Androgen receptor expression inversely correlates with immune cell infiltration in human epidermal growth factor receptor 2-positive breast cancer.

INTRODUCTION: Although targeting human epidermal growth factor receptor 2 (HER2) is a meaningful treatment in HER2-positive breast cancer, ultimately resistance develops. Androgen receptor (AR) expression and immune cell infiltration are thought to be involved in trastuzumab response and may, therefore, be of interest as additional targets for therapy in HER2-positive breast cancer. AIM: To improve insights into the presence among AR expression, immune cell infiltration and HER2, we analysed HER2-positive breast tumours. METHODS: Primary tumours of 221 patients treated with trastuzumab for metastatic disease were selected. HER2 status was centrally confirmed. AR, T-cells (CD3 and CD8), programmed cell death protein 1 (PD-1) and PD-1 ligand 1 immunohistochemical staining and M2 tumour-associated macrophages (TAMs; CD68 and CD163) immunofluorescence were performed. Tumour-infiltrating lymphocytes were evaluated by haematoxylin and eosin staining. RESULTS: Sufficient tumour material was available for 150 patients. Oestrogen receptor was expressed in 51.3% of the tumours and AR in 81.3% of the tumours. AR expression was inversely correlated with M2 TAM (Pearson's r = -0.361, P < 0.001), CD3+ (r = -0.199, P < 0.030) and CD8+ (r = -0.212, P < 0.021) T-cell infiltration. Clustering analysis showed high immune cell infiltration in AR low-expressing tumours, and low immune cell infiltration in AR-high expressing tumours. CONCLUSION: AR expression inversely correlates with immune cell infiltration in HER2-positive breast cancer.

Bevacizumab combined with docetaxel, oxaliplatin, and capecitabine, followed by maintenance with capecitabine and bevacizumab, as first-line treatment of patients with advanced HER2-negative gastric cancer: A multicenter phase 2 study.

BACKGROUND: The current study was a multicenter, single-arm, phase 2 study performed to investigate the feasibility and efficacy of bevacizumab combined with docetaxel, oxaliplatin, and capecitabine (B-DOC) in patients with advanced human epidermal growth factor receptor 2 (HER2)-negative, previously untreated, gastric or gastroesophageal adenocarcinoma. METHODS: Tumor HER2 status was determined centrally. Patients received 6 cycles of bevacizumab at a dose of 7.5 mg/kg, docetaxel at a dose of 50 mg/m(2) , and oxaliplatin at a dose of 100 mg/m(2) (all on day 1) combined with capecitabine at a dose of 850 mg/m(2) twice daily (days 1-14) every 3 weeks followed by maintenance with capecitabine and bevacizumab in patients with disease control. The primary objective was to demonstrate a progression-free survival (PFS) of >6.5 months, according to the 95% confidence interval (95% CI). Secondary endpoints included safety, objective response rate, overall survival (OS), analyses of circulating tumor cells (CTCs), and pharmacogenetic analyses. RESULTS: Sixty eligible patients were enrolled. The median PFS was 8.3 months (95% CI, 7.2-10.9 months). The objective response rate was 70% (95% CI, 55%-83%) and the disease control rate was 96% (95% CI, 85%-99%). The median OS was 12.0 months (95% CI, 10.2-16.1 months). According to CTC-AE v4.0, the most common treatment-related grade ≥3 adverse events were neutropenia (20%), leukocytopenia (18%), diarrhea (15%), and nausea/vomiting (15%). The presence of CTCs at baseline was strongly predictive of PFS (hazard ratio [HR], 3.8; P =.007) and OS (HR, 3.4; P =.014). The methylenetetrahydrofolate reductase (MTHFR) 677C>T genotype was strongly associated with PFS (HR, 4.7 for TT vs CC or CT; P =.0007) and OS (HR, 5.9; P =.0001). CONCLUSIONS: The B-DOC regimen plus maintenance was feasible and active. CTCs were found to be prognostic in patients treated with B-DOC. Docetaxel-based triplet chemotherapy as a backbone for targeted therapies is feasible and deserves further study. Cancer 2016;122:1434-1443. © 2016 American Cancer Society.

Use of trastuzumab for HER2-positive metastatic breast cancer in daily practice: a population-based study focusing on the elderly.

The addition of trastuzumab to chemotherapy in human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer (MBC) prolongs overall survival (OS) in clinical trials. However, treatment patterns and survival in daily practice are unknown. This study aims to compare trastuzumab use and outcome in HER2-positive MBC patients in a population-based cohort with clinical trial cohorts, with a special focus on elderly patients. MBC patients treated with trastuzumab-based chemotherapy in north-east Netherlands between 2005 and 2009 were identified from 23 hospital pharmacies and the Netherlands Cancer Registry. Baseline, treatment, and survival characteristics (Kaplan-Meier analysis) were compared with those found in clinical trials and differences in patients aged less than 65 versus 65 years or more were studied. Of 225 HER2-positive MBC patients (median: 54.8 years), 130 were treated with first-line trastuzumab. In first-line treatment, the median treatment duration was 9 months and the median OS was 30.7 months, which is comparable with the OS of 31.2 months found in a clinical trial with comparable baseline characteristics. In 25 patients aged 65 years or more compared with those aged less than 65 years treated with first-line trastuzumab, patients with a history of early breast cancer had less often been treated with adjuvant chemotherapy (36 vs. 71%; P=0.001). Other baseline characteristics and OS were similar. Patient, treatment, and survival characteristics in a HER2-positive MBC population-based cohort share considerable similarities to those found in clinical trials. The influence of age on trastuzumab treatment was not detected.

Trastuzumab and bevacizumab combined with docetaxel, oxaliplatin and capecitabine as first-line treatment of advanced HER2-positive gastric cancer: a multicenter phase II study.

OBJECTIVE: To investigate the efficacy of bevacizumab and trastuzumab combined with docetaxel, oxaliplatin, and capecitabine (B-DOCT) as first-line treatment of advanced human epidermal growth factor receptor 2 (HER2)-positive gastric cancer (GC). METHODS: In this multicentre, single-arm, phase II study, tumor HER2 status was determined centrally prior to treatment. Patients with advanced HER2-positive adenocarcinoma of the stomach or gastroesophageal junction (immunohistochemistry 3+ or immunohistochemistry 2+/silver in-situ hybridization positive) were treated with six cycles of bevacizumab 7.5 mg/kg (day 1), docetaxel 50 mg/m(2) (day 1), oxaliplatin 100 mg/m(2) (day 1), capecitabine 850 mg/m(2) b.i.d. (days 1-14), and trastuzumab 6 mg/kg (day 1) every three weeks, followed by maintenance with bevacizumab, capecitabine, and trastuzumab until disease progression. The primary objective was to demonstrate an improvement of progression-free survival (PFS) to >7.6 months (observed in the ToGA trial) determined according to the lower limit of the 95 % confidence interval (CI). Secondary endpoints were safety, objective response rate (ORR), and overall survival (OS). RESULTS: Twenty-five patients with HER2-positive tumors were treated with B-DOCT between March 2011 and September 2014. At a median follow-up of 17 months, median PFS was 10.8 months (95%CI: 9.0-NA), OS was 17.9 months (95%CI: 12.4-NA). One-year PFS and OS were 52 % and 79 %, respectively. The ORR was 74 % (95%CI: 52-90 %). Two patients became resectable during treatment with B-DOCT and achieved a pathological complete response. The most common treatment-related grade ≥ 3 adverse events were: neutropenia (16 %), diarrhoea (16 %), and hypertension (16 %). CONCLUSIONS: B-DOCT is a safe and active combination in HER2-positive GC, supporting further investigations of DOC with HER2/vascular endothelial growth factor (VEGF) inhibition in HER2-positive GC.

Ultrasound-Guided Tissue Core Biopsies in Supraclavicular Lymph Nodes in Patients with Suspected Thoracic Malignancies.

BACKGROUND: Supraclavicular (SC) lymph node metastases are important in the analysis of thoracic malignancies for staging as well as for diagnosing purposes. Ultrasound (US) guidance visualises lesions very precisely, enabling tissue biopsies in real-time mode. OBJECTIVES: To report on the diagnostic qualities of SC tissue core biopsies (TCB). METHODS: A retrospective database analysis was performed in TCB performed under US guidance in SC nodes in patients suspected of having a thoracic malignancy. Clinical characteristics and results of diagnostic evaluations were analysed. RESULTS: Between October 2008 and October 2014, 67 sessions for TCB in 65 patients were performed. The diagnostic accuracy of TCB for all diagnoses is 90%, with a sensitivity of 89%. For malignant diagnoses, the sensitivity of US-guided TCB is 93%. In 20 patients, molecular analysis for EGFR and KRAS was performed, with a diagnostic success rate of 95%. One patient suffered a moderate haemorrhage after TCB. CONCLUSION: TCB of SC nodes in the analysis of suspected thoracic malignancy is safe and has a high diagnostic accuracy in determining tumour subtype as well as molecular analysis.

Fully automated fluorescent in situ hybridization (FISH) staining and digital analysis of HER2 in breast cancer: a validation study.

HER2 assessment is routinely used to select patients with invasive breast cancer that might benefit from HER2-targeted therapy. The aim of this study was to validate a fully automated in situ hybridization (ISH) procedure that combines the automated Leica HER2 fluorescent ISH system for Bond with supervised automated analysis with the Visia imaging D-Sight digital imaging platform. HER2 assessment was performed on 328 formalin-fixed/paraffin-embedded invasive breast cancer tumors on tissue microarrays (TMA) and 100 (50 selected IHC 2+ and 50 random IHC scores) full-sized slides of resections/biopsies obtained for diagnostic purposes previously. For digital analysis slides were pre-screened at 20x and 100x magnification for all fluorescent signals and supervised-automated scoring was performed on at least two pictures (in total at least 20 nuclei were counted) with the D-Sight HER2 FISH analysis module by two observers independently. Results were compared to data obtained previously with the manual Abbott FISH test. The overall agreement with Abbott FISH data among TMA samples and 50 selected IHC 2+ cases was 98.8% (κ = 0.94) and 93.8% (κ = 0.88), respectively. The results of 50 additionally tested unselected IHC cases were concordant with previously obtained IHC and/or FISH data. The combination of the Leica FISH system with the D-Sight digital imaging platform is a feasible method for HER2 assessment in routine clinical practice for patients with invasive breast cancer.

Analysis of "dry" mesothelioma with ultrasound guided biopsies.

BACKGROUND: Image-guided sampling of the thickened pleura is a sensitive approach in patients with malignant pleural mesothelioma with pleural effusion. Malignant pleural mesothelioma presenting without effusion however is more of a diagnostic challenge. In this study we report the diagnostic yield and complications of ultrasound-guided cutting needle biopsies in this particular category of patients. METHODS: A retrospective database analysis from September 2007 until January 2012 was performed in 56 patients with malignant pleural mesothelioma. Clinical characteristics and results of diagnostic evaluations were analysed. RESULTS: Of the 56 patients with malignant pleural mesothelioma, 20 patients presented without pleural effusion of with locular effusion. Ultrasound-guided cutting needle biopsy was performed in 14/20 patients with a diagnostic accuracy of 80%. Only 1 patient had mild haemoptysis immediately following biopsies. CONCLUSION: Diagnosing patients with pleural thickenings suspect for malignant mesothelioma without pleural effusion or with loculated pleural effusion is effective and safe with ultrasound-guided cutting needle biopsies.

Determining sensitivity and specificity of HER2 testing in breast cancer using a tissue micro-array approach.

INTRODUCTION: Overexpression of the human epidermal growth factor receptor 2 (HER2) as a result of HER2 gene amplification is associated with a relatively poor prognosis in breast cancer and is predictive of HER2-targeting therapy response. False-positive rates of up to 20% for HER2 testing have been described. HER2-testing laboratories are therefore encouraged to participate in external quality control schemes in order to improve HER2-testing standardization. METHODS: This study investigated the feasibility of retesting large numbers of invasive breast cancers for HER2 status on tissue micro-array (TMA) as part of a quality control scheme. For this assessment different HER2 testing methods were used including HER2 detecting antibodies SP3, 4B5, Herceptest and mono color silver in situ hybridization (SISH) and dual color SISH. Final HER2 status for each tumor on the TMA was compared to the local testing result for the same tumor. Discordances between these two results were investigated further by staining whole tumor sections. RESULTS: For this study, 1,210 invasive breast carcinomas of patients treated in six hospitals between 2006 and 2008 were evaluated. Results from the three immunohistochemistry (IHC) and two in situ hybridization (ISH) assays performed on the TMAs were compared. The final HER2 status on TMA was determined with SP3, 4B5 and mono color SISH. Concordance between local HER2 test results and TMA retesting was 98.0%. Discordant results between local and TMA retesting were found in 20 tumors (2.0%). False positive HER2 IHC results were identified in 13 (1.3%) tumors; false negative IHC results in seven (0.7%) tumors. CONCLUSIONS: Retesting large volumes of HER2 classified breast carcinomas was found to be feasible and can be reliably performed by staining TMAs with SP3, 4B5 and mono color SISH in combination with full-sized slides for discordant cases. The frequency of false-positive results was lower than previously reported in the literature. This method is now offered to other HER2-testing laboratories.

A diagnostic program for patients suspected of having lung cancer.

BACKGROUND: A standardized diagnostic program, initiated to reduce the length of the diagnostic track and to improve application of diagnostic tools for patients referred with suspicious abnormalities on standard chest radiographs, was evaluated. METHODS: The findings on integrated positron emission tomography/computed tomography (PET-CT) determined the choice of invasive investigations to be performed the same day. Diagnostic results, time courses, and number and sorts of applied invasive investigations were assessed. RESULTS: In 297 eligible patients, malignant disease was diagnosed in 72% and benign disease was diagnosed in 26% of patients. One percent of the patients had no abnormalities at all. For 85% of patients with malignancy, investigations were completed in 1 day, resulting in a diagnosis and definitive clinical disease stage. The median time from start of the analysis to informing the patient about diagnosis and tumor stage was 7 days. One invasive investigation was performed in 53% of patients in the study group, and at least 2 investigations were performed in 33% of patients. Bronchoscopies formed a part of the diagnostic process in 59% of patients. Surgical diagnostic procedures were performed in 8% of patients. CONCLUSION: The diagnostic program resulted in a short time to diagnosis, with finalization of invasive investigations in 1 day in the majority of patients. The imaging-based choice of invasive investigations precluded bronchoscopies in a substantial portion of the patients.

Percutaneous ultrasound-guided biopsies in the evaluation of thoracic tumours after PET-CT: a prospective diagnostic study.

BACKGROUND: Lesions detected by positron emission tomography-computed tomography (PET-CT) during the analysis of thoracic tumours are often impalpable at physical examination, and subsequent ultrasound (US) may aid in finding these lesions for pathologic evaluation. OBJECTIVES: The success rate of percutaneous US-guided biopsies of palpable and non-palpable lesions and the impact on tumour stage were studied prospectively. METHODS: Lesions, significant for diagnosis and disease stage, with metabolic activity on PET-CT and presumed appropriate for percutaneous approach under US guidance were selected for cytologic aspiration or tissue core biopsies. RESULTS: In 127 patients, 134 lesions (subdivided into 24 local thoracic, 74 supraclavicular and 36 distant metastatic lesions) were biopsied percutaneously under US guidance. Malignancy, benign disease and inadequate biopsies were found in 80% (106/134), 14% (19/134) and 7% (9/134), respectively. In 55% (56/102) of patients, biopsies confirmed the disease stage. Fifty-one percent (18/35) of distant lesions and 54% (43/68) of supraclavicular lesions were impalpable on physical examination. CONCLUSIONS: US-guided biopsies in patients with suspected thoracic malignancy on PET-CT provide an excellent tool for obtaining a pathological diagnosis, leading to a definitive disease stage in over half of the patients.

The prognostic role of apoptosis mediators in rectal adenocarcinoma.

BACKGROUND/AIMS: Genes included in apoptosis may be involved in tumor biology and identify specific groups of patients with individual therapy. The aim of this study was to evaluate the prognostic value of some apoptosis markers in conjunction with pathological factors in operable rectal cancer patients. METHODOLOGY: Tumor samples from 87 patients with rectal adenocarcinoma were analyzed retrospectively. The immunohistochemistry from 'tissue array' for the expression of p53, p21, Bcl-2 and cycline D1, combined with pathological features were correlated with the survival data. The mean postoperative follow-up was 48 months. RESULTS: Five-year cancer-specific survival (CSS) was 44.9%. Independent prognostic factors for CSS were the p53 and p21 proteins, whereas for cancer-free survival (CFS) p21 was a positive independent factor. When pathological factors were also introduced in the analysis, the positive prognostic role on CSS was obtained for p21 and lymphatic invasion; CFS was positively influenced by the presence of p21 protein and lymphatic invasion. For patients with pN0, p21 protein was an independent predictive marker for CSS and CFS. CONCLUSIONS: Molecular markers of apoptosis, such as p53 and p21, had significant prognostic role in rectal cancer patients, together with lymphatic invasion. The presence of p21 protein in pN0 patient outcome may influence management, but needs further evaluation.

Mediastinal incidentalomas.

INTRODUCTION: Incidental mediastinal lymphadenopathy challenges pulmonologists to decide on eventual further diagnostic steps. The aim of this study was to characterize unexpected mediastinal findings by imaging and pathologic analysis. METHODS: Entry criterion for this prospective explorative study was mediastinal lymphadenopathy as an incidental finding on computed tomography (CT) scans made for indications other than the analysis and staging of neoplasms. Lymph node dimensions were measured on CT scan. Subsequent diagnostic investigations were positron emission tomography, endoscopic ultrasound- or endobronchial ultrasound-guided punctures, and clinical follow-up. RESULTS: Eighty-three patients from eight hospitals met the entry criteria. The median number of Naruke stations with enlarged nodes was 7 (range 3-9). The median size of all nodes measured varied between 6 and 14 mm. The median number of lymph node stations with nodes of at least 10 mm was 3 (range 0-8). Hilar node enlargement was detected in 77% of patients. No definitive diagnosis was obtained in 7 of 83 (8%) patients. Lymphocytes were found in 55 of 83 (66%) and sarcoidosis in 18 of 83 (22%) of aspirates. Positron emission tomography showed metabolic activity in 87% of patients. Follow-up CT scans were available for 36 of 62 (58%) patients without a classifying diagnosis. Two patients developed lung cancer 2 years after initial analysis. CONCLUSIONS: Incidental mediastinal lymph nodes on CT are characterized by multiplicity, relative small sizes, and coexistence with hilar lymphadenopathy in the majority of patients. These nodes often display increased metabolic activity. The low predictive value for malignancy justifies a restrictive attitude toward invasive diagnostic testing.

HER2 status in gastro-oesophageal adenocarcinomas assessed by two rabbit monoclonal antibodies (SP3 and 4B5) and two in situ hybridization methods (FISH and SISH).

AIMS: HER2 gene amplification has been detected in 10-20% of gastric adenocarcinomas. In view of the recently demonstrated clinical benefit of the anti-human epidermal growth factor receptor 2 (HER2) drug trastuzumab in the treatment of advanced gastric cancer, reliable HER2 testing is of key importance. The aim of this study was to examine HER2 status in gastro-oesophageal adenocarcinomas comparing SP3 and 4B5 immunohistochemistry (IHC) with dual probe HER2 [fluorescence in situ hybridization (FISH) and silver in situ hybridization (SISH)]. METHODS AND RESULTS: IHC and SISH were carried out on biopsy specimens of 146 patients with adenocarcinomas of the oesophagus and stomach. All SP3-IHC-positive cases and 91% of 4B5-IHC-positive cases were amplified. Sensitivity of SP3-IHC-positivity and 4B5-IHC-positivity for amplification was 77% and 96%, respectively. Results of FISH performed in 42 cases were identical to SISH. Amplification was heterogeneous in 73% of the adenocarcinomas; 24% of the oesophago-gastric carcinomas and 7% of distal stomach tumours were amplified. CONCLUSIONS: HER2-positivity is present in a significant proportion of oesophago-gastric adenocarcinomas (24%), but at a lower rate in the distal stomach (7%). Sensitivity for amplification is higher with 4B5 IHC than with SP3. FISH and SISH yield identical results, but assessment is much easier with SISH. Our findings provide important guidance for HER2-testing in gastro-oesophageal adenocarcinomas for patients in whom anti-HER2 treatment is considered.

Comparison of EUS-guided fine needle aspiration and integrated PET-CT in restaging after treatment for locally advanced non-small cell lung cancer.

BACKGROUND: After induction treatment restaging of mediastinal disease in patients with stage III non-small cell lung cancer (NSCLC) may lead to selection of candidates for further surgical treatment. Nodal down-staging is the best predictive characteristic for proceeding with surgery. We report our experience in restaging with endoscopic ultrasound-guided fine needle aspirations (EUS-FNA) and with repeated integrated positron emission tomography and computed tomography (PET-CT). METHODS: Twenty-eight patients with stage III NSCLC were staged with integrated PET-CT, cerebral magnetic resonance imaging (MRI) and pathologically proven nodal disease. Restaging was performed with PET-CT and EUS-FNA on the same nodes that showed initially metastatic disease provided these nodal sites determined the tumor stage. Cerebral MRI was not repeated. When restaging EUS-FNA revealed no malignant cells anymore, patients were operated. The postoperative pathologic results were compared with the preoperative restaging EUS-FNA results. Also, patterns of decreased fluoro-2-deoxyglucose (FDG) uptake were compared with the postoperative pathologic results. RESULTS: Restaging EUS-FNA was well tolerated in all patients even in those with clinical signs of radiation esophagitis. Of the 28 patients 15 were down-staged based on cytologic findings with restaging EUS-FNA and in one patient the cytology was not conclusive. Of these 15 patients, down-staging was histologically confirmed after mediastinal exploration in 11 patients and 1 patient had persistent nodal disease at resection. In 3 patients no mediastinal tissue verification was performed. Two subjects were not fit for operation, and in the other patient intraoperative nodal staging was omitted. The negative predictive value for restaging EUS-FNA was 91.6%. The accuracy of EUS-FNA was 92.3%. Concordance between findings of restaging EUS-FNA and metabolic response of lymph node metastases occurred in 17 out of 27 patients. CONCLUSION: Restaging with EUS-FNA after induction chemo(-radiotherapy) is well tolerated and predicts the absence of nodal metastasis reliably. Although changes in mediastinal FDG-PET uptake show a high concordance with EUS-FNA, pathological confirmation is still superior and therefore necessary. EUS-FNA is the procedure of first choice for mediastinal restaging.

EBV-positive immunodeficiency lymphoma after alemtuzumab-CHOP therapy for peripheral T-cell lymphoma.

Chemotherapy with alemtuzumab and the combination of cyclophosphamide, adriamycin, oncovin, and prednisone (CHOP) has become experimental trial therapy for aggressive T-cell lymphoma. Several multicenter phase 3 trials will incorporate this scheme. As part of an ongoing phase 2 trial in which we recently treated 20 patients with 8 cycles of CHOP every 2 weeks with 3 additional doses of 30 mg alemtuzumab per cycle, we observed the development of Epstein-Barr virus (EBV)-positive lymphoproliferative disease, after completion of the immunochemotherapy in 3 patients with peripheral T-cell lymphoma. Because the occurrence of EBV-positive lymphoproliferative disease is rare after alemtuzumab monotherapy, such as is given for chronic lymphocytic leukemia, we think that early reporting of this potential side effect is warranted. It may be caused by intrinsic T-cell defects in patients with T-cell lymphoma, or by the combination of alemtuzumab with CHOP chemotherapy.

Pathologic examination of sentinel lymph nodes in breast cancer by a single haematoxylin-eosin slide versus serial sectioning and immunocytokeratin staining: clinical implications.

BACKGROUND: The objective was to determine the additional value of pathologic examination using three-level sectioning and immunocytokeratin (ICK) staining of sentinel lymph node (SN) biopsies in cT1-2N0M0 breast carcinoma patients regarding lymph node staging and eligibility of systemic therapy taking primary tumor characteristics in account. METHODS: SN slides of 277 patients out of a total group of 961 patients known to have tumor-positive SNs detected by three-level sectioning and ICK staining were re-examined. Haematoxylin-eosin (HE) slide level three was scanned for tumor deposits, and when present, extra capsular extension, maximum tumor diameter and number of positive SNs was noted. In addition, slides of the axillary dissection of non-SNs were reviewed, with determination of metastasis size and number of positive non-SNs. Primary tumor characteristics (grade, diameter, estrogen receptor) were recorded. RESULTS: In the single-HE examination, 26 cases SN micrometastasis and 6 macrometastasis were missed, 3 cases of micrometastasis were incorrectly classified as isolated tumor cells, and 9 patients with macrometastasis were misclassified as micrometastasis. In addition, in the tumor-negative single-HE examination, additional axillary lymph node dissection (ALND) revealed 6 cases of non-SN metastasis. Taking primary tumor factors into account for adjuvant systemic therapy, 21 patients would have been denied the choice for systemic therapy if single-HE examination was carried out only. CONCLUSIONS: Single-HE examination of SN may result in a reduction of locoregional and systemic treatment according to treatment guidelines then current in the Netherlands.