Indicators of loco-regional recurrence in breast cancer. The South East Swedish Breast Cancer Group.

AIM: The aim of the investigation was to contribute to the identification of patients who have increased or decreased risk of loco-regional recurrence. METHODS: Six hundred and twenty-nine consecutive patients with primary breast cancer diagnosed between 1988 and 1990 were studied. Two-thirds of the patients underwent mastectomy. Radiotherapy was administered if patients were node positive or breast conserved. The Nottingham histological grading protocol was used and presence of lymphovascular invasion was assessed. Investigated parameters were: age, size, grade, steroid receptor content, surgical radicality, vascular invasion and nodal status. Statistically significant risk factors for loco-regional recurrence using univariate or Cox proportional hazard analysis were grade and lymphovascular invasion. RESULTS: Women with grade 1-2, node-negative tumours without vascular invasion had a very low loco-regional recurrence rate-3.1%. Seventeen percent of patients with grade 3 tumours and vessel invasion had loco-regional recurrence. CONCLUSIONS: Our findings, and those of others, indicate that the use of adjuvant radiotherapy should be influenced to a greater extent by grade and lymphovascular invasion.

p53 status: an indicator for the effect of preoperative radiotherapy of rectal cancer.

BACKGROUND: Rectal carcinoma is a common malignancy, with a history of high local recurrence rates following surgery. In recent years. preoperative radiotherapy and refined surgical technique have improved local control rates. AIM: To investigate the relationship between expression of nuclear p53 protein and the outcome in rectal carcinoma, with and without short-term preoperative radiotherapy. MATERIAL: Specimens from 163 patients from the Southeast Swedish Health Care region included in the Swedish rectal cancer trial between 1987-1990. METHOD: New sections from the paraffin blocks of the preoperative biopsy and the surgical specimen were examined immunohistochemically using a p53 antibody (PAb 1801). RESULT: Expression of nuclear p53 protein was seen in 41% of the tumours. The p53 negative patients treated with preoperative radiotherapy had a significant reduction of local failure compared with the non-irradiated p53 negative patients (P = 0.0008). In contrast, p53 positive patients showed no benefit from preoperative radiotherapy. The interaction between p53 status and the benefit of radiotherapy was statistically significant (P = 0.018). CONCLUSION: Expression of nuclear p53 protein in rectal carcinoma seems to be a significant predictive factor for local treatment failure after preoperative radiotherapy. Further investigations are necessary to select patients for preoperative treatment based on analysis of the preoperative biopsies.

Primary radiotherapy of T1 and T2 glottic carcinoma--analysis of treatment results and prognostic factors in 223 patients.

Treatment results in 223 patients with T1 and T2 glottic carcinoma were analysed. A multivariate analysis was performed to evaluate the prognostic significance of factors related to tumour, patient and treatment. Locoregional control after radiotherapy was 90% for 129 patients with T1 tumours and 73% for 94 with T2 tumours. Disease-specific survival was 96% and 81% for patients with T1 and T2 tumours, respectively. In the multivariate analysis of locoregional control, subglottic extension contributed prognostic information to T-stage. In the univariate analysis, number of involved tumour sites, cord mobility and treatment interruption had a significant influence, which was lost in the multivariate analysis. Age gave additional prognostic information in the multivariate analysis of disease-specific survival. Significant adverse effects of radiotherapy were found in 9 patients (4%). Forty-nine patients (22%) had a second malignancy, 11 (5%) diagnosed before the glottic carcinoma.

Determination of urinary 8-hydroxydeoxyguanosine by automated coupled-column high performance liquid chromatography: a powerful technique for assaying in vivo oxidative DNA damage in cancer patients.

An automated analytical method has been developed for determination of the oxidative DNA adduct, 8-hydroxydeoxyguanosine (8OHdG) in human urine, based on coupled-column high performance liquid chromatography with electrochemical detection. Urine is concentrated on Bondelut CH by means of an automated sample processor, and the enriched sample injected on to a polymeric reversed phase column coupled in line with an electrochemical detector and a C18 reversed phase column. By use of the electrochemical detector, a suitable retention time interval is set for collection of the fraction containing 8OHdG from the chromatography on the first column; this fraction is collected in a 2 mL loop and injected onto the C18 column. The system is operated by an automatic valve station controlled by an integrator. The method has a large sample capacity and measures 31.1, 15.7, and 7.43 nmol 8OHdG/L urine with variation coefficients of 8, 8 and 24% within series and 8, 11 and 23% between series. Normal healthy individuals were found to excrete 14.9 +/- 7.8 nmol 8OHdG/24 h, or 1.11 +/- 0.62 mumol 8OHdG per mol creatinine, in their urine, whereas increased levels of 8OHdG were found in 24 h collections from a variety of cancer patients, both in samples taken before onset of oncological therapy (1.84 +/- 1.12 mumol/mol creatinine, P < 0.01 versus healthy individuals) and after therapy onset (2.18 +/- 1.44 mumol/mol creatinine, P < 0.001 versus healthy individuals). Moreover, mean values of 8OHdG in random urinary samples from cancer patients were significantly higher than from healthy individuals (2.42 +/- 2.28 versus 1.19 +/- 0.48 mumol/mol creatinine, P < 0.001), both in samples taken before therapy onset (1.91 +/- 0.96, P < 0.001 versus healthy individuals) and after (2.57 +/- 2.46, P < 0.001 versus healthy individuals). High levels of urinary 8OHdG were found in patients subjected to whole body irradiation, and in patients receiving chemotherapy with various cytostatic agents. The potential use of the method for detecting increased urinary 8OHdG excretion and conditions associated with increased oxidative DNA damage in humans is discussed.

Relationship of DNA ploidy and S-phase fraction to survival after first recurrence of breast cancer.

Flow cytometry was performed on frozen specimens from the primary tumour of 184 women with recurrent breast cancer. No significant association was seen between DNA ploidy and the other prognostic factors investigated. Patients with a high S-phase fraction had more often a negative estrogen receptor (ER) status and a short disease-free interval. A shorter survival after disease recurrence was seen both in patients with DNA aneuploid tumours and among those with a high S-phase fraction. Patients with DNA tetraploid tumours showed the longest survival after recurrence. In this subgroup, half of the patients survived more than 3 years after recurrence and the estimated survival rate at 10 years was 17%. In a Cox's regression analysis including 116 patients, site of recurrence, number of positive nodes at time of primary operation, size and ER content of the primary tumour as well as DNA ploidy showed additional prognostic value.

S-phase fraction is a prognostic factor in stage I breast carcinoma.

PURPOSE AND METHODS: The prognostic significance of cell proliferation, estimated as cytometric S-phase fraction (SPF), was investigated in node-negative breast cancer patients with small tumors (T1, NO). The 219 stage I patients originated from two series and were diagnosed either from 1978 to 1981 or from 1981 to 1985. The tumors were analyzed for estrogen receptors (ERs) by isoelectric focusing and for cellular DNA content by static cytofluorometry or flow cytometry. RESULTS: A high SPF correlated with the absence of ERs and abnormal DNA content, and was less often found in tumors smaller than 11 mm compared with those with a diameter between 11 and 20 mm. Among the variables age, tumor size, DNA ploidy, ER status, and SPF, only SPF showed a significant association with distant recurrence and breast cancer survival in systemically untreated patients. The relative recurrence rate for patients with an SPF of 10% or greater was three times that for patients with lower SPFs. Estimated 8-year breast cancer survival rates for the same groups were 72% and 91%, respectively. CONCLUSION: This study suggests that cytometric SPF has prognostic significance in stage I breast carcinoma.

Squamous cell carcinoma of the oral cavity. A review of 176 cases with application of malignancy grading and DNA measurements.

This retrospective study comprised 176 patients with squamous cell carcinoma of the oral cavity treated at The Linköping University Hospital over a 19-year period. Clinical parameters, microscopic malignancy grading (according to Jakobsson et al. and Glanz and Eichhorn), DNA cytofluorometry, analysis of therapeutic modalities and statistics regarding survival and prognosis are reported. The mean age was 70 years with a male: female ratio of 1.3:1 One hundred and four patients had T1 or T2 tumours and 109 an N0 neck. Cervical lymph node metastases were more frequent in patients with larger tumours (T3 + T4) than in those with smaller (T1 + T2) (P less than 0.01), in tumours with a high malignancy grading compared to those with a low (P less than 0.05) and in DNA non-diploid tumours compared to diploid ones (P less than 0.001). The aneuploid tumours responded better to preoperative radiotherapy than did diploid (P less than 0.01) or polyploid (P less than 0.05) tumours. Eighty-nine per cent of the recurrences occurred within 1 year of initial therapy. Secondary treatment was successful in 15 of 37 (41%) patients in whom the tumour recurred either at the primary site or in regional lymph nodes, but only in 1 of 8 (12%) with recurrences in both locations. Surgery alone or combined with radiotherapy resulted in equivalent survival rates for tumours in stages I and II. In advanced stages combined radiotherapy and surgery gave better survival figures than either modality alone (P less than 0.01; Kaplan-Meier). The presence of lymph node metastases (P less than 0.001), tumour size (P less than 0.01) and tumour ploidy (P less than 0.005) were the only clinical and histological parameters that significantly influenced survival (Cox regression analysis). Twenty-four patients developed a secondary primary malignancy; 21 of these were located in the aerodigestive tract.

Microvascular anastomoses in irradiated vessels: a comparison between the Unilink system and sutures.

A new mechanical device (the Unilink system) was compared to conventional suture anastomoses in irradiated microvessels. Twenty rabbits received a single radiation dose of 20 Gy from a 7-MeV electron source through an anterior neck field. One and 6 months following irradiation, the carotid arteries and facial veins were divided and anastomosed on one side with the Unilink system and on the other side with suture technique. At sacrifice 4 weeks postoperatively, all vessels were evaluated for patency and histologic changes associated with radiation and anastomotic trauma. Histology disclosed severe radiation changes. Also, intimal hyperplasia was consistently found at the anastomotic sites in the arteries, while it was totally absent in the venous anastomoses. Occlusive thrombosis was found in two arteries, one anastomosed with the Unilink system and one sutured. Two other arteries, one from each group, had subtotal occlusions at the anastomotic site. No occlusions occurred in any of the venous anastomoses. The overall patency in this study was 97.5 percent, with no difference between the two techniques.

Prognostic value of DNA ploidy and S-phase fraction in relation to estrogen receptor content and clinicopathological variables in primary breast cancer.

Tumors from 472 women with primary breast cancer were analyzed by flow cytometry. Divided into four categories, DNA ploidy showed significant association with disease recurrence and mortality. When allowance was made for its correlation with nodal status and estrogen receptor (ER) content, DNA ploidy did not add prognostic information. S-phase fraction was estimated in 290 DNA histograms. In contrast, it was significantly related to recurrence and mortality when controlling for nodal status, tumor size and ER content. When the follow-up was divided into two periods DNA ploidy and S-phase fraction showed association with disease recurrence in the first period only (less than 2.5 years), while the association with mortality was valid for both periods. Light scatter was measured in 234 samples. A low light scatter variability for the stemline nuclei was related to a high recurrence rate during the early follow-up period. In conclusion, DNA flow cytometry adds prognostic information concerning breast cancer patients.

Prognostic significance of the receptor for epidermal growth factor in human mammary carcinomas.

125I-EGF binding technique was used to demonstrate high affinity receptor binding for epidermal growth factor (EGF-R) in 72 human mammary carcinomas. 27% of the tumors were EGF-R positive and the presence of this receptor was correlated with receptor levels for estradiol, DNA-pattern, proliferative index, thymidine kinase, tumor size, number of lymph node metastases and 6-year relapse probability. Our results confirm previous reports of an inverse relation between the cellular content of EGF-R and ER. In addition, we could associate EGF-R positivity with an aneuploid DNA-pattern and an increased growth rate, as measured by proliferative index. No correlation was found between EGF-R positivity in the primary tumor and presence of axillary lymph node metastasis at the time of operation. The 6-year relapse rate was somewhat higher for patients whose primary tumors were EGF-R positive. Moreover patients who had lymph node metastases at the time of operation and EGF-R positive tumors experienced a significantly lower 6-year disease free survival rate as compared to those who were node negative and had receptor negative tumors. It remains to be shown whether EGF-R alone can be used as an independent prognostic factor.

Preoperative irradiation in oral cavity carcinoma. A study with special reference to DNA pattern, histological response and prognosis.

The relation between DNA pattern and response to preoperative radiotherapy was studied in 51 patients with oral squamous cell carcinomas operated 4 weeks after irradiation. Small tumors (T1 and T2) showed more pronounced response to radiotherapy than larger ones (T3 and T4), as did DNA aneuploid tumors. Eight of 11 DNA aneuploid tumors showed no remaining tumor in the operation specimen, compared to 6 of 21 DNA polyploid and 2 of 19 DNA diploid tumors. None of 16 patients without demonstrable remaining cancer in the operation specimen had local recurrence. The presence of lymph node metastases was the most important prognostic factor.

Prognostic value of malignancy grading and DNA measurements in small glottic carcinomas.

Thirty-three small glottic carcinomas (T1 and small T2; UICC, 1978) were examined by malignancy grading using the 8-factor system proposed by Jakobsson et al. [Acta radiol. Ther. phys. Biol. 12: 1-8, 1973] and the 4-factor system set up by Glanz and Eichhorn [Hals-Nas.-Ohrenarzt 33: 103-111, 1985]. DNA ploidy, S-phase, and the presence of polyploid cell nuclei were determined. There was no significant difference in malignancy score or DNA values between tumors that recurred after a full course of radiotherapy and those that did not. All but one carcinoma that recurred had polyploid nuclei, in comparison with only 13 of the 22 nonrecurrent tumors. Neither malignancy grading nor DNA measurements seem to predict the clinical course of small glottic carcinomas. Accurate and adequate radiotherapy may be more reliable for local tumor control than tumor character. The extent of the tumor, such as the involvement of the anterior commissure, may be another factor affecting the prognosis.

Receptors for EGF and oestradiol and thymidinekinase activity in different histological subgroups of human mammary carcinomas.

The cellular content of receptors for epidermal growth factor (EGF) was measured in different histological subgroups of human mammary carcinomas. EGF receptors were detected in 36% of the ductal and all the medullary carcinomas. In contrast lobular and pure colloid tumours did not contain measurable amounts of the receptor. The receptor was found both among tumours with an euploid and aneuploid DNA pattern. The EGF receptor is thus found in carcinomas with a varying degree of differentiation as judged by the cellular DNA pattern. There was no correlation between the proliferative activity of the tumours as measured by thymidinekinase activity and the amount of EGF receptors in the tumour. Tumours with detectable EGF receptor often had low levels of oestrogen receptor. This finding could only partly be explained by the menstrual status of the patients.

DNA-measurements on formalin-fixed, paraffin-embedded squamous cell carcinomas from different ENT-regions.

DNA measurements were performed on formalin-fixed, paraffin-embedded squamous cell carcinomas, using the method of Hedley et al. Ultrasonication was sometimes necessary after enzymatic disintegration to dispose of residual cytoplasm. The isolated nuclei were stained with Hoechst 33258. DNA measurements were performed using a rapid system for static fluorometry (FLUORA-programme). The method proved to be highly accurate. The mean CV of the stem cell peaks from different tumours was 5.3. There was no difference between material less or more than 5 years old (mean CVs 5.2 and 5.5 respectively). RNase treatment had no effect and was therefore not considered necessary. The proliferative activity was calculated by computer assuming a rectilinear distribution of S-phase cells. Because routinely paraffin-embedded material can be used, DNA analysis can be performed on tumour material where the clinical outcome is known, in order to evaluate certain DNA variables.

DNA measurement--an objective predictor of response to irradiation? A review of 24 squamous cell carcinomas of the oral cavity.

DNA measurements on biopsy material from 24 squamous cell carcinomas of the oral cavity given preoperative radiotherapy indicate that DNA aneuploid tumours respond better to radiotherapy than do diploid and polyploid tumours. The mean S-phase value was higher (16.1%) for 8 tumours that were eradicated by preoperative radiotherapy than for 13 that did not respond (8.1%). These factors correlated better with the response than did histological and clinical (T) classifications. DNA-ploidy and S-phase estimation can complement the histological diagnosis, and may prove valuable when planning treatment.

A comparison of static cytofluorometry and flow cytometry for the estimation of ploidy and DNA replication in human breast cancer.

332 primary invasive breast carcinomas were analysed by static cytofluorometry and flow cytometry. The ploidy distributions were similar, and 54% of the tumors were judged DNA aneuploid by both methods. The coefficient of variation of the G0-G1 peaks ranged from 2.0 to 8% with both techniques, but the mean was somewhat lower with flow cytometry--4.1%, compared to 4.9% for the static measurements. The proportion of S-phase cells was possible to estimate from 80% of the flow histograms and 70% of the static histograms. S-phase was not estimated from the static histograms if less than 150 tumor cells were measured. With 160 tumors S-phase was measured by both methods. The range was 0 to 27% with the static measurements and 0.7 to 25% with flow cytometry. Corresponding mean values were 7.6% and 8.2%, which are similar to thymidine labeling index results with breast cancers reported in some studies. A close correlation was obtained (r = 0.927) comparing S-phase fractions estimated from aneuploid tumors with flow cytometry and static cytofluorometry if more than 200 cells were measured with the latter. The proportion of S-phase cells was significantly lower for the diploid tumors. We conclude that both techniques can be useful for the estimation of DNA ploidy and replication in human breast cancer.

Proliferative index, cytosol estrogen receptor and axillary node status as prognostic predictors in human mammary carcinoma.

The predictive value of tumor cell estrogen receptor (ER) content, DNA ploidy, proliferative index (PI) and lymph node involvement was examined in 210 women operated on for primary breast cancer in 1979. At a minimum follow-up of 60 months the highest relapse free survival was observed in patients whose tumours had ER greater than 0.30 fmole/micrograms DNA (ER rich) and PI less than 5% (PI low). Subclassification of tumors in euploid and aneuploid groups gave no prognostic information. When examined by axillary node status (no nodal involvement - involved nodes) both ER and PI contributed prognostic information. Multivariate analysis using Cox proportional hazards model showed that nodal involvement was the strongest predictor of recurrence (p = 0.0007). However, ER and PI contributed significant independent prognostic information (p = 0.048 and p = 0.038), respectively.

Receptors for retinoic acid and retinol in human mammary carcinomas.

The cellular content of receptors for retinol (CRBP) and retinoic acid (CRABP) was measured in 148 human mammary carcinomas. High levels of CRABP were found in lobular carcinomas while those of the papillary subgroup had low levels of the receptor. Intermediate values for CRABP were observed for ductal, colloid and medullar carcinomas. The cellular levels of CRBP were high in ductal carcinomas and low in papillar carcinomas. A positive correlation was observed between the receptor for estradiol and CRABP. However, no significant correlation was found between the tumor cell DNA pattern and the content of CRABP and CRBP respectively. Recurrence of disease could be predicted by the nodal status and the level of estradiol receptor while the DNA pattern and the content of receptors for retinoic acid and retinol failed.

DNA distribution, cytosol estrogen receptors and axillary nodes as prognostic predictors in breast carcinoma.

One hundred and fifty patients with breast carcinoma were examined to compare axillary node status, estrogen receptor level and cellular DNA content as prognostic indicators. Seventy-four per cent of the patients were postmenopausal and forty per cent had axillary node metastases. Estrogen receptor was measured by isoelectric focusing in polyacrylamide gel. DNA was measured in individual cell nuclei by means of Feulgen-acriflavine-sulphate stained imprints. Fifty-two per cent of the tumors had diploid and/or tetraploid DNA pattern, and the rest aneuploid pattern. Axillary node metastases, aneuploid DNA pattern and low level of estrogen receptor were related to recurrence. When introduced into Cox's proportional hazards procedure, axillary nodes and estrogen receptor level but not DNA pattern remained as significant predictors of recurrence.

Nasal cuboidal metaplasia with dysplasia. Precursor to adenocarcinoma in wood-dust-exposed workers?

The histological study of the non-tumours nasal mucosa in 22 wood-workers with ethmoidal adenocarcinoma was carried out and special attention was paid to the presence of cuboidal metaplasia with or without dysplasia. The workers had been exposed to wood dust for an average of 38 years (range 18 to 55 years). In 19 cases cuboidal metaplasia was found and 16 of these also had dysplasia. In 10 cases there was a transitional zone with dysplastic cuboidal epithelium in continuity with the tumour. In 5 cases there was squamous metaplasia. The results indicate cuboidal metaplasia with dysplasia being a possible precursor to nasal adenocarcinoma in workers exposed to wood dust, similar to the findings of squamous metaplasia and squamous cell carcinoma in nickel workers.