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Supplementary Table 1 from FBXW7/hCDC4 Is a General Tumor Suppressor in Human Cancer

Histologic grading of breast cancer defines morphologic subtypes informative of metastatic potential, although not without considerable interobserver disagreement and clinical heterogeneity particularly among the moderately differentiated grade 2 (G2) tumors. We posited that a gene expression signature capable of discerning tumors of grade 1 (G1) and grade 3 (G3) histology might provide a more objective measure of grade with prognostic benefit for patients with G2 disease. To this end, we studied the expression profiles of 347 primary invasive breast tumors analyzed on Affymetrix microarrays. Using class prediction algorithms, we identified 264 robust grade-associated markers, six of which could accurately classify G1 and G3 tumors, and separate G2 tumors into two highly discriminant classes (termed G2a and G2b genetic grades) with patient survival outcomes highly similar to those with G1 and G3 histology, respectively. Statistical analysis of conventional clinical variables further distinguished G2a and G2b subtypes from each other, but also from histologic G1 and G3 tumors. In multivariate analyses, genetic grade was consistently found to be an independent prognostic indicator of disease recurrence comparable with that of lymph node status and tumor size. When incorporated into the Nottingham prognostic index, genetic grade enhanced detection of patients with less harmful tumors, likely to benefit little from adjuvant therapy. Our findings show that a genetic grade signature can improve prognosis and therapeutic planning for breast cancer patients, and support the view that low- and high-grade disease, as defined genetically, reflect independent pathobiological entities rather than a continuum of cancer progression. (Cancer Res 2006; 66(21): 10292-301)

Supplementary File 2 from Genetic Reclassification of Histologic Grade Delineates New Clinical Subtypes of Breast Cancer

The ubiquitin-proteasome system is a major regulatory pathway of protein degradation and plays an important role in cellular division. Fbxw7 (or hCdc4), a member of the F-box family of proteins, which are substrate recognition components of the multisubunit ubiquitin ligase SCF (Skp1-Cdc53/Cullin-F-box-protein), has been shown to mediate the ubiquitin-dependent proteolysis of several oncoproteins including cyclin E1, c-Myc, c-Jun, and Notch. The oncogenic potential of Fbxw7 substrates, frequent allelic loss in human cancers, and demonstration that mutation of FBXW7 cooperates with p53 in mouse tumorigenesis have suggested that Fbxw7 could function as a tumor suppressor in human cancer. Here, we carry out an extensive genetic screen of primary tumors to evaluate the role of FBXW7 as a tumor suppressor in human tumorigenesis. Our results indicate that FBXW7 is inactivated by mutation in diverse human cancer types with an overall mutation frequency of ∼6%. The highest mutation frequencies were found in tumors of the bile duct (cholangiocarcinomas, 35%), blood (T-cell acute lymphocytic leukemia, 31%), endometrium (9%), colon (9%), and stomach (6%). Approximately 43% of all mutations occur at two mutational “hotspots,” which alter Arg residues (Arg465 and Arg479) that are critical for substrate recognition. Furthermore, we show that Fbxw7Arg465 hotspot mutant can abrogate wild-type Fbxw7 function through a dominant negative mechanism. Our study is the first comprehensive screen of FBXW7 mutations in various human malignancies and shows that FBXW7 is a general tumor suppressor in human cancer. [Cancer Res 2007;67(19):9006–12]

Supplementary File 3 from Genetic Reclassification of Histologic Grade Delineates New Clinical Subtypes of Breast Cancer

Supplementary File 1 from Genetic Reclassification of Histologic Grade Delineates New Clinical Subtypes of Breast Cancer

A scoring system for judging the aggressiveness of basal cell carcinomas of the head and neck is devised, based upon 258 tumours from 123 patients. This system is discussed, and we suggest using the depth of growth, the degree of palisading and the narrow epithelial strand formation (narrow strands) as a scoring basis. It is shown that tumours located on the ear are more aggressive than tumours in other places. Tumours on the nose often recur, even though their histological picture is not that aggressive. This phenomenon is also discussed. For proper scoring, an excisional biopsy should be performed unless the tumour is very small. Recurrent tumours and tumours treated with X-ray also have a higher score.

The ubiquitin-proteasome system is a major regulatory pathway of protein degradation and plays an important role in cellular division. Fbxw7 (or hCdc4), a member of the F-box family of proteins, which are substrate recognition components of the multisubunit ubiquitin ligase SCF (Skp1-Cdc53/Cullin-F-box-protein), has been shown to mediate the ubiquitin-dependent proteolysis of several oncoproteins including cyclin E1, c-Myc, c-Jun, and Notch. The oncogenic potential of Fbxw7 substrates, frequent allelic loss in human cancers, and demonstration that mutation of FBXW7 cooperates with p53 in mouse tumorigenesis have suggested that Fbxw7 could function as a tumor suppressor in human cancer. Here, we carry out an extensive genetic screen of primary tumors to evaluate the role of FBXW7 as a tumor suppressor in human tumorigenesis. Our results indicate that FBXW7 is inactivated by mutation in diverse human cancer types with an overall mutation frequency of ∼6%. The highest mutation frequencies were found in tumors of the bile duct (cholangiocarcinomas, 35%), blood (T-cell acute lymphocytic leukemia, 31%), endometrium (9%), colon (9%), and stomach (6%). Approximately 43% of all mutations occur at two mutational “hotspots,” which alter Arg residues (Arg465 and Arg479) that are critical for substrate recognition. Furthermore, we show that Fbxw7Arg465 hotspot mutant can abrogate wild-type Fbxw7 function through a dominant negative mechanism. Our study is the first comprehensive screen of FBXW7 mutations in various human malignancies and shows that FBXW7 is a general tumor suppressor in human cancer. [Cancer Res 2007;67(19):9006–12]

Earlier reports of a human exogenous retrovirus (HMTV) related closely to mouse mammary tumor virus (MMTV) led us to search for these viral sequences in breast cancer tissues and normal tissues. A real-time PCR was developed based on MMTV and published HMTV envelope sequences. The real-time PCR method can detect one to ten copies of MMTV target DNA. Tissue samples were collected prospectively from 18 breast cancer patients and 11 non-malignant control cases, as well as peripheral blood leukocytes from the same women. Despite the high sensitivity of the real-time PCR method used, none of the samples were positive for HMTV DNA or RNA. The absence of HMTV DNA in both breast cancer samples and controls indicates either that the concentration of putative HMTV DNA in the breast cancers was too low for detection or that it did not exist there.

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.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).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.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.

To assess the efficacy and safety of epirubicin, capecitabine and cisplatin (EXC) combination therapy in locally advanced breast cancer (LABC) and investigate the predictive value of selected biomarkers.Newly diagnosed LABC patients received four 3-weekly cycles of neoadjuvant EXC (epirubicin 60 mg/m(2) day 1; capecitabine 1000 mg/m(2) bid, days 1-14; cisplatin 60 mg/m(2)day 1) and two cycles of post-operative EXC.Eight (17%) of 48 patients had inflammatory breast cancer. Overall response rate was 74% (95% CI: 59-86%), including complete responses in 13% (95% CI: 5-26%). Nine (22%; 95% CI: 11-38%) of 41 patients undergoing surgery achieved pathologic complete response (pCR), giving a pCR rate of 19% (95% CI: 9-33%) in the intent-to-treat population. Haematological toxicity was manageable. The most problematic toxicities were chemotherapy-induced nausea/vomiting and hypercoagulative disorders. None of the biomarkers investigated, including HER2, predicted response.EXC showed high efficacy in LABC, with high clinical response and pCR rate. Nausea and vomiting were unexpectedly frequent, and more aggressive prophylaxis and management of these side effects is recommended in future studies of this combination.

The aim of this study was to investigate the expression of activated (phosphorylated) ERK1/2, oestrogen receptor alpha phosphorylated at S118 (ERalphaS118), and HER2 in primary breast cancer, and to make correlations with the outcome of tamoxifen therapy. We performed immunohistochemical analysis to determine the expression of HER2, ERalphaS118, and activated ERK1/2 in tumours obtained from 279 women with primary breast cancer. HER2 status was also estimated by fluorescence in situ hybridisation. We identified 108 women with ERalpha-positive tumours who had received adjuvant tamoxifen. Activated ERK1/2 (pERK1/2) and ERalphaS118 were found to be associated with each other and with other factors correlated with good prognosis. HER2 was inversely associated with pERK1/2. Positive staining for pERK1/2 (particularly intense staining) indicated better relapse-free survival (P=0.05) and a trend towards better breast cancer-corrected survival in women treated with tamoxifen. To conclude, this study shows that activated ERK1/2 and ERalphaS118 are associated with improved survival. The poorer outcome in HER2-positive women who receive adjuvant tamoxifen cannot be explained by the crosstalk between HER2 and ERalphaS118 via activated ERK1/2 alone.

Background: Histologic grade in breast cancer provides clinically important prognostic information. However, 30% – 60% of tumors are classifi ed as histologic grade 2. This grade is associated with an intermediate risk of recurrence and is thus not informative for clinical decision making. We examined whether histologic grade was associated with gene expression profi les of breast cancers and whether such profi les could be used to improve histologic grading. Methods: We analyzed microarray data from 189 invasive breast carcinomas and from three published gene expression datasets from breast carcinomas. We identifi ed differentially expressed genes in a training set of 64 estrogen receptor (ER) – positive tumor samples by comparing expression profi les between histologic grade 3 tumors and histologic grade 1 tumors and used the expression of these genes to defi ne the gene expression grade index. Data from 597 independent tumors were used to evaluate the association between relapse-free survival and the gene expression grade index in a Kaplan – Meier analysis. All statistical tests were two-sided. Results: We identifi ed 97 genes in our training set that were associated with histologic grade; most of these genes were involved in cell cycle regulation and proliferation. In validation datasets, the gene expression grade index was strongly associated with histologic grade 1 and 3 status; however, among histologic grade 2 tumors, the index spanned the values for histologic grade 1 – 3 tumors. Among patients with histologic grade 2 tumors, a high gene expression grade index was associated with a higher risk of recurrence than a low gene expression grade index (hazard ratio = 3.61, 95% confi dence interval = 2.25 to 5.78; P

We studied the effect of postoperative radiotherapy (RT) after breast sector resection for ductal carcinoma in situ (DCIS). The study protocol stipulated radical surgery but microscopically clear margins were not mandatory. We randomised 1,046 operated women to postoperative RT or control between 1987 and 1999. The primary endpoint was ipsilateral local recurrence. Secondary endpoints were contralateral breast cancer, distant metastasis and death. After a median follow-up of 5.2 years (range 0.1-13.8) there were 44 recurrences in the RT group corresponding to a cumulative incidence of 0.07 (95% confidence interval (CI) 0.05-0.10). In the control group there were 117 recurrences giving a cumulative incidence of 0.22 (95% CI 0.18-0.26) giving an overall hazard ratio of 0.33 (95% CI 0.24-0.47, p < 0.0001). Twenty two percent of the patients had microscopically unknown or involved margins. We found no evidence for different effects of RT on the relative risk of invasive or in situ recurrence. Secondary endpoints did not differ. Women undergoing sector resection for DCIS under conditions of population based screening mammography benefit from postoperative RT to the breast. Seven patients needed RT-treatment to prevent one recurrence.

The cytochrome P450 mitochondrial enzyme 25-hydroxyvitamin D3 1alpha-hydroxylase (1alpha-hydroxylase) of renal tubule cells hydroxylates the major circulating form of vitamin D (25(OH)D3) to the active systemic hormone 1,25(OH)2D3. Local production of 1,25(OH)2D3 appears to occur also at other sites where 1alpha-hydroxylase is expressed for autocrine/paracrine regulation. To reduce risks of hypercalcemia during treatment with vitamin D, we have previously suggested use of non-1alpha-hydroxylated vitamin D analogues to target tissues where 1alpha-hydroxylase is expressed, including the parathyroid glands in secondary hyperparathyroidism. The present study was undertaken to examine expression of 1alpha-hydroxylase in breast cancer and to investigate whether a non-1alpha-hydroxylated vitamin D analogue displayed biological function. In addition, expression of the 25-hydroxyvitamin D3 24-hydroxylase (24-hydroxylase) and the vitamin D receptor (VDR) was investigated.The expression of 1alpha-hydroxylase, 24-hydroxylase and VDR was investigated in breast cancer specimens (n = 19) and normal breast tissues (n = 10) by immunohistochemistry and/or RT-PCR. Consecutive cryosections of 6 mum essentially free of immune cells were used in the analyses. The effect of vitamin D analogues on transcriptional activation was analyzed in transiently transfected MCF-7 breast cancer cells.1alpha-hydroxylase protein was demonstrated in 79% and 100% of breast cancer specimens and normal breast, respectively. The overall relative mRNA levels of 1alpha-hydroxylase and 24-hydroxylase in normal breast compared to breast tumors were: 1alpha-hydroxylase, 1 +/- 0.07 versus 0.7 +/- 0.05, respectively (p0.001); 24-hydroxylase, 1 +/- 0.08 verus 2.1 +/- 0.2, respectively (p0.001). The VDR was expressed in 95% of the tumors as expected, with mRNA levels of 1 +/- 0.09 and 1.4 +/- 0.12 (p0.05) in breast cancer and normal breast, respectively. The ketoconazole-sensitive transcription activation potential of the non-1alpha-hydroxylated vitamin D analogue prodrug of EB1089 (EB1285) was demonstrated in MCF-7 cells, which express 1alpha-hydroxylase. The activity of EB1285 was about 20% of 1,25(OH)2D3.These results demonstrate nearly normal expression levels of 1alpha-hydroxylase, 24-hydroxylase and VDR in the majority of investigated breast cancer specimens. A non-1alpha-hydroxylated vitamin D analogue displayed activity in breast cancer cells. Such analogues may present future therapeutic options for proliferative disorders where 1alpha-hydroxylase is expressed.

Cyclin E is one of the key regulators of the G(1)/S transition in the cell cycle. Overexpression of cyclin E has been observed in several malignancies and is associated with high proliferation, aberrant expression of other cell cycle regulators and chromosomal instability in vitro. To explore potential associations between cyclin E deregulation and inactivation of the p53 tumor suppressor gene in human breast cancer, we investigated the immunohistochemical expression of cyclin E in paraffin embedded breast cancers from 270 women with known p53 status by cDNA based sequencing of the p53 gene. The breast cancers were divided into three subgroups according to the percentage of cyclin E-positive cells. One hundred and seventy-one patients (63%) had low cyclin E, 72 (27%) medium and 27 (10%) had high cyclin E content. Fifty-six percent (15/27) of the breast cancers with high cyclin E had p53 gene mutations, compared with 14% (24/171) of those with low cyclin E content (P < 0.0001). In p53 mutated breast cancers high cyclin E content was associated with insertions, deletions and nonsense point mutations in the p53 tumor suppressor gene, whereas low cyclin E was linked to p53 missense point mutations. We also observed statistically significant associations between a high cyclin E content and aneuploidy, high S phase, larger tumor size, estrogen receptor negativity, presence of axillary node metastases and high tumor grade. High cyclin E content was associated with poor overall survival in univariate and multivariate analysis (hazard ratio 2.4, 95% confidence interval 1.3-4.5). In summary, our findings demonstrate that overexpression of cyclin E is associated with an aggressive tumor phenotype and specific types of p53 mutations.

Background It is not clear whether risk factors for local recurrence after breast-conserving surgery differ in women having surgery for in situ or invasive cancer. Furthermore, the Nottingham Prognostic Index (NPI) and Nottingham Histological Grade (NHG) have been little studied as determinants of local recurrence risk. Method In a case–control study (491 cases and 1098 controls) nested within a cohort of 7502 women who had surgery for in situ or invasive cancer of the breast, patient characteristics, tumour characteristics and treatment-related variables were evaluated as risk factors for local recurrence. Results Multivariate conditional logistic regression analyses showed that age below 40 years, tumour multicentricity and an unclear or unknown surgical margin were significant risk factors for local recurrence. Radiotherapy to the breast and adjuvant hormone therapy were protective. Cancer in situ was not associated with a higher risk of local recurrence than invasive cancer (odds ratio 1·0, 95 per cent confidence interval 0·8 to 1·3). NHG and NPI were not helpful in determining risk of local recurrence. Conclusion Margin status, age, tumour multicentricity, and use of radiotherapy and adjuvant hormone therapy were important determinants of risk of local recurrence. With the exception of surgical margin, variables related to the quality of surgical management did not predict risk of local recurrence.

In a study of invasive breast cancer, multiple correspondence analysis (MCA) revealed clustering of eight pathobiological variables. Two different phenotypes were distinguished by an index calculated on the basis of the variables (histologic grade, necrosis, lymphoid infiltration, number of mitosis and expression of c-erbB-2, p53, progesterone receptor and Bcl-2). Phenotype A lesions share most of the features of normal breast tissue. Phenotype B looks more malignant, has a higher early recurrence rate and is more frequently seen in younger patients. Our aim was to see if ductal breast carcinoma in situ (DCIS) could be divided into the same phenotypes. One hundred and eighty DCIS were investigated. Association between the eight variables was studied in 2 x 2 models. The phenotype index was calculated by summing weights for the variables in the MCA. All variables were associated, except Bcl-2. DCIS was divided in two phenotypes. Thirty-three tumours were Phenotype A and 147 Phenotype B. The mean age at diagnosis was 65.5 and 58.4 years for Phenotypes A and B, respectively (p = 0.0012). No difference regarding local relapse free survival was seen. Two phenotypes were distinguished in DCIS, similar to invasive breast cancer. In an earlier study, 45% of the invasive cancers were classified as Phenotype B. In this study, 82% of DCIS were Phenotype B. This may indicate that invasive breast cancer of Phenotype B is derived from DCIS of Phenotype B. The distribution of DCIS phenotypes with a small proportion of Phenotype A DCIS may be due to that Phenotype A DCIS is less likely to be detected by mammography, or that some invasive breast cancers of Phenotype A progress to invasiveness without passing the in situ phase.

Vanishing bile duct-like syndrome in a patient with Hodgkin lymphoma – pathological development and restitution

Ductal breast carcinoma in situ (DCIS) is regarded as a precursor to invasive breast cancer. The progression from in situ to invasive cancer is however little understood. We compared some tumour markers in invasive and in situ breast carcinomas trying to find steps in this progression. We designed a semi-experimental setting and compared histopathological grading and tumour marker expression in pure DCIS (n = 194), small invasive lesions (n = 127) and lesions with both an invasive and in situ component (n = 305). Grading was done according to the Elston–Ellis and EORTC classification systems, respectively. Immunohistochemical staining was conducted for p53, c-erbB-2, Ki-67, ER, PR, bcl-2 and angiogenesis. All markers correlated with grade rather than with invasiveness. No marker was clearly associated with the progression from in situ to invasiveness. The expression of tumour markers was almost identical in the 2 components of mixed lesions. DCIS as a group showed a more ‘malignant picture' than invasive cancer according to the markers, probably, due to a higher proportion of poorly differentiated lesions. The step between in situ and invasive cancer seems to occur independently of tumour grade. The results suggest that well-differentiated DCIS progress to well-differentiated invasive cancer and poorly differentiated DCIS progress to poorly differentiated invasive cancer. © 2001 Cancer Research Campaign http://www.bjcancer.com

Purpose: To evaluate the risk of needle track seeding or tumor cell implantation as the cause of locally recurrent breast cancer after breast conserving surgery. Material and Methods: We reviewed recurrences from a consecutive series of 303 clinically nonpalpable breast cancers treated with breast conserving surgery after pre-operative localization. The median mammographic follow-up was 5.4 years. The suspicion of seeding or implantation was based on the location of the recurrent lesion in comparison with the needle path in two orthogonal mammographic projections. Pre-operative percutaneous biopsies had been done in 71% (214/303) of the cases. Postoperative radiotherapy was administered to 82% (194/238) of the invasive cancers and to 28% (18/65) of the ductal cancers in situ (DCIS). Results: Locally recurrent cancer occurred in 11% (33/303) of the cases. Radiotherapy demonstrated a protective effect from relapse among invasive cancers but not for DCIS. Seeding or implantation was suspected in 3 recurrent invasive cancers which had not been subject to radiotherapy. The histopathological diagnosis of the primary cancer and the recurrent cancer were the same in these cases: adenoid cystic, mucinous and tubuloductal cancer. Conclusion: Seeding or implantation was suspected as the cause of local recurrence in 7% (3/44) of the invasive cancers which did not receive radiotherapy.

The present report shows that the human renal cell carcinoma (RCC) cell lines, A498 and CAKI-2, express the estramustine-binding protein (EMBP). The RCC cell lines investigated were highly sensitive for estramustine, with cell arrest in atypical metaphase. In vitro experiments using a fluorimetric cytotoxicity assay (FMCA) showed a pronounced cytotoxic effect mediated by estramustine. Immunohistochemical analysis of tumour specimens from patients with RCC showed positive staining for EMBP in 12/16 cases. Immunoscintigraphy was performed in an experimental system in nude mice, heterotransplanted with the CAKI-2 cell line. A radiolabelled monoclonal anti-EMBP antibody was used. The results show a specific uptake of the antibody in the RCC tumour, expressed as a percentage of the injected dose per gram tissue, which ranged from 4.03 to 6.9. The results obtained form the basis for clinical studies on the feasibility of utilizing estramustine in the management of RCC. Immunoscintigraphy using the monoclonal anti-EMBP antibody is of potential use for in vivo characterization of the malignancy and in the selection patients suitable for treatment with estramustine.

Biopsies from the subacromial bursa were obtained from 30 patients with an impingement syndrome and from an autopsy series of 13 "normal" shoulders. Bursal fibrosis seemed to be a characteristic of the patient group; inflammatory cells, found in 7/30 specimens from the patients, were not found in the autopsy series. The microscopic findings could not be predicted from peroperative observations. There was an association between a poor outcome of surgery and absence of bursal fibrosis.

Members of the epidermal growth factor receptor, EGFR, family are interesting as targets for radionuclide therapy using targeting agents labeled with α- or β-emitting radionuclides, especially when EGFR-positive colorectal carcinomas, CRC, are resistant to EGFR inhibiting agents like cetuximab and various tyrosine kinase inhibitors. The expression of EGFR, HER2 and HER3 was therefore analyzed in CRC samples from primary tumors, corresponding lymph node metastases and, in a few cases, liver metastases. The expression of HER2 and EGFR was scored from immunohistochemical preparations using the HercepTest criteria 0, 1+, 2+ or 3+ for cellular membrane staining while HER3 expression was scored as no, weak or strong cytoplasm staining. Material from 60 patients was analyzed. The number of EGFR 2+ or 3+ positive primary tumors was 16 out of 56 (29%) and for lymph node metastases 8 out of 56 (14%) whereas only one out of nine (11%) liver metastases were positive. Thus, there was lower EGFR positivity in the metastases. Only one among 53 patients was strongly HER2 positive and this in both the primary tumor and the metastasis. Eight out of 49 primary tumors (16%) were strongly HER3 positive and the corresponding numbers for lymph node metastases were 9 out of 49 (18%) and for liver metastases 2 out of 9 (22%). The observed number of strongly EGFR positive cases was somewhat low but EGFR might be, for the cases with high EGFR expression in metastases, a target for radionuclide therapy. HER2 seems not to be of such interest due to rare expression, neither HER3 due to mainly expression in the cytoplasm. The requirements for successful EGFR targeted radionuclide therapy are discussed, as well as patient inclusion criteria related to radionuclide therapy.

A 23-year-old woman was presented with a prolonged history of a small lump in the buccal mucosa. A local excision was performed. The morphology diagnosed a Spitz nevus and she underwent supplementary excision of scar tissue. Two years later a submandibular lump appeared on the ipsilateral side of the neck. Cytology from fine needle aspiration indicated spread of a melanocytic tumor and she underwent a modified supraomohyiod neck dissection. One of the lymph nodes showed an inclusion of cells in the deep layers with epitheloid and spindle cells in a pattern similar to that of the primary oral lesion. The finding suggests a mechanical spread of melanocytes from the Spitz nevus to the regional lymph node. After more than 3 years of follow-up there is no further manifestation of disease. It is believed that this may be an example of how a Spitz tumor, although inherently benign, can spread along lymphatics in a pseudometastatic fashion. To our knowledge this is the first report of an oral Spitz melanoma with metastatic behavior.

There are several substances available to target members of the epidermal growth factor receptor (EGFR) family, both for imaging in nuclear medicine and for various forms of therapy. The level and stability of expression in both primary tumors and corresponding metastases is crucial in the assessment of a receptor as a target in systemic tumor therapy. To date, the expression of EGFR family members has only been determined in primary laryngeal carcinomas, and we have not found published data regarding the receptor status in corresponding metastatic lesions.Expression of EGFR, HER2, and HER3 was investigated immunohistochemically in both lymph node metastases and corresponding primary laryngeal squamous carcinomas (n = 40).EGFR overexpression (2+ or 3+) was found in 87.5% (35/40) of the laryngeal primary tumors and 82.5% (33/40) of the corresponding lymph node metastases. There was a good agreement between the primary tumors and the paired metastases regarding EGFR expression. HER2 overexpression was found in only four cases (10.5%) of the studied primary tumors and in all cases the HER2 expression was retained in the paired metastases. Another two metastases gained HER2 status when compared to the corresponding primary tumors. Strong HER3 staining was found in 26.7% of both the primary tumors and the corresponding metastases.The high frequency and stability in EGFR expression is encouraging for efforts to use EGFR targeting agents (e.g. Iressa, Tarceva, Erbitux or radiolabeled antibodies) for therapy of laryngeal carcinoma. For a few laryngeal carcinoma patients with HER2 overexpression, anti-HER2 agents could possibly be used.

The expression of EGFR, HER2 and HER3 receptors were analyzed in immunohistochemical preparations from primary esophageal tumours and corresponding lymph node metastases. The goal was to evaluate whether any of these receptors are suitable as targets for radionuclide based imaging and therapy. The receptor expressions were evaluated in parallel samples, primary tumour and metastasis, from each patient (n=51). The majority of the cases were esophageal squamous cell carcinomas, ESCC (n=40). The HercepTest scoring was used for the analysis of both HER2 and EGFR expression (0, 1+, 2+ or 3+). HER3 was only evaluated as negative, weak or strong staining. EGFR overexpression (2+/3+) was found in 67.5% (27/40) of both the ESCC primary tumours and the corresponding lymph node metastases. There were only a few changes in these EGFR-scores: two cases from 2+/3+ to 0/1+ when the primary tumours were compared to the corresponding metastases and 2 changes the other way around. HER2 overexpression (2+/3+) was found in only 3 of the primary ESCC tumours and 2 of the lymph node metastases. EGFR and HER2 stainings were found mainly in the cell membranes. The HER3 staining (weak or strong) was mainly cytoplasmic and granular and was observed in about half (20/39) of the cases, for both the ESCC primary tumours and the corresponding lymph node metastases. It was concluded that ESCC lymph node metastases generally have a strong expression of EGFR in their cell membranes and to the same extent as in the primary tumours. The stability in EGFR expression is encouraging for efforts to develop radionuclide based EGFR imaging agents. It is also possible that EGFR targeting agents (e.g. Iressa, Tarceva, Erbitux or radiolabelled antibodies) can be applied for therapy of ESCC.

Pertuzumab (Omnitarg) is a novel antibody against HER-2, domain II. HER-2 is a tyrosine kinase receptor that is overexpressed in several carcinomas, especially breast cancer. Pertuzumab, labeled with the low-energy β emitter 177Lu, might be a candidate for targeted radiotherapy of disseminated HER-2–positive micrometastases. The radiolabeled antibody [177Lu]pertuzumab showed favorable targeting properties in BALB/c (nu/nu) mice with HER-2–overexpressing xenografts. The absorbed dose in tumors was more than five times higher than the absorbed dose in blood and more than seven times the absorbed dose in any other normal organ. Experimental therapy showed that [177Lu]pertuzumab delayed tumor progression compared with controls (no treatment, P < 0.0001; nonlabeled pertuzumab antibody, P < 0.0001; and 177Lu-labeled irrelevant antibody, P < 0.01). No adverse side effects of the treatment could be detected. Thus, the experimental results support the planning of clinical studies applying [177Lu]pertuzumab for therapy. [Cancer Res 2007;67(1):326–31]

Aim: The primary aims were to study risk factors for an ipsilateral breast event (IBE) after sector resection for ductal carcinoma in situ of the breast (DCIS) in a trial comparing adjuvant radiotherapy to no therapy and to assess predictive factors for response to radiotherapy Secondary aims were to analyse reproducibility of the histopathological evaluation and to estimate correctness of diagnosis in the trial. Setting: A randomised trial in Sweden (the SweDCIS trial), including 1046 women with a median of 5.2 years of follow-up in a population, offered routine mammographic screening. Methods: A case-cohort design with a total of 161 cases of IBE (42 of those being members of the subcohort) and 284 sampled for the sub-cohort. Ninety five percent of the participants' slides could be retrieved and were re-evaluated by three experienced pathologists. Results: Low nuclear grade (NG 1-2) and absence of necrosis halves the risk of IBE in both irradiated and non-irradiated patients. Lesion size, margins of excision and age at diagnosis did not modify these associations. The presence of necrosis modified the effect of radiotherapy: relative risk was 0.40 with necrosis present and 0.07 with necrosis absent (p-value for interaction 0.068). In all subsets of prognostic factors, radiotherapy conferred a substantial benefit. The risk factors for in situ and invasive IBE were similar. The agreement between pathologists was moderate (kappa = 0.486). Correctness of diagnosis in the subcohort of SweDCIS was 84.8%. Conclusion: Although nuclear grade and necrosis carry prognostic information, we could not define a group with very low risk after sector resection alone. Radiotherapy has a protective effect in all substrata of risk factors studied. The interaction between the presence of necrosis and radiotherapy is a clinically and biologically relevant research area.

Histologic grading of breast cancer defines morphologic subtypes informative of metastatic potential, although not without considerable interobserver disagreement and clinical heterogeneity particularly among the moderately differentiated grade 2 (G2) tumors. We posited that a gene expression signature capable of discerning tumors of grade 1 (G1) and grade 3 (G3) histology might provide a more objective measure of grade with prognostic benefit for patients with G2 disease. To this end, we studied the expression profiles of 347 primary invasive breast tumors analyzed on Affymetrix microarrays. Using class prediction algorithms, we identified 264 robust grade-associated markers, six of which could accurately classify G1 and G3 tumors, and separate G2 tumors into two highly discriminant classes (termed G2a and G2b genetic grades) with patient survival outcomes highly similar to those with G1 and G3 histology, respectively. Statistical analysis of conventional clinical variables further distinguished G2a and G2b subtypes from each other, but also from histologic G1 and G3 tumors. In multivariate analyses, genetic grade was consistently found to be an independent prognostic indicator of disease recurrence comparable with that of lymph node status and tumor size. When incorporated into the Nottingham prognostic index, genetic grade enhanced detection of patients with less harmful tumors, likely to benefit little from adjuvant therapy. Our findings show that a genetic grade signature can improve prognosis and therapeutic planning for breast cancer patients, and support the view that low- and high-grade disease, as defined genetically, reflect independent pathobiological entities rather than a continuum of cancer progression. (Cancer Res 2006; 66(21): 10292-301)

Background Postmenopausal hormone-replacement therapy (HRT) increases breast-cancer risk. The influence of HRT on the biology of the primary tumor, however, is not well understood. Methods We obtained breast-cancer gene expression profiles using Affymetrix human genome U133A arrays. We examined the relationship between HRT-regulated gene profiles, tumor characteristics, and recurrence-free survival in 72 postmenopausal women. Results HRT use in patients with estrogen receptor (ER) protein positive tumors (n = 72) was associated with an altered regulation of 276 genes. Expression profiles based on these genes clustered ER-positive tumors into two molecular subclasses, one of which was associated with HRT use and had significantly better recurrence free survival despite lower ER levels. A comparison with external data suggested that gene regulation in tumors associated with HRT was negatively correlated with gene regulation induced by short-term estrogen exposure, but positively correlated with the effect of tamoxifen. Conclusion Our findings suggest that post-menopausal HRT use is associated with a distinct gene expression profile related to better recurrence-free survival and lower ER protein levels. Tentatively, HRT-associated gene expression in tumors resembles the effect of tamoxifen exposure on MCF-7 cells.

Introduction Adjuvant breast cancer therapy significantly improves survival, but overtreatment and undertreatment are major problems. Breast cancer expression profiling has so far mainly been used to identify women with a poor prognosis as candidates for adjuvant therapy but without demonstrated value for therapy prediction. Methods We obtained the gene expression profiles of 159 population-derived breast cancer patients, and used hierarchical clustering to identify the signature associated with prognosis and impact of adjuvant therapies, defined as distant metastasis or death within 5 years. Independent datasets of 76 treated population-derived Swedish patients, 135 untreated population-derived Swedish patients and 78 Dutch patients were used for validation. The inclusion and exclusion criteria for the studies of population-derived Swedish patients were defined. Results Among the 159 patients, a subset of 64 genes was found to give an optimal separation of patients with good and poor outcomes. Hierarchical clustering revealed three subgroups: patients who did well with therapy, patients who did well without therapy, and patients that failed to benefit from given therapy. The expression profile gave significantly better prognostication (odds ratio, 4.19; P = 0.007) (breast cancer end-points odds ratio, 10.64) compared with the Elston–Ellis histological grading (odds ratio of grade 2 vs 1 and grade 3 vs 1, 2.81 and 3.32 respectively; P = 0.24 and 0.16), tumor stage (odds ratio of stage 2 vs 1 and stage 3 vs 1, 1.11 and 1.28; P = 0.83 and 0.68) and age (odds ratio, 0.11; P = 0.55). The risk groups were consistent and validated in the independent Swedish and Dutch data sets used with 211 and 78 patients, respectively. Conclusion We have identified discriminatory gene expression signatures working both on untreated and systematically treated primary breast cancer patients with the potential to spare them from adjuvant therapy.

The expressions of all four receptors in the epidermal growth factor receptor family, EGFR. HER2, HER3, and HER4 were evaluated by immunohistochemistry in 19 cases of metastatic squamous cell carcinoma of the oral cavity and base of tongue. EGFR had a similar and high expression in both primary tumours and the corresponding metastases, while the expression in normal epithelium was lower in most cases. HER2 was not expressed to the same extent as EGFR. However, when HER2 was well expressed, it was in most cases expressed to the same extent and intensity in the primary tumours, metastases, and normal epithelium. The expression of HER3 and HER4 varied and was mainly cytoplasmic in all cases studied. No overexpression of HER3 and HER4 in tumours was seen as compared to normal epithelium. In order to further investigate the distribution of HER3, two HER3 expressing cell lines originating from tongue cancer were analysed in vitro, using radiolabelled anti-HER3 antibodies directed to the extracellular domains of the receptor. The results indicated that HER3 was not present in measurable amounts in the cellular membrane. There is a need for improved diagnostics and therapy for the studied type of tumours, e.g. using radiolabelled antibodies or ligands, and EGFR seemed suitable as target since the expression was high, membrane associated and similar in the primary tumours and the corresponding metastases.

The aim of this study was to evaluate whether the HER2 expression in breast cancer is retained in metastases. The HER2 expression in primary tumours and the corresponding lymph node metastases were evaluated in parallel samples from 47 patients. The HercepTest was used for immunohistochemical analyses of HER2 overexpression in all cases. CISH/FISH was used for analysis of gene amplification in some cases. HER2 overexpression (HER2-scores 2+ or 3+) was found in 55% of both the primary tumours and of the lymph node metastases. There were only small changes in the HER2-scores; six from 1+ to 0 and one from 3+ to 2+ when the metastases were compared to the corresponding primary tumours. However, there were no cases with drastic changes in HER2 expression between the primary tumours and the corresponding lymph node metastases. The literature was reviewed for similar investigations, and it is concluded that breast cancer lymph node metastases generally overexpress HER2 to the same extent as the corresponding primary tumours. This also seems to be the case when distant metastases are considered. It has been noted that not all patients with HER2 overexpression respond to HER2-targeted Trastuzumab treatment. The stability in HER2 expression is encouraging for efforts to develop complementary forms of therapy, for example, therapy with radionuclide-labelled Trastuzumab.

The incidence of lung cancer is increasing throughout the world and is the most common cause of cancer-related death. Early detection followed by surgery has a reasonable, curative potential, but 30-50% of patients experience relapses. The immunohistochemical expressions of HER-2, EGFR and COX-2 were investigated in 53 resected non-small cell lung carcinomas and correlated to microvessel density and clinical data. HER-2, EGFR and COX-2 overexpressions were demonstrated in 15%, 30% and 40% of the tumours, respectively. In adenocarcinomas, HER-2 and COX-2 overexpression were more common, whereas in squamous cell carcinomas, EGFR overexpression was more common. COX-2 expression correlated with HER-2 expression (p = 0.002), and demonstrated a trend towards a correlation with microvessel density (p = 0.10). None of the markers alone had any impact on survival. However, HER-2+/EGFR- tumours proved to have a poor prognosis. In conclusion, adjuvant treatment with HER-2 antagonists might be a future treatment option in resected non-small cell lung cancer patients, especially when HER-2 is overexpressed without a concomitant overexpression of EGFR.

The monoclonal antibody E4 (IgG2a, kappa) was raised by immunizing mice with dispersed cells obtained from human benign prostatic hyperplasia (BPH). The antibody identifies an antigen abundantly expressed in normal prostate epithelial cells, in benign epithelial prostatic cells and in well- and moderately well differentiated adenocarcinomas of the prostate, whereas poorly differentiated prostatic adenocarcinomas display somewhat less expression. Investigation of the human prostatic adenocarcinoma cell line DU 145 revealed E4 immunoreactivity localized to the cell surface. SDS-PAGE analysis under reducing conditions demonstrated an approximate molecular weight of 70 000 for the antigen. The highly specific reactivity with prostate tissue, as well as intense surface staining, especially in well- and moderately well differentiated prostatic adenocarcinomas, makes the E4 antibody a useful immunohistochemical marker and a possible candidate for future immunoscintigraphy and/or targeted radiotherapy.

The expression of the proliferation-associated antigen Ki-67 was studied in human prostatic cancer. The antigen was analyzed with an immuno-histochemical technique in TUR specimens. A correlation was seen between Ki-67 positivity and differentiation grade. All TUR specimens (15/15) with poorly differentiated carcinomas expressed the antigen. Moderately differentiated carcinomas constituted an intermediate group and slightly less than half of the cancers (12/27) were positive for the antigen. Only one of the highly differentiated carcinomas (1/12) expressed the antigen. All TUR specimens from patients with benign prostatic hyperplasia (8/8) were negative. The effect on Ki-67 positivity was also investigated in a human prostatic cancer heterotransplanted to nude mice and subjected to ionizing irradiation with or without concomitant estramustine treatment. The antigen expression was compared with that seen in tumour tissues from untreated mice and from mice treated with estramustine alone. A pronounced effect was seen in the combination treatment group with an approximately 50% reduction of the Ki-67 positive cells. The results are discussed in relation to prognosis and follow-up after radiation therapy and the possible use of estramustine in combination with radiation therapy.

PURPOSE: To study the long-term effectiveness of postoperative radiotherapy after sector resection for breast cancer in a randomized trial in which mammography is a major pathway to diagnosis. PATIENTS AND METHODS: Three hundred eighty-one women with a unifocal breast cancer ≤ 20 mm in diameter on the preoperative mammogram and without histopathologic signs of axillary metastases were treated by sector resection plus axillary dissection. Of these patients, 184 women were randomized to receive postoperative radiotherapy to the breast (XRT group), and 197 women received no further treatment (non-XRT group). RESULTS: The local recurrence rate was 8.5% (95% confidence interval [CI], 3.9% to 13.1%) in the XRT group and 24.0% (95% CI, 17.6% to 30.4%) in the non-XRT group (P = .0001). Survival free from regional and distant recurrence was 83.3% in the XRT group (95% CI, 77.5% to 89.1%) and 80.0% in the non-XRT group (95% CI, 73.9% to 86.1%) (P = .23). Overall survival was 77.5% in the XRT group (95% CI, 70.9% to 84.1%) and 78% in the non-XRT group (95% CI, 71.7% to 84.3%) (P = .99). A subgroup analysis suggested that women older than 55 years of age without comedo or lobular carcinomas had a low risk of local recurrence of 6.1% (95% CI, 0.1% to 9.1%) in the XRT-group and 11.0% (4.0% to 18.0%) in the non-XRT group (P = .16). CONCLUSION: Sector resection plus radiotherapy resulted in an absolute reduction in local recurrence of 16% at 10 years compared with surgery alone. Women older than 55 years of age without comedo or lobular carcinomas may have a low risk of local recurrence. Postoperative radiotherapy was not shown to reduce distant recurrences or improve overall survival.

The increased incidence of ductal carcinoma in situ of the breast (DCIS) in the era of mammography screening requires a deeper knowledge of the biology of the disease and calls for a suitable classification system to optimise therapy. Our aim was to evaluate the correlation to prognosis for two new classification systems of DCIS. The histopathological specimens from 195 women consecutively diagnosed between 1986 and 1994 with a primary DCIS were re-classified by two separate observers using the system proposed by an European Organization for Research and Treatment of Cancer (EORTC) working group and the Van Nuys system. The relapse-free survival (RFS) by histopathological subgroup and by nuclear grade only was estimated for women treated with breast conserving surgery (n = 149). Thirty-two local recurrences occurred among 149 women (mean follow-up time 59 months). No distant recurrences or breast cancer deaths were reported. The women in the group with the highest differentiation according to the EORTC classification had no recurrences. RFS did not differ appreciably between the two other groups. This was true also after stratification for radiotherapy. We found no statistically significant difference in RFS between the three groups in the Van Nuys classification. There was an overall agreement between the observers in 79% and 64% of the cases, according to the EORTC and Van Nuys systems, respectively. We were able to define one group with highly differentiated lesions and an excellent prognosis with the EORTC classification. Further classification into intermediate and low differentiated lesions did not help predict RFS.

Between 1982 and 1992, 73 patients received radical external beam radiotherapy (EBR) for prostatic adenocarcinoma at the Central Hospital, Västerås, with an average dose of 68.1 Gy (range 64.5-72.5 Gy) and an average cumulative radiation effect (CRE) of 18.2 (range 17.1-19.9) All patients had a negative bone scan prior to treatment and presented with clinical stage T1-T4,Nx,Mo. Twenty-four out of 36 surviving patients consented to participate in a follow-up study which included transrectal, ultrasonically guided prostate biopsies. The average follow-up from EBR to biopsy was 5.5 years (range 3.6-11.1 years). Sixteen (67%) of the patients had a positive biopsy. The average PSA level in the biopsy-positive group was 35.7 ng/mL (range 3.9-200 ng/mL). In the biopsy-negative group, the average PSA was 3.7 ng/mL (range 0.2-13.0 ng/mL). The conclusion is that the degree of local cure achieved in patients with prostatic cancer treated with external beam radiotherapy is low (33%), and that a higher radiation dose may be required to eradicate this malignancy.

Risk factors for local recurrence after breast-conserving treatment of early breast cancer have not previously been evaluated in settings where mammography has been a major pathway to diagnosis of both primary tumour and recurrences or in patients treated surgically by a formal sector resection.Three hundred eighty-one women with stage 1 primary breast cancer were randomised after a standardised sector resection to either a course of postoperative radiotherapy to 54 Gy to the breast (XRT group) or to surgery alone (non XRT group). At five years, 43 local recurrences, six of them in the XRT group, appeared. Patient characteristics collected from the medical records, histopathological characteristics determined by re-examination of slides, and mammographic characteristics from the pre-operative mammograms were evaluated as risk factors for recurrence by univariate and multivariate Cox proportional hazards models. Results are reported as relative hazards (RH) with 95% confidence intervals (95% CI).In the univariate analysis comedo cancer, RH 3.5 (95%, CI 18 6.7), lobular cancers RH 2.8 (95% CI 1.1 7.1), mammographic appearance as circular/oval shaped density, RH 2.3 (95% CI 1.1 4.51, and mammographic appearance as a stellate lesion with microcalcifications inside the lesion, RH 3.8 (95% CI 1.1-13.0) were identified as risk factors for local recurrence. Age, with a RH of 0.97 (95% CI 0.94 0.99) for each increasing year was inversely associated with risk. A multivariate analysis, which also took postoperative radiotherapy into account, only showed comedo cancers with a RH 2.6 (95% CI 1.3-5.0) and mammographic appearance of a stellate lesion with microcalcification inside the lesion RH 4.5 (95% CI 1.1-17.6) to be statistically significant. The estimates for age RH 0.98 (95% CI 0.95 1.01 and lobular cancers RH 2.5 (95%, CI 0.98 6.6) were marginally changed, with widened CIs. Patients60 years of age, without comedo or lobular carcinomas were found to be at low risk 15.9% at five years in Kaplan Meyer estimate) of local recurrence, even without postoperative radiotherapy.Low age, comedo and lobular cancers and mammographic appearance of the tumour as a stellate lesion with microcalcifications inside the lesion indicate an increased risk for local recurrence after sector resection in stage 1 tumours at five years. Patients60 years of age without comedo or lobular cancers are at low risk for local recurrence at five years even without postoperative radiotherapy.

Background: Mutations in the p53 tumor suppressor gene (also known as TP53) have been detected in a wide variety of human cancers. In breast cancer, the presence of p53 gene alterations has been associated with worse prognosis. Purpose: We compared a complementary DNA (cDNA)-based sequencing method and an immunohistoch emical (IHC) method for their abilities to detect p53 mutations in breast cancer specimens. In addition, we determined the prognostic value of information obtained when these two methods were used. Methods: Specimens from 316 primary breast tumors were evaluated for the presence of mutant p53 protein by use of the mouse monoclonal antibody Pab 1801 (that recognizes both wild-type and mutant forms of p53) and standard IHC methods. In addition, the entire coding region of p53 genes expressed in these tumors was screened for mutations by combining reverse transcription, the polymerase chain reaction, and DNA sequencing. Probabilities for overall survival (OS), breast cancer-corrected survival (BCCS; death from breast cancer is the considered event), and relapse-free survival (RFS) were estimated by use of the Kaplan-Meier method, and survival curves for different patient subgroups were compared by use of the logrank method. All reported P values are from two-sided tests. Results: Sixty-nine (22%) of 316 tumors had p53 gene mutations detected by the cDNAbased sequencing method; only 31 (45%) of these mutations were located in evolutionary conserved portions of the p53 coding region. Sixty-four tumors (20% of the total) had elevated levels of p53 protein as detected by IHC, suggesting the presence of mutations. Of the sequencing-positive tumors (i.e., p53 mutant), 23 exhibited negative IHC reactions, indicating that IHC failed to detect 33% of the mutations. Furthermore, 19 of the IHC-positive tumors were sequencing negative (i.e., p53 wild-type), suggesting a 30% false-positive frequency with IHC. Four tumors (1.3% of the total) could not be analyzed by the cDNA-based sequencing method, and three tumors (1% of the total) could not be analyzed by IHC. The 5-year estimates for RFS, BCCS, and OS were significantly shorter for patients with p53 sequencing-positive tumors than for patients with sequencing-negative tumors (P = .001, P = .01, and P - .0003, respectively). Patients with IHC-positive tumors showed reduced survival in all three categories when compared with those with IHCnegative tumors, but the differences were not statistically significant. Conclusions: Use of a cDNA-based sequencing method to determine the status of the p53 gene in primary breast cancers yielded better prognostic information than IHC performed with the Pab 1801 monoclonal antibody. [J Natl Cancer Inst 1996;88:173-82]

A long list of potential prognostic markers has been analysed for breast cancer, some of them will be reviewed in this article. The lymph node status is still the best prognostic marker. The lymph node status combined with information on tumour size, receptor- and proliferation status of the tumour should be analysed as standard for all breast cancer patients. Prognostic information for breast cancer patients has also been described for the membrane protein c-erbB2, the protease cathepsin D, plasminogen activators and inhibitors, certain oncogenes and tumour suppressor genes. Some of these factors also give potential additional information on the response to different oncological therapies, and are better denoted predictive factors. In this overview we shortly describe the above mentioned prognostic factors with major focus on the tumour suppressor gene p53 and its prognostic value and potential predictive value.

In this study, we have investigated the model DNA values and the expression of estramustine-binding protein (EMBP) in formalin-fixed and paraffin-embedded TUR specimens from 76 untreated patients with prostatic cancer. In addition, specimens from 13 patients were analyzed for tumour EMBP expression only. Ploidy was measured as diploid, tetraploid and non-tetraploid aneuploid or aneuploid in the near-diploid region. All patients had been referred during 1978-1981, and were subjected to TUR due to urinary obstruction. Survival data were obtained for all patients through March 1988. Statistical analyses were performed using a Cox's regression model with respect to survival and cause specific survival and correlated to the DNA pattern and the expression of EMBP. The existence of a near-diploid aneuploid cell population as well as poor differentiation grade were both statistically significantly correlated with poor survival. Near-diploid aneuploid cell lines were seen in 9/76 (12%) of the patients and were also seen in well differentiated cancers (4/17). The expression of EMBP was most abundant in the moderately differentiated cancers. However, all prostatic cancer specimens investigated were positive for the antigen. Patients with poorly differentiated carcinomas and high EMBP expression showed a tendency towards better prognosis than those with poorly differentiated carcinomas and low EMBP expression. The present patient material was, however, too small to show a statistically significant correlation between EMBP and survival.

All cases of squamous-cell carcinoma of the external ear that were treated at the ENT Clinic, University Hospital, Lund, Sweden, between 1970 and 1977 were analyzed retrospectively to determine parameters for predicting the development of metastases. These cases included 62 males and 3 females, with a mean age of 77 years. The frequency of metastases for these patients was 16.4%. The carcinomas were graded according to four parameters: depth of growth, mode of invasion, cellular differentiation, and cellular plasmolymphocytic response. We concluded that depth of growth and mode of invasion were the most useful of these parameters and that prophylactic lymph node dissection should be performed in cases of tumors larger than 4 cm in diameter, of tumors infiltrating the cartilage, and of smaller tumors with maximum scores for depth of growth and mode of invasion.

Single cell proteins prepared from yeast or bacterial cells grown on a chemically pure hydrocarbon fraction were fed to male and female rats either as the sole sources of protein at a dietary protein level of 20 % or as supplements to a commercial stock diet at two levels providing 15 and 25 %, respectively, of single cell protein. Two control diets were used. One was a synthetic casein containing diet and the other a commercial stock diet. With the exception of an increased occurrence of renal calcification yeast diets did not affect mortality, general condition or behaviour of the animals. Yeast as the sole source of protein produced a lower growth rate than the casein and stock diets. This could be due to a deficit in sulfur-containing amino acids. Adverse effects on the health of rats fed bacterial single cell protein as the sole source of protein were observed. These were arrested growth, weight losses, bleedings at nose and eyes and increased mortality. At the histological examination of organs from these rats, severe pyelonephritis, nephrocalcinosis and testicular atrophies were seen. Blood from these rats showed a changed neutrophil: lymphocyte ratio.

A lipoprotein concentrate was obtained by extraction from sunflower seeds with a water-ethanol-salt solution. This contained about 50% protein and 20% fat. Chemical score was 51, based on analysis of available lysine. For a 3-week period the protein efficiency ratio (PER) was 2.16 and the productive protein value (PPV) 37.6. After addition of lysine the PER increased to about 3.3 and the PPV to about 58. A 90-day toxicological study was carried out on rats fed a diet containing 11 % protein from the lipoprotein concentrate as the sole source of protein. Histological examinations showed a testicular atrophy but otherwise mainly normal conditions. Hematological findings were normal.