Tumour epidermal growth factor receptor, erbB-2 and cathepsin D in node-negative invasive breast cancer: their impact on the selection of patients for systemic adjuvant therapy.

OBJECTIVE: To determine the feasibility and the economic impact of tumour EGFR, erbB-2 and cathepsin-D measurements in women with node-negative breast cancer. DESIGN: Consecutive tumour samples received at a regional steroid receptor laboratory from patients with node-negative breast cancer were evaluated with commercially available kits to determine EGFR, erbB-2 and cathepsin-D levels. SETTING: All node-negative patients whose tumours were submitted to the steroid receptor laboratory from November 1992 to March 1994 were included (n = 142). A control group of concurrent node-negative breast cancer patients from the London Regional Cancer Centre (LRCC) database were also evaluated to determine the representativeness of our sample. MAIN OUTCOME MEASURE: To determine the proportion of patients who were positive for the 3 newer prognostic factors relative to their risk of relapse. RESULTS: We found 75 positive values in 69 patients (48.6%). We demonstrated that each factor identified a different high-risk subgroup. Epidermal growth factor receptor (EGFR) positivity (> 10 fmol/mg protein) was found in 16.3% of patients, with 19.9% of patients positive for erbB-2 (> 250 units/mg protein) and 17.3% positive for cathepsin D (> 70 pmol/mg protein). Between 10% and 23.2% more node-negative patients currently seen in a regional cancer centre could be offered systemic adjuvant chemotherapy based on a single positive new factor. CONCLUSIONS: These tumour evaluations are straightforward using material already available in a regional steroid receptor laboratory or on tumour tissue available to pathologists. The economic impact is minimal; the 1995 cost of performing all 3 evaluations is Can$425-616 (US$304-440) per patient treated depending on the number of assays per run. Prospective clinical trials incorporating tumour EGFR, erbB-2 and cathepsin D are feasible and economically viable.

Osteopontin expression in a group of lymph node negative breast cancer patients.

The aim of this study was to examine the cellular distribution of osteopontin (OPN) protein [by immunohistochemical (IHC) analysis] and mRNA [by in situ hybridization (ISH)] in the primary tumors of lymph node negative (LNN) breast cancer patients and to determine whether the level of immunodetectable OPN may be associated with tumor aggressiveness. We examined OPN levels in tumors from 154 patients with LNN breast cancer who were followed for a median of 7 years (range 1.7-16.3 years). IHC staining for OPN was seen in tumor infiltrating macrophages and lymphocytes in 70% of these tumors, and in the carcinoma cells themselves in 26%. ISH was performed to determine cellular distribution of OPN mRNA expression in sections from selected tumors. OPN mRNA was detected in groups of tumor cells, individual tumor cells and tumor infiltrating macrophages and lymphocytes. Matched sections showed that some tumor cells with IHC staining for OPN protein were also positive for OPN mRNA by ISH, in contrast with previous studies which have shown OPN mRNA expression only in tumor infiltrating inflammatory cells. Our results thus indicate that OPN protein can be produced by breast cancer cells in vivo and suggest that it may also be taken up from the environment (i.e., secreted by inflammatory cells or other tumor cells). Tumor cell IHC staining intensity was then assessed using a semiquantitative scoring system. Univariate analysis showed tumor cell OPN positivity above an optimized cutpoint to be significantly associated with decreased disease-free survival (DFS) and overall survival (OS). The results of this pilot study thus suggest that the ability of breast cancer cells to either synthesize OPN or to bind and sequester OPN from the microenvironment may be associated with tumor aggressiveness and poor prognosis.

Axillary node dissection in patients with breast cancer diagnosed through the Ontario Breast Screening Program: a need for minimally invasive techniques.

OBJECTIVE: To determine the role of axillary node dissection by studying patient and tumour characteristics of invasive breast cancer through the Ontario Breast Screening Program (OBSP). DESIGN: A retrospective evaluation. SETTING: The London, Ont., branch of the OBSP. PATIENTS: Three groups of women seen were studied: 50 women with screen-detected breast cancers, which were palpable and detected by the nurse-examiner, 62 women with occult screen-detected breast cancers and 353 age matched women with invasive breast cancer from the LRCC prospective database, who served as controls. MAIN OUTCOME MEASURE: The proportion of involved axillary nodes within the 3 groups based on patient and tumour characteristics. RESULTS: Twenty-five (22.3%) of the 112 women had screen-detected tumours less than 1 cm in dimension, but only 1 had an involved axillary node. Twelve (19%) of the 62 women with occult screen-detected tumours had involved lymph nodes compared with 17 (34%) of the 50 women with palpable screen-detected cancers (NS). In the control group tumour dimension less than 1 cm versus 1 cm or larger had a marked effect on the probability of axillary node involvement (12.5% v. 40.7%, p = 0.001). In the palpable screen-detected group 3 times as many women with outer quadrant or central lesions had involved nodes as those with inner quadrant lesions (38% v. 12%) and for those with a family history of breast cancer almost twice as many had involved axillary nodes. In occult screen-detected patients there was more nodal involvement in patients aged 60 years or less than in those older than 60 years (35% v. 10%, p = 0.042); only 4 of 41 patients older than 60 years had involved nodes at surgery. A significant difference in nodal involvement was found with respect to high or intermediate grade versus low grade lesions in the occult group (44% v. 12%, p = 0.033). In the control group, tumour grade (intermediate and high [45.3%] v. low [20.0%]) and hormone replacement therapy (HRT) (current or recent use [56.5%] v. no use [34.5%]) were significant findings (p < 0.001 and p = 0.005 respectively). CONCLUSIONS: Women older than 60 years with tumours smaller than 1 cm had a low probability of nodal positivity (0% to 8.7%), but there is insufficient information in this group to give a 95% or better prediction of nodal status at the time of surgery. Studies of minimally invasive techniques such as sentinel node biopsy are needed in this group to minimize surgical morbidity in these women who, as a result of early diagnosis, have an excellent long-term outlook.

Osteopontin and p53 expression are associated with tumor progression in a case of synchronous, bilateral, invasive mammary carcinomas.

OBJECTIVE: To examine the association between expression of osteopontin (OPN), p53, other molecular markers (Ki-67, c-erb B2, and estrogen receptor protein) and tumor progression in a case of synchronous, bilateral, invasive mammary carcinomas of the same histology. DESIGN: Immunohistochemistry was performed on formalin-fixed, paraffin-embedded tissue sections. Plasma OPN level was determined by a quantitative antigen capture assay. SETTING: The patient was seen, treated, and followed up for a period of 5 years at the London Regional Cancer Centre, Ontario, Canada. PATIENT: A 60-year-old woman presented with bilateral infiltrating mammary carcinomas of the same histologic type and grade. Bilateral mastectomy and axillary node dissection showed involvement of 3 of 12 right axillary and 0 of 11 left axillary lymph nodes. She later developed a right chest wall recurrence, followed by widespread metastatic disease to the skull, liver, and left femur. RESULTS: The primary tumor of the right breast was OPN- and p53-positive, whereas the tumor of the left breast was negative for both markers. The development of right axillary lymph node metastases, chest wall recurrence, and distant metastases was associated in all instances with an immunohistochemical profile of high level expression of OPN and p53. Plasma assay for OPN at the time of last admission showed a markedly elevated OPN level. CONCLUSIONS: Increased p53 expression was found to be associated with increased tumor aggressiveness. The association of increased OPN expression with increased malignancy in breast cancer is a novel finding and raises the possibility of a role for OPN in tumor progression, as well as the potential for this marker in predicting clinical aggressiveness.

Elevated plasma osteopontin in metastatic breast cancer associated with increased tumor burden and decreased survival.

Osteopontin (OPN) is a secreted, integrin-binding phosphoprotein that has been implicated in both normal and pathological processes; qualitative increases in OPN blood levels have been reported in a small number of patients with metastatic tumors of various kinds. We measured plasma OPN levels in 70 women with known metastatic breast carcinoma, 44 patient controls who were on follow-up after completion of adjuvant treatment for early breast cancer, and 35 normal volunteers. The median plasma OPN of patients with metastatic disease was 142 microgram/liter (range, 38-1312 microgram/liter) and was significantly different (P < 0.0001, Mann Whitney U test) from both control groups (medians, 60 and 47 microgram/liter; ranges, 15-117 and 22-122 microgram/liter). Furthermore, we found that increasing plasma OPN is associated with shorter survival (P < 0.001) when patients were grouped in terciles for plasma OPN. This was also demonstrated when using a Cox proportional hazards model. Median plasma OPN levels were significantly increased for three or more sites of involvement (median, 232 microgram/liter; n = 13) versus 1 or 2 metastatic sites (medians, 129 and 130 microgram/liter; n = 29 and 28, respectively). Plasma OPN levels were correlated with other biochemical markers related to the extent of disease, such as serum alkaline phosphatase, aspartate succinate aminotransaminase, and albumin (r = 0.81, 0.62, and -0.56, respectively; all P < 0.001). This study demonstrates a statistically significant elevation in plasma OPN in the majority ( approximately 70%) of a large series of patients with metastatic breast cancer when compared (95th percentile) to healthy women or patients who had completed adjuvant treatment for early-stage breast cancer. Furthermore, this is the first study to demonstrate that higher OPN levels in patients with metastatic breast cancer may be associated with an increased number of involved sites and decreased survival.

Quantification of osteopontin in human plasma with an ELISA: basal levels in pre- and postmenopausal women.

OBJECTIVES: To develop an immunoassay for osteopontin (OPN), a secreted phosphoglycoprotein that is implicated in a number of human diseases, and establish basal plasma OPN levels in healthy women. DESIGN AND METHODS: An antigen-capture ELISA was developed to quantity OPN in plasma using a combination of mouse monoclonal and rabbit polyclonal antibodies. Basal OPN levels were determined in blood plasma of 21 pre- and 14 postmenopausal women obtained at 7-day intervals over a 4-week period. RESULTS: A group of 35 healthy women had a median OPN level of 31 micrograms/L (range = 14-64 micrograms/L). Comparison between pre- and postmenopausal women showed that their 4-week average OPN levels did not differ significantly (p > 0.16, Mann-Whitney test), and that levels in each premenopausal individual remained constant during the menstrual cycle, unaffected by cyclical levels of leuteinizing hormone and progesterone. CONCLUSION: Systematic quantification of plasma OPN can now be done by ELISA, which was used to establish basal plasma OPN levels in a group of healthy women. Levels in pre- and postmenopausal women appeared relatively stable over a 4-week period.

Neither toxicity nor dose intensity of carboplatin is affected by glomerular filtration rate versus body surface area dose calculation in gynecologic malignancy.

Twelve patients were given 31 courses of carboplatin using a glomerular filtration rate (GFR)-based area under the curve (AUC) dose schedule, and nine patients were given 35 cycles at a body surface area (BSA) dose of 350 mg m-2 every 3 weeks. The GFR was determined using technetium-99m-DTPA. The dose given was calculated according to AUC, 5 for previously treated and 7 for previously untreated patients x GFR + 25. Patients treated using the GFR had a 22% lower projected dose intensity (DI) and a 15% lower received DI compared with controls. The percentage difference between the received and projected DI was not different between the two groups of patients. In 11 of 12 patients treated according to the GFR, if the BSA calculation dose had been used it would have resulted in a higher dose of carboplatin. Twenty per cent (six of 30 courses) of GFR-based doses were delayed compared to 29% (10 of 35) of the BSA-calculated control groups. We conclude that giving a dose according to a BSA of 350 mg m-2 leads to a higher DI and total dose and does not substantially effect toxicity. It is also cost effective as it eliminates the need for unnecessary radiometric GFR determination.

HTLV-1 associated T-cell lymphoma in a patient with a 10-year history of non-epidermotropic T-cell skin infiltrates.

We report a patient who had non-epidermotropic T-cell infiltrates of the skin for 10 years prior to the development of a HTLV-1 associated disseminated T-cell lymphoma. The presentation and initial course of the lymphoma were unusual and we believe this is the first report of such an association. We therefore feel that patients of Caribbean origin with non-Hodgkin's lymphoma should be tested for HTLV-1 so that appropriate chemotherapy can be instituted at the outset in this poor prognosis group.

Successful treatment of patients in whom germ cell tumour masses enlarged on chemotherapy while their serum tumour markers decreased.

Enlargement of tumour masses with a fall or no change in tumour marker levels was noted in eight of 287 patients during chemotherapy for NSGCT at the Charing Cross and Mount Vernon Hospitals between 1977 and 1988. These eight patients had elements of differentiated teratoma in the primary specimen and in five the enlarging masses showed cystic change on CT scan. The increase in tumour mass occurred within 6 months of starting treatment. At surgery, four patients were found to have differentiated teratoma and have been followed for 15 months to 5 years without relapse. Two patients who also had some areas of embryonal carcinoma in the resected specimens had post-operative chemotherapy and are alive disease free at 8 and 24 months respectively. Two patients died: one post-operatively of uncontrolled haemorrhage secondary to aortic rupture and the second of acute myeloid leukaemia following 8 years of intermittent therapy for unresectable differentiated teratoma. The successful outcome in six of these eight patients suggests that enlarging teratomatous masses on chemotherapy can be managed by surgical resection and, when active tumour is present, by the use of post-operative chemotherapy.

A comparison of the in vivo and in vitro radiation response of three human cervix carcinomas.

The radiation response of three carcinoma of the cervix tumours has been compared in vivo in xenografts using growth delay and in vitro by means of a monolayer-based clonogenic assay. Tumours have been irradiated with 60Co gamma-rays at both high (70-100 cGy/min) and continuous low dose rates (3-5 cGy/min) in order to determine the relative in vivo and in vitro sparing effects associated with lowering radiation dose rate. In vitro, two of the lines (HX155c and HX156c) showed significant low dose rate sparing when compared to the HX160c line (which showed little sparing) (p = 0.012). Despite greater scatter for the in vivo data, there was a general tendency for the in vivo results to follow that predicted from the in vitro experiments. In vivo, HX156 exhibited significant sparing (p = 0.011) whereas HX160 again exhibited no significant sparing (p = 0.15). Although the HX155 line did show some sparing in vivo this did not reach significance (p = 0.111). All three lines showed less actual specific growth delay in vivo than that predicted from in vitro data. These in vitro/in vivo findings lead to the conclusion that rapid predictive testing of radiosensitivity (ideally utilising low dose rate irradiation) would be beneficial in determining the choice of radiotherapy regimens for the treatment of this disease.

In vitro chemosensitivity of four new carcinoma of the cervix cell lines: relationship to radiosensitivity.

Using a clonogenic cell survival assay, the chemosensitivity of four recently established cervix carcinoma cell lines to four drugs used in the treatment of this disease (bleomycin, cisplatin, etoposide and methotrexate) has been determined. Exposure was for 1 h except for methotrexate where 24 h was used. Results showed that, on a molar basis, bleomycin was most cytotoxic against each cell line (doses to produce 10% cell survival from 10 to 40 mciroM). However, if the clinical area under the curve (AUC) values are taken into consideration, the cell lines appeared to be relatively resistant to cisplatin (surviving fraction range of 0.9-0.6) and methotrexate but sensitive to bleomycin and etoposide (surviving fraction range of 0.18-0.012). Among this small group of lines, there was some evidence to suggest a positive correlation between chemo- and radiosensitivity; the HX/151c line was significantly more radiosensitive than the other three lines (P = 0.03) and clearly more chemosensitive, particularly against bleomycin (correlation coefficient of 0.81) and etoposide (correlation coefficient of 0.80).

The effect of 3-aminobenzamide in the radiation response of three human cervix carcinoma xenografts.

By means of growth delay measurements, the in vivo radiation response of three recently established human cervix carcinoma xenograft lines has been determined at both high (70 cGy/min) and low dose rate (5 cGy/min). In addition, we have investigated the role of the polyADP-ribosylation inhibitor, 3-aminobenzamide, (3-AB, administered at 450 mg/kg) in modifying the xenograft response to continuous low dose rate irradiation (5 cGy/min). For all three lines, less growth delay was observed for 9 Gy administered at 5 cGy/min compared to 70 cGy/min; in terms of the times for tumours to reach twice their original weight (T2 values), low dose rate effect sparing ratios of 2.1 for HX 155, 1.5 for HX 156 and 2.4 for HX 160 were observed. 3-AB exerted no significant effect on the growth of unirradiated tumours. When 3-AB was given during irradiation at 5 cGy/min to 9 Gy, an enhancement in growth delay was observed for each line. However, the degree of radioenhancement by 3-AB varied among the lines; significant enhancement (p less than 0.01) at all time points tested was observed for HX 156, while for the other two lines, the degree of enhancement was not significant. Enhancement effect ratios (in terms of T2 values) were 1.37 for HX 155, 1.55 for HX 156 and 1.02 for HX 160. Similar differential radiosensitizing effects with 3-AB have been observed previously in vitro. More potent poly(ADP-ribosylation) inhibitors are probably required along with additional normal tissue studies before such an approach may be proposed at the clinical level.

Chemotherapy-radiation interactions in human cervix carcinoma xenografts.

The combination of irradiation and four agents of clinical interest in the treatment of cervix carcinoma (bleomycin, etoposide, cisplatin and ifosfamide) have been investigated using two human cervix carcinoma xenografts in nude mice. As a model of clinical brachytherapy regimes, radiation was administered at a continuous low dose rate of 5 cGy min-1 to a total dose of 9 or 12 Gy. No substantial enhancement in tumour growth delay over that observed for radiation alone was observed with bleomycin, etoposide or cisplatin. Ifosfamide, however, led to substantial additional growth delay, an effect which was lost when irradiation was administered at a higher dose rate of 70 cGy min-1. As dose-rates of around 5 cGy min-1 allow greater repair of radiation damage than at the higher dose-rate without significant cell cycling or repopulation, it is possible that ifosfamide may act as an inhibitor of repair processes in this model. It would be of interest to evaluate the role of ifosfamide and brachytherapy regimes in the clinical treatment of carcinoma of the cervix.