, Kappa statistic, sensitivity, specificity, positive and negative predictive values were obtained on the cross-tabulation of results. RESULTS: Comparison of both MSI and MMR status was complete for 89 cases. Overall agreement between methods (concordance) was 93.3% (95% CI[85.9%-97.5%]). A one-sided test to determine whether the accuracy is better than the "no information rate," which is taken to be the largest class percentage in the data, is significant (p<0.00001). Unweighted Kappa was 0.84, along with the sensitivity (88.5%), specificity (95.2%), PPV (88.5%), and NPV (95.2%). The balanced accuracy (i.e. the average between sensitivity and specificity) was 92%. DISCUSSION: We show the equivalence of MSI testing and MMR IHC. We advocate the implementation of MMR IHC in future EC classification schemes, enabling stratification of cases for future clinical trials as well as assisting identification of Lynch syndrome, so that screening and risk reducing interventions can be undertaken.

Exploring palliative treatment outcomes in women with advanced or recurrent ovarian clear cell carcinoma.

OBJECTIVE: Clear cell carcinoma (CCC) of the ovary is increasingly recognized as responding poorly to chemotherapy (CT). This review examines the outcomes achieved with a variety of CT regimens, looking for evidence of activity that might guide the development of more effective treatments. METHODS: A retrospective chart review of all cases of CCC referred to the BC Cancer Agency (BCCA) between 2000 and 2008 was conducted. Data were collected from those with primarily advanced disease and from those who recurred after adjuvant treatment. Outcomes were measured using broad definitions of treatment benefit (any objective or subjective evidence of disease control) in order to reflect the real-life use of palliative therapy. RESULTS: There were 158 women with pure CCC. First-line therapy for advanced disease was delivered to 33 patients. Second- and third-line treatment was delivered to 47 and 25 patients, respectively. The total number of treatment courses was 105: 88 CT-alone courses, 14 radiation therapy (RT)-alone and 3 combined modality. Treatment benefit was recorded in 24% of patients receiving CT, 64% of patients receiving RT, and each who received combined modality treatment. There was no CT drug class identified as obviously efficacious. CONCLUSION: Most patients with advanced or recurrent CCC have a low benefit-to-failure ratio from palliative CT. The role of RT and targeted agents must be explored to improve clinical outcomes for such patients.

Autofluorescence imaging can identify preinvasive or clinically occult lesions in fallopian tube epithelium: a promising step towards screening and early detection.

BACKGROUND: Optical imaging systems are robust, portable, relatively inexpensive, and have proven utility in detecting precancerous lesions in the lung, esophagus, colon, oral cavity and cervix. We describe the use of light-induced endogenous fluorescence (autofluorescence) in identifying preinvasive and occult carcinomas in ex vivo samples of human fallopian tube (FT) epithelium. METHODS: Women undergoing surgery for an i) ovarian mass, ii) a history suggestive of hereditary breast-ovarian cancer, or iii) known serous ovarian cancer following neoadjuvant chemotherapy (NAC) were approached for informed consent. Immediately following surgery, FT's were photographed in reflectance and fluorescence at high resolution. Images included: (1) white-light reflectance of luminal/epithelial surface; (2) narrow-band green reflectance (570 nm) (3) green autofluorescence (405/436 nm excitation); and (4) blue autofluorescence (405 nm excitation). Areas revealing a loss of natural tissue fluorescence or marked increase in tissue microvasculature were recorded and compared to final histopathologic diagnosis (SEE-FIM protocol). RESULTS: Fifty-six cases involving one or both fallopian tubes underwent reflectance and fluorescence visualization. Nine cases were excluded, either secondary to non-ovarian primary pathology (7) or excessive trauma (2) rendering tissue interpretation impossible. Of the 47 cases remaining, there were 11 high grade serous (HGS) and 9 non-serous ovarian carcinomas undergoing primary debulking surgery, 5 serous carcinomas having received NAC, 8 benign ovarian tumors, and 14 women undergoing risk-reducing bilateral salpingo-oophorectomy (RRBSO). Methodology was feasible, efficient, and reproducible. TIC or carcinoma was identified in 7/11 HGS, 3/5 NAC, and 1/14 RRBSO. Optical images were reviewed to determine test positive or negative based on standardized criteria. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated for the entire cohort (73%; 83%; 57%; 91%) and in a subgroup that excluded non-serous histology (87.5%; 92%; 78%; 96%). CONCLUSIONS: Abnormal FT lesions can be identified using ex vivo optical imaging technologies. With this platform, we will move towards genomic interrogation of identified lesions, and developing in vivo screening modalities via falloposcopy.

Histotype predicts the curative potential of radiotherapy: the example of ovarian cancers.

BACKGROUND: To explore the influence of ovarian cancer histotype on the effectiveness of adjuvant radiotherapy (RT). METHODS: A review of a population-based experience included all referred women with no reported macroscopic residuum following primary surgery who underwent adjuvant platin-based chemotherapy (CT), with or without sequential RT, and for whom it was possible to assign histotype according to the contemporary criteria. RESULTS: Seven hundred and three subjects were eligible, of these 351 received RT. For those with apparent stage I and II tumors, the cohort with clear cell (C), endometrioid (E), and mucinous (M) disease who additionally received RT exhibited a 40% reduction in disease-specific mortality and a 43% reduction in overall mortality. CONCLUSIONS: The curability of those with stage I and II C-, E-, and M-type ovarian carcinomas was enhanced by RT-containing adjuvant therapy. This benefit did not extend to those with stage III or serous tumors. These findings necessitate reassessments of the role of RT and of the nonselective surgical and CT approaches that have characterized ovarian cancer care.

New cutpoints to identify increased HER2 copy number: analysis of a large, population-based cohort with long-term follow-up.

BACKGROUND: HER2 gene amplification and/or protein overexpression in breast cancer is associated with a poor prognosis and predicts response to anti-HER2 therapy. We examine the natural history of breast cancers in relationship to increased HER2 copy numbers in a large population-based study. PATIENTS AND METHODS: HER2 status was measured by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) in approximately 1,400 breast cancer cases with greater than 15 years of follow-up. Protein expression was evaluated with two different commercially-available antibodies. RESULTS: We looked for subgroups of breast cancer with different clinical outcomes, based on HER2 FISH amplification ratio. The current HER2 ratio cut point for classifying HER2 positive and negative cases is 2.2. However, we found an increased risk of disease-specific death associated with FISH ratios of >1.5. An 'intermediate' group of cases with HER2 ratios between 1.5 and 2.2 was found to have a significantly better outcome than the conventional 'amplified' group (HER2 ratio >2.2) but a significantly worse outcome than groups with FISH ratios less than 1.5. CONCLUSION: Breast cancers with increased HER2 copy numbers (low level HER2 amplification), below the currently accepted positive threshold ratio of 2.2, showed a distinct, intermediate outcome when compared to HER2 unamplified tumors and tumors with HER2 ratios greater than 2.2. These findings suggest that a new cut point to determine HER2 positivity, at a ratio of 1.5 (well below the current recommended cut point of 2.2), should be evaluated.

Serous borderline ovarian tumors in long-term culture: phenotypic and genotypic distinction from invasive ovarian carcinomas.

Serous borderline ovarian tumors (SBOTs) are differentiated, slow growing, noninvasive, and have a better prognosis than their invasive counterparts, but recurrence and progression to invasive carcinomas are common, and unlike high-grade serous carcinomas, they tend to be nonresponsive to chemotherapy. However, due to a lack of culture systems and animal models, information about the properties of SBOT and their changes with neoplastic progression is extremely limited. Our objective was to establish a cell culture model for SBOTs and to characterize their phenotype and genotype. We compared cultures derived from two SBOTs, one of which was a short-term culture containing a BRAF mutation but few other cytogenetic changes while the other culture developed into a spontaneously immortalized permanent cell line and had numerical and structural chromosomal abnormalities but lacked RAS/BRAF mutations. Both cultures formed whorl-like epithelial colonies and resembled low-grade invasive carcinomas by their secretion of CA125 and oviduct-specific glycoprotein, production of matrix metalloproteinases, E-cadherin expression, and telomerase activity. Other characteristics associated with neoplastic transformation, including invasiveness, anchorage-independent growth, and tumorigenicity, were not observed. Importantly, cell motility was reduced in both lines, likely contributing to the lack of invasiveness. The results reveal a striking phenotypic similarity between the two cell lines, regardless of their cytogenetic diversity, which suggests that their characteristic phenotype is regulated to a large degree by epigenetic and environmental factors. In conclusion, we have established the first permanent SBOT cell line, which provides a new model to elucidate the undefined relationship of SBOTs to invasive ovarian carcinomas.

The role of immunohistochemistry for smooth-muscle actin, p63, CD10 and cytokeratin 14 in the differential diagnosis of papillary lesions of the breast.

BACKGROUND: Histological differentiation of mammary papillary lesions can be difficult. The evaluation of myoepithelial cells can be helpful, with benign papilloma showing a continuous myoepithelial cell layer, which becomes attenuated or absent in malignant papillary lesions. METHODS: A large series of 100 papillomas (28 papillomas with florid epithelial hyperplasia) and 68 papillary carcinomas (9 invasive, 44 in situ, and 15 ductal carcinomas in situ (DCIS) involving papillomas) of the breast were stained for myoepithelial cells by immunohistochemistry using antibodies to smooth-muscle actin (SMA), p63, CD10 and cytokeratin (CK) 14. RESULTS: In the papillomas, using these four antibodies, myoepithelial cells were positive in 88%, 99%, 91% and 95% of cases, respectively, with SMA showing marked stromal component cell staining and CD10 showing epithelial and stromal staining. CK14 also showed epithelial staining in 71% of papillomas and 96% of papillomas with florid epithelial hyperplasia. In the papillary carcinomas, 36 (53%) cases showed staining of myoepithelial cells that were scattered, discontinuous and diminished in number and the remaining 32 (47%) cases did not show myoepithelial cells. Invasive papillary carcinoma has the lowest proportion (33%) with myoepithelial cells, and DCIS involving papillomas had the highest proportion (87%). CONCLUSIONS: p63 had the highest sensitivity and did not cross-react with stromal cells and only rarely with epithelial cells. CK14 has the added ability to distinguish between florid epithelial hyperplasia involving papilloma and DCIS involving papillomas. CK14 and p63 may be used as an adjunct in assessing difficult papillary lesions of the breast.

CD44s is useful in the differentiation of benign and malignant papillary lesions of the breast.

BACKGROUND/AIMS: CD44s, the standard form of CD44, has been shown to be downregulated during malignant transformation of breast cancers. It has also been reported recently to be a useful marker in differentiating between benign and malignant papillary lesions of the breast, with high expression in the former. CD44s expression in benign and malignant papillary lesions was evaluated. METHODS: CD44s expression was assessed by immunohistochemistry in 101 benign papillomas and 59 papillary carcinomas (seven invasive papillary carcinomas, 41 papillary ductal carcinomas in situ, and 11 ductal carcinomas involving papillomas). RESULTS: Patients' age and tumour size were significantly different between the papilloma and papillary carcinoma groups (p < 0.0001). CD44s showed positive staining in 45 papillomas (45%) and five papillary carcinomas (8%), and the difference was significant (p < 0.0001). The myoepithelial cells, when present, were also positive for CD44s in both groups, with no observable differences. Using CD44s positive staining to differentiate between benign and malignant papillary lesions gives a sensitivity, specificity, and accuracy of 45%, 92%, and 62%, respectively. CONCLUSIONS: CD44s may be useful as an adjunct in the evaluation of morphologically problematic cases of papillary lesion of the breast.

Intratumoral oxygenation of invasive squamous cell carcimoma of the vulva is not correlated with regional lymph node metastasis.

INTRODUCTION: Tumour hypoxia has been found to be associated with tumour aggressiveness. Our primary aim was to explore the relationship between pretreatment tumour oxygenation in primary vulvar carcinoma and nodal status. Our secondary objective was to assess if there was a relationship between the clinical and biological variables. METHODS: 20 women with ISCC of the vulva were assessed with pretreatment primary tumour oxygenation with an Eppendorf pO2 probe. Patients underwent standard surgical management. Pathological assessment of the primary and nodal tissues was then performed. Primary tumour specimens were also stained for microvessel density and carbonic anhydrase IX. The relationship between smoking, preoperative Hgb, tumour CAIX expression, MVD, and Eppendorf pO2 measurements vs nodal metastasis and between these clinical and biological variables was assessed. RESULTS: Seven patients had positive lymph nodes, 13 had negative nodes. While neither current smoking status, tumour size, tumour oxygen measurements, MVD and CAIX expression correlated with metastatic nodal disease, a low preoperative Hgb correlated with pathological nodal status (p < 0.027). CONCLUSIONS: Although this analysis failed to demonstrate a strong correlation between various measures of tumour oxygenation with nodal metastasis, it may be due to the small number of patients. Only preoperative anaemia is correlated with nodal metastasis in early ISCC of the vulva.

P63 expression in lung carcinoma: a tissue microarray study of 408 cases.

p63 is a recently discovered member of the p53 family that has been shown to be important in the development of epithelial tissues. p63 may also play a role in squamous cell carcinomas of the lung, head and neck, and cervix, and its expression is increased in these tumors. The purpose of this study was to investigate the expression of p63 in a broad spectrum of histologic types of lung tumors. A total of 441 cases of primary lung tumors with follow-up data were identified, and the paraffin-embedded tissue blocks were used to construct a duplicate core tissue microarray. After review of the tissue cores, 408 cases, consisting of 123 squamous cell carcinomas, 93 adenocarcinomas, 68 large cell carcinomas, 68 classic carcinoids, 31 atypical carcinoids, 11 large cell neuroendocrine carcinomas, and 14 small cell carcinomas, were adequate for analysis. Immunohistochemistry was performed at 2 different laboratories using monoclonal antibody 4A4 to detect the expression of p63, using different staining protocols. p53 expression was also studied with immunohistochemistry using monoclonal antibody DO-7. Kaplan-Meier curves were plotted to compare the survival of p63-expressing versus nonexpressing tumors. A large proportion of squamous cell carcinomas expressed p63 (96.9%), most showing strong positive nuclear immunoreactivity. Expression in other nonsmall cell lung cancers was also present. Thirty percent of adenocarcinomas and 37% of large cell carcinomas showed p63 expression. In the neuroendocrine tumors, an increasing proportion of tumors stained for p63 as tumor grade increased; 1.9% of classic carcinoids, 30.8% of atypical carcinoids, 50% of large cell neuroendocrine carcinomas, and 76.9% of small cell carcinomas were positive. Approximately half of the positively staining neuroendocrine cases showed strong staining. Expression of p63 was of prognostic significance in neuroendocrine tumors (P < 0.0001), with higher-grade tumors more likely to express p63. Correlation between p63 and p53 expression was not observed (P = 0.18) in nonsmall cell lung cancer; however, a significant correlation between the 2 markers was found in neuroendocrine tumors (P < 0.0001). p63 staining was repeated with a different staining protocol, yielding similar results overall but a lower percentage of positive cases (34.2% vs. 48.4% of tumors positive). In conclusion, p63 expression is consistently expressed in squamous cell carcinoma in the lung, but is also expressed in a subset of adenocarcinomas and large cell carcinomas. Pulmonary neuroendocrine tumors also show p63 staining in some instances, particularly in higher-grade tumors, and the majority of small cell carcinomas are p63-positive. These results suggest that p63 may be involved in oncogenesis in a broader range of tumors than was previously thought.

Evaluation of immunohistochemical markers in non-small cell lung cancer by unsupervised hierarchical clustering analysis: a tissue microarray study of 284 cases and 18 markers.

This study has investigated a panel of immunomarkers in non-small cell lung carcinoma (NSCLC). Unsupervised hierarchical clustering analysis was used to investigate the possibility of identifying different subgroups in NSCLC based on their molecular expression profile rather than morphological features. A tissue microarray consisting of 284 cases of NSCLC was constructed. Immunohistochemistry was used to detect the presence of 18 biomarkers including synaptophysin, chromogranin, bombesin, NSE, GFI1, ASH-1, p53, p63, p21, p27, E2F-1, cyclin D1, Bcl-2, TTF-1, CEA, HER2/neu, cytokeratin 5/6, and pancytokeratin. Univariate analysis of all 18 markers for prognostic significance was performed. Immunohistochemical scoring data for NSCLC were analysed by unsupervised hierarchical clustering analysis. Kaplan-Meier survival curves were plotted for the different cluster groups of lung tumours identified by this method. Analysis of the three different World Health Organization (WHO) subtypes (adenocarcinoma, squamous cell carcinoma, large cell carcinoma) of NSCLC individually showed that different markers were significant in different subtypes. For example, p53 and p63 were significant for squamous cell carcinoma (p = 0.007 and p = 0.03, respectively), whereas cyclin D1 and HER2/neu were significant prognostic markers for adenocarcinoma (p = 0.025 and p = 0.015, respectively). These markers were not significant prognostic predictors for NSCLC as a group. Hierarchical clustering analysis of NSCLC produced four separate cluster groups, although the vast majority of cases were found in two cluster groups, one dominated by squamous cell carcinoma and the other by adenocarcinoma. The clinical outcomes of cases from the four cluster groups were not significantly different. Prognostic indicators vary between different morphological subtypes of NSCLC. Unsupervised hierarchical clustering analysis, based on an extended immunoprofile, identifies two main cluster groups corresponding to adenocarcinoma and squamous cell carcinoma; cases of large cell carcinomas are assigned to one of these two groups based on their molecular phenotype.

Applications of microarrays to histopathology.

High-throughput microarray technologies have the potential to impact significantly on the practice of histopathology over the coming years. Global gene expression profiling allows for a systematic search of all human genes for novel diagnostic and prognostic markers and for potential therapeutic targets. Likewise, gene copy number changes can be determined on a gene-by-gene basis using microarrays. Tissue microarrays are an efficient method to extend and validate the findings obtained from the initial 'discovery' phase of the research, done using cDNA microarrays. In addition, tissue microarrays can be used for quality assurance for immunohistochemical and in situ hybridization procedures. In this review we give a brief overview of microarray technology and research uses, and discuss potential applications of microarrays in the practice of diagnostic histopathology.

Immunoprofile of cervical and endometrial adenocarcinomas using a tissue microarray.

Adenocarcinomas of the uterine cervix show a wide range of morphological features, and can be confused with endometrial adenocarcinoma in biopsy or curetting specimens. The objective of this study was to use tissue microarray technology to evaluate the immunoprofile of a large set of uterine adenocarcinomas with an extended panel of antibodies, comparing the profile of primary cervical and endometrial adenocarcinomas. A tissue microarray was constructed using paraffin-embedded, formalin-fixed tissues from 141 hysterectomy specimens. Duplicate 0.6-mm cores were obtained from 57 cervical adenocarcinomas (16 in situ and 41 invasive) and 84 endometrial adenocarcinomas. Tissue array sections were immunostained with 21 commercially available antibodies [B72.3, CD 99, carcinoembryonic antigen (CEA), c-kit, pancytokeratin, CK 5/6, CK 7, CK8/18, CK19, CK 20, CK 22, EMA, estrogen receptor (ER), KP-1, melan-A, p53, PLAP, S-100, synaptophysin, TTF-1, and vimentin] utilizing the avidin-biotin (ABC) technique. Hierarchical clustering analysis of the tumors was done based on the immunostaining results. Only ER ( P<0.001), CEA ( P=0.04), vimentin ( P<0.001), and CK 8/18 ( P=0.002) showed a significantly different frequency of positivity in endometrial relative to cervical adenocarcinomas. ER, vimentin, and CK 8/18 were more likely to be expressed in endometrial adenocarcinomas, while cervical adenocarcinomas more frequently expressed CEA. We were able to identify immunoprofiles that were highly specific for endocervical adenocarcinoma (ER(-), vimentin(-), CK 8/18(-), CEA(+)) or endometrial adenocarcinoma (ER(+), vimentin(+), CK 8/18(+), CEA(-)), but most tumors showed an intermediate, non-specific immunophenotype. Hierarchical clustering analysis was useful in the interpretation of these intermediate immunophenotypes. Papillary serous adenocarcinoma of the endometrium was less likely to express vimentin ( P=0.002) than endometrioid carcinoma of the endometrium.