Histopathological risk factors for ipsilateral breast events after breast conserving treatment for ductal carcinoma in situ of the breast--results from the Swedish randomised trial.

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.

S-phase fraction and urokinase plasminogen activator are better markers for distant recurrences than Nottingham Prognostic Index and histologic grade in a prospective study of premenopausal lymph node-negative breast cancer.

PURPOSE: Histologic grade, Nottingham Prognostic Index (NPI), estrogen receptor (ER) and progesterone receptor (PgR) status, and tumor size have previously been shown to be important prognostic indicators for distant recurrence of breast cancer. The purpose of this study was to compare the prognostic value of these factors with flow cytometric S-phase fraction (SPF), urokinase plasminogen activator (uPA), and plasminogen activator inhibitor type 1 (PAI-1) in premenopausal patients with lymph node-negative breast cancer. PATIENTS AND METHODS: In 237 consecutive premenopausal patients with lymph node-negative breast cancer and freshly frozen tumor material available, SPF, ER and PgR status, uPA and its inhibitor PAI-1, histologic grade, and NPI were evaluated. RESULTS: SPF was univariately the most powerful prognostic factor for distant recurrence, followed by uPA, histologic grade, PgR, age, ER, NPI, and PAI-1, the latter being nonsignificant. Multivariate analysis revealed that neither NPI nor histologic grade was significant after adjustment for SPF, a fact that may be explained by the strong association between these factors. uPA was, however, an independent prognostic factor in addition to SPF, NPI, or histologic grade. CONCLUSION: In this prospective study, SPF and uPA were found to be independent prognostic factors in premenopausal women with lymph node-negative breast cancer. We suggest that SPF, if performed under standardized conditions, can replace histologic grade as a selection instrument for adjuvant medical treatment. The value of the combination of SPF and uPA needs to be confirmed in an independent prospective trial.

Are cellular polarisation and mitotic frequency prognostic factors for local recurrence in patients with ductal carcinoma in situ of the breast?

There is still no generally accepted histopathological classification system for ductal carcinoma in situ (DCIS) of the breast. Nuclear grade, with or without other histopathological parameters (i.e. comedo-type necrosis and cellular polarisation), has been demonstrated to yield prognostic information. A detailed method for the evaluation of the mitotic frequency in DCIS, based on an approach by Contesso, was used in this study. We also investigated if cellular polarisation and mitotic frequency were important for the ipsilateral local recurrence-free interval (IL-RFI) in 121 DCIS patients who had been operated upon with breast-conserving treatment (BCT) without radiotherapy. Both cellular polarisation and the mitotic frequency were associated with histopathological and cellular biological factors (in previous evaluations), and were of borderline significance for IL-RFI in the univariate analyses. However, when nuclear grade was included in the multivariate analyses (with or without the growth pattern), neither cellular polarisation nor the mitotic frequency were of any independent prognostic value.

Cell biological factors in ductal carcinoma in situ (DCIS) of the breast-relationship to ipsilateral local recurrence and histopathological characteristics.

All cases of ductal carcinoma in situ (DCIS) diagnosed from 1987 to 1991 in the Southern Health Care Region of Sweden, and operated upon with breast conserving treatment (BCT) with (n=66) or without (n=121) postoperative radiation (RT) were clinically followed, morphologically re-evaluated and analysed for cell biological factors (immunohistochemical assays or DNA flow cytometry). Median age at diagnosis was 58 years (range 29--83 years) and median follow-up was 62 months. Oestrogen (ER)- and progesterone receptor (PR)-negativity, c-erbB-2 overexpression, low bcl-2 expression, p53 accumulation, DNA non-diploidy and high Ki67, were strongly associated with high grade DCIS, and comedo-type necrosis. In contrast, significant associations to growth pattern (not diffuse versus diffuse) were seen only for c-erbB-2 and PgR. There was also a strong relationship between the cell biological factors, and a summary cell biological index based on principal component analysis was introduced (CBI-7). In the group that had not received postoperative RT, 31 ipsilateral local recurrences occurred (13 invasive, 18 DCIS). Ipsilateral recurrence-free interval (IL-RFI) was in univariate analyses significantly, or almost significantly, shorter for patients showing p53 accumulation, high Ki67 or low bcl-2, compared with patients with normal p53, low Ki67 and high bcl-2. The prognostic importance of the remaining cell biological factors was less pronounced. On the other hand, the index CBI-7, was a strong predictor for recurrence.

Tumour biological features of BRCA1-induced breast and ovarian cancer.

BRCA1 mutations, although implicated in disease predisposition in a major part of the hereditary breast cancer population, do not seem to be crucially involved in tumorigenesis of sporadic breast and ovarian cancers. This suggests that tumours arising in BRCA1 mutation carriers may differ from BRCA1 negative hereditary and sporadic cancer in genetic and biological features, as well as in clinical behaviour. Prior to BRCA1 analysis, 79 breast and 19 ovarian tumours from 57 breast and breast-ovarian cancer families, and 170 tumours from a comparison group of stage II breast cancers were studied with regard to histopathological features; immunohistochemistry [c-erbB-2, p53, oestrogen receptor (ER) and progesterone receptor (PR)], DNA flow cytometry and S-phase fraction. BRCA1 mutations were found in 40 breast and 15 ovarian tumours. The BRCA1 positive breast tumours were significantly more often of ductal type, histological grade III and manifested a heavy lymphocyte infiltration. Additionally, as compared to BRCA1 negative tumours, the BRCA1 positive tumours were significantly more often ER, PgR and c-erbB-2 negative. Furthermore, they were significantly more often DNA non-diploid, as well as being characterised by higher S-phase fraction values. These results suggest that BRCA1-induced breast cancers may manifest distinct tumour biological features of clinical importance.

Immunocytochemical demonstration of oestrogen receptor beta in blood vessels of the female rat.

The role of oestrogen receptor (ER) beta in vascular function remains unclear. With the use of a specific ERbeta antibody we have now, using immunocytochemistry, visualized ERbeta in different parts of the vascular tree. In about 70% of medial smooth muscle cells of female rat aorta, tail artery and uterine artery, nuclear immunoreactivity to ERbeta was observed. In these vessels endothelial cells also expressed ERbeta. Vascular expression of the ERalpha subtype was lower than that of ERbeta. In aorta and tail artery, no immunoreactivity towards ERalpha was observed, while in uterine vessels occasional medial smooth muscle and endothelial cells expressed this ER subtype. ERbeta and alpha expression in uterine vessels was independent of the stage of the oestrous cycle, suggesting that variations in uterine blood flow occurring during the cycle are independent of ER density. The regional distribution of ERalpha, as determined by immunocytochemistry, was supported by measurements of ERalpha levels by enzyme immunoassay. In the uterine artery, the level of ERalpha was several times higher (P<0.001) than that of aorta and tail artery (10.1+/-1.7 fmol/mg protein in the uterine artery vs 3.3+/-1.0 and 0.5+/-0.5 fmol/mg protein in aorta and tail artery respectively). Thus, a prominent nuclear expression of ERbeta was observed in the vascular wall of several parts of the vascular tree, while ERalpha predominantly was expressed in uterine vessels, suggesting that ERbeta and alpha may have different roles in vascular function.

Clonal chromosome-aberrations in fibrocystic breast disease-associated with increased risk of cancer.

Short-term cultures of 29 samples of fibrocystic breast disease were cytogenetically analyzed. Clonal chromosome aberrations were found in six specimens, whereas the remaining 23 had a normal karyotype. Three of the abnormal samples displayed karyotypic anomalies previously associated with breast cancer, i.e., gain of Iq, trisomy 18 and cytogenetic multiclonality. Furthermore, all cytogenetically aberrant specimens had either proliferative disease without atypia or atypical hyperplasia, features of fibrocystic disease considered risk factors for subsequent breast cancer development. The cytogenetic similarities between breast carcinomas and proliferative fibrocystic breast disease add further support for classifying certain types of fibrocystic disease as a premalignant condition. Whether cytogenetically abnormal fibrocystic lesions are the ones that subsequently progress to cancer remains to be elucidated.

Cytogenetic findings in phyllodes tumors of the breast: karyotypic complexity differentiates between malignant and benign tumors.

Clonal karyotypic abnormalities were detected in short-term cell cultures from six phyllodes tumors of the breast. Whereas all five benign tumors had simple chromosomal changes, the highly malignant one had a near-triploid stemline, indicating that karyotypic complexity is a marker of malignancy in phyllodes tumors. Interstitial deletions of the short arm of chromosome 3, del(3)(p12p14) and del(3)(p21p23),were the only aberrations in two benign tumors. Cytogenetic polyclonality was detected in three benign tumors: two had cytogenetically unrelated clones, whereas the third had three different, karyotypically related cell populations as evidence of clonal evolution. The finding of clonal chromosome abnormalities in both the epithelial and connective tissue components of the phyllodes tumors indicates that they are genuinely biphasic, that is, that both components are part of the neoplastic parenchyma.

Multiple polysomies in breast carcinomas: preferential gain of chromosomes 1, 5, 6, 7, 12, 16, 17, 18, and 19.

Chromosome G-banding analysis of metaphase cells from 16 primary breast carcinomas revealed the presence of multiple polysomies in near-diploid as well as in polyploid cells. Chromosome 17 was preferentially gained in 7 tumors, followed in frequency by chromosomes 1, 12, and 19 (5 tumors each), and chromosomes 5, 6, 7, 16, and 18 (4 tumors each). Eleven of the 16 carcinomas had, apart from the clones exhibiting the numerical gains, other unrelated clones. Nine of these 11 cases had clones with structural chromosome aberrations, 5 of which had structural aberrations involving the short arm of chromosome 3. The biologic significance, if any, of this seemingly nonrandom coexistence of multiple polysomies with structural aberrations of 3p is at present not known. The pattern of numerical chromosome aberrations observed in the present study is comparable to previous results from fluorescence in situ hybridization (FISH) studies, with the use of centromeric probes on interphase cells. However, unlike FISH studies, which have been focused on chromosomes 1, 3, 7, 8, 11, 16, and 17, the cytogenetic results reveal that other chromosomes also may be nonrandomly gained as part of multiple polysomies in breast carcinomas. In addition, the tumors with multiple polysomies were generally of high histologic grade and with metastasis to axillary lymph nodes, suggesting that multiple wholechromosome gains may be a pathway of genetic evolution or progression or both in some breast carcinomas.

Cytogenetic changes in benign proliferative and nonproliferative lesions of the breast.

Cytogenetic analysis of short-term cultures from 69 cases of fibrocystic breast changes and 10 samples of normal mammary tissue revealed clonal chromosome aberrations in six fibrocystic lesions. All the histologically normal tissue samples had a normal karyotype. The frequency of cytogenetically abnormal cases seems to correlate with the degree of histopathologic changes of the tissue; nonproliferative lesions may have clonal chromosome alterations, but at a low frequency. Whether women with karyotypically altered fibrocystic "disease" have a higher risk of developing invasive breast cancer, compared with women without microscopically visible genetic anomalies in fibrocystic lesions, remains unknown.

Ipsilateral local recurrence in relation to therapy and morphological characteristics in patients with ductal carcinoma in situ of the breast.

METHOD AND RESULTS: A standardized histopathological protocol has been designed, in which different histological characteristics of ductal carcinoma in situ (DCIS) are reported: nuclear grade (ng), growth pattern according to Andersen et al., necrosis, size of the lesion, resection margins and focality. Using this protocol a re-evaluation of a population-based consecutive series of 306 cases of DCIS has been done as well as a thorough clinical follow-up. After a median follow-up of 63 months, 13% have developed ipsilateral local recurrences, invasive and/or in situ. Ipsilateral local recurrence-free survival (IL-RFS) was significantly better for patients operated with mastectomy (ME) or breast conserving therapy (BCT) with radiotherapy (RT) than for patients operated with BCT without RT (5-year IL-RFS 96% vs 94% vs 79%, P<0.001). In the subgroup of BCT without RT there were significant differences in IL-RFS between histopathological subgroups: ng 1 + 2 (non-high grade) vs ng 3 (high grade; P=0.014), non-high-grade without comedo-type necrosis vs non-high-grade with comedo-type necrosis vs high-grade (the Van Nuys classification system; P=0.025). Growth pattern (not diffuse vs diffuse) and margins (free vs involved or not evaluated) showed a tendency (P=0.07 and 0.05, respectively) to be associated to IL-RFS. In contrast, no significant differences in IL-RFS were found in subgroups based on mode of detection, focality or size. Ninety-four per cent of the local recurrences after BCT appeared at the previous operation site. CONCLUSIONS: In the BCT without RT group, combinations of either non-high grade and not a diffuse growth pattern or non-high grade and free margins identified groups (constituting approximately 30% of the patients) were at low risk of developing ipsilateral recurrences (6-10%), compared to a 31-37% recurrence risk in the remaining groups during the observed follow-up time. The beneficial effect of post-operative RT for these low-risk groups can be questioned, and should be studied further.

Differential effects of estrogen on DNA synthesis in human periodontal ligament and breast cancer cells.

BACKGROUND: It is important to clarify the biological function of the female sex hormones estrogen and progesterone in periodontal ligament cells, as these hormones may affect periodontal health. We have previously shown that human periodontal ligament cells express estrogen receptor beta (ERbeta) but not ERalpha, whereas human breast cancer cells (MCF7) express both ERalpha and ERbeta. Data on progesterone receptor (PgR) expression in human periodontal ligament cells have not been reported. OBJECTIVES: Determine PgR expression in human periodontal ligament and MCF7 cells and to investigate how estrogen affects DNA and collagen synthesis in these two cell types showing different pattern of expression for ERalpha and beta. METHODS: Periodontal ligament cells were obtained from the periodontal ligament of premolars extracted for orthodontic reasons and MCF7 cells from the American Type Culture Collection (ATCC). PgR expression was determined by immunocytochemistry. DNA and collagen synthesis was determined by [(3)H]thymidine and L-[(3)H]proline incorporation, respectively. RESULTS: PgR immunoreactivity was observed in nuclei of MCF7 but not periodontal ligament cells. Treatment with estrogen (17beta-estradiol, E(2)) at physiological concentrations for 24 h stimulated DNA synthesis by more than two times in MCF7 cells, whereas there was no effect on periodontal ligament cell DNA synthesis. The ER blocker ICI 182780 fully reversed the stimulatory effect of E(2). Not only short-term (24 h) but also long-term (5 days) treatment with E(2) lacked effect on DNA synthesis in periodontal ligament cells. Neither periodontal ligament cell viability nor collagen synthesis was affected by E(2) treatment. Identical results were observed in periodontal ligament cells from male and female subjects. CONCLUSIONS: Human MCF7 but not periodontal ligament cells express PgR, suggesting that progesterone via PgR affects MCF7 but not periodontal ligament cells. Further, estrogen stimulates breast cancer MCF7 cell proliferation, whereas it has no effect on proliferation of periodontal ligament cells, probably reflecting cell type specific ER expression pattern in these two cell types.

The microscopical appearance of the subcutaneously transposed spleen.

The microscopical appearance of rat splenic tissue has been examined after subcutaneous transposition of the spleen and after portal vein ligation in rats subjected to splenic transposition. The histological picture was compared to that of control animals. Apart from congested red pulp and capsular fibrosis in the subcutaneous position did not seem to result in serious histological changes in the spleen. In three patients treated with subcutaneous transposition of resected spleen because of portal hypertension, histological needle biopsies from the splenic tissue were made postoperatively. Principally the same histological changes were found in the clinical as in the experimental material.

Benign solitary neurilemoma (Schwannoma). A correlative cytological and histological study of 28 cases.

A correlative histological and cytological study of 28 cases of solitary neurilemoma is presented. The typical histological mixture of Antoni type A and B tissue correlated well with the cytological findings. In the smears, the Antoni type A tissue was represented by tissue fragments with a fibrillar ground substance and slender, spindle-shaped cells forming obvious Verocay bodies in 24 cases, and the Antoni type B tissue by loose, microcystic fragments with single, elongated nuclei sometimes looking like fish-hooks and with indistinct, slender, cytoplasmic processes. The ancient neurilemomas, a variant of the ordinary neurilemomas, in this series were characterised both histologically and cytologically by nuclear hyperchromasia and polymorphism, which it is important to be aware of in order to avoid an erroneous diagnosis of sarcoma or probable sarcoma. The differential diagnosis is discussed and it is concluded that diagnosis of neurilemoma by fine-needle aspiration biopsy is possible, particularly when Verocay bodies and/or a fibrillar ground substance are present in the smears. It is stressed that fine-needle aspiration biopsy is an important aid to pre-operative diagnosis of neurilemoma, and thus to planning of the operative treatment.

Cytodiagnosis of soft tissue tumors and tumor-like conditions by means of fine needle aspiration biopsy.

In the evaluation of soft tissue tumors primary sarcomas must be differentiated from other malignancies, such as cancer metastases, malignant lymphomas, as well as benign tumors and tumor-like conditions. As far as possible this should be done without open biopsy to avoid local spread. Fine needle aspiration biopsy as opposed to thick needle biopsy utilizes smaller needle diameters up to 0.8 mm with assumed negligible risks of local or distant tumor spread. Such a fine needle aspiration biopsy for cytodiagnosis was supposed to increase the reliability of the preoperative diagnosis. In a consecutive series of 187 patients referred to an orthopedic oncology group because of suspected malignancy fine needle aspiration biopsy was made. In 129 cases with histology and in 49 non-operated cases with at least 2 years of clinical follow-up the reliability of the method is analyzed. In 163 cases the aspirated material was sufficient for a diagnosis and 152 cytological reports were correct regarding the diagnosis of a malignancy contra a benign tumor or tumor-like condition. Thirty-five of 43 malignancies were primary soft tissue sarcomas, 28 of these with a correct cytologic diagnosis of sarcoma. Eight were malignant lymphomas or carcinomas. Fourty-nine patients with 48 benign and one malignant cytological diagnosis were not operated upon. At least 2 years of clinical follow-up confirmed a benign diagnosis in these cases. The total reliability is thus around 85%. Fine needle aspiration biopsy is considered to be a very valuable complement to other investigations in the diagnostic work-up of soft tissue tumors.

Estramustine binding site in human breast cancer biopsy samples. Its relation to estrogen and progesterone receptor levels, age and menopausal status.

Estramustine is a cytotoxic metabolite of estramustine phosphate (Estracyt), which is used in the treatment of prostatic carcinoma. An estramustine binding site (EMBS) at pH 4.8-4.9 was demonstrated in 74 of 306 (24%) breast cancer biopsy samples using isoelectric focusing in polyacrylamide gels. The presence of EMBS was significantly (P less than 0.001) correlated with negative or low estrogen and progesterone receptor values. EMBS positivity was found in 31% of the samples from pre- and perimenopausal patients and in 22% of the samples from postmenopausals. If patients were instead divided into different age groups, EMBS positivity was most frequent in samples from patients between 50 and 59 years of age (42%). With increasing age the percentage of EMBS positivity fell successively. For patients under 50 years, no difference with respect to EMBS positivity between age groups could be demonstrated. The possible value of EMBS determinations in breast cancer tissue specimens for the selection of those patients that will respond to Estracyt therapy should be evaluated.

Estrogen receptor enzyme immunoassay in fine-needle aspirates from human breast cancer.

An enzyme immunoassay was compared with a radioligand assay (isoelectric focusing) for estrogen receptor (ER) determination in 100 preoperative fine-needle aspirates from human breast cancer. A strong correlation (rs = 0.85) between the two estimates was found. However, ER estimates by enzyme immunoassay were 6 times higher than with isoelectric focusing, mainly due to an underestimation of ER content with the latter method in highly blood-contaminated or diluted samples. Enzyme immunoassay provided adequate ER measurement even at very high (50%) blood admixture. To study the reliability of ER measurement with enzyme immunoassay in fine-needle aspirates, a comparison was made with estimates in the corresponding surgical biopsies (65 cases). With DNA as reference parameter a significant correlation (rs = 0.68) was obtained. Excluding aspirates with low cellularity (less than 50,000 cells/ml), only one ER negative fine-needle aspirate was found among 47 ER positive surgical biopsies. A few ER positive aspirates corresponded to ER negative surgical biopsies possibly due to tumour heterogeneity or the fact that fine-needle aspiration sometimes may represent a better sampling technique than surgical biopsy.

Oestrogen receptor analysis of paraffin sections and cytosol samples of primary breast cancer in relation to outcome after adjuvant tamoxifen treatment. The South Sweden Breast Cancer Group.

The aim of the present study was to compare oestrogen receptor (ER) analysis results obtained in cytosols of frozen breast cancer tissue (using biochemical assay) with those obtained in paraffin-embedded tissue (using immunoperoxidase staining with monoclonal antibodies (DAKO-ER, 1D5), and an ER positivity cut-off level of >10% stained nuclei). In 86% (84/98) of the samples the same ER status (28 negative and 56 positive) was obtained with both procedures. In eight cases, the paraffin section was ER positive but the corresponding cytosol sample ER negative, whereas six cases showed the opposite pattern. The ER positive subgroup manifested better outcome after adjuvant treatment than the ER negative subgroup (p = 0.003 (cytosol), and p = 0.004 (paraffin)). As compared with the percentage of stained nuclei, staining intensity yielded no additional information. Although the results of ER analysis of paraffin-embedded material seem promising, it is too early to prefer it to frozen tissue, though this would be useful when no frozen tissue is available.

Aspiration cytology of soft tissue tumours. A prospective study of its influence on choice of surgical procedure.

Tumour cells may be dispersed into the wound when diagnostic or excisional biopsy is undertaken. This risk is reduced by using fine needle aspiration biopsy. The definitive excision of a soft tissue sarcoma may then be carried out in a more limited manner. In selected cases diagnostic and definitive operation may be combined, accepting the risk of excision of a benign lesion with unnecessarily broad margins. Aspiration cytology could possibly help the surgeon to decide on the extent of the excision. One hundred and nine consecutive patients with soft tissue lesions were referred to the Orthopaedic Oncology Group at Lund in Southern Sweden because of suspected malignancy. Biopsy had not been carried out. Further diagnostic and therapeutic procedures were planned as if aspiration cytology was not going to be used. The procedure was then performed with a fine needle and the cytodiagnosis was used to modify the plan. Sixty-seven tumours were examined histologically. Ten were sarcomas, two metastatic carcinomas and one a malignant lymphoma. The surgical plan was changed after the cytology was known in 33 of the 109 cases. Compartmental or wide excisions which had been planned became unnecessary in 20 patients. No operation was done in 6 patients and in 14 others a more limited excision was carried out. It was possible to avoid planned incisional biopsy in 3 patients with sarcoma.