Contribution of germline MLH1 and MSH2 mutations to lobular carcinoma in situ of the breast.

Lobular carcinoma in situ (LCIS) is an unusual histological pattern of non-invasive neoplastic disease of the breast occurring predominantly in women aged between 40 and 50 years. LCIS is frequently multicentric and bilateral suggesting a genetic basis to the disease. The high frequency of microsatellite instability in lobular breast cancers, coupled with increased risk of breast cancer associated with germline mismatch repair gene mutations raises the possibility that mutations MSH2 or MLH1 might confer susceptibility to LCIS. To explore this possibility we have examined a series of 71 LCIS patients for germline MSH2 and MLH1 mutations. No mutations were detected in MSH2. Two sequence variants were identified in MLH1. The first was a CTT-->CAT substitution, codon 607 (exon 16) changing leucine to histidine. The other mutation detected in MLH1 was a TAC-->TAA substitution codon 750 (exon 19) creating a stop codon, predicted to generate a truncated protein. These findings suggest that mutations in MLH1 may underlie a subset of LCIS cases.

Lobular carcinoma in situ of the breast is not caused by constitutional mutations in the E-cadherin gene.

Lobular carcinoma in situ (LCIS) is an unusual histological pattern of non-invasive neoplastic disease of the breast occurring predominantly in women aged between 40 and 50 years. LCIS is frequently multicentric and bilateral, and there is evidence that it is associated with an elevated familial risk of breast cancer. Although women with LCIS suffer an increased risk of invasive breast disease, this risk is moderate suggesting that LCIS may result from mutation of a gene or genes conferring a high risk of LCIS, but a lower risk of invasive breast cancer. The high frequency of somatic mutations in E-cadherin in LCIS, coupled with recent reports that germline mutations in this gene can predispose to diffuse gastric cancer, raised the possibility that constitutional E-cadherin mutations may confer susceptibility to LCIS. In order to explore this possibility we have examined a series of 65 LCIS patients for germline E-cadherin mutations. Four polymorphisms were detected but no pathogenic mutations were identified. The results indicate that E-cadherin is unlikely to act as a susceptibility gene for LCIS.

Induction of transforming growth factor beta 1 in human breast cancer in vivo following tamoxifen treatment.

We have investigated the ability of tamoxifen to regulate members of the transforming growth factor beta (TGF-beta) family in human breast cancers in vivo. Using immunohistochemical techniques, we find that 3 months of tamoxifen treatment causes a consistent induction of extracellular TGF-beta 1 in breast cancer biopsies, compared with matched pretreatment samples from the same patient. The induced TGF-beta is localized between and around stromal fibroblasts and appears to be derived from these cells. Lower levels of TGF-beta 1,-beta 2, and -beta 3 seen in epithelial cells were not altered by tamoxifen treatment. The increased stromal staining of TGF-beta 1 occurred in estrogen receptor-negative as well as estrogen receptor-positive tumors. These results provide in vivo evidence for a novel, estrogen receptor-independent mechanism of action for tamoxifen, involving the stromal induction of a potent growth inhibitor for epithelial cells.

Chemoprevention of breast cancer.

The hypothesis that oestrogen is an important promoter of human breast cancer raises the possibility that endocrine intervention could prevent the disease. Various methods of reducing oestrogenic activity have been proposed including dietary control, progestin therapy, and ovarian ablation. Tamoxifen is a synthetic anti-oestrogen of low toxicity with proven anti-proliferative activity in endocrine sensitive breast cancer which makes it an attractive alternative for a trial of endocrine prevention. We have undertaken a double-blind placebo-controlled pilot study to assess the feasibility of mounting a large multicentre study of tamoxifen in the prevention of breast cancer in high risk women. Two hundred women were randomised to tamoxifen or placebo during an 18 month accrual period. Acute toxicity was mild and serial studies of blood lipids, clotting factors, and bone mineral density suggested that no long-term deleterious effects are likely to be seen. It is estimated that 10,000 women would be required with a 10-15 year follow up in order to detect a 25% prevention effect.

Chemoprevention of breast cancer.

Epidemiological and experimental evidence indicate that oestrogenic activity plays an important role in the promotion of human breast cancer. This raises the possibility that anti-oestrogenic intervention could prevent the development of this disease. In order to detect a 25% reduction in incidence of breast cancer between a treatment and control population, at least 300 breast cancers would need to develop. With a high-risk group of women, such as those with a family history, between 40 and 60 years old, 10,000 women would be required with a 10-year follow-up for 250-300 cancer to develop. This would indicate that to have a reasonable chance of detecting a significant prevention of breast cancer, by an anti-oestrogenic intervention, 5,000 treatment and 5,000 control women with a high risk of developing breast cancer would be needed. However, before such a major trial could be attempted it was essential to evaluate the ethics, logistics, patient and doctor acceptability, acute toxicity, patient accrual and compliance of tamoxifen, in a prevention context, in a small feasibility trial. We have therefore started a double-blind placebo controlled feasibility trial designed to accrual 200 women aged between 35 and 65 with a family history of breast cancer. Between October 1986 and July 1987 a total of 124 patients were randomised to receive either tamoxifen or placebo. With these patients as background the present paper will outline the various problems, including acute toxicity, compliance and patient acceptability for tamoxifen versus placebo in a prevention trial.

Immunohistochemical distribution of c-erbB-2 in infiltrating and in situ breast cancer.

An immunohistochemical study of c-erbB-2 expression was performed on invasive and in situ breast cancer. Strong membrane staining was seen in 16% of the infiltrating ductal carcinomas and 44% of the in situ lesions. c-erbB-2 was overexpressed in ductal rather than lobular tumours. Our results indicate that a small sub-group of breast carcinomas are associated with over-expression of this oncogene which may define an important subgroup of in situ and infiltrating ductal carcinomas.

Patterns of breast relapse after local excision +/- radiotherapy for early stage breast cancer.

One hundred patients with breast recurrence have been identified from patients at the Royal Marsden Hospital, treated by local excision +/- radiotherapy for early stage primary invasive breast cancer between 1961 and 1985. The mean follow-up was 58 months (range 1 month - 19 years). In 74/100 patients, breast recurrence occurred within the breast parenchyma, was not associated with systemic relapse and carried a relatively good prognosis with a median survival of 77 months from the time of breast relapse. In 67 patients with parenchymal relapse in whom the site of relapse could be reliably compared with that of the original tumour, 60 (90%) patients developed recurrent tumours at or close to the primary site. In 24/100 patients, breast recurrence occurred in the overlying skin and in only two of these patients (2% of total) did recurrence actually occur within the scar tissue. Skin relapse was associated with systemic relapse and carried a relatively poor prognosis with a median survival of 36 months from the time of recurrence. The pattern of breast relapse was similar in irradiated and unirradiated patients. Skin relapse appears to be a manifestation of metastatic disease while parenchymal relapse may represent regrowth of primary tumour. This pattern of breast relapse questions the requirement for radiotherapy to the whole breast after local excision for early stage breast cancer.

Micrometastases in bone marrow in patients with primary breast cancer: evaluation as an early predictor of bone metastases.

The bone marrow of 307 patients with primary breast cancer was examined for tumour cells by immunocytochemistry using an antiserum to epithelial membrane antigen. Micrometastases were found in 81 cases (26.4%) and their presence was related to various poor prognostic factors: spread to lymph nodes, vascular invasion, T stage, and pathological size. The median duration of follow up was 28 months. Seventy five patients relapsed, 60 at distant sites. Of these 60 patients, 26 had micrometastases detected at presentation and 34 were free of micrometastases initially. The relapse free interval was significantly shorter for patients with micrometastases, and these patients had a shorter survival. Analysis of the sites of relapse showed that the test predicted bone metastases only. Thus 10 out of 19 patients (53%) who developed bone metastases at first relapse had micrometastases at presentation compared with only 41 out of 288 patients (14%) who remained free of bone metastases or relapsed in non-skeletal sites. The presence of micrometastases detected at the time of initial surgery in a patient with primary breast cancer is a useful predictor of early relapse in bone and may help in selecting patients for subsequent systemic treatment.

Adjuvant aminoglutethimide therapy for postmenopausal patients with primary breast cancer.

Three hundred and twenty-two postmenopausal patients with primary breast cancer and ipsilateral axillary node involvement were randomized to receive aminoglutethimide and hydrocortisone or placebo for 2 years in a double blind randomized trial between April 1980 and March 1985. Two hundred and eighty-six patients were eligible for the study of whom 145 received active drug and 141 received placebo. At the present time significantly fewer patients have relapsed or died without previous relapse in the treatment arm (P = 0.002); 43 of 145 (30%) patients receiving aminoglutethimide have relapsed or died compared with 63 of 141 (40%) of those receiving placebo. Local recurrence is also significantly reduced (P = 0.002) since only 6 patients receiving active treatment developed local recurrence compared to 21 receiving placebo. Side effects were severe enough to necessitate complete withdrawal or reduction of therapy in 27 of 145 (19%) in the treatment arm of the study compared with 21 of 141 (15%) in the placebo arm. A single treatment-related death occurred, due to agranulocytosis. Aminoglutethimide and hydrocortisone therefore delay relapse after surgery for primary breast cancer in postmenopausal women. It is too early to assess any effect on overall survival.

Prognostic significance of micrometastases in bone marrow in patients with primary breast cancer.

Metastatic breast cancer cells were found in the bone marrow of 60 (23%) of 269 patients with primary breast cancer, none of whom had metastatic disease disclosed by any other investigation, including bone scanning and radiological skeletal survey. We estimated the number of cancer cells as less than or more than 20 cancer cells seen. Twenty-six patients had less than 20 cancer cells present, and 34 had 20 or more. At a median follow-up time of 22 months, 53 patients had relapsed, 19 of 60 (31.7%) in the group found to have micrometastases and 34 of 195 (17.2%) in the group that had normal bone marrow. Patients with micrometastases are relapsing at a faster rate than those without micrometastases (P = less than 0.05). Patients with less than 20 cancer cells present are relapsing faster than those with no cancer cells but slower than those with 20 or more cancer cells (P = less than 0.01). We conclude that the presence of cancer cells in the marrow at primary diagnosis is a prognostic factor in patients with primary breast cancer.

Ifosfamide plus mesna with and without adriamycin in soft tissue sarcoma.

Early results with ifosfamide plus mesna in soft tissue sarcoma showed an initial response rate of 38% in 42 patients. All these patients treated at The Royal Marsden Hospital plus 30 more (total 67) have now been analysed. Single doses of 5 or 8 g/m2 ifosfamide were given over 24 h by infusion in dextrose saline together with 400 mg/m2 or 600 mg/m2, respectively, of mesnum every 4 h to give a total of 9 doses. A diuresis of 200 ml/hour was maintained during therapy. Treatment was repeated 3-weekly. CR was seen in 6 and PR in 10 patients. More recently doxorubicin was added to ifosfamide therapy in an attempt to improve on these results. At first only 20 mg/m2 doxorubicin was given but this was escalated to 40 mg/m2 and 60 mg/m2. Mesna has been given in higher dosage (5 g/m2 over 24 h), but otherwise the schedule is as above. In all 60 patients have been treated and most are now evaluable for response. Encephalopathy has been seen with both regimens. The incidence and patient characteristics are reported.

Eye signs in 611 cases of posterior fossa aneurysms: their diagnostic and prognostic value.

This review of 611 cases of aneurysms in the posterior fossa managed by one neurosurgeon between 1965 and 1980 shows that eye signs are important indications for investigation and may lead to earlier detection and treatment of the aneurysm. They were present in 50% of the patients. Major defects in ocular motility or the visual field were present preoperatively in 57 of the 64 patients with unfavourable surgical results--postoperative death or total physical dependency. Of the same 64 patients 55% (35) had "giant" aneurysms, as compared with 17% (102) of the entire group of patients. Hypertension was present in 50% of the patients who died and 25% of those with poor functional results after surgery. Computer-assisted tomography and four-vessel angiography are essential for accurate localization of the aneurysm and for blood-flow information important in determining the best surgical approach.

Screening for metastases in breast cancer: an assessment of biochemical and physical methods.

Ten tumor markers were measured in serum or urine at approximately three month intervals in patients with breast cancer following mastectomy but before development of overt metastatic disease. In 23 patients who later had metastases, only three markers, alkaline phosphatase, carcinoembryonic antigen (CEA), and gamma-glutamyl transpeptidase (gamma-GT) were consistently abnormal prior to the development of detectable metastases in more than one patient. In half the patients, a "lead interval" of three months or more was obtained using these three markers and little advantage was obtained by the addition of any other biochemical marker. The value of these three measurements was then assessed in a larger group of patients and compared with other tests for metastases. Alkaline phosphatase, CEA, gamma-GT, clinical examination, and chest x-ray were the best indices of the metastatic state in breast cancer, being collectively abnormal in 98% of patients at first presentation with metastases. The authors recommend screening patients postoperatively with these five tests for metastases; more detailed tests should only be carried out if results of one or more these are abnormal.

Assessment of biochemical tests to screen for metastases in patients with breast cancer.

Of ten tumour markers measured every 3 months after mastectomy in patients with apparently localised primary breast cancer, plasma levels of alkaline phosphatase, carcinoembryonic antigen, and gamma-glutamyl transpeptidase were the most useful in detecting metastatic disease. With these three tests a "lead interval" of 3 months or more was obtained in about half the 23 patients who developed overt metases. Clinical examination, chest X-ray, and these three markers proved the most useful combination of tests in screening for metastases, since at least one test was abnormal in 46 of 47 patients at the time of the development of metastases as judged by more detailed physical tests.

Globular bodies: a primary cause of the opacity in senile and diabetic posterior cortical subcapsular cataracts?

We examined 9 cataracts from maturity onset diabetics and 4 senile posterior subcapsular cataracts by scanning electron microscopy, transmission electron microscopy, immunofluorescence for crystallin proteins and actin, histochemical methods and x-ray diffraction. The cataractous regions contained spherical globules up to 20 mu in diameter, often in a fibrous matrix. Some were extracellular Morgagnian globules, apparently formed by blebbing from the cell surface; others appeared to have been formed intracellularly. The area of globular degeneration was usually 300 mu deep, but had deeper fusiform extensions. Morphological changes in the cell cytoplasm varied according to their depth in the cataract. Electron microscopy showed intracellular and extracellular globules, many of them were bounded by lipid bilayer membranes. Immunofluorescent staining showed that all the globules contained gamma-crystallin; some contained alpha- and beta-crystallins and actin. All the globules contained higher concentrations of cysteine or cystine than the surrounding lens tissue but they did not react to stains for carbohydrate or calcium. X-ray diffraction studies showed that crystalline calcium salts were absent. Globules and cavities averaged 45% of the total area in cross section. Assuming an area of cataract to be 300 micron thick and that globules 1 mu in diameter scattered, while 2--20 mu in diameter reflected light, we calculated that light passing through such a thickness would be reduced by 65%. Thus the globules could account for most of the opacity of the cataractous area. Presumably the fibrous degeneration of the cells causes enough light scattering to account for the remainder of the reduction. Cataract patients complain of decreased visual acuity, a golden halo around objects, and difficulties when driving while facing oncoming traffic at night. These probably result from light scattering. In our previous experiments, globular bodies containing gamma-crystallin were found in cells grown in tissue culture, and blebs with increased acitn content similar to Morgagnian globules were formed in tissue culture by treating differentiated rat lens cells of stage 2 by cytochalasin D (which impaired microfilament function). These results suggest the possibility of simulating in tissue culture the morphological alterations seen in the cataractous cell.