Differences in body temperature between black-and-white and red-and-white Holstein cows reared on a hot climate using infrared thermography.

Heat stress is one of the greatest issues of the dairy industry in regions with hot climate. Since coat color appears to be related to heat stress adaptiveness, we compared rectal temperatures and surface temperatures of Red-and-white (RW, n = 14) and Black-and-white (BW, n = 16) Holstein cows using infrared thermography in both cold (July; mean temperature: 15.5 °C) and hot (March; mean temperature: 30.5 °C) seasons in Southern Brazil. Thermographic images were taken from the left side of the animal at a distance of 4 m. The images obtained were then analyzed using the software Testo IRSoft. The variables obtained by thermography of the body surface include the temperature of non-pigmented patches, obtained using the average of five spots on white patches in a rectangle drawn on the body of the cow from the scapula to the ilium of the cow until the middle of the ribs; the temperature of pigmented patches, obtained using an average of 5 pigmented spots on the same rectangle; the temperature at the hottest spot and the temperature at the coldest spot, within the same rectangle. Rectal temperature measures were taken by a mercury thermometer during milkings. In our findings, during the cold season, RW cows had lower temperatures on the surface of pigmented spots (p = 0.01) but did not differ from BW animals when comparing rectal temperatures (p = 0.70). During the hot season, however, RW cows had lower temperatures on white spots (p = 0.049) as well as lower rectal temperatures (p = 0.029). These results suggest that the red coat phenotype presents less absorption of solar radiation, retaining less heat.

Assessment of PARP protein expression in epithelial ovarian cancer by ELISA pharmacodynamic assay and immunohistochemistry.

Targeting Poly (ADP-ribose) polymerase 1 (PARP-1) involved in base excision repair (BER) has been shown to be a clinically effective treatment strategy in epithelial ovarian cancer (EOC) defective in homologous recombination (HR). The aim of this study was to evaluate fresh EOC tumor tissue in regard to PAR (Poly (ADP-ribose)) concentration as a surrogate marker for PARP activity and PARP protein expression in archival samples by immunohistochemistry (IHC). The prospective study cohort consisted of 57 fresh tumor samples derived from patients undergoing primary (n = 38) or interval debulking surgery (n = 19) for EOC and parallel archival paraffin-embedded tumor samples. PARP activity in fresh frozen tumor tissue was assessed by an enzymatic chemiluminescence assay and PARP protein expression in paraffin-embedded tumor tissue by IHC. No correlation was detected between PARP enzyme activity and PARP staining by IHC (p = 0.82). High PARP activity was associated with platinum sensitivity both in the entire study cohort (p = 0.022) and in the high-grade subgroup (p = 0.017). High PARP activity was also associated with improved progression-free survival (PFS) (32 vs 14 months, log-rank p = 0.009). However, PARP immunostaining pattern was not predictive of patient survival. In conclusion, we present a novel finding of high PARP activity associated with platinum sensitivity and improved PFS in EOC. There was no association between PARP IHC and pharmacodynamic assay, and the correlation of PARP IHC with clinico-pathological characteristics and patient survival was poor. Pharmacodynamic assay rather than IHC seems to reflect better biologically significant PARP.

Correction of rectal sacculation through lateral resection in dogs with perineal hernia - technique description / Correção de saculação retal por meio de ressecção lateral em cães com hérnia perineal - descrição da técnica

The occurrence of perineal hernias in dogs during routine clinical surgery is frequent. The coexistence of rectal diseases that go undiagnosed or are not correctly treated can cause recurrence and postoperative complications. The objective of this report is to describe a surgical technique for treatment of rectal sacculation through lateral resection in dogs with perineal hernia, whereby restoring the rectal integrity.

A ocorrência de hérnias perineais em cães na rotina clínica cirúrgica é frequente. A coexistência de doenças do reto não diagnosticadas ou não tratadas corretamente pode causar recidiva e complicações pós-operatórias. Este relato tem como objetivo descrever uma técnica cirúrgica para o tratamento de saculação retal por meio de ressecção lateral em cães com hérnia perineal, com restabelecimento da integridade retal.

Very high quantitative tumor HER2 content and outcome in early breast cancer.

BACKGROUND: It is unknown how a very high tumor total HER2 (human epidermal growth factor receptor-2) content (H2T) influences outcome in early breast cancer treated with adjuvant trastuzumab plus chemotherapy. PATIENTS AND METHODS: H2T was measured using a novel quantitative assay (HERmark(®)) from formalin-fixed tumor tissue of 899 women who participated in the FinHer trial (ISRCTN76560285). In a chromogenic in situ hybridization (CISH) test, 197 (21.9%) patients had HER2-positive cancer and were randomly assigned to receive trastuzumab or control. RESULTS: Cancer H2T levels varied 1808-fold. High H2T levels were correlated with a positive HER2 status by CISH (P < 0.0001). A nonlinear association was present between H2T and the hazard of distant recurrence in a subpopulation treatment effect pattern plot analysis in CISH-positive disease. Patients with very high H2T (defined by ≥22-fold the median of HER2-negative cancers; 13% of CISH-positive cancers) did not benefit from adjuvant trastuzumab [hazard ratio (HR) 1.23; 95% confidence interval (CI) 0.33-4.62; P = 0.75], whereas the rest of the patients with HER2-positive disease by CISH (87%) did benefit (HR 0.52; 95% CI 0.28-1.00; P = 0.050). CONCLUSION: Patients with HER2-positive breast cancer with very high tumor HER2 content may benefit less from adjuvant trastuzumab compared with those whose cancer has more moderate HER2 content.

Exogenous sex hormone use and risk of meningioma: a population-based case-control study in Finland.

BACKGROUND: Previous studies on association of exogenous female sex hormones and risk for meningioma have yielded conflicting results. The aim of this study was to evaluate the potential relation between prior use of menopausal hormone therapy or oral contraception and risk of meningioma. METHODS: This population-based case-control study was conducted during years 2000-2002 in Finland. All women aged 20-69 years with meningioma diagnosis were identified from five university hospitals, and frequency-matched controls were randomly chosen from population register. A total of 264 cases and 505 controls were interviewed on their use of menopausal hormone therapy, oral and other contraception, fertility treatment, treatment for gynecological problems, age at menarche, and number of children. We also analyzed separately tumors expressing progesterone or estrogen receptors. Of the successfully stained tumor specimens, 86.3% were positive for progesterone receptor and 50% for estrogen receptor. RESULTS: Postmenopausal hormonal treatment, use of contraceptives, or fertility treatment did not influence the risk of meningioma. In further analysis by hormone receptor status, there was some indication for an increased risk of progesterone receptor-positive meningiomas associated with oral contraceptive use (OR 1.39, 95% confidence interval 0.92-2.10) and other hormonal contraception (OR 1.50, 95% CI 0.95-2.36). CONCLUSIONS: Overall, we found little indication that reproductive factors or use of exogenous sex hormones affect meningioma risk.

A quality assurance exercise to evaluate the accuracy and reproducibility of chromogenic in situ hybridisation for HER2 analysis in breast cancer.

BACKGROUND: Chromogenic in situ hybridisation (CISH) is an alternative to immunohistochemistry or FISH for the assessment of HER2 oncogene status in breast cancer. Although CISH is being used increasingly in routine diagnostics, there are no established inter-laboratory quality assurance programmes for this test. METHODS: The reproducibility of HER2 CISH analysis was assessed when performed by seven different centres that use the test routinely in diagnostic service. RESULTS: The results from 28 cases showed overall concordance of 98.5% (192/195 tests; kappa coefficient 0.91). One of the discrepancies was due to the invasive carcinoma having been cut out in the sections received by two of the centres, and the other two were in the non-amplified/equivocal/low-amplified category. CONCLUSION: This is believed to be the first report of a quality assurance study assessing laboratories that use HER2 CISH routinely in clinical diagnostics. The results show that CISH is a robust technique providing a suitable assay for the frontline testing of HER2 status in breast cancer.

A public-domain image processing tool for automated quantification of fluorescence in situ hybridisation signals.

AIMS: To develop and evaluate an automated method for quantification of HER2 fluorescence in situ hybridisation (FISH) signals. METHODS: Using a popular, open source image manipulation tool, ImageJ, a macro for FISH signal assessment was created. A comparison against traditional manual counting was performed in breast cancer specimens from 42 patients. The tumour specimens were hybridised with probes for HER2 and chromosome 17 centromere (CEP17) and selected areas were digitised for image processing. Hybridisation signals were calculated both manually and automatically with the ImageJ custom macro. RESULTS: The correlation coefficient between the automatic and manual HER2/CEP17 ratios was 0.98. The corresponding percentage agreement was 90% and the kappa value was 0.82. CONCLUSIONS: This study shows that it is possible to automate the determination of HER2 amplification by the use of open-source software, with results comparable to manual counting. The automated counting decreases the time needed for sample analysis and provides possibilities to enhance inter- and intralaboratory reproducibility of results. The FISH quantification tool (FishJ) is available for download as an ImageJ macro or alternatively it can be utilised through a web interface with an option of uploading FISH images for hybridisation signal counting. Combined with digitisation of FISH samples, the FishJ macro enables gene copy number to be assessed and re-evaluated on any area of a digitised specimen.

Gluten affects epithelial differentiation-associated genes in small intestinal mucosa of coeliac patients.

In coeliac disease gluten induces an immunological reaction in genetically susceptible patients, and influences on epithelial cell proliferation and differentiation in the small-bowel mucosa. Our aim was to find novel genes which operate similarly in epithelial proliferation and differentiation in an epithelial cell differentiation model and in coeliac disease patient small-bowel mucosal biopsy samples. The combination of cDNA microarray data originating from a three-dimensional T84 epithelial cell differentiation model and small-bowel mucosal biopsy samples from untreated and treated coeliac disease patients and healthy controls resulted in 30 genes whose mRNA expression was similarly affected. Nine of 30 were located directly or indirectly in the receptor tyrosine kinase pathway starting from the epithelial growth factor receptor. Removal of gluten from the diet resulted in a reversion in the expression of 29 of the 30 genes in the small-bowel mucosal biopsy samples. Further characterization by blotting and labelling revealed increased epidermal growth factor receptor and beta-catenin protein expression in the small-bowel mucosal epithelium in untreated coeliac disease patients compared to healthy controls and treated coeliac patients. We found 30 genes whose mRNA expression was affected similarly in the epithelial cell differentiation model and in the coeliac disease patient small-bowel mucosal biopsy samples. In particular, those genes involved in the epithelial growth factor-mediated signalling pathways may be involved in epithelial cell differentiation and coeliac disease pathogenesis. The epithelial cell differentiation model is a useful tool for studying gene expression changes in the crypt-villus axis.

Quantitative real-time PCR analysis and microarray-based RNA expression of HER2 in relation to outcome.

BACKGROUND: Our aim was to use quantitative real-time PCR (Q-PCR) and RNA expression profiles (RNA-EPs) to investigate HER2 status in relation to outcome. PATIENTS AND METHODS: Cut-off levels for Q-PCR and RNA-EP were established in relation to immunohistochemistry (IHC) validated by FISH in a test set of frozen tissue samples from 40 primary breast cancers. The HER2 status was subsequently studied in another validation set of 306 tumors, where Q-PCR and RNA-EP results were compared with previously carried out IHC that we had validated by chromogenic in situ hybridization (CISH). RESULTS: Q-PCR and RNA-EP offered similar sensitivity (90% versus 77%), specificity (93% versus 95%), and negative (99% versus 98%) and positive (63% versus 61%) predictive values for HER2 determinations. Analyses of relapse-free survival (RFS) and overall survival on the basis of 5 and 10 years of follow-up indicated equivalent hazard ratios for all three techniques. In contrast to IHC/CISH, both Q-PCR and RNA-EP analyses of HER2 also gave statistically significant results regarding RFS and breast cancer-corrected survival after 10 years of follow-up. CONCLUSION: The use of RNA-EP and Q-PCR to analyze HER2 in frozen and formalin-fixed breast cancer samples may be an alternate approach to IHC in combination with FISH/CISH.

p-53 gene mutations as a predictive marker in a population of advanced breast cancer patients randomly treated with doxorubicin or docetaxel in the context of a phase III clinical trial.

BACKGROUND: Preclinical data indicate that p-53 gene mutations predict resistance to doxorubicin (A) but not to docetaxel (Taxotere) (T). In the TAX 303 trial, A and T have been compared with advanced breast cancer patients. PATIENTS AND METHODS: Primary tumor samples from patients participating in the TAX 303 trial were collected. p-53 gene mutations were evaluated by denaturing high-performance liquid chromatography (DHPLC) and confirmed by sequencing. Topoisomerase II alpha (topo II alpha) protein levels were evaluated by immunohistochemistry. Clinical and biological data were correlated. RESULTS: Tumor samples for DHPLC analysis were available for 108 of 326 patients from the clinical trial. p-53 gene mutations were observed in 20% of patients. In patients with a mutated p-53 gene, a trend for a lower percentage of responders was observed in the A arm (17%) compared with the T arm (50%). In the wild-type p-53 cohort, response rates to A and T were 27% and 36%, respectively. Of the 16 patients carrying wild-type p-53- and topo II protein-positive tumors, seven (44%) responded to anthracyclines, while response rate to the same drug was 13% in the remaining cohorts [odds ratio 5.06 (95% confidence interval 1.19-21.41), P = 0.03]. The combination of the two markers had no predictive value in patients treated with docetaxel. CONCLUSIONS: (i) p-53 gene analysis indicates that gene mutations may compromise the efficacy of A while they do not interfere with the antitumor activity of T; and (ii) the evaluation of multiple molecular markers including p-53 and proliferation markers as topo II protein levels looks more promising in predicting response to anthracyclines.

Amplification of the epidermal growth factor receptor in astrocytic tumours by chromogenic in situ hybridization: association with clinicopathological features and patient survival.

Chromogenic in situ hybridization (CISH) was used to detect amplification of the epidermal growth factor receptor (EGFR) gene in tissue microarrays of tumours derived from 287 patients with grade II-IV diffuse astrocytomas. Amplification was found in 32% of the tumours with a highly significant association with histological grade (4% in grade II, 21% in grade III and 39% in grade IV; P < 0.001). Amplification of the EGFR gene was more common in primary than in secondary glioblastomas (41%vs. 16%, P = 0.033). Overexpression of EGFR mRNA and protein (wild-type and vIII variant) was found to correlate with EGFR gene amplification (P = 0.028, P = 0.035 and P = 0.014 respectively), but wild-type EGFR protein was also frequently overexpressed in tumours without EGFR gene amplification. Patients with older age (P < 0.001) and tumours with lack of p53 overexpression (P = 0.03) and higher apoptosis rate (P < 0.001) had significantly more EGFR gene amplifications than their counterparts. No such correlation with apoptosis was found in glioblastomas. The survival of patients with EGFR gene-amplified grade III tumours was significantly shorter than in those with grade III non-amplified tumours (P = 0.03). No such difference was noted in glioblastomas (grade IV tumours). Our data verify the central role of EGFR in the pathobiology of astrocytic tumours, and highlight the advantages of CISH as a simple and practical assay to screen for EGFR gene amplification in astrocytic tumours.

Dual-colour chromogenic in situ hybridization for testing of HER-2 oncogene amplification in archival breast tumours.

Chromogenic in situ hybridization (CISH), which uses an enzymatic reaction to detect the hybridized DNA probe, is a new alternative to fluorescence in situ hybridization (FISH) for the assessment of HER-2 oncogene amplification status in breast cancer. The main advantage of CISH over FISH is the use of bright-field microscopy, which is rapid and allows the histopathological evaluation of tumour tissue sections. The main disadvantage of CISH has been the use of a single probe, thereby making it necessary to hybridize the control probe (chromosome 17 centromere) on an adjacent tissue section. The present paper presents an efficient protocol for dual-colour CISH (dc-CISH) based on the co-hybridization of probes to the HER-2 oncogene and chromosome 17 centromere. The probes were detected sequentially with antibodies to digoxigenin and biotin and with secondary antibody polymers labelled with horseradish peroxidase and alkaline phosphatase. The peroxidase reaction was visualized with tetramethyl benzidine (green reaction product) and the alkaline phosphatase reaction with New Fuchsin (red reaction product). The accuracy of the method was verified by comparing the results for four cell lines and 40 tumour samples with those obtained using FISH (Vysis Inc.). The results of FISH and dc-CISH showed high concordance (91%, Kappa coefficient = 0.82). It is concluded that dual-colour CISH, which is a new alternative to FISH enables the assessment of copy number ratio (HER-2/17 centromere) in conjunction with proper histopathological evaluation and the ease of bright-field microscopy.

Epithelial syndecan-1 expression is associated with stage and grade in colorectal cancer.

OBJECTIVES: Loss of epithelial heparan sulfate proteoglycan syndecan-1 has been associated with a more aggressive behavior in various cancer forms, but the prognostic significance of syndecan-1 expression in colorectal cancer is unclear. The aim of this study was to evaluate the prognostic value of immunohistochemical syndecan-1 expression in a series of 237 patients with colorectal cancer. METHODS: Paraffin-embedded formalin-fixed specimens were stained with a syndecan-1-specific monoclonal antibody, and both the epithelial and stromal expression were analyzed. RESULTS: Epithelial expression of syndecan-1 was seen in 222 tumors (94%), and it was associated with low stage of disease (p = 0.002) and low histological differentiation grade (p = 0.048). The cumulative 5-year survival of patients with weak and strong syndecan-1 expression was 49 and 54 %, respectively (p = 0.234). Syndecan-1 stromal immunoreactivity was observed in 138 tumors (58%), but lacked prognostic significance. Staining pattern and distribution can be viewed from digitized representative microscope slides (virtual slides) at http://www.webmicroscope.net/supplements/syndecan. CONCLUSIONS: The results are in line with previous reports in that low epithelial syndecan-1 expression was associated with a higher histological grade and a more advanced clinical stage of the patients. This study shows that syndecan-1 is expressed also in stromal tissue of colorectal cancer, but it does not support the proposed role of stromal syndecan-1 expression as a marker of poor clinical outcome.

No GIST-type c-kit gain of function mutations in neuroblastic tumours.

AIMS: Neuroblastic tumours (NTs) have been shown to respond to imatinib treatment in vivo and in vitro, possibly via inactivating the c-kit receptor. The purpose of this study was to identify gastrointestinal stromal tumour (GIST)-type c-kit gene associated mutations in exons 9, 11, 13, and 17 in NTs to recognise a subset of tumours that would probably respond to imatinib treatment. METHODS: Expression of the c-kit protein was detected immunohistochemically in a total of 37 archival paraffin wax embedded NTs using polyclonal rabbit antihuman c-kit antibody. After immunohistochemistry, c-kit gene associated chromosomal mutations in all cases of NT were detected with denaturing high performance liquid chromatography (HPLC). RESULTS: Denaturing HLPC analysis did not reveal GIST-type mutations in four immunohistochemically detected c-kit positive or in 33 c-kit negative NTs. CONCLUSIONS: c-kit receptor expression and GIST-type c-kit gene mutations are rare events in NTs. Oncogenic activation of c-kit in NTs presumably differs from that of GISTs, which may influence their responsiveness to imatinib treatment. Whether c-kit has an essential role in the pathogenesis of NTs remains to be investigated.

Ultrarapid Ki-67 immunostaining in frozen section interpretation of gliomas.

BACKGROUND: Astrocytic tumours, the most common gliomas, are often classified intraoperatively using standard morphological staining. The final diagnosis and grading of gliomas on paraffin wax sections is often assisted by Ki-67 immunohistochemistry, but standard immunostaining protocols take too long to be used intraoperatively. AIMS: To investigate a new rapid Ki-67 immunohistochemical test for its use in an intraoperative setting. METHODS: The new Ki-67 immunostaining (Ultrarapid-Ki67) method on frozen sections can be carried out in 10 minutes. Thirty four pilocytic and diffuse astrocytomas were immunostained by rapid Ki-67 and results were compared with corresponding MIB-1 staining, histological grading, and prognosis. RESULTS: The staining protocol was practical to perform and the results were morphologically and quantitatively indistinguishable from those after immunostaining with MIB-1, an antibody recognising Ki-67 in paraffin wax embedded tissue. A comparison of Ultrarapid-Ki67 and MIB-1 immunostaining of paraffin wax sections showed almost identical quantitative correlation in astrocytic gliomas (r = 0.916; p<0.001). The Ultrarapid-Ki67 indices (percentage of positive cells) of low grade (I/II) astrocytomas ranged from 0% to 6.1%, whereas those of representative high grade (III/IV) tumours were significantly higher (range, 5.6-45%; p<0.001). The best prognostic cutoff point for Ultrarapid-Ki67 was 7.5%, which divided diffuse grade II-IV astrocytomas into significantly differing subsets (p = 0.0008). CONCLUSION: Ultrarapid-Ki67 immunostaining is a useful adjunct to morphological diagnosis and grading of astrocytic tumours, and as a fast test (approximately 10 minutes for staining plus three to four minutes for scoring), it could be used in routine intraoperative diagnosis of gliomas and other neoplastic diseases.

Amplification of HER-2 in gastric carcinoma: association with Topoisomerase IIalpha gene amplification, intestinal type, poor prognosis and sensitivity to trastuzumab.

BACKGROUND: HER-2/neu gene amplification has predictive value in breast cancer patients responding to trastuzumab. We wanted to investigate the frequency and clinical significance of HER-2/neu amplification in gastric carcinoma. PATIENTS AND METHODS: The frequency of HER-2/neu and Topoisomerase IIalpha gene amplification was studied in adenocarcinomas of the stomach (n=131) and the gastroesophageal junction (n=100) by chromogenic in situ hybridization (CISH). Sensitivity of a gastric cancer cell line N87 with HER-2/neu amplification to trastuzumab was studied by a cell viability assay and compared with that of a HER-2 amplified breast cancer cell line SKBR-3. Growth inhibition of N87 cells was also verified in vivo in N87 xenograft tumors. RESULTS: HER-2/neu amplification was present in 16 (12.2%) of the 131 gastric and in 24 (24.0%) of the 100 gastroesophageal adenocarcinomas. Co-amplification of Topoisomerase IIalpha was present in the majority of gastric (63%) and esophagogastric junction cancers (68%) with HER-2/neu amplification. HER-2/neu amplification was more common in the intestinal histologic type of gastric cancer (21.5%) than in the diffuse (2%) or the mixed/anaplastic type (5%, P=0.0051), but it was not associated with gender, age at diagnosis or clinical stage. Presence of HER-2/neu amplification was associated with poor carcinoma-specific survival (P=0.0089). HER-2/neu targeting antibody trastuzumab inhibited the growth of a p185(HER-2/neu) overexpressing gastric and breast carcinoma cell lines (N87 and SKBR-3) with equal efficacy. CONCLUSIONS: HER-2/neu amplification is common in the intestinal type of gastric carcinoma, and it is associated with a poor outcome. HER-2 might be a useful target in this disease, and this hypothesis deserves to be investigated in clinical trials.

A digital atlas of breast histopathology: an application of web based virtual microscopy.

AIMS: To develop an educationally useful atlas of breast histopathology, using advanced web based virtual microscopy technology. METHODS: By using a robotic microscope and software adopted and modified from the aerial and satellite imaging industry, a virtual microscopy system was developed that allows fully automated slide scanning and image distribution via the internet. More than 150 slides were scanned at high resolution with an oil immersion x 40 objective (numerical aperture, 1.3) and archived on an image server residing in a high speed university network. RESULTS: A publicly available website was constructed, http://www.webmicroscope.net/breastatlas, which features a comprehensive virtual slide atlas of breast histopathology according to the World Health Organisation 2003 classification. Users can view any part of an entire specimen at any magnification within a standard web browser. The virtual slides are supplemented with concise textual descriptions, but can also be viewed without diagnostic information for self assessment of histopathology skills. CONCLUSIONS: Using the technology described here, it is feasible to develop clinically and educationally useful virtual microscopy applications. Web based virtual microscopy will probably become widely used at all levels in pathology teaching.

HER-2/neu as a predictive marker in a population of advanced breast cancer patients randomly treated either with single-agent doxorubicin or single-agent docetaxel.

PURPOSE: To evaluate the predictive value of HER-2 in a population of advanced breast cancer patients randomly treated either with single-agent doxorubicin (A) or with single-agent docetaxel (T). EXPERIMENTAL DESIGN: Patients from this study participated in a phase III clinical trial in which doxorubicin or docetaxel was administered for advanced disease. HER-2 was evaluated by IHC. In all positive cases, FISH was used to confirm the HER-2 positive status. The different cohorts of patients identified by HER-2 were examined to assess a possible relationship between HER-2 status and treatment effect. RESULTS: Tumor samples were available for 176 of the 326 patients entered in the clinical trial (54%). HER-2 positivity was observed in 20% of the study population. A statistically significant interaction was found between response rates to the study drugs and HER-2 status, with HER-2 positive patients deriving the highest benefit from the use of T (odds ratio for HER-2 positive patients treated with T = 3.12 (95% CI 1.11-8.80), p = 0.03). The interaction between HER-2 and response rates to A and T was also confirmed by a multivariate analysis. No statistically significant interaction was found between HER-2 and drugs efficacy evaluated in terms of time to progression and overall survival, although in the HER-2 negative cohort A was at least as effective as T in term of overall survival. CONCLUSIONS: These results suggest that in HER-2 positive breast cancer patients docetaxel might be more active than doxorubicin, while in HER-2 negative patients doxorubicin might be at least as effective as docetaxel. Although the present results cannot have an impact on current practice, they allow us to formulate the hypothesis that HER-2 positive breast cancer is a heterogeneous disease with regard to sensitivity to anthracyclines and taxanes, and that this might be dependent upon other molecular markers including the p-53 and topoisomerase II alpha genes. This hypothesis is currently being tested prospectively in two different 'bench to bed-side' clinical trials.

HER2 oncogene amplification in extramammary Paget's disease.

AIMS: To study HER2 oncogene amplification and over-expression in skin samples of 23 patients with extramammary Paget's disease (EMP). EMP is a rare intra-epidermal adenocarcinoma, which has been reported to over-express the HER2 oncoprotein. METHODS AND RESULTS: HER2 gene amplification, detected by chromogenic in-situ hybridization, was found in 43% (10/23) of the lesions. HER2 protein over-expression (3+ immunostaining intensity) was found in 12 tumours (52%), including all 10 tumours with gene amplification. Two tumours showed low-level (2+) HER2 immunostaining. Mammary Paget's lesions, which were used as controls, showed HER2 amplification and over-expression in all 10 cases studied. CONCLUSIONS: These results indicate that HER-2 protein over-expression in EMP is common and due exclusively to gene amplification. They open up the possibility of HER2-targetted immunotherapy for patients with HER2+ disease.