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Purpose: In this study, we aimed to explore if the combination of tumor infiltrating lymphocytes (TILs) and change in tumor load on dynamic contrast-enhanced magnetic resonance imaging leads to better assessment of response to neoadjuvant chemotherapy (NAC) in patients with breast cancer, compared to either alone., Methods: In 190 NAC treated patients, MRI scans were performed before and at the end of treatment. The percentage of stromal TILs (%TILs) was assessed in pre-NAC biopsies according to established criteria. Prediction models were developed with linear regression by least absolute shrinkage and selection operator and cross validation (CV), with residual cancer burden as the dependent variable. Discrimination for pathological complete response (pCR) was evaluated using area under the receiver operating characteristic curves (AUC). We used Cox regression analysis for exploring the association between %TILs and recurrence-free survival (RFS)., Results: Fifty-one patients reached pCR. In all patients, the %TILs model and change in MRI tumor load model had an estimated CV AUC of 0.69 (95% confidence interval (CI) 0.53-0.78) and 0.69 (95% CI 0.61-0.79), respectively, whereas a model combining the variables resulted in an estimated CV AUC of 0.75 (95% CI 0.66-0.83). In the group with tumors that were ER positive and HER2 negative (ER+/HER2-) and in the group with tumors that were either triple negative or HER2 positive (TN&HER2+) separately, the combined model reached an estimated CV AUC of 0.72 (95% CI 0.60-0.88) and 0.70(95% CI 0.59-0.82), respectively. A significant association was observed between pre-treatment %TILS and RFS (hazard ratio (HR) 0.72 (95% CI 0.53-0.98), for every standard deviation increase in %TILS, p = 0.038)., Conclusion: The combination of TILs and MRI is informative of response to NAC in patients with both ER+/HER2- and TN&HER2+ tumors., (© 2024. The Author(s).)

Purpose: To analyze concordance rates between individual foci of bifocal BC for histological grade, type and intrinsic subtype based on immunohistochemical (IHC) and mRNA-testing using MammaTyper., Methods: We evaluated histological grade and type as well as intrinsic subtype based on IHC status for estrogen and progesterone receptors, HER2 and the mitotic activity index in 158 individual foci of 79 bifocal BC. A subgroup of 31 cases additionally underwent mRNA-based subtyping using the MammaTyper (MT) test. We calculated concordance rates between individual foci, as well as Cohen's Kappa (K)., Results: For 79 bifocal BC, concordance rates between individual foci for grade, histological type, and IHC-based subtype were 69.6% (K=0.53), 92.4% (K=0.81), and 74.7% (K=0.62), respectively. In the MT subgroup of 31 bifocal BC, concordance rates between individual foci for grade, histological type, IHC-based and mRNA-based intrinsic subtype were 87.1% (K=0.78), 90.3% (K=0.73), 87.1% (K=0.82), and 87.1% (K=0.7), respectively. Overall concordance between IHC- and mRNA-based subtype in the MT subgroup was 79% (K=0.7). In 6/79 cases (7.6%), testing of the smaller focus added clinically relevant information either on IHC- or mRNA-level: four cases showed high hormonal receptor expression while the expression in the larger focus was negative or low, warranting additional endocrine treatment; two cases presented with higher proliferative activity in the smaller focus, warranting additional chemotherapy., Conclusion: In bifocal BC, intertumoral heterogeneity on the morphological, immunohistochemical and molecular level is common, with discordant intrinsic subtype in up to 25% between individual foci, with about 8% clinically relevant discordances., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: Guidelines recommend endocrine treatment for estrogen receptor-positive (ER+) breast cancers for up to 10 years. Earlier data suggest that the 70-gene signature (MammaPrint) has potential to select patients that have an excellent survival without chemotherapy and limited or no tamoxifen treatment. The aim was to validate the 70-gene signature ultralow-risk classification for endocrine therapy decision making., Methods: In the IKA trial, postmenopausal patients with non-metastatic breast cancer had been randomized between no or limited adjuvant tamoxifen treatment without receiving chemotherapy. For this secondary analysis, FFPE tumor material was obtained of ER+HER2- patients with 0-3 positive lymph nodes and tested for the 70-gene signature. Distant recurrence-free interval (DRFI) long-term follow-up data were collected. Kaplan-Meier curves were used to estimate DRFI, stratified by lymph node status, for the three predefined 70-gene signature risk groups., Results: A reliable 70-gene signature could be obtained for 135 patients. Of the node-negative and node-positive patients, respectively, 20% and 13% had an ultralow-risk classification. No DRFI events were observed for node-negative patients with an ultralow-risk score in the first 10 years. The 10-year DRFI was 90% and 66% in the low-risk (but not ultralow) and high-risk classified node-negative patients, respectively., Conclusion: These survival analyses indicate that the postmenopausal node-negative ER+HER2- patients with an ultralow-risk 70-gene signature score have an excellent 10-year DRFI after surgery with a median of 1 year of endocrine treatment. This is in line with published results of the STO-3-randomized clinical trial and supports the concept that it is possible to reduce the duration of endocrine treatment in selected patients., (© 2022. The Author(s).)

Purpose: Pregnancy-associated breast cancer, although most commonly defined as breast cancer diagnosed during pregnancy or ≤1 year following delivery, knows a variety of definitions, likely related to the diversity of reported clinicopathological features and prognosis. More insight into the different breast cancer subgroups during pregnancy, time after delivery and the postpartum period is therefore warranted., Methods: Patients with breast cancer diagnosed during pregnancy or ≤6 months postdelivery were included, and subdivided according to gestational trimester, and postpartum patients according to lactational status. Subgroups were compared to matched non-PABC patients, to investigate the influence of pregnancy and lactation on clinical course and outcome., Results: Overall, 662 PABC patients were included (median age 34 years, median follow-up 6.5 years). PABC patients showed an advanced stage at diagnosis and an inferior 5-years-OS (75.4% vs. 83.2%, p = 0.000) compared to 1392 matched non-PABC patients. In subgroup analysis, first trimester PABC patients showed a significantly lower tumor size and stage as compared to other trimesters. Patients diagnosed during the first trimester and postpartum non-lactating patients had a relatively good OS (81.3% and 77.9%, respectively) versus patients diagnosed during the second and third trimesters and during lactation (OS 60.0%, 64.9% and 65.6%, respectively, p = 0.003)., Conclusion: In this large (uniquely specified) PABC cohort, an inferior outcome was found for patients diagnosed within the second and third gestational trimesters and during lactation. These findings indicate that PABC is clinically a heterogeneous group of breast cancer patients that should be redefined based on trimester of diagnosis and lactational status., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: The large variation in histologic grading of invasive breast cancer (IBC) that has been reported likely influences tailoring adjuvant therapy. The role of grading in therapeutic decision-making in daily practice, was evaluated using the Dutch national guidelines for IBC-management., Methods: Synoptic reports of IBC resection-specimens, obtained between 2013 and 2016, were extracted from the nationwide Dutch Pathology Registry, and linked to treatment-data from the Netherlands Cancer Registry. The relevance of grading for adjuvant chemotherapy (aCT) was quantified by identifying patients for whom grade was the determinative factor. In addition, the relation between grade and aCT-administration was evaluated by multivariate logistic regression for patients with a guideline-aCT-indication., Results: 30,843 patients were included. Applying the guideline that was valid between 2013 and 2016, grade was the determinative factor for the aCT-indication in 7744 (25.1%) patients, a percentage that even increased according to the current guideline where grade would be decisive for aCT in 10,869 (35.2%) patients. Also in current practice, the indication for adjuvant endocrine therapy (aET) would be based on grade in 9173 (29.7%) patients. Finally, as patients with lower-grade tumors receive aCT significantly less often, grade was also decisive in tailoring aCT de-escalation., Conclusions: In the largest study published so far we illustrate the increasing importance of histologic grade in tailoring adjuvant systemic breast cancer therapy. Next to playing a key-role in aCT-indication and de-escalation, the role of grading has expanded to the indication for aET. Optimizing histologic grading by pathologists is urgently needed to diminish the risk of worse patient outcome due to non-optimal treatment.

Introduction: Pathological nipple discharge (PND) is a common breast-related complaint for referral to a surgical breast clinic because of its association with breast cancer. The aim of this meta-analysis was to compare the diagnostic efficacy of magnetic resonance imaging (MRI) and ductoscopy in patients with PND. Additionally, we determined the most cost-efficient strategy for the treatment of PND and the detection of breast cancer in PND patient without radiological suspicion for malignancy., Materials and Methods: PubMed and EMBASE were searched to collect the relevant literature from the inception of both diagnostic methods until January 27th 2020. The search yielded 815 original citations, of which 10 studies with 894 patients were finally included for analysis. Costs of ductoscopy, MRI and duct excision surgery were obtained from the UMC Utrecht as established in the year 2019. These costs included: medical personnel, overhead costs, material costs and sterilisation costs., Results: The meta-analysis showed no significant difference in sensitivity between ductoscopy (44%) and MRI (76%) for the detection of malignancy in patients with PND. However, ductoscopy (98%) had a statistically significantly higher specificity than MRI (84%). Individual costs were €1401.33, €822.13 and €6494.27 for ductoscopy, MRI and duct excision surgery, respectively. Full diagnostic strategy involving ductoscopy was on average €1670.97, while with MRI it was €2070.27., Conclusion: Patients undergoing MRI are more often (false) positive which more often leads to duct excision surgery referrals compared to ductoscopy. This makes ductoscopy significantly more cost-effective compared MRI in patients with PND without radiological suspicion for malignancy.

Purpose: Breast cancer is the most common type of malignancy in pregnant women, occurring approximately once in every 3000 pregnancies. Pregnancy-associated breast cancer (PABC) is commonly defined as breast cancer diagnosed during or within one year after pregnancy, and it accounts for up to 6.9% of all breast cancers in women younger than 45 years old. Whether these cancers arise before or during pregnancy, and whether they are stimulated by the high hormonal environment of pregnancy, is currently unknown. This study assesses the histopathological profile of PABC in a large Dutch population-based cohort., Methods: We identified 744 patients with PABC (in this cohort defined as breast cancer diagnosed during or within 6 months after pregnancy) diagnosed between 1988 and 2019, in the nationwide Dutch Pathology Registry (PALGA). An age-matched PALGA cohort of unselected breast cancer patients (≤ 45 years), diagnosed between 2013 and 2016, was used as a control. Histopathologic features of both cohorts were compared., Results: The median age of PABC patients was 34.3 years old (range 19-45 years) and most breast cancers were diagnosed during pregnancy (74.2%). As compared to age-matched controls, PABC patients had tumors of higher Bloom-Richardson grade (grade I: 1.5% vs. 12.4%, grade II: 16.9% vs. 31.3%, grade III: 80.3% vs. 39.5%, p < 0.0001). Furthermore, estrogen (ER)- and progesterone (PR)-receptor expression was less frequently reported positive (ER: 38.9% vs. 68.2% and PR: 33.9% vs. 59.0%, p < 0.0001), while a higher percentage of PABC tumors overexpressed HER2 (20.0% vs. 10.0%, p < 0.0001). The most observed intrinsic subtype in PABC was triple-negative breast cancer (38.3% vs. 22.0%, p < 0.0001), whereas hormone-driven cancers were significantly less diagnosed (37.9% vs. 67.3%, p < 0.0001)., Conclusion: This study, based on a large population-based cohort of 744 PABC Dutch patients, underlines the more aggressive histopathologic profile compared to age-matched breast cancer patients ≤ 45 years. Further in-depth genetic analysis will be performed to unravel the origin of this discriminating phenotype. It definitely calls for timely detection and optimal treatment of this small but delicate subgroup of breast cancer patients.

Purpose: The addition of trastuzumab to adjuvant chemotherapy has improved the outcome of human epidermal growth-factor receptor 2 (HER2)-positive breast cancer. Uncertainty remains about the optimal timing of trastuzumab treatment. Therefore, we compared long-term outcome after concurrent versus sequential treatment, in a population-based setting, using data from the nationwide Netherlands Cancer Registry., Methods: We identified 1843 women diagnosed in The Netherlands from January 1st 2005 until January 1st 2008 with primary, HER2-positive, T 1-4 N any M 0 breast cancer who received adjuvant chemotherapy and trastuzumab. Kaplan-Meier survival estimates and Cox regression were used to compare recurrence-free survival (RFS) and overall survival (OS) between women who received trastuzumab concurrently with versus sequentially after chemotherapy. Hazard ratios (HR) were adjusted for age, year of diagnosis, grade, pathological T-stage, number of positive lymph nodes, ER-status, PR-status, socio-economic status, radiotherapy, hormonal therapy, ovarian ablation, and type of chemotherapy., Results: After a median follow-up of 8.2 years, RFS events had occurred in 224 out of 1235 (18.1%) concurrently treated women and 129 out of 608 (21.2%) sequentially treated women (adjusted-HR 0.91; 95% confidence interval (CI) 0.67-1.24; P = 0.580). Deaths occurred in 182/1235 (14.7%) concurrently treated women and 104/608 (17.1%) sequentially treated women (adjusted-HR 0.92; 95% CI 0.65-1.29; P = 0.635)., Conclusions: The results of this population-based study are consistent with earlier randomized trials, demonstrating a non-significant difference in outcome for concurrently treated women compared to those who were treated sequentially, suggesting both options are justified.

Purpose: For optimal management of ductal carcinoma in situ (DCIS), reproducible histopathological assessment is essential to distinguish low-risk from high-risk DCIS. Therefore, we analyzed interrater reliability of histopathological DCIS features and assessed their associations with subsequent ipsilateral invasive breast cancer (iIBC) risk., Methods: Using a case-cohort design, reliability was assessed in a population-based, nationwide cohort of 2767 women with screen-detected DCIS diagnosed between 1993 and 2004, treated by breast-conserving surgery with/without radiotherapy (BCS ± RT) using Krippendorff's alpha (KA) and Gwet's AC2 (GAC2). Thirty-eight raters scored histopathological DCIS features including grade (2-tiered and 3-tiered), growth pattern, mitotic activity, periductal fibrosis, and lymphocytic infiltrate in 342 women. Using majority opinion-based scores for each feature, their association with subsequent iIBC risk was assessed using Cox regression., Results: Interrater reliability of grade using various classifications was fair to moderate, and only substantial for grade 1 versus 2 + 3 when using GAC2 (0.78). Reliability for growth pattern (KA 0.44, GAC2 0.78), calcifications (KA 0.49, GAC2 0.70) and necrosis (KA 0.47, GAC2 0.70) was moderate using KA and substantial using GAC2; for (type of) periductal fibrosis and lymphocytic infiltrate fair to moderate estimates were found and for mitotic activity reliability was substantial using GAC2 (0.70). Only in patients treated with BCS-RT, high mitotic activity was associated with a higher iIBC risk in univariable analysis (Hazard Ratio (HR) 2.53, 95% Confidence Interval (95% CI) 1.05-6.11); grade 3 versus 1 + 2 (HR 2.64, 95% CI 1.35-5.14) and a cribriform/solid versus flat epithelial atypia/clinging/(micro)papillary growth pattern (HR 3.70, 95% CI 1.34-10.23) were independently associated with a higher iIBC risk., Conclusions: Using majority opinion-based scores, DCIS grade, growth pattern, and mitotic activity are associated with iIBC risk in patients treated with BCS-RT, but interrater variability is substantial. Semi-quantitative grading, incorporating and separately evaluating nuclear pleomorphism, growth pattern, and mitotic activity, may improve the reliability and prognostic value of these features.

Purpose: Little is known about the impact of 70-gene signature (70-GS) use on patients' chemotherapy decision-making. The primary aim of this study was to evaluate the impact of 70-GS use on patients' decisions to undergo chemotherapy. The perceived decision conflict during decision-making was a secondary objective of the study., Methods: Patients operated for estrogen receptor positive early breast cancer were asked to fill out a questionnaire probing their inclination to undergo chemotherapy before deployment of the 70-GS test. After disclosure of the 70-GS result patients were asked about their decision regarding chemotherapy. Patients' decisional conflict was measured using the 16-item decisional conflict scale (DCS); scores < 25 are associated with a persuaded decision while a score > 37.5 implies that one feels unsure about a choice., Results: Between January 1th 2017 and December 31th 2018, 106 patients completed both questionnaires. Before deployment of the 70-GS, 58% of patients (n = 62) formulated a clear treatment preference, of whom 21 patients (34%) changed their opinion on treatment with chemotherapy following the 70-GS. The final decision regarding chemotherapy was in line with the 70-GS result in 90% of patients. The percentage of patients who felt unsure about their preference to be treated with chemotherapy decreased from 42 to 5% after disclosure of the 70-GS. The mean total DCS significantly decreased from pre-test to post-test from 35 to 23, irrespective of the risk estimate (p < 0.001)., Conclusion: Deployment of the 70-GS changed patients' inclination to undergo adjuvant chemotherapy in one third of patients and decreased patients' decisional conflict.

Purpose: Patient management of invasive breast cancer (IBC) is to a large extent based on hormone- and HER2-receptor assessment. High-quality, reliable receptor assessment is of key importance as false results may lead to under- or overtreatment of patients. Surveillance of case-mix adjusted positivity rates has been suggested as a tool to identify laboratories with insufficient testing assays, as this covers the whole process of receptor assessment and enables laboratories to benchmark their positivity rates against other laboratories. We studied laboratory-specific variation in hormone- and HER2 positivity rates of 33,046 breast cancer patients using real-life nationwide data., Methods: All synoptic pathology reports of IBC resection-specimens, obtained between 2013 and 2016, were retrieved from the nationwide Dutch pathology registry (PALGA). Absolute and case-mix adjusted receptor positivity rates were compared to the mean national proportion and presented in funnel plots in separate analyses for estrogen (ER), progesterone (PR) and HER2. Case-mix adjustment was performed by multivariable logistic regression., Results: 33,794 IBC lesions from 33,046 patients of 39 pathology laboratories were included. After case-mix adjustment, mean positivity rates were 87.2% for ER (range 80.4-94.3), 71.3% for PR (62.5-77.5%), and 9.9% for HER2 (5.5-12.7%). Overall, 14 (35.9%), 17 (43.6%) and 11 (28.2%) laboratories showed positivity rates outside the 95% confidence interval for ER, PR and HER2, respectively., Conclusion: This nationwide study shows that absolute variation in hormone- and HER2-receptor positivity rates between Dutch pathology laboratories is limited. Yet, the considerable number of outlying laboratories shows that there is still need for improvement. Continuous monitoring and benchmarking of positivity rates may help to realize this.

Purpose: A considerable part of ductal carcinoma in situ (DCIS) lesions may never progress into invasive breast cancer. However, standard treatment consists of surgical excision. Trials aim to identify a subgroup of low-risk DCIS patients that can safely forgo surgical treatment based on histologic grade, which highlights the importance of accurate grading. Using real-life nationwide data, we aimed to create insight and awareness in grading variation of DCIS in daily clinical practice., Methods: All synoptic pathology reports of pure DCIS resection specimens between 2013 and 2016 were retrieved from PALGA, the nationwide Dutch Pathology Registry. Absolute differences in proportions of grade I-III were visualized using funnel plots. Multivariable analysis was performed by logistic regression to correct for case-mix, providing odds ratios and 95% confidence intervals for high-grade (III) versus low-grade (I-II) DCIS., Results: 4952 DCIS reports from 36 laboratories were included, of which 12.5% were reported as grade I (range 6.1-24.4%), 39.5% as grade II (18.2-57.6%), and 48.0% as grade III (30.2-72.7%). After correction for case-mix, 14 laboratories (38.9%) reported a significantly lower (n = 4) or higher (n = 10) proportion of high-grade DCIS than the reference laboratory. Adjusted ORs (95%CI) ranged from 0.52 (0.31-0.87) to 3.83 (1.42-10.39). Significant grading differences were also observed among pathologists within laboratories., Conclusion: In this cohort of 4901 patients, we observed substantial inter- and intra-laboratory variation in DCIS grading, not explained by differences in case-mix. Therefore, there is an urgent need for nationwide standardization of grading practices, especially since the future management of DCIS may alter significantly depending on histologic grade.

Purpose: Breast cancer in men is uncommon; it accounts for 1 % of all patients with primary breast cancer. Its treatment is mostly extrapolated from its female counterpart. Accurate predictions are essential for adjuvant systemic treatment decision-making and informing patients. Several predictive models are available for female breast cancer (FBC) including the Morphometric Prognostic Index (MPI), Nottingham Prognostic Index (NPI), Adjuvant! Online and Predict. The aim of this study was to examine and compare the prognostic performance of these models for male breast cancer (MBC)., Methods: The population of this study consists of 166 MBC patients. The prognostic scores of the patients are categorized by good, (moderate) and poor, defined by the test itself (MPI and NPI) or based on tertiles (Adjuvant! Online and Predict). Survival according to prognostic score was compared by Kaplan-Meier analysis and differences were tested by logRank. The prognostic performances were evaluated with C-statistics. Calibration was done with the aim to estimate to what extent the survival rates predicted by Predict were similar to the observed survival rates., Results: All prediction models were capable of discriminating between good, moderate and poor survivors. P-values were highly significant. Comparison between the models using C-statistics (n = 88) showed equal performance of MPI (0.67), NPI (0.68), Adjuvant! Online (0.69) and Predict (0.69). Calibration of Predict showed overestimation for MBC patients., Conclusion: In conclusion, MPI, NPI, Adjuvant! and Predict prognostic models, originally developed and validated for FBC patients, also perform quite well for MBC patients., Competing Interests: The authors declare that they have no conflict of interest.

Ki67 has been proposed as prognostic proliferation marker in luminal breast cancer (BC), but little is known on the influence of Ki67 assessment methods on subtyping into luminal A- and B-like tumors. Our aim was to study the influence of different Ki67-labeling index (Ki67-LI) assessment methods on the proportion of BCs classified as luminal A-like. 280 early BCs were subtyped according to the St Gallen 2015 definitions into 71 % luminal (HER2 negative), 6 % luminal B-like (HER2 positive), 13 % triple negative, 1 % HER2 positive (nonluminal), and 9 % special type. Digitized whole slides were counted manually on the screen. We used nine defined counting methods to assess the Ki67-LI (including the International Ki67 in Breast Cancer Working Group recommendations), and compared the resulting medians and the proportions of cancers classified as luminal A-like according to the formerly used cut-off <20 %. Methods assessing hot spots and tumor periphery resulted in significantly higher Ki67-LI medians than those measuring an average proliferation (27.45 % vs 16.96 %, p < 0.0001). Substantially lower median Ki67-LI were found when assessing 1020 compared to counting 100, 200, 300 cells (17.65 vs 33 %, vs 28 %, vs 24.33 %, respectively; p < 0.0001), or 510 cells (20.59 %, p = 0.019). Applying a standard Ki67-LI cut-off <20 % to define low proliferation for all methods, the proportion of luminal A-like cancers varied between 13 and 44 %. The proportion of BCs classified as luminal A-like is highly influenced by the Ki67-LI assessment method. As a consequence, the selection of a specific Ki67-LI assessment method may have a direct effect on the proportion of patients considered having low-risk disease and thus influence therapeutic decision making. This calls for a standardized assessment method.

Promoter hypermethylation of tumor suppressor genes seems to be an early event in breast carcinogenesis and is potentially reversible. This makes methylation a possible therapeutic target, a marker for treatment response and/or a prognostic factor. Methylation status of 40 tumor suppressor genes was compared between 53 primary breast tumors and their corresponding metastases to brain, lung, liver, or skin. In paired analyses, a significant decrease in methylation values was seen in distant metastases compared to their primaries in 21/40 individual tumor suppressor genes. Furthermore, primary tumors that metastasized to the liver clustered together, in line with the finding that primary breast carcinomas that metastasized to the brain, skin, or lung, showed higher methylation values in up to 27.5 % of tumor suppressor genes than primary carcinomas that metastasized to the liver. Conversion in methylation status of several genes from the primary tumor to the metastasis had prognostic value, and methylation status of some genes in the metastases predicted survival after onset of metastases. Methylation levels for most of the analyzed tumor suppressor genes were lower in distant metastases compared to their primaries, pointing to the dynamic aspect of methylation of these tumor suppressor genes during cancer progression. Also, specific distant metastatic sites seem to show differences in methylation patterns, implying that hypermethylation profiles of the primaries may steer site-specific metastatic spread. Lastly, methylation status of the metastases seems to have prognostic value. These promising findings warrant further validation in larger patient cohorts and more tumor suppressor genes.

Ductal carcinoma in situ (DCIS) of the breast is difficult to remove completely during surgery as it is not palpable and can therefore require re-excision. Real-time visualization of DCIS using near-infrared fluorescent probes could help the surgeon during surgery as well as the pathologist post-operatively to distinguish the tumor from healthy tissue. As hypoxia-induced necrosis is a common phenomenon in DCIS, we investigated the molecular imaging of DCIS using a fluorescent antibody targeting a hypoxia marker, carbonic anhydrase IX (CAIX), in a preclinical mouse model. A monoclonal antibody against human CAIX was fluorescently labeled with the near-infrared dye IRDye800CW and characterized in vitro. An in vivo study was performed in SCID/Beige mice that were orthotopically transplanted with human breast cancer cells mimicking human DCIS (MCF10DCIS) and MCF10DCIS stably expressing CAIX. A clinically approved fluorescence imaging system was used to monitor probe uptake and to determine tumor-to-normal tissue ratios (TNR). Mean in vivo TNR of CAIX-transduced (CAIX+) tumors was 7.5 ± 0.5. Mean in vivo TNR of DCIS tumors with hypoxic areas reached a plateau level at 48 h after injection of 2.1 ± 0.1 (mean ± SEM) compared to 1.7 ± 0.1 in DCIS without hypoxic areas. Mean intra-operative TNR of DCIS tumors with necrotic regions was higher than that of DCIS tumors without necrotic regions 96 h after injection-2.9 ± 0.1 and 1.5 ± 0.1, respectively-while the TNR of CAIX+ tumors was 11.2 ± 1.0. Specific tumor uptake of MabCAIX-IRDye800CW was confirmed by a biodistribution assay, and immunofluorescence imaging on tumor sections showed specific uptake in hypoxic tumor regions, with higher contrast than conventional chromagen-based immunohistochemistry. Molecular fluorescence imaging with MabCAIX-IRDye800CW can be successfully used to detect hypoxic DCIS before and during surgery to facilitate radical resection. Furthermore, it allows for sensitive CAIX-specific immunofluorescence microscopy of tumor sections, thereby introducing the concept of molecular fluorescence pathology.

Estrogen catabolism is a major function of CYP2C19. The effect of CYP2C19 polymorphisms on tamoxifen sensitivity may therefore not only be mediated by a variation in tamoxifen metabolite levels but also by an effect on breast cancer risk and molecular subtype due to variation in lifelong exposure to estrogens. We determined the association between these polymorphisms and tamoxifen sensitivity in the context of a randomized trial, which allows for the discernment of prognosis from prediction. We isolated primary tumor DNA from 535 estrogen receptor-positive, stages I-III, postmenopausal breast cancer patients who had been randomized to tamoxifen (1-3 years) or no adjuvant therapy. Recurrence-free interval improvement with tamoxifen versus control was assessed according to the presence or absence of CYP2C19 2 and CYP2C19 17. Hazard ratios and interaction terms were calculated using multivariate Cox proportional hazard models, stratified for nodal status. Tamoxifen benefit was not significantly affected by CYP2C19 17. Patients with at least one CYP2C19 2 allele derived significantly more benefit from tamoxifen (HR 0.26; p = 0.001) than patients without a CYP2C19 2 allele (HR 0.68; p = 0.18) (p for interaction 0.04). In control patients, CYP2C19 2 was an adverse prognostic factor. In conclusion, breast cancer patients carrying at least one CYP2C19 2 allele have an adverse prognosis in the absence of adjuvant systemic treatment, which can be substantially improved by adjuvant tamoxifen treatment.

Promoter hypermethylation of several tumour suppressor genes often occurs during breast carcinogenesis, but little is known about epigenetic silencing in the possible precursor columnar cell lesion (CCL). Promoter hypermethylation of 50 different tumour suppressor genes was assessed in normal breast tissue (N = 10), CCL (N = 15), ductal carcinoma in situ (DCIS) grade I originating in CCL (N = 5) and paired CCL (N = 15) with DCIS (N = 7) and/or invasive carcinoma (N = 14) by Methylation-specific multiplex ligation-dependent probe amplification. Increasing mean cumulative methylation levels were found from normal breast tissue to CCL to DCIS and invasive carcinoma (P < 0.001) with similar methylation levels in DCIS and invasive carcinoma. Methylation levels and frequencies (in the overall analysis and analysis of only the synchronous lesions) were the highest for RASSF1, CCND2, ID4, SCGB3A1 and CDH13. The methylation levels of ID4, CCND2, and CDH13 increased significantly from normal breast tissue to CCL and to DCIS/invasive carcinoma. RASSF1, SCGB3A1 and SFRP5 had significant higher methylation levels in CCL compared to normal breast tissue, but showed no significant differences between CCL, DCIS and invasive carcinoma. Also, no difference was found between CCLs with and without atypia, or CCLs with or without synchronous cancer. In conclusion, promoter hypermethylation for several established tumour suppressor genes is already present in CCLs, underlining that promoter hypermethylation is an early event in breast carcinogenesis. Atypia in CCL or the presence of synchronous more advanced lesions does not seem to be accompanied by higher methylation levels.

For the management of non-palpable breast cancer, accurate pre-operative localisation is essential to achieve complete resection with optimal cosmetic results. Radioguided occult lesions localisation (ROLL) uses the radiotracer, injected intra-tumourally for sentinel lymph node identification to guide surgical excision of the primary tumour. In a multicentre randomised controlled trial, we determined if ROLL is superior to the standard of care (i.e. wire-guided localisation, WGL) for preoperative tumour localisation. Women (>18 years.) with histologically proven non-palpable breast cancer and eligible for breast conserving treatment with sentinel node procedure were randomised to ROLL or WGL. Patients allocated to ROLL received an intra-tumoural dose of 120 Mbq technetium-99 m nanocolloid. The tumour was surgically removed, guided by gamma probe detection. In the WGL group, ultrasound- or mammography-guided insertion of a hooked wire provided surgical guidance for excision of the primary tumour. Primary outcome measures were the proportion of complete tumour excisions (i.e. with negative margins), the proportion of patients requiring re-excision and the volume of tissue removed. Data were analysed according to intention-to-treat principle. This study is registered at ClinincalTrials.gov, number NCT00539474. In total, 314 patients with 316 invasive breast cancers were enrolled. Complete tumour removal with negative margins was achieved in 140/162 (86 %) patients in the ROLL group versus 134/152 (88 %) patients in the WGL group (P = 0.644). Re-excision was required in 19/162 (12 %) patients in the ROLL group versus 15/152 (10 %) (P = 0.587) in the WGL group. Specimen volumes in the ROLL arm were significantly larger than those in the WGL arm (71 vs. 64 cm(3), P = 0.017). No significant differences were seen in the duration and difficulty of the radiological and surgical procedures, the success rate of the sentinel node procedure, and cosmetic outcomes. In this first multicentre randomised controlled comparison of ROLL versus WGL in patients with histologically proven breast cancer, ROLL is comparable to WGL in terms of complete tumour excision and re-excision rates. ROLL, however, leads to excision of larger tissue volumes. Therefore, ROLL cannot replace WGL as the standard of care.

Recent molecular data pointed towards the possibility of a stepwise dedifferentiation in a subgroup of invasive breast cancer (BC) cases. It was hypothesized that oestrogen receptor positive (ER+) grade 3 (G3) ductal invasive BCs are the end stage of a dedifferentiation process of luminal BC. A progression of luminal A towards luminal B BCs associated with a 'progression through grade' and an increased cell proliferation seemed the obvious explanation. In order to verify this hypothesis on a morphological and immunohistochemical level, we investigated 865 invasive BC cases. All cases were reviewed for the presence of intratumoural heterogeneity in grade of the invasive cancer and the presence of associated ductal carcinoma in situ (DCIS). With the use of tissue microarrays, the molecular subtype was determined and correlated with clinico-pathological features. In addition, all cases were stained for p21, p27, Ki-67, Cyclin D1, bcl-2, p53, and p16 and the results subjected to a biomathematical dependency analysis. The frequency of ER-positivity decreased with tumour size. The frequency of luminal A BC decreased as well, whereas the number of luminal B BCs remained constant. A gradual increase of the frequency of basal-like, HER2-driven and non-expressor BCs with tumour size was seen. In only 1 out of 865 BC cases, both a G1 and a G3 invasive cancer component was seen within the same BC. In two cases, a ductal invasive G1 carcinoma was associated with a poorly-differentiated DCIS. The frequency of columnar cell lesions was evenly distributed over ER+ and ER- ductal invasive G3 carcinomas. The biomathematical analysis gave striking hints against an obligate progression of BC trough grade. In conclusion, our results show that a morphological recognizable striking 'progression through grade' at least in its extreme form from G1 towards G3 is a very rare event in the natural course of invasive BC, including luminal BC.

Gene amplification is an important mechanism for oncogene activation, a crucial step in carcinogenesis. Compared to female breast cancer, little is known on the genetic makeup of male breast cancer, because large series are lacking. Copy number changes of 21 breast cancer related genes were studied in 110 male breast cancers using multiplex ligation-dependent probe amplification. A ratio of >1.3 was regarded indicative for gene copy number gain and a ratio >2.0 for gene amplification. Data were correlated with clinicopathological features, prognosis and 17 genes were compared with a group of female breast cancers. Gene copy number gain of CCND1, TRAF4, CDC6 and MTDH was seen in >40 % of the male breast cancer cases, with also frequent amplification. The number of genes with copy number gain and several single genes were associated with high grade, but only CCND1 amplification was an independent predictor of adverse survival in Cox regression (p = 0.015; hazard ratio 3.0). In unsupervised hierarchical clustering a distinctive group of male breast cancer with poor prognosis (p = 0.009; hazard ratio 3.4) was identified, characterized by frequent CCND1, MTDH, CDC6, ADAM9, TRAF4 and MYC copy number gain. Compared to female breast cancers, EGFR (p = 0.005) and CCND1 (p = 0.041) copy number gain was more often seen in male breast cancer, while copy number gain of EMSY (p = 0.004) and CPD (p = 0.001) and amplification in general was less frequent. In conclusion, several female breast cancer genes also seem to be important in male breast carcinogenesis. However, there are also clear differences in copy number changes between male and female breast cancers, pointing toward differences in carcinogenesis between male and female breast cancer and emphasizing the importance of identifying biomarkers and therapeutic agents based on research in male breast cancer. In addition CCND1 amplification seems to be an independent prognosticator in male breast cancer.

In breast cancer, it has been shown that pN0(i+) and pN1mi have a comparable negative impact on disease-free survival, compared with pN0. However, pN0(i+) is considered to be a heterogeneous group. We determined the effect of metastatic size and microanatomic location within the pN0(i+) group on breast cancer recurrence. We included all Dutch breast cancer patients diagnosed in 1998-2005 with favorable primary tumor characteristics and a final nodal status of pN0(i+). For this analysis, only patients without adjuvant systemic therapy were eligible (n = 513). Presence of single tumor cells versus cell clusters, metastatic size and microanatomic location were recorded. Primary endpoint was disease-free survival. Analyses were adjusted for age at diagnosis, tumor size, tumor grade, axillary treatment and hormone receptor status. The 5-year disease-free survival of patients with single tumor cell(s) (n = 93) was 78.6% and with tumor cell cluster(s) (n = 404) 77.1%. The hazard ratio for disease events was 1.05 (95% CI 0.63-1.76) for cell cluster(s) compared with single cell(s). In a Cox regression model, doubling of metastatic tumor size corresponded to a hazard ratio of 1.21 (95% CI 1.02-1.43). The adjusted hazard ratio was 0.90 (95% CI 0.54-1.50) for parenchymal (n = 112) versus sinusoidal location (n = 395). Single tumor cells bear similar prognostic information as small tumor cell clusters, even though results do suggest that within the pN0(i+) group, increasing size of nodal involvement is associated with reduced survival. Microanatomic location does not seem to have prognostic relevance.

In early-stage breast cancer and DCIS patients, breast-conserving therapy is today's standard of care. The purpose of this study was to evaluate the relation between the microscopic tumor diameter (mTD), the excised specimen (ES) volume, and the irradiated postoperative complex (POC) volume, in patients treated with breast-conserving therapy. In 186 patients with pTis-2N0 breast cancer, the mTDs, ES, and POC volumes (as delineated on the radiotherapy-planning CT scan), were retrospectively determined. Linear regression analysis was performed to study the association between the mTD, and the ES and POC volumes. The explained variance (r (2)) was calculated to establish the proportion of variation in the outcome variable that could be explained by the determinant (P ≤ 0.05). Moreover, the influence of tumor characteristics, age, surgical procedures, and breast size was studied. Median mTD was 1.2 cm (range 0.1-3.6 cm), median ES volume was 60 cm(3) (range 6-230 cm(3)) and median POC volume was 15 cm(3) (range 0.5-374 cm(3)). The POC was not clearly visible on the majority of the CT scans, based on a median assigned cavity visualization score of 3 (range 1-5). The explained variance for the mTD on the ES volume was low (r(2) = 0.08, P < 0.001). A slightly stronger association was observed in palpable tumors (r(2) = 0.23, P < 0.001) and invasive lobular carcinomas (r(2) = 0.39, P = 0.01). Furthermore, weak associations were observed between POC volume and mTD (r(2) = 0.04, P = 0.01), and POC and ES volume (r(2) = 0.23, P < 0.001). A weak association was observed between breast volume and ES volume (r(2) = 0.27, P < 0.001). In conclusion, both the excised and the irradiated POC volumes did not show a clinically relevant association with the mTD in women with early-stage breast cancer treated with breast-conserving therapy. Future studies should focus on improvement of surgical localization, development of image-guided, minimally invasive operation techniques, and more accurate image-guided target volume delineation in radiotherapy.

Physicians are moving away from routine axillary lymph node dissection (ALND) in clinically node-negative breast cancer. We conducted a systemic review on the safety of this policy. Pubmed and Cochrane library were searched for. Sixty-eight studies were included: studies of clinically node-negative patients in the pre-sentinel node (SN) era; observational studies of SN-negative patients, without ALND; comparative studies of SN-negative patients, with a non-ALND and an ALND group; SN-positive studies, of patients without ALND. Primary endpoint was the pooled axillary recurrence rate (ARR) of each category; secondary endpoint was overall survival (OS) rate. In pre-SN studies, with larger tumors and less systemic therapy, ARR without ALND after 5-10 years follow-up was 12-18%, with 5% reduced OS. In the observational SN-negative studies, with median follow-up of 36 months, the pooled ARR was 0.6% (95% CI 0.6-0.8). In the comparative SN-negative studies, pooled ARR was 0.4% (95% CI 0.2-0.6) without ALND versus 0.3% (95% CI 0.1-0.6) with ALND at 31 and 47 months, respectively, and no survival disadvantage. In SN-positive studies, ARR was up to 1.7% (95% CI 1.0-2.7) at 30 months. For patients with an H&E positive SN the ARR without ALND was 5% after 23 months, which may imply rates as high as 13 and 18% after 5 and 8 years. In conclusion, this systematic review confirms the safety of omitting ALND in SN-negative patients. There is a potential role for avoiding ALND in selected SN-positive patients, but eligibility criteria and the role of systemic therapy need further to be elucidated.

Evaluation of prognostic factors in lymph node negative (LNneg) invasive lobular cancers (ILCs). Prospective analysis of proliferation and other prognosticators in 121 LNneg ILCs (119 months median follow-up, range 19-181), without adjuvant chemotherapy. ILC subtype was assessed in accordance with WHO-2003 criteria. Immunohistochemical E-cadherin and estrogen receptor were used. With a median follow up time of 83 months (range 19-181), 30 of the 121 (25%) ILC patients developed distant metastases and 27 (22%) died. None of the cases classified as solid/pleomorphic lobular were E-cadherin or estrogen receptor positive, contrasting the other ILCs. The solid/alveolar ILCs (n = 17) had a worse survival (50%) than the other ILCs (n = 104; 83%, P < 0.0001). Mitotic activity index (MAI) (but not nuclear grade or tubule formation) was prognostic with a threshold 0-5 versus >5 (=MAI-5) (contrasting MAI < 10 vs. > or = 10 in breast cancers in general; 85 and 54% survival, P < 0.0001). In multivariate analysis only subtype and MAI but none of the other characteristics had independent prognostic value. Histologic subtype and MAI have independent prognostic value in node negative invasive lobular cancers.

Hypoxia is a hallmark of cancer. Hypoxia inducible factor-1alpha (HIF-1alpha) is the key regulator of the hypoxia response. HIF-1alpha is overexpressed during sporadic breast carcinogenesis and correlated with poor prognosis. Little is known on the role of HIF-1alpha in hereditary breast carcinogenesis. A recent study suggests a role for BRCA1 in HIF-1alpha regulation. We therefore examined the expression of HIF-1alpha in BRCA1 related breast cancers. By immunohistochemistry we studied expression of HIF-1alpha and some of its downstream targets in 30 hereditary invasive breast cancers in comparison with 200 sporadic controls. HIF-1alpha overexpression was significantly more frequent in BRCA1 related breast cancers (27/30, 90%) than in sporadic controls (88/200, 44%) (P < 0.0001). 19/30 (63%) of BRCA1 tumors showed perinecrotic (hypoxia induced) and 8/30 (27%) a diffuse HIF-1alpha overexpression pattern, the latter more likely related to genetic alterations in oncogenes and tumor suppressor genes. In contrast, sporadic breast cancer HIF-1 expressing tumors showed an inverse ratio of perinecrotic/diffuse expression (31 vs. 69%, P = 0.0002). Glut-1 and CAIX, downstream HIF1 targets, were expressed in 27/30 (90%) and 15/21 (71%) of hereditary cases versus 54/183 (29%) and 24/183 (13%) in sporadic cases. p300 levels, necessary for HIF-1 downstream activation, were significantly higher in hereditary cases (20/21, 95%) compared to sporadic cases (91/183, 50%, P = 0.0001). In conclusion, in BRCA1 germline mutation related breast cancer, functional HIF-1alpha overexpression is seen at a much higher frequency than in sporadic breast cancer, mostly hypoxia induced. This points to an important role of hypoxia and its key regulator HIF-1alpha in hereditary breast carcinogenesis.

Fanconi anemia (FA) is a recessive disorder associated with progressive pancytopenia, multiple developmental defects, and marked predisposition to malignancies. FA is genetically heterogeneous, comprising at least 12 complementation groups (A-M). Activation of one of the FA proteins (FANCD2) by mono-ubiquitination is an essential step in DNA damage response. As FANCD2 interacts with BRCA1, is expressed in proliferating normal breast cells, and FANCD2 knockout mice develop breast tumors, we investigated the expression of FANCD2 in sporadic and hereditary invasive breast cancer patients to evaluate its possible role in breast carcinogenesis. Two tissue microarrays of 129 and 220 sporadic breast cancers and a tissue microarray containing 25 BRCA1 germline mutation-related invasive breast cancers were stained for FANCD2. Expression results were compared with several clinicopathological variables and tested for prognostic value. Eighteen of 96 (19%) sporadic breast cancers and two of 21 (10%) BRCA1-related breast cancers were completely FANCD2-negative, which, however, still showed proliferation. In the remaining cases, the percentage of FANCD2-expressing cells correlated strongly with mitotic index and percentage of cells positive for the proliferation markers Ki-67 and Cyclin A. In immunofluorescence double staining, coexpression of FANCD2 and Ki-67 was apparent. In survival analysis, high FANCD2 expression appeared to be prognostically unfavorable for overall survival (p = 0.03), independent from other major prognosticators (p = 0.026). In conclusion, FANCD2 expression is absent in 10-20% of sporadic and BRCA1-related breast cancers, indicating that somatic inactivating (epi)genetic events in FANCD2 may be important in both sporadic and hereditary breast carcinogenesis. FANCD2 is of independent prognostic value in sporadic breast cancer.

Specific mutations in BRCA1 and BRCA2 genes have been identified in specific populations and ethnic groups. However, little is known about the contribution of BRCA1 and BRCA2 mutations to breast cancers in the Indonesian population. One hundred-twenty moderate to high risk breast cancer patients were tested using PCR-DGGE, and any aberrant band was sequenced. Multiplex ligation-dependent probe amplification (MLPA) was performed on all samples to detect large deletions in the two genes. Twenty-three different mutations were detected in 30 individuals, ten were deleterious mutations and 20 were "unclassified variants" with uncertain clinical consequences. Three of seven (c.2784&#95;2875insT, p.Leu1415X and del exon 13-15) and two of four (p.Glu2183X and p.Gln2894X) deleterious mutations that were found in BRCA1 and BRCA2 respectively, are novel. Several novel, pathogenic BRCA1 and BRCA2 germline mutations are found in early onset Indonesian breast cancer patients, these may therefore be specific for the Indonesian population.

Certain nuclear morphometric features measured in breast tumor tissue have been shown to predict the prognosis of breast cancer patients. However, the application of these features to predicting risk of breast cancer development has received little attention. We conducted a case-control study to evaluate nuclear morphometric features in benign breast tissue in association with subsequent breast cancer risk. The study was nested within a cohort of 4,888 women with a histopathologic diagnosis of benign breast disease (BBD) and involved 61 cases and 71 controls, amongst whom there were 53 matched case-control sets. Conditional logistic regression models were fitted to assess various measurements of nuclear size and nuclear shape factors in relation to subsequent breast cancer risk. In multivariate analysis, subsequent breast cancer risk was positively associated with a nuclear shape factor that takes the shortest nuclear axis and the longest nuclear axis into consideration simultaneously (highest quartile versus lowest 3 quartiles: odds ratio = 3.07, 95% confidence limits = 1.61, 5.84). In contrast, there was no alteration in subsequent breast cancer risk in association with nuclear size features and other shape factors. In conclusion, our study results suggest that the shape factor that takes both the shortest nuclear axis and the longest nuclear axis into consideration might be of value to predict subsequent development of breast cancer among women with BBD.

Objective: The increased incidence of breast cancer in the southeastern Netherlands was accompanied by markedly improved relative survival and stable mortality. We investigated whether the average aggressiveness of tumors changed over time in a population-based study, before the introduction of mass screening., Methods: The mitotic activity index (MAI) was determined retrospectively for 1051 consecutive patients diagnosed with invasive, non-metastatic breast cancer in 1975, 1981, 1988, and 1989. Trends over time, and effects of age, tumor size and lymph node status were examined by univariate and multivariate regressions., Results: Age-adjusted incidence of low MAI tumors changed from 35/100,000 in 1975 to 45/100,000 in 1988-89, an increase of 30% (P = 0.01), the incidence of tumors with a high MAI increased about 20% (P = 0.28), from 25 to 29/100,000. For small tumors (T1) the odds for a high MAI was lower in 1981 (OR: 0.80; 95% CI: 0.37-1.73) and 1988-89 (OR: 0.66; 95% CI: 0.35-1.23) compared to 1975. Among T3 and T4 tumors the odds increased to 2.03 (95% Cl: 0.71-5.86) in 1981 and 2.16 (0.76-6.18) in 1988-89., Conclusion: Although the incidence of tumors with low aggressive potential increased, the incidence of high MAI tumors also increased. Stable breast cancer mortality rates in the face of increasing incidence rates during the period 1975-89 cannot be attributed solely to changes in tumor aggressiveness; early diagnosis and better treatment may also have contributed.

Purpose: To assess the relation between growth factors, growth-factor receptors, p53, bcl-2 and bax expression, and the rate of apoptosis in invasive breast cancer patients., Materials and Methods: Tumors from 45 patients were assessed by immunohistochemistry for expression patterns of five growth factors and their receptors; platelet-derived growth factor A chain (PDGF-AA) and PDGF-receptor alpha (PDGFalphaR), PDGF-BB and PDGFbetaR, transforming growth-factor alpha (TGFalpha) and its receptor-epidermal growth factor receptor (EGFR) and vascular-endothelial growth factor (VEGF) and its receptors vascular-endothelial growth factor receptor I (FLT-1) and vascular-endothelial growth factor receptor II (FLK-1/KDR), two growth-inhibiting factors; transforming-growth factor beta I (TGFbeta1) and TGFbeta2 and their receptor couple TGFbeta receptor I (TGFbetaR-I) and TGFbetaR-II, and basic-fibroblast growth factor (bFGF). Besides, the expression patterns of the bcl-2, bax and p53 gene products were investigated. Expression patterns were correlated to the number of apoptotic cells assessed by light microscopy., Results: PDGF-BB and bFGF showed a positive correlation with the AI (p = 0.006 and p = 0.030, respectively). EGFR expression was associated with a high number of apoptotic cells but did not reach significance (p = 0.10). None of the other individual growth factors, growth-inhibiting factors or receptors showed a significant relation with the AI. The presence of a possible auto- or paracrine loop of the TGFalpha/EGFR combination was associated with a high number of apoptotic cells but did not reach significance (p = 0.20). PDGF-AA, bFGF and EGFR expression showed a significant relation to p53 overexpression. TGFbeta2 expression showed an inverse correlation with p53 overexpression., Conclusion: We found an association between several growth factors and growth-factor receptors with number of apoptotic cells. This underlines the importance of growth factors and their receptors not just in proliferation, but also, directly and/or indirectly, in regulating the rate of apoptosis in invasive breast cancer. Growth factors and their receptors may therefore be useful as targets of anti-cancer therapy by inducing apoptosis or increasing the sensitivity of cells for chemo- or hormonal therapy induced apoptosis.

We determined the mitotic and apoptotic index through the spectrum of pre-invasive ductal breast lesions to invasive carcinoma in search of disturbances in the proliferation/cell death balance in breast carcinogenesis. Seventy-two pure pre-invasive ductal breast lesions (without invasive carcinoma) and 103 invasive breast carcinomas were used. The numbers of mitotic and apoptotic cells were microscopically counted in hematoxylin and eosin stained sections (MI and AI, respectively), and the ratio of the values of MI and AI was calculated for each individual case (M/A index). A distinction was made between well differentiated and poorly differentiated breast lesions, based on histological type and nuclear grade, to arrive at two plausible progression models for breast carcinogenesis. For the well differentiated breast lesions, the MI was rather equal for hyperplasias and well differentiated DCIS, but increased 6-fold from DCIS to well differentiated invasive carcinoma. The AI remained in the same range, resulting in a 4-fold increase of the M/A index. For the poorly differentiated breast lesions, a significant increase in MI and AI was found from hyperplasia to poorly differentiated DCIS. From DCIS to poorly differentiated invasive carcinoma, the MI increased significantly and the AI decreased 2-fold (n.s.), resulting in a 2.5-fold significant increase of the M/A index. In conclusion, the net increase of the number of cells in the transition from well differentiated pre-invasive to well differentiated invasive carcinoma is accompanied by an increase of cell proliferation rather than decrease in apoptosis, suggesting that in these lesions, proliferation related mechanisms are most important in carcinogenesis and progression. In contrast, in poorly differentiated breast lesions, decreased apoptosis seems to be also important in carcinogenesis and progression. At present, we are gathering patients with invasive breast cancer who had a previous biopsy with a pre-invasive lesion to obtain further more direct evidence for this hypothesis.

This study compares the influence of two different nuclear sampling methods on the prognostic value of assessments of mean and standard deviation of nuclear area (MNA, SDNA) in 191 consecutive invasive breast cancer patients with long term follow up. The first sampling method used was 'at convenience' sampling (ACS); the second, systematic random sampling (SRS). Both sampling methods were tested with a sample size of 50 nuclei (ACS-50 and SRS-50). To determine whether, besides the sampling methods, sample size had impact on prognostic value as well, the SRS method was also tested using a sample size of 100 nuclei (SRS-100). SDNA values were systematically lower for ACS, obviously due to (unconsciously) not including small and large nuclei. Testing prognostic value of a series of cut off points, MNA and SDNA values assessed by the SRS method were prognostically significantly stronger than the values obtained by the ACS method. This was confirmed in Cox regression analysis. For the MNA, the Mantel-Cox p-values from SRS-50 and SRS-100 measurements were not significantly different. However, for the SDNA, SRS-100 yielded significantly lower p-values than SRS-50. In conclusion, compared with the 'at convenience' nuclear sampling method, systematic random sampling of nuclei is not only superior with respect to reproducibility of results, but also provides a better prognostic value in patients with invasive breast cancer.