How reliable is Ki-67 immunohistochemistry in grade 2 breast carcinomas? A QA study of the Swiss Working Group of Breast- and Gynecopathologists.

UNLABELLED: Adjuvant chemotherapy decisions in breast cancer are increasingly based on the pathologist's assessment of tumor proliferation. The Swiss Working Group of Gyneco- and Breast Pathologists has surveyed inter- and intraobserver consistency of Ki-67-based proliferative fraction in breast carcinomas. METHODS: Five pathologists evaluated MIB-1-labeling index (LI) in ten breast carcinomas (G1, G2, G3) by counting and eyeballing. In the same way, 15 pathologists all over Switzerland then assessed MIB-1-LI on three G2 carcinomas, in self-selected or pre-defined areas of the tumors, comparing centrally immunostained slides with slides immunostained in the different laboratoires. To study intra-observer variability, the same tumors were re-examined 4 months later. RESULTS: The Kappa values for the first series of ten carcinomas of various degrees of differentiation showed good to very good agreement for MIB-1-LI (Kappa 0.56-0.72). However, we found very high inter-observer variabilities (Kappa 0.04-0.14) in the read-outs of the G2 carcinomas. It was not possible to explain the inconsistencies exclusively by any of the following factors: (i) pathologists' divergent definitions of what counts as a positive nucleus (ii) the mode of assessment (counting vs. eyeballing), (iii) immunostaining technique, and (iv) the selection of the tumor area in which to count. Despite intensive confrontation of all participating pathologists with the problem, inter-observer agreement did not improve when the same slides were re-examined 4 months later (Kappa 0.01-0.04) and intra-observer agreement was likewise poor (Kappa 0.00-0.35). CONCLUSION: Assessment of mid-range Ki-67-LI suffers from high inter- and intra-observer variability. Oncologists should be aware of this caveat when using Ki-67-LI as a basis for treatment decisions in moderately differentiated breast carcinomas.

Validation of a postfixation tissue storage and transport medium to preserve histopathology and molecular pathology analyses (total and phosphoactivated proteins, and FISH).

Tumor biomarker studies are integral to oncology clinical trials but may yield artifactual results owing to variation in sample procurement and processing. Ethanol, 70% vol/vol, was validated as a sample transport medium using markers of the PI3K/Akt/mTOR pathway. BT474 tumor xenografts were excised and slices were immediately placed into formaldehyde and fixed for 24 hours. Fixed tissue slices were immediately processed into paraffin or transferred to 70% vol/vol ethanol and stored at room temperature for 1, 2, and 4 weeks before further processing. Freshly cut tissue sections were evaluated for pAKT(S473), HER2, pHER-2(Y1248), pS6(S235/236), and pS6(S240/244), Ki-67, and HER2 by fluorescence in situ hybridization and stained with H&E and Masson trichrome. No significant changes were observed when comparing samples stored in 70% ethanol for up to 4 weeks with immediately processed tissue. Ethanol, 70% vol/vol, provides a safe storage medium for formaldehyde-fixed tumor tissue, facilitating sample transport during multicenter clinical trials.

HER2 gene status in primary breast cancers and matched distant metastases.

INTRODUCTION: The status of the gene encoding human EGF-like receptor 2 (HER2) is an important prognostic and predictive marker in breast cancer. Only breast cancers with HER2 amplification respond to the targeted therapy with trastuzumab. It is controversial to what degree the primary tumour is representative of distant metastases in terms of HER2 status. Discrepancies in HER2 status between primary tumours and distant metastases have been described, but their reasons remain unclear. Here, we compared HER2 status on cytological specimens of distant metastases with the result from the primary carcinomas, and explored the prevalence of and the reasons for discrepant results. METHODS: HER2 status was determined by fluorescence in situ hybridisation. HER2 gene amplification was defined as a HER2/chromosome 17 signal ratio of 2 or more. HER2 results from cytological specimens of matched distant metastases were compared with the results from the corresponding primary tumours (n = 105 patients). In addition, lymph node metastases were analysed in 31 of these patients. RESULTS: HER2 amplification was found in 20% of distant metastases. HER2 status was discordant between the primary tumour and distant metastasis in 7.6% of the 105 patients. Re-evaluation revealed that in five patients (4.7%), discrepancies were due to interpretational difficulties. In two of these patients, focal amplification had initially been overlooked as a result of heterogeneity in the primary tumours or in the metastases, respectively. A further three patients had borderline amplification with a ratio close to 2. Discrepancy remained unexplained in three patients (2.9%). CONCLUSION: HER2 gene status remains highly conserved as breast cancers metastasise. However, discrepant results do occur because of interpretational difficulties and heterogeneity of HER2 amplification. Cytological specimens from distant metastases are well suited for HER2 fluorescence in situ hybridisation analysis.

Expression and localization of melanocortin-1 receptor in human adipose tissues of severely obese patients.

OBJECTIVE: The melanocortin system is a key regulator in the hypothalamus of energy intake and expenditure. It is frequently linked with obesity and apparently modulates sympathetic outflow to white adipose tissues. The role of the melanocortins within adipose tissues, however, is not entirely clear. This study was aimed at determining the quantitative expression of the five melanocortin receptors (MC1-R to MC5-R) in subcutaneous and omental fat of obese patients and non-obese subjects. RESEARCH METHODS AND PROCEDURES: Expression of MC1-R to MC5-R, proopiomelanocortin, agouti signaling protein, leptin, leptin receptor, and uncoupling protein-1 was investigated in human fat samples by quantitative reverse transcription-polymerase chain reaction. MC1-R expression was also studied in preadipocytes, adipocytes, and monocytic THP-1 cells and by immunohistochemical localization in adipose tissues. RESULTS: Notable expression was found for MC1-R, whereas no mRNA for MC2-R and MC3-R was detected; MC4-R and MC5-R mRNA was occasionally detectable but at very low levels. MC1-R mRNA in subcutaneous fat was increased in obese patients as compared with controls; omental fat of both groups had slightly higher MC1-R expression than subcutaneous fat and did not differ between patient groups. Immunohistochemical analysis of the MC1-R in adipose tissue sections showed that MC1-R expression was higher in macrophages but also present in adipocytes. DISCUSSION: The expression of MC1-R and the lack of MC2-R in human adipose tissues indicate that the melanocortins may regulate cell proliferation and/or inflammatory signals rather than lipolysis. Also, the increased expression of MC1-R in subcutaneous fat of obese subjects may reflect one aspect of the pathophysiology of obesity.

Generation and analysis of melanoma SAGE libraries: SAGE advice on the melanoma transcriptome.

In this study, we generated three SAGE libraries from melanoma tissues. Using bioinformatics tools usually applied to microarray data, we identified several genes, including novel transcripts, which are preferentially expressed in melanoma. SAGE results converged with previous microarray analysis on the importance of intracellular calcium and G-protein signaling, and the Wnt/Frizzled family. We also examined the expression of CD74, which was specifically, albeit not abundantly, expressed in the melanoma libraries using a melanoma progression tissue microarray, and demonstrate that this protein is expressed by melanoma cells but not by benign melanocytes. Many genes involved in intracellular calcium and G-protein signaling were highly expressed in melanoma, results we had observed earlier from microarray studies (Bittner et al., 2000). One of the genes most highly expressed in our melanoma SAGE libraries was a calcium-regulated gene, calpain 3 (p94). Immunohistochemical analysis demonstrated that calpain 3 moves from the nuclei of non-neoplastic cells to the cytoplasm of malignant cells, suggesting activation of this intracellular proteinase. Our SAGE results and the clinical validation data demonstrate how SAGE profiles can highlight specific links between signaling pathways as well as associations with tumor progression. This may provide insights into new genes that may be useful for the diagnosis and therapy of melanoma.

Candidate genes for testicular cancer evaluated by in situ protein expression analyses on tissue microarrays.

By the use of high-throughput molecular technologies, the number of genes and proteins potentially relevant to testicular germ cell tumor (TGCT) and other diseases will increase rapidly. In a recent transcriptional profiling, we demonstrated the overexpression of GRB7 and JUP in TGCTs, and confirmed the reported overexpression of CCND2. We also have recent evidences for frequent genetic alterations of FHIT and epigenetic alterations of MGMT. To evaluate whether the expression of these genes is related to any clinicopathological variables, we constructed a tissue microarray with 510 testicular tissue cores from 279 patients diagnosed with TGCT, covering various histological subgroups and clinical stages. By immunohistochemistry, we found that JUP, GRB7, and CCND2 proteins were rarely present in normal testis, but frequently expressed at high levels in TGCT. Additionally, all premalignant intrabular germ cell neoplasias were JUP-immunopositive. MGMT and FHIT were expressed by normal testicular tissues, but at significantly lower frequencies in TGCT. Except for CCND2, the expressions of all markers were significantly associated with various TGCT subtypes. In summary, we have developed a high-throughput tool for the evaluation of TGCT markers, and utilized this to validate five candidate genes whose protein expressions were indeed deregulated in TGCT.

HER-2 and TOP2A coamplification in urinary bladder cancer.

HER-2/NEU is one of the most frequently amplified oncogenes and a potential therapeutic target in bladder cancer. In breast cancer, the adjacent TOP2A gene, the molecular target for several anticancer drugs, is frequently coamplified together with HER-2. To study the amplification and expression of TOP2A and HER-2 and associations with tumor phenotype and clinical outcome in bladder cancer, a tissue microarray containing 2,317 bladder tumor samples was analyzed by FISH and immunohistochemistry. Overall amplification frequencies were 6.3% for HER-2 and 1.5% for TOP2A. Amplifications were most frequently seen in advanced-stage (pT2-4) tumors (HER-2 13.8%, TOP2A 3.4%). Of HER-2-amplified tumors, 56% also had alterations of TOP2A, including 14.7% coamplifications, 33.3% gains and 8% deletions. Only 17.6% of TOP2A amplifications occurred independently of HER-2 alterations. Both HER-2 and TOP2A amplifications were significantly associated with advanced tumor stage (HER-2 p < 0.0001, TOP2A p = 0.0218), high grade (p < 0.0001 for both) and protein overexpression (p < 0.0001 for both). TOP2A amplification and overexpression were linked to shortened survival in muscle-invasive tumors (p = 0.0042 and 0.0077, respectively). In summary, our data suggest that HER-2 amplifications are frequently linked to alterations of the TOP2A gene in bladder cancer. The anatomy of the 17q12-q21 amplicon may be important for response to therapies targeting HER-2 or TOP2A.

High frequency of BRAF mutations in nevi.

To evaluate the timing of mutations in BRAF (v-raf murine sarcoma viral oncogene homolog B1) during melanocytic neoplasia, we carried out mutation analysis on microdissected melanoma and nevi samples. We observed mutations resulting in the V599E amino-acid substitution in 41 of 60 (68%) melanoma metastases, 4 of 5 (80%) primary melanomas and, unexpectedly, in 63 of 77 (82%) nevi. These data suggest that mutational activation of the RAS/RAF/MAPK pathway in nevi is a critical step in the initiation of melanocytic neoplasia but alone is insufficient for melanoma tumorigenesis.

High-throughput copy number analysis of 17q23 in 3520 tissue specimens by fluorescence in situ hybridization to tissue microarrays.

The chromosomal region 17q23 has been shown to be commonly amplified in breast tumors, especially those with poor prognosis. In addition to breast cancer, studies by comparative genomic hybridization have implicated the involvement of 17q23 in other tumor types as well. Here we performed a large-scale survey on the distribution and frequency of the 17q23 copy number increases across different tumor types using fluorescence in situ hybridization on tissue microarrays containing 4788 specimens. A total of 4429 tumor samples representing 166 different tumor categories and 359 normal tissue samples from 40 different tissue categories were analyzed. Successful hybridizations were observed in 3520 of the 4788 specimens (74%). Increased 17q23 copy number was detected in 15% of the evaluable specimens with tumors originating from the lung, mammary gland, and soft tissue being most frequently affected. Interestingly, high-level amplification was detected only in 2% of the tumors and was generally restricted to mammary tumors. In addition, we observed an association between the frequency of increased 17q23 copy number and tumor progression in various tumor types. These results indicate that increased 17q23 copy number occurs frequently in several different tumor types suggesting that increased dosage of genes in this region might play a role in development and progression of many tumor types.

Amplification pattern of 12q13-q15 genes (MDM2, CDK4, GLI) in urinary bladder cancer.

The chromosomal region 12q13-q15 is recurrently amplified in bladder cancer. Putative target genes located in this region include MDM2, CDK4, and GLI. To evaluate the involvement of these genes in bladder cancer, we screened a tissue microarray (TMA) containing 2317 samples by fluorescence in situ hybridization (FISH). Amplification was found for MDM2 in 5.1%, for CDK4 in 1.1%, and for GLI in 0.4% of interpretable tumors. Among tumors having amplification of at least one of these 12q13-q15 genes, 76.6% had amplification of MDM2 alone and 6.4% had amplification of CDK4 alone. Coamplifications were seen of MDM2 and CDK4 in 10.6%, and of CDK4 and GLI in 6.4%. Neither coamplifications of all three genes nor isolated GLI amplifications were found. These data suggest a prominent role of MDM2 as a 12q13-q15 amplification target in bladder cancer. However, independent CDK4 amplifications do also occur suggesting either two non-overlapping amplification sites or else a minimal overlapping region between MDM2 and CDK4 perhaps containing another yet unknown oncogene. The frequency of amplification increased significantly from stage pTa to pT1-4 (P<0.04) and from low to high grade (P<0.005). These data are consistent with a high level of genetic instability in invasively growing and high-grade bladder tumors.

Clinical validation of candidate genes associated with prostate cancer progression in the CWR22 model system using tissue microarrays.

To explore molecular mechanisms of prostate cancer progression, we applied tissue microarrays (TMAs) to analyze expression of candidate gene targets discovered by cDNA microarray analysis of the CWR22 xenograft model system. A TMA with 544 clinical specimens from different stages of disease progression was probed by mRNA in situ hybridization and protein immunohistochemistry. There was an excellent correlation (r = 0.96; n = 16) between the expression levels of the genes in the xenografts by cDNA microarray and mRNA in situ hybridization on a TMA. One of the most highly overexpressed genes in hormone-refractory CWR22R xenografts was the S100P gene. This gene, coding for a calcium signaling molecule implicated in the loss of senescence, was also significantly associated with progression in clinical tumors by TMA analysis (P < 0.001), suggesting dysregulation of this pathway in hormone-refractory and metastatic prostate cancers. Conversely, two genes that were down-regulated during tumor progression in the CWR22 model system were validated in vivo: crystallin mu (CRYM) and a LIM-domain protein LMO4 both showed significantly lower mRNA levels in hormone-refractory tumors as compared with primary tumors (P < 0.001). These results illustrate a strategy for rapid clinical validation at the mRNA and protein level of gene targets found to be differentially expressed in cDNA microarray experiments of model systems of cancer.