Does sterilization with fractionated electron beam irradiation prevent ACL tendon allograft from tissue damage?

PURPOSE: Allografts are frequently used for anterior cruciate ligament (ACL) reconstruction. However, due to the inherent risk of infection, a method that achieves complete sterilization of grafts is warranted without impairing their biomechanical properties. Fractionation of electron beam (FEbeam) irradiation has been shown to maintain similar biomechanical properties compared to fresh-frozen allografts (FFA) in vitro. Therefore, aim of this study was to evaluate the biomechanical properties and early remodelling of grafts that were sterilized with fractionated high-dose electron beam irradiation in an in vivo sheep model. METHODS: ACL reconstruction was performed in 18 mature merino mix sheep. Sixteen were reconstructed with allografts sterilized with FEbeam irradiation (8 × 3.4 kGy) and two with FFA. Eight FFA from prior studies with identical surgical reconstruction and biomechanical and histological analyzes served as controls. Half of the animals were sacrificed at 6 and 12 weeks, and biomechanical testing was performed. Anterior-posterior laxity (APL) was assessed with an AP drawer test at 60° flexion, and load to failure testing was carried out. Histological evaluation of mid-substance samples was performed for descriptive analysis, cell count, crimp and vessel density. For statistical analysis a Kruskal-Wallis test was used for overall group comparison followed by a Mann-Whitney U test for pairwise comparison of the histological and biomechanical parameters. RESULTS: Biomechanical testing showed significantly decreased stiffness in FEbeam compared to FFA at both time points (p ≤ 0.004). APL was increased in FEbeam compared to FFA, which was significant at 6 weeks (p = 0.004). Median of failure loads was decreased in FEbeam grafts, with 12 reconstructions already failing during cyclic loading. Vessel density was decreased in FEbeam compared to FFA at both time points, with significant differences at 12 weeks (p = 0.015). Crimp length was significantly shorter in FEbeam compared to FFA at both time points (p ≤ 0.004) and decreased significantly in both groups from 6 to 12 weeks (p ≤ 0.025). CONCLUSION: ACL reconstruction with fractionated Ebeam sterilization significantly alters the biomechanical properties and the early remodelling process of treated grafts in vivo. Therefore, this sterilization method cannot be recommended for clinical application. As substantial changes in the remodelling are inherent in this study, care in the rehabilitation of even low-dose sterilized allografts, used for ACL reconstruction, is recommended.

Targeted sequencing of BRCA1 and BRCA2 across a large unselected breast cancer cohort suggests that one-third of mutations are somatic.

BACKGROUND: A mutation found in the BRCA1 or BRCA2 gene of a breast tumor could be either germline or somatically acquired. The prevalence of somatic BRCA1/2 mutations and the ratio between somatic and germline BRCA1/2 mutations in unselected breast cancer patients are currently unclear. PATIENTS AND METHODS: Paired normal and tumor DNA was analyzed for BRCA1/2 mutations by massively parallel sequencing in an unselected cohort of 273 breast cancer patients from south Sweden. RESULTS: Deleterious germline mutations in BRCA1 (n = 10) or BRCA2 (n = 10) were detected in 20 patients (7%). Deleterious somatic mutations in BRCA1 (n = 4) or BRCA2 (n = 5) were detected in 9 patients (3%). Accordingly, about 1 in 9 breast carcinomas (11%) in our cohort harbor a BRCA1/2 mutation. For each gene, the tumor phenotypes were very similar regardless of the mutation being germline or somatically acquired, whereas the tumor phenotypes differed significantly between wild-type and mutated cases. For age at diagnosis, the patients with somatic BRCA1/2 mutations resembled the wild-type patients (median age at diagnosis, germline BRCA1: 41.5 years; germline BRCA2: 49.5 years; somatic BRCA1/2: 65 years; wild-type BRCA1/2: 62.5 years). CONCLUSIONS: In a population without strong germline founder mutations, the likelihood of a BRCA1/2 mutation found in a breast carcinoma being somatic was ∼1/3 and germline 2/3. This may have implications for treatment and genetic counseling.

Pathological non-response to chemotherapy in a neoadjuvant setting of breast cancer: an inter-institutional study.

To identify markers of non-response to neoadjuvant chemotherapy (NAC) that could be used in the adjuvant setting. Sixteen pathologists of the European Working Group for Breast Screening Pathology reviewed the core biopsies of breast cancers treated with NAC and recorded the clinico-pathological findings (histological type and grade; estrogen, progesterone receptors, and HER2 status; Ki67; mitotic count; tumor-infiltrating lymphocytes; necrosis) and data regarding the pathological response in corresponding surgical resection specimens. Analyses were carried out in a cohort of 490 cases by comparing the groups of patients showing pathological complete response (pCR) and partial response (pPR) with the group of non-responders (pathological non-response: pNR). Among other parameters, the lobular histotype and the absence of inflammation were significantly more common in pNR (p < 0.001). By ROC curve analyses, cut-off values of 9 mitosis/2 mm(2) and 18% of Ki67-positive cells best discriminated the pNR and pCR + pPR categories (p = 0.018 and < 0.001, respectively). By multivariable analysis, only the cut-off value of 9 mitosis discriminated the different response categories (p = 0.036) in the entire cohort. In the Luminal B/HER2- subgroup, a mitotic count <9, although not statistically significant, showed an OR of 2.7 of pNR. A lobular histotype and the absence of inflammation were independent predictors of pNR (p = 0.024 and <0.001, respectively). Classical morphological parameters, such as lobular histotype and inflammation, confirmed their predictive value in response to NAC, particularly in the Luminal B/HER2- subgroup, which is a challenging breast cancer subtype from a therapeutic point of view. Mitotic count could represent an additional marker but has a poor positive predictive value.

The role of AIB1 and PAX2 in primary breast cancer: validation of AIB1 as a negative prognostic factor.

Background The steroid-receptor coactivator amplified in breast cancer one (AIB1) is implicated to be a prognostic factor, although the results are not unanimous. Recently its effect was suggested to be modified by paired box 2 gene product (PAX2). Patients and methods Using immunohistochemistry (IHC) AIB1 and PAX2 were investigated in two cohorts of early breast cancer, including systemically untreated premenopausal lymph-node-negative women and pre- and postmenopausal women receiving tamoxifen. Results AIB1 scores were available for 490 patients and PAX2 scores were available for 463 patients. High AIB1 was a negative prognostic factor for distant disease-free survival (DDFS, P = 0.02) and overall survival (OS, P < 0.001) in systemically untreated women, while no prognostic effect was seen in the tamoxifen-treated cohort, indicating AIB1 to be a predictor of tamoxifen response. In systemically untreated patients, PAX2 was not a prognostic factor, nor did it modify the effect of AIB1. However, in ER-positive patients receiving tamoxifen, PAX2 appeared to be a positive prognostic factor in premenopausal patients, while a negative factor in postmenopausal. The interaction between the menopausal status and PAX2 was significant (P = 0.01). Conclusions In an independent cohort of low-risk premenopausal patients, we validate AIB1 as a negative prognostic factor, indicating AIB1 to be an interesting target for new anti-cancer therapies. The effect of PAX2 warrants further studies.

Comparison of the oestrogen and progesterone receptor status in primary breast carcinomas as evaluated by immunohistochemistry and immunocytochemistry: a consecutive series of 267 patients.

OBJECTIVE: The use of cytological specimens to evaluate tumour biomarkers in metastatic breast cancer lesions has attracted increased interest because of the considerable number of reports that have shown discordance between the primary tumour and metastatic lesion. Oestrogen receptor (ER) and progesterone receptor (PgR) assays are crucial for the management of patients with breast cancer, in both adjuvant and palliative settings. The aim of this study was to compare the ER and PgR immunocytochemical analysis of fine needle aspiration (FNA) samples with the immunohistochemistry (IHC) of surgical specimens and core biopsies from primary breast cancers. METHODS: The FNA specimens were prepared as cell blocks (n = 25) or ThinPreps (n = 258) for the immunocytochemistry (IC) ER and PgR analyses. Sixteen patients were excluded because of lack of follow-up (n = 1), neoadjuvant therapy (n = 3) or cell counts in their fine needle aspirates that were too low (n = 12). The results of IC on 25 cell blocks and 242 ThinPreps were compared with IHC on the corresponding core needle biopsies (n = 16) or excised tumours (n = 251). The ER and PgR status was defined as negative (when less than 10% of the nuclei were stained) or positive (when equal or more than 10% of the nuclei were stained). Kappa statistics were used to evaluate the concordance. RESULTS: The ER concordance was 98% with ThinPrep (κ = 0.93) and 92% with cell block (κ = 0.82). The corresponding values for PgR were 96% (κ = 0.91) and 96% (κ = 0.92). CONCLUSIONS: Our results confirm that, in cases in which biopsies or surgical specimens are not available, IC (with either cell block or ThinPrep techniques) is a reliable method for the determination of the ER and PgR status performed under strict conditions using primary breast carcinomas, and is therefore potentially useful in metastatic settings.

AIB1 is a predictive factor for tamoxifen response in premenopausal women.

BACKGROUND: Clinical trials implicate the estrogen receptor (ER) coactivator amplified in breast cancer 1 (AIB1) to be a prognostic and a treatment-predictive factor, although results are not unanimous. We have further investigated this using a controlled randomised trial of tamoxifen versus control. MATERIALS AND METHODS: A total of 564 premenopausal women were entered into a randomised study independent of ER status. Using a tissue microarray, AIB1 and ER were analysed by immunohistochemistry. RESULTS: AIB1 scores were obtained from 349 women. High AIB1 correlated to factors of worse prognosis (human epidermal growth factor receptor 2, Nottingham histological grade 3, and lymph node metastases) and to ER negativity. In the control arm, high AIB1 was a negative prognostic factor for recurrence-free survival (RFS) (P = 0.02). However, ER-positive patients with high AIB1 responded significantly to tamoxifen treatment (P = 0.002), increasing RFS to the same level as for systemically untreated patients with low AIB1. Although ER-positive patients with low AIB1 had a better RFS from the beginning, this was not further improved by tamoxifen (P = 0.8). CONCLUSIONS: In the control group, high AIB1 was a negative prognostic factor. However, ER-positive patients with high AIB1 responded significantly to tamoxifen. This implicates high AIB1 to be an independent predictive factor of improved response to tamoxifen and not, as has previously been discussed, a factor predicting tamoxifen resistance.

Breast cancer patients with micrometastases only: is a basis provided for tailored treatment?

Women with breast cancer and micrometastases only constitute a treatment dilemma. If only a micrometastasis is found in a sentinel lymph node, an axillary lymph node dissection may be considered to be overtreatment and perhaps could be avoided. However, studies have shown decreased survival in patients with micrometastases only. This paper focuses on the pathological work-up behind the classification of breast cancer patients having micrometastases only and on the most recent literature concerning prognosis for breast cancer patients with micrometastases. The goal was to determine if studies to date have been able to define a population of breast cancer patients with micrometastases where the size of the metastasis could indicate whether an axillary lymph node dissection should be undertaken. Tailored surgical treatment of breast cancer patients with micrometastases only is not possible at the present time, due to lack of standardization in the pathological work-up of lymph nodes, which implies that this group of breast cancer patients cannot be delimited with sufficient precision. Tailored systemic therapy is also impossible due to lack of a precisely defined target for a feasible therapy.

Axillary lymph node micrometastases in invasive breast cancer: national figures on incidence and overall survival.

The purpose of this study was to estimate the incidence and prognostic value of axillary lymph node micrometastases (Nmic) of 2 mm or less in breast carcinomas. Results are based on data from the Danish Breast Cancer Cooperative Group (DBCG). The study was carried out as a nationwide, population-based trial with a study series consisting of 6,959 women under 75 years of age registered in the national DBCG data base from 1 January 1990 to 31 October 1994. All patients had contracted operable primary breast carcinoma, stage I-III, classified according to the TNM system as T1-T3, N0-N1, M0. Women with four or more metastatic axillary lymph nodes were excluded. All patients were treated systematically according to approved national guidelines and treatment protocols. Metastases were recognized microscopically on haematoxylin and eosin-stained sections. In case of doubt immunohistochemical staining for cytokeratin was performed. There was no serial sectioning. Micrometastases were tumour deposits of 2 mm or smaller, and accordingly included deposits of 0.2 mm and smaller. With a median observation time of 10 years and 2 months, women with Nmic (N=427) experienced a significantly worse overall survival (OS) compared with node-negative (Nneg) women (N=4,767) (relative risk (RR)=1.20, 95% CI: 1.01-1.43), irrespective of menopausal status. Women with macrometastases (Nmac) (N=1,765) had significantly worse final outcome than women with Nmic (RR=1.54, 95% CI: 1.29-1.85), irrespective of menopausal status. Multivariate analysis adjusted for patient-, histopathologic-, and loco-regional therapeutic variables showed that cases with Nmic had a significantly higher risk of death relative to Nneg cases (adjusted RR=1.49, 95% CI: 1.18-1.90). Interaction analysis showed that the number of nodes examined had a significant impact on adjusted relative risk of death according to axillary status. Furthermore, the number of nodes involved significantly influenced adjusted risk of death in the Nmic compared to the Nmac series. In conclusion, the results of the present study revealed worse final outcome in women with Nmic compared with Nneg, where all Nmic cases received adjuvant systemic treatment. Interaction analysis showed that the number of retrieved axillary nodes and the number of affected nodes had a different influence on survival related to axillary status. The different risk pattern in Nmic vs Nmac patients indicates that Nmic cases do not show the traditional risk pattern as revealed by the Nmac cases, in which increasing number of positive nodes is associated with an orderly increasing adjusted RR.

Consistency in recognizing microinvasion in breast carcinomas is improved by immunohistochemistry for myoepithelial markers.

Microinvasion is the smallest morphologically identifiable stage of invasion. Its presence and distinction from in situ carcinoma may have therapeutic implications, and clinical staging also requires the recognition of this phenomenon. Microinvasion is established on the basis of several morphological criteria, which may be difficult and not perfectly reproducible among pathologists. The aim of this study was to assess the consistency of diagnosing microinvasion in the breast on traditional haematoxylin and eosin (HE) stained slides and to evaluate whether immunohistochemistry (IHC) for myoepithelial markers could improve this. Digital images were generated from representative areas of 50 cases stained with HE and IHC for myoepithelial markers. Cases were specifically selected from the spectrum of in situ to microinvasive cancers. Twenty-eight dedicated breast pathologists assessed these cases at different magnifications through a web-based platform in two rounds: first HE only and after a washout period by both HE and IHC. Consistency in the recognition of microinvasion significantly improved with the use of IHC. Concordance rates increased from 0.85 to 0.96, kappa from 0.5 to 0.85, the number of cases with 100% agreement rose from 9/50 to 25/50 with IHC and the certainty of diagnosis also increased. The use of IHC markedly improves the consistency of identifying microinvasion. This corroborates previous recommendations to use IHC for myoepithelial markers to clarify cases where uncertainty exists about the presence of microinvasion. Microinvasive carcinoma is a rare entity, and seeking a second opinion may avoid overdiagnosis.

Intraoperative frozen section examination of axillary sentinel lymph nodes in breast cancer.

The study presents the results from intraoperative frozen section assessment of axillary sentinel lymph nodes (SLNs) in breast cancer. Routine histological frozen sections from one level were used, two sections stained with haematoxylin and eosin. Immunohistochemistry for cytokeratins was applied to the permanent SLN paraffin sections only. Axillary dissection was performed on all SLN-positive cases regardless of the size of the metastatic deposits. With a detection rate of 83%, 272 patients entered the study over a period of 46 months. A total of 61 cases were SLN positive by frozen section analysis. The paraffin sections gave an additional 23 SLN-positive cases. The false-negative rate for frozen sections was then 27% (23/84). Micrometastases were found in 28 of 84 cases, and macrometastases in 56. The false-negative rate of frozen sections for micrometastases was 71% (20/28), and for macrometastases 5% (3/56). A total of 73% (61/84) of the patients underwent axillary surgery as a one-step procedure.

The prognostic value of angiogenesis by Chalkley counting in a confirmatory study design on 836 breast cancer patients.

This study addresses the prognostic value of estimating angiogenesis by Chalkley counting in breast cancer. A population-based group consisting of 836 patients with operated primary, unilateral invasive breast carcinomas was included from a predefined region and period of time. The median follow-up time was 11 years and 4 months. The microvessels were immunohistochemically stained by antibodies against CD34. The Chalkley count was obtained by a 25-point grid within three, subjectively selected, vascular tumor areas of highest microvessel density. The Chalkley count was analyzed in three categories using predefined Chalkley cutoff points at five and seven. There were significant correlations between high Chalkley counts and axillary lymph node metastasis, large tumor size, high histological malignancy grade, and histological type. A high Chalkley count showed lower probabilities of recurrence-free survival (P < 0.0001) and overall survival (P < 0.0001). In the Cox multivariate analysis, the hazard ratio (and 95% confidence interval) showed that the increased risk to die were: 1.55 (1.19-2.03) with Chalkley counts between 5 and 7; 2.26 (1.72-2.98) with counts > or =7 compared with counts < or =5; and 1.46 (1.14-1.87) with counts > or =7 compared with counts between 5-7. The study confirmed that estimation of angiogenesis by Chalkley counting had independent prognostic value in breast cancer patients. The Chalkley count could be useful to stratify node-negative patients for adjuvant treatment.

Immunohistochemical assessment of oestrogen and progesterone receptors: correlations with the DCC method and clinical outcome in primary breast cancer patients.

Two different methods to determine steroid receptors were analysed with respect to their ability to estimate prognosis in primary breast cancer patients. The immunohistochemical assay (IHA) was compared with the dextran-coated charcoal (DCC) method of receptor determination. A random sample of 281 patients with invasive ductal carcinoma was drawn from 841 consecutive patients with primary breast carcinoma treated at Odense University Hospital between 1 January 1980 and 31 December 1990. Receptor determination by the DCC method had been carried out previously in 164 patients for the oestrogen receptor and in 132 patients for the progesterone receptor. The former group was reassessed by IHA with the antibody ER1D5, and the latter with the antibody PgR-ICA. The median follow-up time was 8.3 years (range 2.9-12.9 years). A cutoff of zero was used for the DCC method. Immunohistochemical results were quantified by counting in systematically random sampled fields of vision and values above zero were considered to be positive. Overall agreement of positive and negative cases was 86% for the oestrogen receptor and 83% for the progesterone receptor. Although the study included a limited number of patients, receptor positive cases fared better than negative cases in all situations. Investigation of the prognostic power revealed that classification based on IHA allowed better discrimination of patients than classification based on the DCC method. The reason for this difference might be because distinction between benign and malignant tissue is possible using the IHAmethod. Thus, IHAresults appear to be more clinically relevant.

Needle biopsy of breast cancer. Appearance of tumour cells along the needle track.

Appearance of tumour cells along the needle track in patients with breast cancer diagnosed by SURECUT needle biopsy is found in two out of 47 consecutive cases. It is not known whether these cells are early implantation metastases or are harmless. To avoid any unnecessary risk to the patients, we recommend that needle biopsy is so performed, that it is possible to remove the track during the definite surgical procedure, and that penetration into the muscles of the thoracic wall during the biopsy procedure is avoided.

A tumour in the breast: vaccination granuloma as a differential diagnosis.

AIMS: Breast cancer is the most frequent type of cancer in women; special attention is therefore paid to tumours in the breast region. Vaccination granuloma is a differential diagnosis of tumours in the upper part of the breast. METHODS: All granulomatous lesions in the breast region since 1970 were retrieved from the files of the Departments of Pathology at Odense University Hospital and Svendborg Hospital. RESULTS: Fourteen cases with a histology compatible with vaccination granuloma were found. Eight patients had a known history of tetanus vaccination. The tumours were ovoid, or even rod-shaped, and measured between 8x3 mm and 12x10 mm. Histologically, there were necrotic foci surrounded by histiocytes, lymphocytes, and plasma cells. Aluminium was detected by staining with solochrome azurine. CONCLUSIONS: The possibility of a vaccination granuloma should be kept in mind in patients with a palpable tumour in the upper part of the breast, as well as in mammography screening conditions and in follow-up patients after previous treatment for breast cancer.

The importance of surgery and accurate axillary staging for survival in breast cancer.

AIMS: The purpose of this study was to investigate, within the context of the Danish Breast Cancer Cooperative Group (DBCG) programmes, whether a dedicated surgical approach had a significant bearing on the outcome of breast cancer treatment. METHODS: From 1 January 1980 to 31 December 1990, patients below 70 years of age with operable breast cancer from Odense University Hospital (n=743) were compared with those from the rest of Denmark (denoted rest-DK) (n=15,419). All patients were treated according to nationwide DBCG guidelines and reported to the DBCG Data Centre. The potential median observation time was 11.2 years (range 6.0-16.9). Patients underwent mastectomy or breast conserving therapy, and high risk lymph-node positive patients had adjuvant systemic therapy with or without radiotherapy. RESULTS: Comparing total patients series, overall survival (OS) was significantly superior in patients from Odense compared with rest-DK (P=0.02), with 10-year OSs of 62% (95% CI: 58-65%) and 56% (55-57%), respectively. In subgroups, the OS of low-risk node negative patients (protocol A) in Odense compared with rest-DK was significantly better (P=0.02); 10-year OS was 78% (73-84%) versus 72% (70-73%). Among the high-risk pre-menopausal patients (protocol B), the OS was significantly better in Odense (P=0.009); 10-year OS was 67% (60-75%) versus 53% (51-55%) in rest-DK. Post-menopausal high-risk patients (protocol C) did not differ significantly in OS between Odense and rest-DK (P=0.61). Locoregional control in the Odense series was superior compared with rest-DK. More lymph nodes were recovered and examined from the axilla in the Odense series than in rest-DK, a median of 10 vs. 6 nodes. In the Odense series, a significantly higher proportion of pre-menopausal patients had positive lymph nodes, predominantly one to three positive nodes, and subsequently a lower proportion of pre-menopausal patients had negative lymph nodes compared with rest-DK (P=0.02), indicating a more accurate staging in Odense vs. rest-DK. The survival benefit among the patients from Odense cannot be explained by stage migration alone, but seems to represent a true survival advantage. Overall mortality was significantly lower in the Odense series compared with rest-DK. Whether or not this difference could be explained by lower background mortality in the Odense series or was caused by superior treatment is discussed. CONCLUSIONS: The extent of surgery seems important for locoregional tumour control and accurate axillary lymph-node staging. In combination, these might lead to superior recurrence-free and overall survival, although differences in background mortality cannot be ignored. Surgery, therefore, might represent a risk factor by itself.

Completion axillary dissection can safely be omitted in screen detected breast cancer patients with micrometastases. A decade's experience from a single institution.

BACKGROUND: The need for completion axillary lymph node dissection (ALND) in breast cancer patients with micrometastases in the sentinel nodes (SNs) is controversial. The aim of this retrospective observational study is to determine if the method of detection of early breast cancer is predictive for additional positive nodes in patients with micrometastases in the SNs. METHODS: Between 2001 and 2011 a total of 1993 women with primary unilateral breast cancer had surgery at Skåne University Hospital, Lund. Of 1993 patients, 1458 had an SN biopsy and nearly all patients with micro- and macrometastases had ALND. RESULTS: Micrometastases defined as >0.2 mm/>200 cells and ≤2.0 mm were found in 62 of 757 screen-detected patients and in 81 of 701 patients with symptomatic breast cancer. Only 3 of the screen-detected patients with micrometastases, all with tumour size >15 mm (range 18-39 mm), had metastases in the completion ALND whereas this was found in 18 of the symptomatic patients with micrometastases (p = 0.01), (tumour size, range 10-30 mm). Logistic regression analysis adjusted for method of detection, tumour size and histological grade showed 5 times higher odds for further metastases in ALND in patients with symptomatic presentation vs. screen-detected breast cancer. CONCLUSION: Despite the small number of patients with micrometastases in this large cohort of breast cancer patients, these results support the contention that completion ALND can safely be omitted in screen-detected breast cancer patients with micrometastases in the SNs.

[Cruciate ligament injuries under gender aspects]. / Gendermedizinische Aspekte einer Kreuzbandruptur.

An injury of cruciate ligament is one the most common knee injuries. This accident happens mostly without external impact and towards the end of training and competition sessions. Women, especially athletes playing team sports ball games such as soccer or disciplines such as tennis, are affected 2 to 8 times more often than men. Anatomic, biomechanical and endocrinological differences are currently discussed as potential risk factors. In terms of prevention, biomechanical impact is of greatest importance given its influenceability through various training opportunities. Training programs including endurance aspects, strengthening knee musculature, balance as well as plyometric trainings were most effective. Further studies should focus more on concomitants of course of injuries.

Vascular grading of angiogenesis: prognostic significance in breast cancer.

The study aimed to evaluate the prognostic value of angiogenesis by vascular grading of primary breast tumours, and to evaluate the prognostic impact of adding the vascular grade to the Nottingham Prognostic Index (NPI). The investigation included 836 patients. The median follow-up time was 11 years and 4 months. The microvessels were immunohistochemically stained by antibodies against CD34. Angiogenesis was graded semiquantitatively by subjective scoring into three groups according to the expected number of microvessels in the most vascular tumour area. The vascular grading between observers was moderately reproduced (kappa = 0.59). Vascular grade was significantly associated with axillary node involvement, tumour size, malignancy grade, oestrogen receptor status and histological type. In univariate analyses vascular grade significantly predicted recurrence free survival and overall survival for all patients (P < 0.0001), node-negative patients (P < 0.0001) and node-positive patients (P < 0.0001). Cox multivariate regression analysis showed that vascular grading contributed with independent prognostic value in all patients (P < 0.0001). A prognostic index including the vascular grade had clinical impact for 24% of the patients, who had a shift in prognostic group, as compared to NPI, and implied a better prognostic dissemination. We concluded that the angiogenesis determined by vascular grading has independent prognostic value of clinical relevance for patients with breast cancer.

Angiogenesis in breast cancer: a comparative study of the observer variability of methods for determining microvessel density.

The purpose of this study was to evaluate the reliability of different methods for estimating neovascularization in breast cancer and to compare them in terms of observer variability. The microvessel endothelium was stained immunohistochemically by antibodies against CD34. The investigated methods included Chalkley counting, estimation of intratumoral microvessel density (MVD) by one hot-spot, MVD by the mean value of three hot-spots, and the highest value of MVD in three hot-spots. In addition, we applied stereology in the quantification of angiogenesis in the whole tumor section by random and systematically distributed sampling fields. Each of forty tumors was measured with all methods, twice by the same observer and once by another observer. Observer variation was analyzed by orthogonal regression, estimating the slope and intercepts with 95% confidence intervals (CI), and by analysis of agreement using difference plots. Intraobservationally, the methods had variations of the same magnitude (coefficient of variation [CV] approximately 20%). Interobservationally, the stereologic estimate of vessel profiles, Q(A), from the whole tumor section and the Chalkley counting method had the lowest variation (CV approximately 21%), with a small contribution by observers alone (CV 8% to 9%). Interobservationally, the MVD methods had considerable variation with a large contribution by observers alone (CV approximately 30%), which was lowest using the mean of three hot-spots. Correlation slope and 95% CI of Chalkley were 1.18 (0.95, 1.48), CV 20%; slope of MVD (mean) was 1.14 (0.91, 1.43), CV 31%; and slope of MVD (max) was 1.15 (0.92, 1.45), CV 36%. The slope of MVD on one hot-spot was 1.33 (1.08, 1.63); CV 38%. Additional measurements performed using a conference microscope, eliminating subjectivity in hot-spot selection and field sampling, optimized the reproducibility: slope was 1.02 (0.99, 1.04); CV of differences, 3.5%. On the other hand, reproducibility was not necessarily optimized by choosing the same hot-spot area, because variation in selecting a microscopic field could yield different counting numbers. The stereologic estimation of QA based on the whole tumor section had a high reproducibility, with low variation due to observers. The Chalkley and MVD methods had moderate reproducibility, and the Chalkley method had low variation due to observers alone. For all methods, the biologic variation among patients was the major contributor to the total variation. The Chalkley and MVD methods have been published to provide significant prognostic estimates in breast cancer, but the Chalkley method has less observer variation and may be superior from a methodologic point of view.