Is digitalis a therapy for breast carcinoma?

We have previously reported effects on breast carcinoma by digitalis on patients in vivo with significant effects on cytometric features and recurrence rate. Increased attention is now paid to the anti-proliferative and apoptosis inducing effect on cancer cells in vitro by glycosides from foxglove as well as by some other glycosides. The present study is a long-term follow-up (22.3 years) of 175 patients with breast carcinoma, of which 32 were on digitalis treatment, when they acquired their breast carcinoma. There was a lower death rate (6%) from breast carcinoma among the patients on digitalis, when compared with patients not on digitalis (34%). Also proliferation/aneuploidy was less pronounced of the tumors in patients on digitalis. These observations were statistically significant although the statistical analysis was hampered in the life-table analysis by the fact that only 2/32 patients on digitalis died from breast cancer. Serious consideration should be given to the effects of digitalis derivatives on cancer cells in cancer drug design. This field of research is not sufficiently explored and holds promise to contain drugs superior to present-day adjuvant therapy both with respect to effects and side-effects.

C-erb-b2/int-2 amplification appears faster in breast-cancer patients receiving second-line endocrine treatment.

We have examined the appearance of c-erb-b2 and int-2 amplification in 2 different groups of breast-cancer patients. The groups differed with regard to their clinical status in that one group was receiving first-line endocrine treatment (tamoxifen) whereas the second was receiving second-line endocrine treatment (after failing on tamoxifen). The latter group of patients showed clinicallya a more advanced disease (higher frequency of stage-IV as compared to stage-III disease). Consecutive tumor samples were obtained using fine-needle biopsies from individual tumor lesions of each patient every second or third month. Median time from diagnosis to the last biopsy for patients receiving tamoxifen was 25 months and, for patients receiving second-line treatment, 55 months. The presence of amplification was determined using semi-quantificative PCR. We found that both genes developed amplification during tumor progression. The appearance of amplification was more pronounced in the clinically more advanced patients receiving second-line treatment (p = 0.018).

Prognostic significance of c-erb-B2 amplification in fine-needle biopsies of breast cancer patients not operated at diagnosis.

Prognostication of breast cancer patients, not operated at diagnosis, poses a clinically difficult problem. To use gene amplification we examined cytological samples and determined c-erb-B2 gene copy number with semi-quantitative PCR. Control experiments showed the same gene-copy number in aliquots that were either air-dried (and MGG-stained), fixed in methanol (and air-dried), or snap-frozen in liquid nitrogen. Therefore we examined the prognostic value of c-erb-B2 amplification in 95 breast cancer patients that had not been operated at diagnosis (up to 12 years previously). Tumor cells were obtained from routine archival cytological smears. 15 patients (16%) had developed amplification. Univariate and multivariate analysis showed that c-erb-B2 amplification is a significant prognostic factor (p < 0.0001). Hence routine cytological MGG smears can be used for prognostic determination.

Higher frequency of gene amplification in breast cancer patients who received adjuvant chemotherapy.

BACKGROUND: Amplification of certain genes is involved in resistance to chemotherapy. The development of such amplification in patients by drug treatment has not yet been established. We have assessed the appearance of gene amplification in breast cancer patients with recurrent disease. One group of patients had previously received adjuvant chemotherapy (cyclophosphamide, methotrexate, 5-fluorouracil [CMF]) after surgery. The second group had not. All of these patients had developed recurrent disease and were receiving first-line endocrine treatment (tamoxifen). METHODS: Tumor cells were obtained from the recurrent carcinoma using fine-needle biopsies. Gene copy number was determined for dihydrofolate reductase and thymidylate synthase with semiquantitative polymerase chain reaction. Dihydrofolate reductase is involved in resistance to methotrexate (M) and thymidylate synthetase in resistance to 5-fluoropyrimidines (F), two targets for the CMF regime. RESULTS: We found that amplification of the examined genes develops in higher frequency among patients who previously received chemotherapy (p = 0.002). CONCLUSIONS: These findings provide strong evidence for gene amplification induced by drug treatment.

Reasons for cervical cancer despite extensive screening.

OBJECTIVE: To explain an age adjusted incidence rate of cervical cancer of 10.1 and 10.5 per 100,000 women, despite extensive screening. SETTING: The Swedish county of Gävleborg in 1986 and 1987. METHODS: Thirty eight patients with "cervical cancer" reported to the central cancer registry in Sweden were investigated. The patients and their diagnoses were scrutinised in a double blind manner. RESULTS: Eighteen per cent (7/38) of cases were shown to be mistakes in data transfer; 11% (4/38) of cases were endocervical adenocarcinomas; 13% (5/38) were histopathological misinterpretations and should have been reported as carcinoma in situ. Of the remaining 22 patients with invasive squamous cancer, 12 (55%) had not participated in the gynaecological health control programme. Of the 10 participants with invasive squamous cancer despite this participation, eight (80%) had repeatedly had abnormal Papanicolaou smears without further gynaecological/histopathological examination and treatment. There was no evidence of cases of carcinoma in situ or endometrial cancer diagnosed in 1986-87 being squamous cervix cancer. The true incidence of squamous cervical cancer among participants was 3.0 per 100,000 for the two years scrutinised. If all the patients with Papanicolaou smear abnormalities had been properly managed at the right time, and the treatment had been successful, the incidence of invasive squamous cancer would have been 0.8 per 100,000 women among participants as opposed to 38.2 per 100,000 among non-participants. CONCLUSION: The evidence strongly suggests overascertainment of cervical cancer, which conceals the success of screening, and also suggests that much attention must be given to clinical management of detected lesions in cervical screening. Care is needed in applying accurate histopathological criteria when making a diagnosis of invasive squamous cancer, to separate squamous cancer from other malignant tumours of the cervix, and in data transfer to cancer registries.

Prognostic value of amplification of c-erb-B2 in bladder carcinoma.

Amplification of c-erb-B2 is examined in patients with transitional cell bladder carcinoma. In a pilot study we found that the amplification correlated with high tumor grade. Tumor grade is a known prognostic factor. Therefore, we next examined the prognostic value of c-erb-B2 amplification in patients with >16 years of clinical follow-up. The gene copy number was determined, using semiquantitative PCR, in archival formalin-fixed tissues. Twenty-three percent (37/163 patients) showed the amplification. The amplification correlated with grade and stage. Moreover, we found that tumor grade (P < 0.001) and c-erb-B2 amplification (P < 0.001) showed prognostic information for survival. Patients with grade 3 tumors and concomitant c-erb-B2 amplification showed the worst prognosis. Multivariate analysis indicates that grade and c-erb-B2 amplification are independent prognostic factors.

Prognostic value of erb-B2 and myc amplification in breast cancer imprints.

BACKGROUND: Amplification of erb-B2 and myc shows prognostic value in patients with operable breast cancer. Amplification is usually detected in tumor samples remaining after pathologic work-up, preventing the examination of small tumors. METHODS: Tumor imprints that contained low numbers of contaminating normal cells were obtained from small tumors. The prognostic value of erb-B2 and myc amplification in imprint breast preparations was examined, using the polymerase chain reaction (PCR) to determine gene copy number. Tumor material was obtained from 162 patients with breast cancer operated 1975-1976. RESULTS: Amplification of erb-B2 existed in 21% and myc in 16% of the patients. Both erb-B2 and myc amplification showed prognostic significance in univariate analysis for survival and relapse free survival. In multivariate analysis, erb-B2 was a significant factor. In small tumors less than 13 mm in greatest dimension, erb-B2 showed prognostic significance for survival but not for relapse free survival. CONCLUSIONS: The use of imprints/PCR allows the detection of gene amplification in breast cancer. The procedure is suitable for the analysis of small tumors and can be used to examine very small tumors, such as those detected mammographically.

Intratumoral heterogeneity for amplified genes in human breast carcinoma.

Intratumoral heterogeneity was studied in human breast cancer by examining separate tumor lesions of individual patients. Tumor samples were obtained from each patient by fine-needle biopsies from 2 to 4 separate tumor lesions. We used a semi-quantitative PCR to distinguish between samples with gene amplification and single gene copy samples. Five genes were analyzed in each biopsy: MDR-1, dihydrofolate reductase, thymidylate synthase, c-erb-B2 and int-2. Three groups of patients were examined: those awaiting initiation of treatment; those receiving first-line endocrine therapy; and those receiving second-line endocrine treatment. A pattern of intratumoral heterogeneity for gene amplification was clearly apparent. The frequency of amplification was lowest before initiating therapy and highest in patients receiving second-line treatment (p = 0.023).

Clinical utility of cellular DNA measurements in prostate carcinoma. Consensus Conference on Diagnosis and Prognostic Parameters in Localized Prostate Cancer. Stockholm, Sweden, May 12-13, 1993.

At a WHO consensus conference on Early Diagnosis and Prognostic Parameters in Localized Prostate Cancer, a working group discussed the clinical utility of DNA measurements in stages T2 and T3 prostate carcinoma. Incidentally discovered prostate cancer of stage T1 was excluded. The members of the working group, representing various clinical and laboratory disciplines, discussed technical considerations of DNA measurements by flow and image analysis, pretreatment prediction of prognosis, and posttreatment clinical relevance. The group agreed to subdivide tumors into diploid, tetraploid and non-tetraploid aneuploid, expressing various degrees of aggressiveness and gave guidance for the definition of limits of these groups. The panel agreed that knowledge on DNA ploidy prior to treatment is of value in treatment decisions, particularly when surveillance is a treatment option. Aneuploid tumors can be expected to respond very poorly to either irradiation or endocrine therapy, and the presence of aneuploid tumor, either on pretreatment biopsies or in radical prostatectomy specimens, is an ominous sign. The identification of a group of patients with a uniformly poor prognosis should encourage medical oncologists and basic scientists to develop adequate treatment options for this particular group. The panel expressed a strong opinion that DNA ploidy should be uniformly studied in clinical trials, particularly in patients with localized prostate cancer.

Demonstration of gene-amplification by PCR in archival paraffin-embedded breast cancer tissue.

The polymerase chain reaction (PCR) is a powerful tool to examine genetic alterations in tumor samples. We describe a simple, rapid, nonisotopic PCR method to semi-quantitatively determine the number of gene copies in human formalin-fixed paraffin-embedded tissue. The procedure is exemplified by analysis of 15 years old breast cancer samples to determine the presence of amplification of c-erb-B2. The samples were obtained from routine specimens kept in pathological archives. Patients with amplified samples showed a poor prognosis, both for recurrences and death in breast cancer.

Breast cancer: prognostic significance of c-erb-B2 and int-2 amplification compared with DNA ploidy, S-phase fraction, and conventional clinicopathological features.

The prognostic value of oncogene amplification and conventional clinicopathological features was determined in consecutive breast cancers detected during 5 months in 1975-1976 in 4 Swedish counties. Material was collected from 162 of the 179 patients and tumor size, nodal status, FSH, estrogen/progesterone receptor status, DNA ploidy and S-phase fraction determined. Tissues remaining from 80 patients were stored frozen until 1991, when amplification of the oncogenes c-erb-B2 and int-2 was determined. We show that c-erb-B2 amplification (but not int-2 amplification) and positive axillary nodal status show prognostic significance for both survival and relapse-free survival in univariate and multivariate analysis. The other examined factors showed no significance.

Gene amplification detected in carcinoma cells from human urinary bladder washings by the polymerase chain reaction method.

BACKGROUND: Urinary bladder carcinoma often is diagnosed from malignant cells in bladder washings obtained at cystoscopic examination. In some cases, there are difficulties distinguishing between cytologic Grades 1 and 2. Detection of genetic alterations in combination with morphologic analysis may facilitate the diagnosis. METHODS: The presence of amplified c-erb-B2 and epidermal growth factor receptor (EGF-R) was examined in tumor cells present in bladder washings. The gene copy number was determined with the polymerase chain reaction (PCR). RESULTS: The authors detected amplified c-erb-B2 and EGF-R in cancer cells of cytologic Grade 2 and 3 tumors. They did not detect amplification in cytologic Grade 1 tumor cells or in cells from bladders, without known malignant neoplasms. CONCLUSIONS: Examination of genetic alterations, in combination with morphologic analysis, facilitates the diagnosis of carcinoma.

Appearance of amplified thymidylate synthase or dihydrofolate reductase genes in stage-IV breast-cancer patients receiving endocrine treatment.

We have examined the appearance of amplification of 2 genes involved in resistance to chemotherapy (thymidylate synthase, dihydrofolate reductase) in patients receiving endocrine treatment. Chronological tumor samples were obtained from breast-cancer patients with clinical stage-IV disease, using fine-needle biopsies. The presence of amplification of thymidylate synthase and dihydrofolate reductase was determined using PCR in 185 fine-needle biopsies from 37 patients. None of the initial samples of each patient showed amplification. However, in 7 of the 37 patients (19%) we detected development of gene amplification: in 2 cases of thymidylate synthase and in 5 cases of dihydrofolate reductase. Five of the 7 patients with amplification were receiving second-line endocrine treatment after failing to respond to tamoxifen.

Reduced occurrence of mdr-1 amplification in stage-IV breast-cancer patients treated with tamoxifen compared with other endocrine treatments.

Amplification of mdr-1 occurs in breast cancer patients receiving endocrine treatment. We have compared a group of patients receiving tamoxifen and a group receiving second-line endocrine treatment after failure of tamoxifen treatment. Chronological tumor samples were obtained by fine-needle biopsies from the same tumor lesion of each patient and PCR was used to detect the appearance of gene amplification. Three of 20 patients receiving tamoxifen developed amplified mdr-1 whereas 7 of 17 patients receiving second-line endocrine treatment developed mdr-1 amplification.

Appearance and detection of multiple copies of the mdr-1 gene in clinical samples of mammary carcinoma.

To examine the presence of multiple copies of the mdr-I gene in clinical tumor samples we have developed an approach where the cells are obtained by fine-needle biopsies and the number of gene copies determined by PCR. The temporal appearance of amplified mdr-I was examined in 20 breast-cancer patients with clinical stage-IV disease receiving endocrine treatment. Tumor samples were obtained every 2nd to 3rd month from the same tumor lesion. None of the initial samples from each patient contained multiple copies of mdr-I. Of 16 patients who showed increased tumor size, 4 developed multiple gene copies, showing that the event occurs without cytotoxic selection of cells with chemotherapy.

Morphometry of nuclei of the normal and malignant prostate in relation to DNA ploidy.

Fine needle biopsies from 70 patients with prostate carcinoma and 10 patients with benign hyperplasia were used to study area, variation in size and form factors of the nuclei by image analysis. The results were related to DNA ploidy of the cell populations as measured by flow cytometry, cytologic grade and patients' survival. Nuclear area differed significantly between benign lesions and tumors. It increased in diploid low-grade tumors from a normal value of 54.2 +/- 3.1 microns2 to 75.6 +/- 5.3 microns2. In aneuploid tumors with an increase in the chromosome number, the nuclear size further increased to about twice that of benign nuclei. Variation in size also differed between benign and malignant epithelium, with a further increase between diploid and gross aneuploid tumors. While nuclear size and variation in nuclear size made it possible to discriminate malignant from benign lesions, form factor did not differ between benign and malignant lesions. In follow-up, however, none of these factors reached significance for predicting survival.

Detection and temporal appearance of multiple copies of c-erb-B2 genes in advanced mammary carcinoma using fine needle biopsies and the polymerase chain reaction.

Aspiration of tumor cells by the fine-needle biopsy method yields only a small number of cells, which hampers conventional molecular analysis for the presence of multiple copies of oncogenes. We have therefore adopted the polymerase chain reaction (PCR) method to study semi-quantitatively the level of the c-erb-B2 gene in human breast tumor samples. Of 39 patients with mammary carcinoma, 7 (19%) contained multiple copies of c-erb-B2 genes, whereas only two samples failed to give informative data. Next the multiple copies of c-erb-B2 genes, whereas only two samples failed to give informative data. Next the temporal appearance of multiple gene copies was examined in 20 patients with clinical stage IV disease. Tumor samples were obtained every second to third month from the same tumor lesion of each patient. None of the initial samples from each patient contained multiple copies of c-erb-B2. Of 16 patients that showed progressive clinical disease, 5 developed multiple gene copies, showing that the event occurs in clinical stage IV disease.