Clinically decisive (dis)agreement in multidisciplinary team assessment of esophageal squamous cell carcinoma; a prospective, national, multicenter study.

BACKGROUND: Decisions regarding tumor staging, operability, resectability, and treatment strategy in patients with esophageal cancer are made at multidisciplinary team (MDT) conferences. We aimed to assess interobserver agreement from four national MDT conferences and whether this would have a clinical impact. METHODS: A total of 20 patients with esophageal cancer were included across all four upper gastrointestinal (GI) cancer centers. Fully anonymized patient data were distributed among the MDT conferences which decided on TNM category, resectability, operability, curability, and treatment strategy blinded to each other's decisions. The interobserver agreement was expressed as both the raw observer agreement and with Krippendorff's α values. Finally, a case-by-case evaluation was performed to determine if disagreement would have had a clinical impact. RESULTS: A total of 80 MDT evaluations were available for analysis. A moderate to near-perfect observer agreement of 79.2%, 55.8%, and 82.5% for TNM category was observed, respectively. Substantial agreement for resectability and moderate agreement for curability were found. However, an only fair agreement was observed for the operability category. The treatment strategies had a slight agreement which corresponded to disagreement having a clinical impact in 12 patients. CONCLUSIONS: Esophageal cancer MDT conferences had an acceptable interobserver agreement on resectability and TM categories; however, the operability assessment had a high level of disagreement. Consequently, the agreement on treatment strategy was reduced with a potential clinical impact. In future MDT conferences, emphasis should be on prioritizing the relevant information being readily available (operability, T & M categories) to minimize the risk of disagreement in the assessments and treatment strategies, and thus, delayed or suboptimal treatment.

Results after surgical treatment of liver metastases in patients with high-grade gastroenteropancreatic neuroendocrine carcinomas.

BACKGROUND: Gastroenteropancreatic neuroendocrine carcinomas (GEP-NEC) are generally characterized by synchronous metastases, high aggressiveness and a dismal prognosis. Current international guidelines do not recommend surgical treatment of liver metastases, however the existing data are scarce. The aim of this study was to evaluate the results of curatively intended resection/radiofrequency ablation (RFA) of liver metastases in patients with metastatic GEP-NEC. METHODS: 32 patients with a diagnosis of high-grade gastroenteropancreatic neuroendocrine neoplasm (Ki-67 > 20%) and with intended curative resection/RFA of liver metastases, were identified among 840 patients from two Nordic GEP-NEC registries. Tumor morphology (well vs poor differentiation) was reassessed. Overall survival (OS) and progression-free survival (PFS) was assessed by Kaplan-Meier analyses for the entire cohort and for subgroups. RESULTS: Median OS after resection/RFA of liver metastases was 35.9 months (95%-CI: 20.6-51.3) with a five-year OS of 43%. The median PFS was 8.4 months (95%-CI: 3.9-13). Four patients (13%) were disease-free after 5 years. Two patients had well-differentiated morphology (NET G3) and 20 patients (63%) had Ki-67 ≥ 55%. A Ki-67 < 55% and receiving adjuvant chemotherapy were statistically significant factors of improved OS after liver resection/RFA. CONCLUSION: This study shows a long median and long term survival after liver surgery/RFA for these selected metastatic GEP-NEC patients, particularly for the group with a Ki-67 in the relatively lower G3 range. Our findings indicate a possible role for surgical treatment of liver metastases in the management of this patient population.

Tumour-associated CD66b+ neutrophil count is an independent prognostic factor for recurrence in localised cervical cancer.

BACKGROUND: The prognostic impact of tumour-promoting immune cells in cervical cancer is unclear. METHODS: Federation of Gynaecology and Obstetrics (FIGO) stage IB and IIA cervical cancer patients (N=101) were assessed for tumour-associated CD66b(+) neutrophils and CD163(+) macrophages by immunohistochemistry in whole tissue sections using stereology. Results were correlated with previous results on tumour-infiltrating CD3(+), CD4(+), and CD8(+) lymphocytes in the same cohort with recurrence-free survival (RFS) as end point. RESULTS: The highest densities of CD66b(+) neutrophils and CD163(+) macrophages were observed in the peritumoural compartment (median 53.1 cells mm(-2) and 1.3% area fraction, respectively). Above median peritumoural and stromal CD66b(+) neutrophils and peritumoural CD163(+) macrophages were significantly associated with short RFS. Multivariate analysis identified high peritumoural neutrophils (HR 2.27; 95% CI 1.09-4.75; P=0.03), low peritumoural CD8(+) lymphocytes (HR 3.67; 95% CI 1.63-8.25; P=0.002), and lymph node metastases (HR 2.70; 95% CI 1.26-5.76; P=0.01) as independent prognostic factors for short RFS, whereas CD163(+) macrophages were not significant. An index of combined intratumoral and peritumoral CD66b(+) neutrophils to CD8(+) lymphocytes had good discriminatory power for each quartile with 5-year RFS of 92%, 80%, 62%, and 44% (P=0.001). CONCLUSION: Tumour-associated neutrophil count is an independent prognostic factor for short RFS in localised cervical cancer. Combining CD66b and CD8 may further improve prognostic stratification. These findings require prospective validation.

Low density of CD3+, CD4+ and CD8+ cells is associated with increased risk of relapse in squamous cell cervical cancer.

The purpose of this study was to investigate the prognostic value of the primary in situ cellular immune response in cervical squamous cell carcinoma. A study of 102 women treated for stage IB and IIA disease, between 1990 and 2000, was performed. Paraffin-embedded cervical tissue processed at the time of diagnosis was immunostained for CD3+ (T cells), CD4+ (T helper/regulatory T cells) and CD8+ (cytotoxic T cells) cells. Immune cell profile densities were estimated using stereology. Both intra- and peritumoural cell densities were estimated. Using Cox's proportional hazards regression modelling we found an increase in cell density to decrease the risk of relapse for all three cell types. The density of peritumoural CD3+ cells seems to have the strongest potential for predicting relapse. An increase in CD3+ cell density from 795 to 2043 cells per mm(2) (25-75 percentile) reduced the hazard ratio to 0.27.

[New therapeutic perspectives in breast ductal carcinoma in situ]. / Nye behandlingsperspektiver ved duktalt carcinoma in situ mammae.

Management of ductal carcinoma in situ (DCIS) of the breast has suffered from a lack of consensus. The results of recent studies may create the basis for a more rational treatment strategy. Surgical treatment of DCIS with mastectomy is curative, but often unnecessary. Excision, in contrast, carries a recurrence risk of 10-63%, and the recurrence is invasive in half the patients. Two large studies have shown that irradiation after breast conservation reduces the recurrence risk to almost half. Thus, adjuvant radiotherapy should, in principle, be offered. Retrospective analyses indicate, however, that patients having tumours removed with a > 10 mm free margin or with small, low-grade tumours may be considered to be adequately treated by surgery alone. A recent study showed that adjuvant antihormone therapy further reduces the risk of recurrence. The absolute benefit seems low, however, and additional investigations are required.

The effect of combretastatin A-4 disodium phosphate in a C3H mouse mammary carcinoma and a variety of murine spontaneous tumors.

PURPOSE: To investigate the activity of combretastatin A-4 disodium phosphate in a transplanted C3H mouse mammary carcinoma and several murine spontaneous tumors. METHODS AND MATERIALS: The C3H mammary carcinoma was grown in the right rear foot of female CDF1 mice and treated when 200 mm3 in size. Spontaneous tumors (341-1437 mm3 in size) arose at different sites in female CDF1 mice that, 19-21 months earlier, had been irradiated. Oxygen partial pressure (pO2) distributions in the C3H tumors were measured with an Eppendorf oxygen electrode at various times after injecting combretastatin (100 mg/kg, i.p.) in restrained, nonanesthetized mice. Immediately after measurement, tumors were excised and necrotic fraction determined from histological sections. In the spontaneous tumors, pO2 was measured before and 3 h after giving combretastatin. The location of these spontaneous tumors required that measurements be made in anesthetised animals, achieved by injecting a mixture of hypnorm and diazepam. RESULTS: In untreated C3H tumors, the mean (+/- 1 SE) percentage of pO2 values < or = 2.5 mmHg was 32% (+/- 11). This was significantly (Student's t-test; p < 0.05) increased to 74% (+/- 4) within 1 h after injecting combretastatin, and remained at this level for at least 6 h, although some recovery was seen at 12 and 24 h. The necrotic fraction in control tumors was 1.9% (+/- 0.4) and this was significantly increased to 16.1% (+/- 3.7) 24 h after drug administration. In spontaneous tumors, the pO2 measurements indicated that 5 of 6 showed some response to combretastatin, although the degree of change was variable. CONCLUSIONS: Combretastatin increased tumor hypoxia and necrosis in the C3H mammary carcinoma, consistent with the induction of vascular damage. Drug-induced changes in pO2 were also found in spontaneous tumors, suggesting that the activity of this drug is not restricted to transplanted tumors alone.

Proliferative activity (MIB-1 index) is an independent prognostic parameter in patients with high-grade soft tissue sarcomas of subtypes other than malignant fibrous histiocytomas: a retrospective immunohistological study including 216 soft tissue sarcomas.

AIMS: To evaluate the prognostic value of tumour proliferative activity, p53 accumulation and bcl-2 expression in a retrospective series of 216 patients with soft tissue sarcomas (STS). METHODS AND RESULTS: The immunohistochemical analyses were performed on formalin-fixed, paraffin-embedded tissue. The proliferative activity was assessed by use of the monoclonal antibody MIB-1 and evaluated in multiple, random systematic sampled fields of vision. The percentage of proliferating cells (the MIB-1 index) ranged between 1% and 85% (median 12%). A significant increase in mean MIB-1 index was seen with increasing histological malignancy grade. Variation in the incidence of p53 accumulation and bcl-2 positivity among different histological subtypes was observed. p53 accumulation was frequent in synovial sarcomas and leiomyo- and rhabdomyosarcomas, whereas bcl-2 preferentially was expressed in synovial sarcomas. Univariate analysis identified patient age, tumour size, histological grade of malignancy, MIB-1 index and p53 accumulation as significant prognostic parameters. Multivariate Cox analysis, including tests for interaction terms between histological subtypes and MIB-1 index, showed independent prognostic effect of MIB-1 index and tumour size in patients with high-grade tumours of other subtypes than malignant fibrous histiocytoma (MFH). CONCLUSIONS: Histopathological malignancy grading is the most important single prognostic factor for overall survival in STS, but estimation of MIB-1 index is useful for identifying the least favourable subgroup of high grade STS of other subtypes than MFH, for whom adjuvant therapy may be indicated.

Objective malignancy grading: a review emphasizing unbiased stereology applied to breast tumors.

Low reproducibility reduces the clinical value of morphologic grading of malignant tumors, and the replacement of subjective classification by objective quantification has been suggested. Simple mitosis counting has been employed for objective malignancy grading most frequently and has proved its prognostic significance in, e.g., sarcomas and carcinomas of the breast and ovary. These and other measurements of morphometry are, however, obtained in two dimensions only, introducing bias due to ignorance of the fact that biologic structures are three-dimensional. Stereologic estimators are, to that end, well-suited, because they enable the assessment of spatial structure from sections. Studies addressing the impact of stereology in tumor pathology are the subject of the current review. Details of estimation are provided of stereologic variables of tumor size, numbers and densities of cancer cell nuclei and mitoses, mean size and size variability of cancer cell nuclei and variables of tissue architecture. Besides a description of their practical estimation the influence on variables of sampling method, tissue processing and observer variability is assessed, and estimator efficiency and measuring equipment is evaluated. Exemplifying the clinical importance of objective grading, results are summarized of prognostic studies of quantitative histopathology in women with breast cancer. It has been shown that many stereologic estimators are applicable to ordinary histologic sections processed under routine conditions. If a systematic random scheme of sampling is employed then the efficiency of estimation is usually high, and reproducible, accurate and representative results are ensured. For objective malignancy grading of breast cancer especially the volume-weighted mean nuclear size, vv (nuc), seems valuable, and the variable usually provides independent information to that of staging parameters. The prognostic value of vv (nuc) seems greatest in lymph node positive subsets, whereas the importance in lymph node negative patients should be further investigated. The clinical significance of some stereologic variables may be restricted due to relatively time consuming measurement procedures. However, the unbiased technique may provide precise measures of basic parameters like "tumor burden" and tumor growth pattern, and thereby be highly useful in experimental oncology. In conclusion, stereology is of great value for quantifying tumor elements. For objective malignancy grading especially assessment of the three-dimensional mean nuclear size seems useful. Prognostic significance of this variable has been demonstrated in, e.g., malignant melanoma and carcinomas of the breast, lung, bladder, prostate and uterine cervix. To determine the real clinical value of the measurements, further evaluation in a routine setting is necessary. In case such prospective studies confirm previous findings, the future replacement of subjective grading techniques by reproducible, objective variables seems feasible.

Total number of cancer cell nuclei and mitoses in breast tumors estimated by the optical disector.

OBJECTIVE: The total number of cancer cell nuclei, N(nuc), and of mitoses, N(mit), in the primary lesion are potentially important indicators of tumor biology. In the present study, such estimates were obtained on breast cancers by an unbiased stereologic method. STUDY DESIGN: The total number estimates are the product of two variables: (1) the volume of tumor, V(T), estimated by the Cavalieri principle, and (2) the densities of cancer cell nuclei and of mitoses obtained in small, three-dimensional samples (i.e., optical disectors) of 40-micron-thick methacrylate sections, which were selected systematically at random from the whole specimen. RESULTS: In 93 prospectively collected tumors, N(nuc) ranged from 0.06 to 7.9 10(9) (median, 0.6 10(9)), and N(mit) ranged from 0.02 to 64 10(6) (median, 1.5 10(6)). Both N(nuc) and N(mit) correlated significantly with V(T) (r = .77 and .60, respectively); however, the steep slopes of the regression lines indicated that densities of nuclei and mitoses increased as a function of tumor size. On average, N(mit) and estimates of mitotic frequency tended to be larger in lymph node-positive patients as compared with lymph node-positive patients as compared with lymph node-negative ones (2P < or = .08), whereas no such relation was found for nuclear counts (2P > or = .40). By counting a median number of 195 nuclei and 28 mitoses per tumor, the average coefficients of error of N(nuc) and N(mit) were 17% and 32%, respectively; this gave seemingly sufficient precision as compared with the huge interpatient variation in estimates, 180% and 490%. Moreover, the intraobserver reproducibility of density estimates was excellent (r > or = .88). CONCLUSION: The present study showed the feasibility, efficiency and reproducibility of the unbiased optical disector principle applied to human breast cancer and provided data on new parameters of biologic relevance. The technique seems suitable for use in experimental oncology, but further studies are needed to investigate its clinical value.

[Objective histological grading of squamous and adenosquamous lung cancer]. / Objektiv histologisk gradering af planocellulaer og adenoskvamøs lungecancer.

The prognostic value of quantitative histopathological parameters was evaluated in 55 consecutively treated patients with operable lung carcinoma of squamous and adenosquamous cell type. Using a projection microscope, estimates were obtained of mean nuclear volume (vV(nuc)), mean nuclear profile area, nuclear profile density, nuclear volume fraction, and mitotic profile frequency. Patient sex, age, and clinical stage was recorded. Single-factor analyses showed a prognostic significance of clinical stage and patient age (2p < or = 0.03), whereas sex was marginally significant (2p = 0.09). Of the quantitative histopathological parameters only estimates of vV(nuc) were of prognostic significance (2p = 0.02), in that small nuclear volumes were associated with the worst prognosis. In a multivariate Cox analysis, clinical stage, age, and vV(nuc) were found to be of significant, independent prognostic value. Thus, the present study indicates that vV(nuc) is of prognostic value, independent of clinical stage. The parameter is highly reproducible and easily obtained using unbiased stereology, and may be of future importance in the management of lung cancer patients.

Stereologic estimation of breast tumor size.

OBJECTIVE: The largest tumor diameter, D(T), is a variable of great clinical value in breast cancer but a biased and imprecise estimator of real tumor size. Three-dimensional, shape-independent estimates would more realistically reflect the tumor bulk and provide more accurate clinical staging. For experimental oncology, the measurements may be useful for precise assessment of tumor burden. STUDY DESIGN: In 64 prospectively collected breast cancers, unbiased stereology was used for estimating the gross tumor volume, V(T), cutting specimens into parallel, equally thick sections with subsequent determination of total tumor sectional area. For comparison, the volume of invasive tumor epithelium, "V"(epi), was obtained by microscopic examination of systematically sampled tissue fractions of the same tumors, embedded in both methacrylate and paraffin. RESULTS: The median D(T) was 2.2 cm, and the median V(T) was 6.72 cm3. The correlation between these variables was not very close (r = .77), and the slope of the regression line was steeper than expected, presumably reflecting a change in tumor shape with growing size. The sampling scheme used for estimation of V(T) proved highly efficient, yielding a mean error coefficient of 9%. The median "V"(epi) in methacrylate was 1.19 cm3, 21% larger than in paraffin. Estimates of "V" (epi) were highly reproducible (r = .97) and correlated highly with point counting-based estimates of the feature (r = .96). "V"(epi) correlated with V(T) (r > or = .75), but the slopes of the regression lines were steeper than expected, corresponding with the correlation found between epithelial volume fraction and tumor size (r = .26). On average, about 25% of the gross tumor was composed of invasive epithelium, but with a wide range. CONCLUSION: In breast cancer, realistic estimates of tumor volume, volume of invasive epithelium and epithelial volume fraction can be obtained by efficient stereologic techniques, which seem useful for clinical and experimental oncology. In the present methodologic study, baseline data were generated. Further studies are needed to assess the clinical value of stereologic tumor size estimates as compared with traditional staging parameters.

Significance of variations in section mounting technique for nuclear stereology and morphology in urinary bladder neoplasms.

Owing to its toxicity it would be desirable to avoid xylene in the processing of histological tissue. Consequently, modifications of the section mounting technique excluding xylene have recently been suggested. Changes in tissue preparation might, however, influence histopathological structures used for malignancy grading of tumours. In the current study, we investigated the impact of alterations in the mounting process on the subjectively evaluated quality of nuclear morphology and on the stereologically obtained mean nuclear volume, vv(nuc), of urothelial neoplasms. Paraffin sections from 14 tumours were, after the haematoxylin-eosin staining, mounted with DPX and a coverslip either, 1) from the water bath, 2) after dehydration in ethanol but without clearing, or, 3) after dehydration and xylene clearing. We found that the nuclear morphology of ethanol-treated and xylene-cleared specimens was somewhat more brilliant than that of sections mounted from water. The vv(nuc) of xylene-cleared sections ranged from 142 to 751 microns3, and the mean value of 350 microns3 was not significantly different from that of ethanol-treated sections of 367 microns3 (2p = 0.67). In contrast, the mean vv(nuc) of sections mounted from water was 459 microns3, approximately 30% larger (2p < or = 0.02). Thus, although previous studies have demonstrated the prognostic value of vv(nuc) in patients with bladder cancer, the present study shows that modifications in section mounting technique may significantly influence the results, underscoring the need for standardization of tissue processing. Sections mounted from ethanol seem to be as good as routine xylene-cleared sections, whereas sections mounted from water have a less brilliant morphology and results of nuclear stereology different from those of routine sections.

Objective malignancy grading of squamous cell carcinoma of the lung. Stereologic estimates of mean nuclear size are of prognostic value, independent of clinical stage of disease.

BACKGROUND: The prognostic value of quantitative histopathologic parameters was evaluated in 55 consecutively treated patients with operable lung carcinoma of squamous (N = 39) and mixed, adenosquamous (N = 16) cell type. Patients alive were followed for at least 12 years. METHODS: Using a projection microscope and a simple test system in fields of vision systematically selected from the whole tumor area of one routine section, five quantitative histopathologic variables were estimated: the mean nuclear volume, the mean nuclear profile area, the density of nuclear profiles, the volume fraction of nuclei to tissue, and the number of mitotic profiles per 10(3) nuclear profiles. For each patient, information was recorded regarding sex, age at diagnosis, and clinical stage of disease. RESULTS: Single-factor analyses showed that a favorable prognosis was associated with early clinical stages (Stages I and II) and young age (P < or = 0.03), and that females tended to do better than males (P = 0.09). Estimates of the mean nuclear volume were of prognostic significance (P = 0.02), small nuclei being associated with the worst prognosis. In a multivariate Cox analysis, clinical stage, age, and mean nuclear volume were found to be parameters of significant, independent prognostic value. CONCLUSIONS: The present feasibility study indicates that estimates of the mean nuclear volume are of prognostic value, independent of the clinical stage of disease. This quantitative histopathologic variable is highly reproducible and easily obtained using an unbiased stereologic method. Thus, the mean nuclear volume may be a parameter of future importance in the clinical management of patients with carcinoma of the lung.

The prognostic value of oncogenic antigen 519 (OA-519) expression and proliferative activity detected by antibody MIB-1 in node-negative breast cancer.

The prognostic value of oncogenic antigen 519 (OA-519) expression and tumour proliferative activity was evaluated in a retrospective series of 118 patients with low-risk breast cancer. Low risk was defined as negative axillary nodes, tumour diameter < or = 50 mm, and no histological evidence of invasion of skin or deep fascia (= T1N0M0 and T2N0M0). The median follow-up time was 104 months (range 5-143 months). Immunohistochemical analysis of OA-519 expression was performed on formalin-fixed, paraffin-embedded tissue. The proliferative activity was estimated using a Ki-67 equivalent monoclonal antibody (MIB-1), which is applicable on formalin-fixed, paraffin-embedded tissue after microwave pretreatment. OA-519 was expressed in about one-third of the tumours and the percentage of proliferating cells (the MIB-1 index) ranged between 1 and 72 per cent (median 17 per cent). Using multivariate Cox analysis, both the MIB-1 index and OA-519 expression were of independent prognostic value (2p < or = 0.01), and the combined immunohistological approach may therefore be useful in selecting patients with node-negative breast cancer who might benefit from adjuvant therapy.

Immunohistochemical quantitation of oestrogen receptors and proliferative activity in oestrogen receptor positive breast cancer.

AIM: To evaluate the effect of the duration of formalin fixation and of tumour heterogeneity on quantitative estimates of oestrogen receptor content (oestrogen receptor index) and proliferative activity (MIB-1 index) in breast cancer. METHODS: Two monoclonal antibodies, MIB-1 and oestrogen receptor, were applied to formalin fixed, paraffin wax embedded tissue from 25 prospectively collected oestrogen receptor positive breast carcinomas, using a microwave antigen retrieval method. Tumour tissue was allocated systematically to different periods of fixation to ensure minimal intraspecimen variation. The percentages of MIB-1 positive and oestrogen receptor positive nuclei were estimated in fields of vision sampled systematically from the entire specimen and from the whole tumour area of one "representative" cross-section. RESULTS: No correlation was found between the oestrogen receptor and MIB-1 indices and the duration of formalin fixation. The estimated MIB-1 and oestrogen receptor indices in tissue sampled systematically from the entire tumour were closely correlated with estimates obtained in a "representative" section. The intra- and interobserver correlation of the MIB-1 index was good, although a slight systematical error at the second assessment of the intraobserver study was noted. CONCLUSION: Quantitative estimates of oestrogen receptor content and proliferative activity are not significantly influenced by the period of fixation in formalin, varying from less than four hours to more than 48 hours. The MIB-1 and the oestrogen receptor indices obtained in a "representative" section do not deviate significantly from average indices determined in tissue samples from the entire tumour. Finally, the estimation of MIB-1 index is reproducible, justifying its routine use.

Quantitative histopathology in ductal carcinoma of the breast. Prognostic value of mean nuclear size and mitotic counts.

BACKGROUND: The prognostic value of quantitative histopathology was investigated in a retrospective study of 71 patients with ductal carcinoma of the breast. All patients were treated according to a standardized protocol. The median follow-up was 6 years. METHODS: Measurements were performed in microscopic fields that were sampled systematically from the whole tumor area of a routine histologic section. The unbiased stereologic method of point-sampled intercepts was used to estimate the mean nuclear volume, [vv(nuc)]. Using a test system with points and counting frames, estimates were obtained of the mean nuclear profile area, [aH(nuc)], the nuclear volume fraction, [Vv(nuc/tis)], the nuclear profile density (ND), the mitotic profile frequency (MF), and the mitotic profile density (MD). Traditional clinicopathologic parameters and biochemical estrogen receptor status were recorded. RESULTS: Single-factor survival analyses were significant regarding regional lymph node status, tumor dimension, clinical stage, age, aH(nuc), and vv(nuc) (P < or = 0.03). A tendency for prognostic value of MF was found (P = 0.10), whereas Vv(nuc/tis), ND, MD, histologic grade, and estrogen receptor status were insignificant. In a multivariate Cox analysis of patients with positive lymph nodes including the variables of tumor dimension, age, ND, vv(nuc), and MF, only vv(nuc) (P = 0.01) or MF (P = 0.004) were parameters of independent prognostic value. CONCLUSION: The present feasibility study suggests that stereologic estimates of the mean nuclear volume and morphometric estimates of the mitotic profile frequency are of independent prognostic value for patients with ductal breast cancer with positive axillary lymph nodes. The prognostic information resulting from the two variables are correlated closely and cannot be separated in this study. Consequently, larger studies are needed. In addition, the independent value of quantitative histopathology in patients with lymph node negative breast cancer should be assessed.

Quantitative histopathology in lymph node-negative breast cancer. Prognostic significance of mitotic counts.

Reliable prognostic factors are needed to improve the stratification of patients with lymph node-negative breast cancer to different therapy modalities. We investigated the prognostic value of quantitative histopathology in a retrospective study of 98 "low-risk" breast cancer patients (T1+2N0M0) with a median follow-up of 9 years. An interactive video system and stereological and morphometric techniques were used to obtain estimates of four nuclear features (mean volume, mean profile area, volume fraction, and profile density), and two mitotic counts [mitotic profile frequency (MF) and mitotic profile density (MD)]. All measurements were performed in fields of vision sampled systematically from the whole tumour area of a routine histological section. Histological grade, histological type, and oestrogen receptor (ER) status was reassessed, whereas tumour diameter and age at diagnosis were recorded from the files. We found that all quantitative histopathological variables and ER status were correlated with histological grade. Single-factor prognostic analyses showed a highly significant value of MF (2p = 0.001) and a marginally significant value of MD (2p = 0.09), whereas no other variable approached statistical significance (2p > or = 0.25). In a multivariate Cox analysis, MF was the only parameter of significant independent prognostic value (2p = 0.03). Thus, the prognostic value of nuclear features found in previous studies could not be reproduced, whereas the marked value of mitotic counts for prediction of the outcome in patients with breast cancer was confirmed. Mitotic counts are easily obtained and may be of clinical value for identification of high-risk cases among patients with lymph node-negative breast cancer.

The influence of tissue processing on quantitative histopathology in breast cancer.

Objective grading of breast cancer by morphometry has been suggested for improving the precision of the prognostic prediction. However, the tissue components evaluated might be influenced by variations in the processing, reducing the clinical value. In the present study, the impact of the period of fixation, of the acidity of the fixative and of the embedding medium was investigated by allocating tissue samples from 27 surgical breast cancer specimens systematically randomly to different modes of processing. The volume-weighted mean volume of cancer cell nuclei, vu v(nuc), was estimated using the method of point-sampled intercepts on vertical sections. In addition, estimates of the mean nuclear profile area, alpha H(nuc), the nuclear volume fraction, Vv(nuc), the nuclear profile density, ND, and the mitotic profile frequency, MF, were obtained. The quantitative histopathological estimates were stable with respect to the investigated variables of the tissue processing. No significant differences were found when comparing the estimates obtained in samples from five tumours fixed in formalin at pH 5.0, 6.0, 7.0, 7.4 and 8.0, respectively. Similarly, no significant correlations between the estimates and the period of formalin fixation (24 h, 3 days and 3 months) were found in samples from five other tumours. However, the nu v(nuc) was 13% larger (2p = 0.004) and the mean ND 17% smaller (2p = 0.04) in hydroxyethyl-methacrylate-embedded samples from 17 tumours as compared to paraffin-embedded samples. Thus, the shrinkage observed in paraffin seems to affect nuclei less than tissue.

Reproducibility of mean nuclear volume and correlation with mean nuclear area in breast cancer: an investigation of various sampling schemes.

Previous studies have shown that quantitative, histopathologic features obtained from a carefully selected area in the tumor section ("selective" approach) have a strong prognostic value in breast cancer. On the other hand, it was found that mean nuclear volume estimation in the whole area of the tumor section by means of "unbiased" stereologic techniques is of great value in predicting the clinical outcome as well. In the present study the results of the two different (ie, selective and random, systematic) sampling methods in assessing mean nuclear volume have been compared as to their intraobserver and interobserver reproducibility in 22 invasive breast cancer cases. The mean nuclear volume (nuclear vv) was assessed both in the most atypical area (AREA) (selected on morphologic criteria) and in the whole tumor section (TOTAL). Furthermore, the correlation with mean nuclear (profile) area (MNA) was studied. Mean nuclear (profile) area was determined in the AREA only. With bivariate correlation analysis the two sampling methods showed high correlation for the nuclear vv values (range of the correlation coefficient, 0.92 to 0.97). There were no systematic intraobserver differences between the different sampling methods. The results of observer 1 showed higher values, both with the selective and random systematic sampling methods. However, these systematic interobserver differences were small (< 9% of the average value of nuclear vv), much smaller than the variation between the tumors (which was > 60%). The time required for assessments in the AREA was less than that required for the determinations in the TOTAL (average, 10 v 20 minutes) in spite of the similar sample size. This is understandable, as in a sclerotic tumor many fields of vision do not contain cancer nuclei. The time required for MNA determinations in the AREA was longer than for nuclear vv assessments in the AREA (15 v 10 minutes). Nuclear vv and MNA (both assessed in the AREA) were (log distributed) significantly correlated (r = .77). Thus, nuclear vv determination in the AREA is the fastest method, and it is also well reproducible and strongly correlated with nuclear vv assessed in the TOTAL. In invasive breast cancer assessments in the whole tumor section can be used if delineation of the measurement area cannot be done easily. In small areas with a limited number of nuclei (eg, microinvasive parts) MNA can be easier to assess than nuclear vv. Further studies are required to compare and evaluate the prognostic value of nuclear vv and MNA.

Quantitative histopathological variables in in situ and invasive ductal and lobular carcinomas of the breast.

This study was carried out to compare quantitative histopathological estimates obtained in normal breast epithelium (N = 15), lobular carcinoma in situ (N = 29), ductal carcinoma in situ (N = 24), invasive lobular carcinoma (N = 39), and invasive ductal carcinoma (N = 71) of the female breast. Using unbiased stereology, the three-dimensional mean nuclear size, v v(nuc), was estimated in routine histological sections, along with morphometric point-counting based estimates of the mean nuclear profile area, aH(nuc), and estimates of the nuclear density index, NI, the mitotic index, MI, and the nuclear volume fraction, Vv(nuc/tis). The vv(nuc), aH(nuc), and MI were, on average, larger in ductal than in lobular carcinomas (2p < or = 0.01), whereas the mean NI was smaller in ductal carcinomas (2p = 3.10(-4). Comparing estimates obtained in tumors of pure ductal carcinoma in situ (N = 11) with those obtained in tumors of pure lobular carcinoma in situ (N = 7), only the difference in mean NI reached statistical significance (2p = 0.001). Several significant differences were found between means of quantitative histopathological estimates obtained in normal breast epithelium, pure in situ lesions, and invasive carcinomas. Overlaps were, however, evident among the groups. There were no significant differences between means of the quantitative variables obtained in carcinoma in situ of the ductal and the lobular type with or without accompanying invasive carcinoma (2p > or = 0.22). A close correlation was found between estimates of vv(nuc) obtained in the in situ component and the invasive part of ductal carcinomas (r = 0.86, 2p = 2.10(-4). Previous studies have shown prognostic value of quantitative histopathological variables in breast carcinomas. The present study points to an additional value of the investigated variables in the diagnostic separation of normal breast epithelium, in situ lesions, and invasive carcinomas. The quantitative variables obtained in the situ lesions did not indicate whether an accompanying invasive tumor was present or not.