Non-Hodgkin's lymphoma of the testis: a retrospective study of 84 patients treated in the French anticancer centres.

Primary non-Hodgkin's lymphoma of the testicle is rare. We analysed cases treated in French anticancer centres from 1969 to 1995. All cases were reviewed and classified according to the R.E.A.L. Classification. Eighty-four cases were included in this study. The median age was 67 years (17-85). Disease was classified as stages I in 42 cases, stages II in 19 and stages III-IV in 23. Diffuse large B-cell lymphoma was diagnosed in 75% of cases. Treatment included orchidectomy and radiotherapy and/or chemotherapy. A complete response was obtained in 72.6% of the patient population and in 100%, 68% and 33% of stage I, II and III-IV disease respectively. Recurrence occurred in 32 cases and the most frequent site was the central nervous system: six of these patients presented stage I disease. Median overall survival was 32 months for the entire population, 52 months for stage I, 32 months for stage II, and 12 months for stage III-IV cases (P < 0.0001). Among patients presenting stage I disease, no difference was found between those treated with combined surgery and chemotherapy or surgery followed or not followed by radiotherapy. This study confirms that non-Hodgkin's lymphoma of the testicle carries a poor prognosis. Systemic adjuvant chemotherapy should be discussed because of the high recurrence rate. Inclusion of these cases in large co-operative prospective studies is recommended.

Improvement of nonviral p53 gene transfer in human carcinoma cells using glucosylated polyethylenimine derivatives.

Polyethylenimine (PEI) derivatives are potent polycationic nonviral vectors for gene transfer. The gene transfer efficiency of glucosylated and galactosylated PEI derivatives was optimized using green fluorescent protein gene as reporter gene in FaDu and PANC3 human carcinoma cell lines. Glucosylated or galactosylated PEI derivatives were found to be slightly less cytotoxic than unsubstituted PEI. Gene transfer efficiency was found to be related to DNA/cell number ratio and optimal gene transfer efficiency was achieved at 4 microg DNA/10(5) cells. PEI-DNA complexes were found to enter cells rapidly and were detected into cytoplasmic vesicles 2 hours post-transfection. Green fluorescent protein gene expression was detected 4-6 hours after transfection and reached maximal value 24 hours post-transfection. The results achieved demonstrated that glucosylated PEI yield higher and longer gene transfer efficiency than unsubstituted PEI. Using glucosylated PEI allowed to achieve significant gene transfer in more than 10% of the total cell population for more than 4 days. These data were then applied to p53 gene transfer in PANC3 cells bearing p53 gene deletion and consequently unable to initiate apoptosis. Using glucosylated PEI, p53 gene transfer was successfully achieved with subsequent recovery of p53 mRNA expression and transient P53 protein expression. P53 protein functionality was further demonstrated because transfected cells underwent apoptosis.

Effects of fluence rate on cell survival and photobleaching in meta-tetra-(hydroxyphenyl)chlorin-photosensitized Colo 26 multicell tumor spheroids.

We report the influence of fluence rate on the photobleaching and cell survival in Colo 26 multicell spheroids photosensitized by meta-tetra-(hydroxyphenyl)chlorin (mTHPC). Photosensitizer degradation and therapeutic efficacy increased dramatically and progressively when the fluence rate was reduced over the range from 90 to 5 mW cm-2. These experimental results were compared to a mathematical model of photobleaching based on self-sensitized singlet oxygen reactions with the photosensitizer ground state. This model incorporates photophysical parameters obtained from microelectrode measurements of oxygen depletion at the surface of mTHPC-sensitized spheroids and was refined by including the inhomogeneous distribution of mTHPC in spheroids and oxygen depletion in the bulk medium. Since the model is consistent with the experimental data we conclude that the fluence rate dependence of the cell survival and of mTHPC photobleaching is due to photochemical oxygen consumption and a predominantly singlet oxygen-mediated mechanism of mTHPC photobleaching. The threshold dose of reacting singlet oxygen was calculated to be 7.9 +/- 2.2 mM in this system.

Detection by immunohistochemistry of c-erbB2 oncoprotein in breast carcinomas and benign mammary lesions.

The presence of the c-erbB2 oncoprotein was demonstrated by immunohistochemistry in a study involving 173 mammary lesions. The lesions included infiltrating cancers, non-invasive neoplasia, as well as atypical and benign lesions. Our aim was to investigate the correlation between the c-erbB2 oncoprotein overexpression and the morphological features of the different mammary tissues analyzed to obtain a better characterization for the growth potential of certain lesions, with emphasis placed on the non-invasive neoplasia and the atypical lesions. Nearly 30% of infiltrating ductal carcinomas (27/89 cases) and 2 out of 24 infiltrating lobular carcinomas were positive. The comedocarcinomas were mostly stained (83%). In contrast, the intraductal carcinomas of cribriform or papillar patterns were consistently negative. No staining was observed in the atypical epithelial hyperplasia located in the vicinity of positive cancers for anti-oncoprotein c-erbB2 antibody. Furthermore, the only 5 positive cases for c-erbB2 out of 32 cancer-free cases were three fibroadenomas and two fibrocystic diseases with atypical ductal hyperplasia. A close correlation was thus observed between c-erbB2 oncoprotein overexpression and cancerous cell morphology, characterized by a marked nuclear hypertrophy often associated with cellular pleomorphism. However, predictive abnormalities of malignant transformation in non-neoplastic epithelial proliferation was difficult to identify, considering only the c-erbB2 expression. A group of tumors with little nuclear abnormalities were found positive for c-erbB2 immunostaining. These probably corresponded to a particular cellular phenotype. Further studies involving other oncogenes should lead to a better characterization of the different tumor phenotypes and help to clarify breast carcinogenesis.

[Breast-like carcinoma of the vulva]. / Carcinome vulvaire de type mammaire.

Breast-like carcinomas of the vulva is a rare finding. Only 11 cases are reported in the literature. This article reports a painful tumor of the left vulvar labia in a 62 year-old woman. Excisional biopsy showed an infiltrating adenocarcinoma, histologically similar to a breast tumor with positive hormonal receptors. The origin of this tumor, ectopic mammary tissue or specific adnexal genito-anal gland, is discussed. From data of the literature, we offer guidelines for diagnosis, treatment and origin of this rare tumor.

[Endocervical glial implant]. / Implant glial endocervical.

We report a new case of glial tissue in uterine cervix found in a 63 year-old woman with an abortion 30 years ago. The lesion, measuring 0.5 cm in diameter, showed a typical feature of mature glial tissue immunoreactive for the glial fibrillary acid protein. Hypothesis concerning its histogenesis (fetal graft, tumor, metaplasia) are discussed. Fetal implantation is likely as in the majority of published cases.

Human haptocorrin in hepatocellular carcinoma.

This study aimed to determine whether haptocorrin (HC), a vitamin B12 binder, is stored in hepatic cells and whether this storage is modified by hepatic carcinogenesis. It was carried out using immunohistochemistry on different liver tissues (normal liver and steatosis, N = 22; cirrhosis, N = 13; and hepatocellular carcinoma, N = 31). No significant immunostaining of HC was detected in noncancerous biopsies with the exception of in one case of cirrhosis. Hepatocellular carcinoma (HCC) sections showed a weak to moderate cytoplasmic staining of cancerous cells (93% of cases) and of noncancerous hepatocytes surrounding the tumor (95%) of cases. Sections with pseudoglandular structures showed a moderate to strong staining of their secretion products. These results and previous studies would seem to confirm the hypothesis that the raised HC serum level observed in HCC is due both to the increased hepatic synthesis of HC and to a decreased uptake by the liver of the particular isoform of this glycoprotein present in the serum of HCC patients.

Determination of endogenous porphyrins and the maximal HpD tumor/normal skin ratio in SKH-1 hairless mice by light induced fluorescence spectroscopy.

The treatment of skin tumors is an application of photochemotherapy (PCT) which involves an initial administration of a photosensitizer (PS) followed by irradiation with a light beam that causes the PS to produce cytotoxic oxygen species within the tumors. As the PS is also present in normal skin, it is necessary to know how it is distributed between the two tissues. In this study, we have used SKH-1 hairless mice bearing papillomas or carcinomas chemically induced. The biodistribution of hematoporphyrin derivative (HpD) and the tissue autofluorescence measurements were studied by light induced fluorescence spectroscopy. The tumor and normal autofluorescence spectra measured on control mice with papillomas or carcinomas had a very similar shape. However, the principal endogenous porphyrin peak at about 630 nm showed a fluorescence signal amplitude 2 (for papilloma) and 1.5 (for carcinoma)-fold higher than the one found for the normal skin. Moreover, the fluorescence intensity of carcinoma spectrum is 1.4-fold lower than the one of papilloma spectrum at 630 nm. The tissue autofluorescence can be used to distinguish tumor from normal skin and benign from malignant tumor. This difference in fluorescence intensity at 630 nm was directly related to the concentration of endogenous porphyrins in the tumor. Fluorescence intensity ratios between tumor and normal skin were measured 4, 8, 24, 48, 72 and 96 hours after intraperitoneal injection of HpD (5 mg/kg body weight). The best tumor/normal skin ratio was 6.2 for HpD and the time required to reach this ratio was 48 h. HpD showed a moderate selectivity since the ratio was higher than 1 during the four first days. Photodynamic therapy with the same dose of HpD used in this biodistribution study must also be carried out to verify that the maximal tumor/skin ratio corresponds to the maximal efficiency of HpD.

Immunohistochemical characterization of the differentiation state of basal cell carcinomas with special interest for infiltrating relapsing tumors.

A retrospective histological and immunohistochemical study was performed on 66 basal cell carcinomas (BCC). To determine the differentiation stages of epithelial cells in these BCC, three monoclonal antibodies directed against cytokeratins K1, K2, K9 and K10-11 (EE21-06), to cytokeratins K1 to K19 (F12-19), and to corneodesmosin (G36-19) were used in indirect immunofluorescence on paraffin-embedded sections. Three histological groups of BCC with specific cytokeratin immunohistochemical features were distinguished: (1) superficial BCC were unlabelled, (2) nodular and variant (keratotic, adenoid) BCC showed an homogeneous labelling, and (3) infiltrative aggressive-type BCC showed a heterogeneous cell to cell labelling. Some nodular BCC cells presented characteristics of granular keratinocytes, i.e. they were labelled by the anticorneodesmosin antibody. All the clinically recurrent tumors were found to be of the infiltrative aggressive-type. If these aggressive forms of BCC were not identified by specific marker, their topographic patterns of labeling with antibodies directed to cytokeratins allowed them to be distinguished. We suggest that an immunohistochemical analysis with antibodies specific for different stages of keratinocyte differentiation is an efficient complement to histological diagnosis of BCC.

Iatrogenic risks of endometrial carcinoma after treatment for breast cancer in a large French case-control study. Fédération Nationale des Centres de Lutte Contre le Cancer (FNCLCC).

Since tamoxifen is widely used in breast cancer treatment and has been proposed for the prevention of breast cancer, its endometrial iatrogenic effects must be carefully examined. We have investigated the association between endometrial cancer and tamoxifen use or other treatments in women treated for breast cancer in a case-control study. Cases of endometrial cancer diagnosed after breast cancer (n = 135) and 467 controls matched for age, year of diagnosis of breast cancer and hospital and survival time with an intact uterus were included. Women who had received tamoxifen were significantly more likely to have endometrial cancer diagnosed than those who had not (crude relative risk = 4.9, p = 0.0001). Univariate and adjusted analyses showed that the risk increased with the length of treatment (p = 0.0001) or the cumulative dose of tamoxifen received (p = 0.0001), irrespective of the daily dose. Women who had undergone pelvic radiotherapy also had a higher risk (crude relative risk = 7.8, p = 0.0001). After adjusting for confounding factors, the risk was higher for tamoxifen users (p = 0.0012), treatment for more than 3 years (all p < 0.03) and pelvic radiotherapy (p = 0.012). Women who had endometrial cancer and had received tamoxifen had more advanced disease and poorer prognosis than those with endometrial cancer who had not received this treatment. Our results suggest a causal role of tamoxifen in endometrial cancer, particularly when used as currently proposed for breast cancer prevention. Pelvic radiotherapy may be an additional iatrogenic factor for women with breast cancer. Endometrial cancers diagnosed in women treated with tamoxifen have poorer prognosis. Women who receive tamoxifen for breast cancer should be offered gynaecological surveillance during and after treatment. A long-term evaluation of the risk-benefit ratio of tamoxifen as a preventive treatment for breast cancer is clearly warranted.

Intracellular distribution of tamoxifen in resistant human breast adenocarcinoma cells using tamoxifen-eosin association.

A tamoxifen-resistant cell line (MCF7TAM) was established from tamoxifen-sensitive MCF-7 human breast cancer cells expressing estrogen receptors. Though the resistant cell line grows in the presence of tamoxifen, estrogen receptors continue to be expressed at similar levels as in the parental cell line. However, estrogen receptors appeared to be altered in the resistant cell line since important discrepancies are observed between results obtained with ligand binding assays and immunoenzymatic assays, tending to show modifications of estrogen receptor ligand binding capacity. The intracellular distribution of tamoxifen in sensitive and resistant cell lines was investigated using fluorescence of eosin-tamoxifen ionic association. Fluorescence emission spectra of eosin, tamoxifen and eosin-tamoxifen complex (lambda(ex)=480 nm) were analyzed and the maximal fluorescence intensity found for the complex (lambda(em)=540 nm) was four times higher than that of eosin alone, while tamoxifen alone did not emit any fluorescence in this spectral range. In MCF-7 cells, tamoxifen was found to be mainly located surrounding the nucleus, although nuclear fluorescence intensity was significantly lower. No highly fluorescent granules were observed in the resistant cell lines as opposed to sensitive cells. Improvement of this fluorescence microscopy methodology could appear of interest, taking into account the complexity of tamoxifen resistance molecular pathways.

Combined flow cytometry and immunohistochemistry analyses for the assessment of nasopharyngeal carcinoma cell kinetics by in vivo bromodeoxyuridine infusion.

Thirty-eight previously untreated patients with a histologically proved diagnosis of nasopharyngeal carcinoma were evaluated for in vivo cell kinetics before treatment by conventional radiation therapy. Thirty-seven tumors were analyzed by flow cytometry. Values of median potential doubling time (Tpot), labelling index (LI), and duration of S phase (Ts) were, respectively, 10.9 days, 3.8%, and 10.8 hours. In 35 cases, the results obtained from two biopsies of the same tumor were compared. A good reproducibility was obtained for LI and Tpot (P < 0.01). Thirty-one tumors were analysed by immunohistochemistry and labelling index (HLI) was determined in 24 tumors with a percentage of labelled cells varying from 6.1% to 39.2% (median value = 18.5%). No correlation was found between LI and HLI, but when observations were focused on the restricted group of DNA aneuploid samples, mean values of LI and HLI were closer (respectively, 12.8 +/- 4.5% and 18.3 +/- 7.7%) and a good correlation was obtained (P = 0.01). Moreover, no difference in proliferation was found between diploid and aneuploid tumors. Considering these results, a combined Tpot was calculated that allowed classification of tumors as highly or slowly proliferative.

[A comparative study of hormone receptors in breast cancer with quantitative immunocytochemistry and biochemistry]. / Etude comparative de la quantification des récepteurs hormonaux dans les cancers du sein par immunocytochimie et biochimie.

As the biochemical assay, the measurement of hormone receptors by immunocytochemistry in invasive breast cancers may predict the ability of patients to respond to hormone therapy. The objective of this work is to determine the reliability of hormone receptors analysis on cytologic spreadings. Estrogen and progesterone receptors analysis was carried out in 109 invasive breast carcinomas: (1) with a SAMBA 2005 image analysis system on frozen cytologic spreadings (ER/PR-ICA, Abbott); (2) by visual evaluation on paraffin sections (ER-1D5, Dako; PR-ICA, Abbott); (3) by biochemistry (EIA, Abbott). There is a significant correlation between the three methods of hormone receptors analysis (0.716 to 0.842). The sensitivity of immunocytochemical methods ranges from 88.0 to 94.3%, and the specificity from 70.0 to 94.7%. The minimum concordance is 87.2%. This study demonstrates that immunocytochemistry of hormone receptors is a good alternative to biochemical measurement, especially when applied to cytologic spreadings. Therefore, immunocytochemistry can be used, when conventional biochemical assay cannot be performed for hormone receptors evaluation, particularly on cytoponctions.

Determination of the maximal tumor/normal skin ratio after HpD or m-THPC administration in hairless mouse (SKh-1) by fluorescence spectroscopy--a non-invasive method.

Two major steps in our study on the treatment of skin tumors by photochemotherapy (PCT) were the development of a skin tumor model in hairless mice by chemical carcinogenesis and by the use fluorescence spectroscopy, a semi-quantitative and non-invasive method, to determine the time after i.p. injection of photosensitizer when the tumor/normal skin ratio is the highest. Carcinogenesis provided mice bearing many benign papillomas and these were used to determine the tumor/normal skin ratios of two photosensitizers by fluorescence spectroscopy. Hematoporphyrin derivative (HpD) (5 mg/kg body weight) and m-tetra(hydroxyphenyl)-chlorin (m-THPC) (0.3 mg/kg body weight) were injected, and fluorescence measured at 4, 8, 24, 48, 72 and 96 h after injection. The tumor/normal skin ratio was 6.2 for HpD and 5.1 for m-THPC. The times required to reach these ratios were 48 h for HpD and 72 h for m-THPC. Published reports indicate that m-THPC gives a much higher tumor/normal skin ratio than HpD. These results must be confirmed by organic extraction. Photodynamic therapy with the same doses of HpD and m-THPC used in this pharmacokinetic study must also be carried out to compare the toxicities of the two photosensitizers and to determine which is best for this type of tumor.

Sensitivity to taxoid derivatives of a newly established human endometrioid ovarian adenocarcinoma radioresistant cell line.

An endometrioid ovarian adenocarcinoma cell line CAVEOC-2 was characterized. Maintained in monolayered culture, CAVEOC-2 cells exhibited a 33-hr doubling time. When xenografted into nude mice, these cells produced fast growing tumors. Colony-forming efficiency in agar was 50%. DNA index was 1.5 and cytogenetic analysis showed a triploid karyotype. CAVEOC-2 cells did not express mdr-1 gene and were chemosensitive to doxorubicin (IC50 = 1.82 +/- 0.76 mumol/l), paclitaxel (IC50 = 3.33 +/- 0.26 nmol/l) and docetaxel (IC50 = 0.68 +/- 0.28 nmol/l), while they showed an intermediate sensitivity to cisplatin (IC50 = 9.40 +/- 1.02 mumol/l). CAVEOC-2 cells seemed highly radioresistant (SF2 = 0.81, alpha = 0.02 Gy-1, beta = 0.025 Gy2, and MID = 4.31 Gy). Activities of glutathione S transferase and gamma-glutamyl transpeptidase were respectively 23.5- and 3.4- fold higher than those of sensitive A2780 cell line. These characteristics make the CAVEOC-2 cells a suitable model for the study of human endometrioid ovarian adenocarcinoma.

Determination of amplification level of the c-erbB-2 proto-oncogene in human breast carcinomas: a comparative study between non-radioactive and radioactive labelling.

A quantitative method of polymerase chain reaction (PCR) using both digoxigenin and radioactive labelled probes has been used for the detection of the c-erbB-2 proto-oncogene amplification in breast carcinomas with formalin-fixed paraffin-embedded tissue sections. c-erbB-2 proto-oncogene amplification has been demonstrated in 14 infiltrating ductal carcinomas. The technique consisted of the co-amplification of c-erbB-2 and IFN-gamma (interferon-gamma) genes. The latter was considered as a single copy gene per genome-equivalent. The aim of this study was to compare two quantitative PCR techniques based on the incorporation of either digoxigenin-11-dUTP or 32P-dCTP, during amplification. For the colorigenic method, using the Dig system, after electrophoresis and transfer, the specific bands were revealed with a chromogenic substrate of phosphatase. Their intensity estimated by scanning photometry following blot transparisation. After electrophoresis, the radioactive gel was submitted to radioautography and the band intensities evaluated by scanning spectrophotometry. For the 14 samples, a good agreement between both methods was noted. The colorigenic method is a valuable alternative to radiolabelling due to: i) time saving, ii) reagent conservation, iii) safe manipulation and iv) sensitivity of the same order for both methods.

[Expression of the c-erbB-2 oncoprotein in mammary Paget's disease. Immunohistochemical study by using 3 antibodies]. / Expression de l'oncoprotéine c-erbB-2 dans la maladie de Paget mammaire. Etude immunohistochimique à l'aide de trois anticorps.

An overexpression of the c-erbB-2 oncoprotein has been demonstrated in the breast cancer and has been associated with a poor prognosis. Our study involved 23 cases of mammary Paget's disease which was found to be associated with the intraductal carcinomas in 13 cases, the intraductal carcinomas supposed micro-invasive in 2 cases, the infiltrating ductal carcinomas with predominant intraductal component in 6 cases and the infiltrating ductal carcinomas in 2 cases. The presence of c-erbB-2 oncoprotein has been determined immunohistochemically with 3 different antibodies: rabbit anti-human c-erbB-2 oncoprotein A485 (Dako), c-erbB-2 oncoprotein (internal domain) mouse monoclonal antibody NCL-CB11 (Novocastra), and c-erbB-2 oncoprotein (external domain) mouse monoclonal antibody NCL-CBE1 (Novocastra). An overexpression of the c-erbB-2 oncoprotein has been observed in 21 of the 23 studied cases. We noted an intense membrane staining in the intraepidermal or intraglandular tumour cells of mammary Paget's disease. Any staining has been observed in 2 cases with glandular component of pure intraductal type. These results are identical whatever the antibody used. In a previous study concerning mammary Paget's disease, it has been noted a correlation between this overexpression and presence of large malignant cells. We also have found this notion in mammary Paget's disease where the c-erbB-2 positive neoplastic cells in the different tumour components were large with prominent cytoplasm. The obtained results argue strongly for adenocarcinomatous origin for mammary Paget's disease and exhibit that the overexpression of c-erbB-2 oncoprotein was not constantly in correlation with a poor prognosis.

Radiosensitivity of multicellular tumour spheroids obtained from human ovarian cancers.

The radioresponsiveness of immunologically characterised (KL1, antivimentin and OC125) human ovarian carcinoma cells, obtained from effusions or solid tumours, was assayed in vitro using the multicellular tumour spheroids (MTS) three-dimensional model. Great interspecimen variabilities were observed in MTS doubling time (1.0-8.5 days), as well as in the doses inducing a 50% decrease in the MTS individual volume (ID50) (0.56-9.15 Gy), or in the overall population MTS number (SCD50) (1.9-15.7 Gy) and the residual/initial MTS individual volume ratio after 2 Gy irradiation (RSV2) (10-88%). The doubling time, DNA-ploidy and S-phase fraction did not correlate with the ID50. Significant correlations were found between the new parameters defined (RSV2 and ID50) and the SCD50, a well-accepted local control parameter. These parameters demonstrated their usefulness for studying the radiosensitivity of MTS prepared from human ovarian tumour biopsies.

Determination of estrogen and progesterone receptors in endometrial adenocarcinomas. Comparison between dextran-coated charcoal and immunoenzymatic methods on curettage biopsies.

Estrogen receptors (ER) and progesterone receptors (PR) were determined on curettages from women with endometrial adenocarcinoma. The results obtained with the enzyme immunoassay (EIA) and the dextran-coated charcoal (DCC) assay were compared. A highly significant correlation was obtained between these methods for the ER measurement (Rs = 0.91). For PR determination, the Rs value between EIA and DCC assay was 0.57 and the mean value of PR-DCC is significantly higher than the mean value of PR-EIA. These results suggest that EIA is a suitable method for ER measurement. For PR determination on curettage material the DCC assay seems more accurate than EIA.

Peroxisomal enzymes in normal and tumoral human breast.

The presence of peroxisomes and their enzymatic content were investigated and compared in healthy and neoplastic human breast epithelial cells using cytochemical studies at the ultrastructural level as well as Western blot and biochemical analyses. Ultrastructural cytochemistry revealed the presence of these organelles in both normal and neoplastic breast tissues. Their mean diameter was 0.27 +/- 0.11 micron. No significant difference was noted between numbers of peroxisomes in normal and neoplastic breast epithelia. Catalase, D-amino acid oxidase, and urate oxidase were found to be expressed in mammary carcinoma and in surrounding non-malignant tissue when the postnuclear supernatant fractions prepared from homogenates were assessed by Western blot techniques. Their specific activities and that of fatty acyl CoA oxidase as determined spectrophotometrically were found to be diminished in the tumour when compared with the control tissue. On the other hand, no significant difference was found in the specific activity of the L-alpha-hydroxy acid oxidase of normal and neoplastic human breast tissues. Investigations of the relationship between peroxisomal enzymes and tumour grade revealed that catalase, urate oxidase, and fatty acyl CoA oxidase activities in breast neoplastic tissues belonging to grade III were significantly lower than in the adjacent normal tissues.