PD-L1 diagnostics in the neoadjuvant setting: implications of intratumoral heterogeneity of PD-L1 expression in triple negative breast cancer for assessment in small biopsies.

PURPOSE: PD-L1 expression is a predictive biomarker for anti-PD-L1 immunotherapy in triple negative breast cancer (TNBC). In the neoadjuvant setting, immunohistochemical (IHC) evaluation of PD-L1 expression can only be performed on small tissue biopsies. In our study we investigated heterogeneity of PD-L1 expression in TNBC, and how reliably PD-L1 expression in small tissue samples reflects PD-L1 expression in larger tumor sections in TNBC. METHODS: Tissue microarrays (TMAs) were constructed from surgical specimens of 110 patients with TNBC. TMAs contained 4 cores (1 mm in diameter) per patient. To evaluate PD-L1 expression, TMAs were stained with PD-L1 IHC 22C3 PharmDx. Single-core PD-L1 expression was compared to overall PD-L1 expression of each patient's tumor, to ascertain how often small samples of tumor tissue show the same PD-L1 expression as larger tumor samples. RESULTS: Our study found substantial heterogeneity of PD-L1 expression between different TMA cores from the same patient. Heterogeneity was greater in immune cells (ICs) than in tumor cells, in large part due to the uneven distribution of ICs in the tumor. For IC PD-L1 expression, we found that sensitivity can be as low as 0.81 for detecting PD-L1 expression at the 1% threshold most commonly used in breast cancer. Negative predictive value for ICs was 0.7. CONCLUSIONS: There is substantial heterogeneity of PD-L1 expression between small tissue samples from the same TNBC tumor, especially for IC expression. This poses challenges for evaluation of PD-L1 expression in the neoadjuvant setting. Negative biopsies should prompt further investigation, and multiple biopsies might be necessary.

An interobserver reproducibility analysis of size-set semiautomatic counting for Ki67 assessment in breast cancer.

PURPOSE: The proliferation marker Ki67 has prognostic and predictive values in breast cancer, and the cutoff of the Ki67 label index (LI) is a key index for chemotherapy. However, poor interobserver consistency in Ki67 assessment has limited the clinical use of Ki67, especially in luminal cancers. Here, we reported a modified Ki67 assessment method, size-set semiautomatic counting (SSSAC) and investigated its interobserver reproducibility. METHODS: One hundred invasive breast cancer tissues were set immunostained for Ki67 in one laboratory, scanned as digital slides, and sent to 41 pathologists at the laboratories of 16 hospitals for Ki67 LI assessment using size-set semiautomatic counting (SSSAC), size-set visual assessment (SSVA) and size-set digital image analysis (SSDIA) with a specific image viewing software (Aperio Image Scope, Leica, Germany). The intraclass correlation coefficient (ICC) and Bland-Altman plot were used to evaluate interobserver reproducibility. The Wilcoxon signed-rank test was used to analyze the difference in the Ki67 values assessed by SSSAC and SSDIA. RESULTS: SSSAC demonstrated better interobserver reproducibility (ICC = 0.942) than SSVA (ICC = 0.802). The interobserver reproducibility was better in Ki67 homogeneously stained slides and centralized hot-spot slides than in scattered hot-spot slides. The Ki67 value assessed with SSSAC was obviously higher than that assessed with SSDIA (negative ranks (SSDIA < SSSAC): N = 80, sum of ranks = 4274.50; positive ranks (SSDIA > SSSAC): N = 17, sum of ranks = 478.50; Z = -6.837; P < 0.001). CONCLUSION: SSSAC shows satisfactory interobserver reproducibility in the Ki67 assessment of breast cancer and may be a candidate standard method for Ki67 LI assessment in breast cancer and other malignancies.

Radiomic Signatures Derived from Diffusion-Weighted Imaging for the Assessment of Breast Cancer Receptor Status and Molecular Subtypes.

PURPOSE: To compare annotation segmentation approaches and to assess the value of radiomics analysis applied to diffusion-weighted imaging (DWI) for evaluation of breast cancer receptor status and molecular subtyping. PROCEDURES: In this IRB-approved HIPAA-compliant retrospective study, 91 patients with treatment-naïve breast malignancies proven by image-guided breast biopsy, (luminal A, n = 49; luminal B, n = 8; human epidermal growth factor receptor 2 [HER2]-enriched, n = 11; triple negative [TN], n = 23) underwent multiparametric magnetic resonance imaging (MRI) of the breast at 3 T with dynamic contrast-enhanced MRI, T2-weighted and DW imaging. Lesions were manually segmented on high b-value DW images and segmentation ROIS were propagated to apparent diffusion coefficient (ADC) maps. In addition in a subgroup (n = 79) where lesions were discernable on ADC maps alone, these were also directly segmented there. To derive radiomics signatures, the following features were extracted and analyzed: first-order histogram (HIS), co-occurrence matrix (COM), run-length matrix (RLM), absolute gradient, autoregressive model (ARM), discrete Haar wavelet transform (WAV), and lesion geometry. Fisher, probability of error and average correlation, and mutual information coefficients were used for feature selection. Linear discriminant analysis followed by k-nearest neighbor classification with leave-one-out cross-validation was applied for pairwise differentiation of receptor status and molecular subtyping. Histopathologic results were considered the gold standard. RESULTS: For lesion that were segmented on DWI and segmentation ROIs were propagated to ADC maps the following classification accuracies > 90% were obtained: luminal B vs. HER2-enriched, 94.7 % (based on COM features); luminal B vs. others, 92.3 % (COM, HIS); and HER2-enriched vs. others, 90.1 % (RLM, COM). For lesions that were segmented directly on ADC maps, better results were achieved yielding the following classification accuracies: luminal B vs. HER2-enriched, 100 % (COM, WAV); luminal A vs. luminal B, 91.5 % (COM, WAV); and luminal B vs. others, 91.1 % (WAV, ARM, COM). CONCLUSIONS: Radiomic signatures from DWI with ADC mapping allows evaluation of breast cancer receptor status and molecular subtyping with high diagnostic accuracy. Better classification accuracies were obtained when breast tumor segmentations could be performed on ADC maps.

Practical approaches to automated digital image analysis of Ki-67 labeling index in 997 breast carcinomas and causes of discordance with visual assessment.

The Ki-67 labeling index (LI) is an important prognostic factor in breast carcinoma. The Ki-67 LI is traditionally calculated via unaided microscopic estimation; however, inter-observer and intra-observer variability and low reproducibility are problems with this visual assessment (VA) method. For more accurate assessment and better reproducibility with Ki-67 LI, digital image analysis was introduced recently. We used both VA and automated digital image analysis (ADIA) (Ventana Virtuoso image management software) to estimate Ki-67 LI for 997 cases of breast carcinoma, and compared VA and ADIA results. VA and ADIA were highly correlated (intraclass correlation coefficient 0.982, and Spearman's correlation coefficient 0.966, p<0.05). We retrospectively analyzed cases with a greater than 5% difference between VA and ADIA results. The cause of these differences was: (1) tumor heterogeneity (98 cases, 56.0%), (2) VA interpretation error (32 cases, 18.3%), (3) misidentification of tumor cells (26 cases, 14.9%), (4) poor immunostaining or slide quality (16 cases, 9.1%), and (5) Estimation of non-tumor cells (3 cases, 1.7%). There were more discrepancies between VA and ADIA results in the group with a VA value of 10-20% compared to groups with <10% and ≥20%. Although ADIA is more accurate than VA, there are some limitations. Therefore, ADIA findings require confirmation by a pathologist.

Assessment of expression of interferon γ (IFN-G) gene and its antisense (IFNG-AS1) in breast cancer.

BACKGROUND: The role of long non-coding RNAs has been extensively appreciated in the contexts of cancer. Interferon γ-antisense RNA1 (IFNG-AS1) is an lncRNA located near to IFN-γ-encoding (IFNG) gene and regulates expression of IFNG in Th1 cells. METHODS: In the present study, we evaluated expression of IFNG and IFNG-AS1 in 108 breast samples including tumoral tissues and their adjacent non-cancerous tissues (ANCTs) using real-time PCR. IFNG-AS1 was significantly upregulated in tumoral tissues compared with ANCTs (expression ratio = 2.23, P = 0.03). RESULTS: Although the expression of IFNG was higher in tumoral tissues compared with ANCTs (relative expression = 1.89), it did not reach the level of significance (P = 0.07). IFNG expression was significantly higher in HER2-negative tumoral tissues compared with HER2-positive ones (P = 0.01) and in grade 1 samples compared with grade 2 ones (P = 0.03). No other significant difference was found in expressions of genes between other groups. CONCLUSION: Significant strong correlations were detected between expression of IFNG and IFNG-AS1 in both tumoral tissues and ANCTs. The present study provides evidences for participation of IFNG and IFNG-AS1 in the pathogenesis of breast cancer and warrants future studies to elaborate the underlying mechanism.

Magnetic Resonance Imaging (MRI) Assessment of Residual Breast Cancer After Neoadjuvant Chemotherapy: Relevance to Tumor Subtypes and MRI Interpretation Threshold.

PURPOSE: To investigate the diagnostic performance of magnetic resonance imaging (MRI) for predicting pathologic complete response after neoadjuvant chemotherapy (NAC) depending on subtypes of breast cancer using different interpretation thresholds of MRI negativity. PATIENTS AND METHODS: A total of 353 women with breast cancer who had undergone NAC were included. Pathologic examination after complete surgical excision was the reference standard. Tumors were divided into 4 subtypes on the basis of expression of hormone receptor (HR) and human epidermal growth factor receptor 2 (HER2). Tumor enhancement was assessed on early and late phases of MRI. MRI negativity was divided into radiologic complete response (rCR, complete absence of enhancement on both early and late phases) and near-rCR (no discernible early enhancement but observed late enhancement). RESULTS: Ninety (25.5%) of 353 patients experienced pathologic complete response. When analyzing the data of all patients, sensitivity of MRI was higher for rCR versus near-rCR (97.72% vs. 90.49%, P < .0001), whereas specificity was lower for rCR versus near-rCR (44.44% vs. 72.22%, P < .0001). Accuracy was equivalent (84.14% vs. 85.84%). In HR-HER2+ tumors, 100% sensitivity and negative predictive value were achieved by assessing early enhancement only. In HR+HER2- tumors, sensitivity of MRI was higher for rCR versus near-rCR (96.12% vs. 86.82%, P = .0005). CONCLUSION: Diagnostic performance of MRI after NAC differs in accordance with the subtypes and threshold of MRI negativity. MRI assessment with consideration of tumor subtypes is required, along with standardization of MRI interpretation criteria in the NAC setting.

Comparison of immunohistochemistry with PCR for assessment of ER, PR, and Ki-67 and prediction of pathological complete response in breast cancer.

BACKGROUND: Proliferation may predict response to neoadjuvant therapy of breast cancer and is commonly assessed by manual scoring of slides stained by immunohistochemistry (IHC) for Ki-67 similar to ER and PgR. This method carries significant intra- and inter-observer variability. Automatic scoring of Ki-67 with digital image analysis (qIHC) or assessment of MKI67 gene expression with RT-qPCR may improve diagnostic accuracy. METHODS: Ki-67 IHC visual assessment was compared to the IHC nuclear tool (AperioTM) on core biopsies from a randomized neoadjuvant clinical trial. Expression of ESR1, PGR and MKI67 by RT-qPCR was performed on RNA extracted from the same formalin-fixed paraffin-embedded tissue. Concordance between the three methods (vIHC, qIHC and RT-qPCR) was assessed for all 3 markers. The potential of Ki-67 IHC and RT-qPCR to predict pathological complete response (pCR) was evaluated using ROC analysis and non-parametric Mann-Whitney Test. RESULTS: Correlation between methods (qIHC versus RT-qPCR) was high for ER and PgR (spearman´s r = 0.82, p < 0.0001 and r = 0.86, p < 0.0001, respectively) resulting in high levels of concordance using predefined cut-offs. When comparing qIHC of ER and PgR with RT-qPCR of ESR1 and PGR the overall agreement was 96.6 and 91.4%, respectively, while overall agreement of visual IHC with RT-qPCR was slightly lower for ER/ESR1 and PR/PGR (91.2 and 92.9%, respectively). In contrast, only a moderate correlation was observed between qIHC and RT-qPCR continuous data for Ki-67/MKI67 (Spearman's r = 0.50, p = 0.0001). Up to now no predictive cut-off for Ki-67 assessment by IHC has been established to predict response to neoadjuvant chemotherapy. Setting the desired sensitivity at 100%, specificity for the prediction of pCR (ypT0ypN0) was significantly higher for mRNA than for protein (68.9% vs. 22.2%). Moreover, the proliferation levels in patients achieving a pCR versus not differed significantly using MKI67 RNA expression (Mann-Whitney p = 0.002), but not with qIHC of Ki-67 (Mann-Whitney p = 0.097) or vIHC of Ki-67 (p = 0.131). CONCLUSION: Digital image analysis can successfully be implemented for assessing ER, PR and Ki-67. IHC for ER and PR reveals high concordance with RT-qPCR. However, RT-qPCR displays a broader dynamic range and higher sensitivity than IHC. Moreover, correlation between Ki-67 qIHC and RT-qPCR is only moderate and RT-qPCR with MammaTyper® outperforms qIHC in predicting pCR. Both methods yield improvements to error-prone manual scoring of Ki-67. However, RT-qPCR was significantly more specific.

PD-L1 Expression and Intratumoral Heterogeneity Across Breast Cancer Subtypes and Stages: An Assessment of 245 Primary and 40 Metastatic Tumors.

Tumor expression of programmed cell death ligand 1 (PD-L1) is associated with immune evasion in a variety of malignancies, including a subset of triple-negative breast carcinomas, and may mark cancers as susceptible to PD-1/PD-L1 inhibitor therapies. We herein characterize PD-L1 expression in breast cancers across the full range of histomorphologies and investigate its intratumoral heterogeneity and fidelity across primaries and metastases. A total of 245 primary and 40 metastatic (20 nodal, 20 distant) breast carcinomas were evaluated with PD-L1 immunohistochemistry on tissue microarray. Tumor PD-L1 staining was seen in 12% of all primaries including 32% of triple-negative cancers. Staining was common in ductal cancers with medullary (54%), apocrine (27%), and metaplastic features (40%). However, diffuse (>50%) staining was rare (2% of all cancers and 5% of triple negatives). Immune staining was seen in 29% of all primaries and 61% of triple negatives. Tumor expression of PD-L1 was conserved in 94% of matched primary/metastasis pairs, while immune staining showed fidelity in 71%; the remaining cases acquired PD-L1 immune cell expression in the metastasis. Only half of cases with positive tumor staining showed concordance across all analyzed cores. These data demonstrate that PD-L1 expression is prevalent among high-grade, hormone receptor-negative breast cancers with a range of histomorphologies and shows fidelity between primary and metastatic sites in treatment-naive cancers, although acquisition of immune PD-L1 staining in metastases is not uncommon. There is considerable intratumoral heterogeneity in PD-L1 expression, undermining the suitability of core biopsy in the determination of PD-L1 status. Clinical trials are needed to determine PD-L1 staining thresholds required for therapeutic response, as diffuse staining is rare.

Standardization for Ki-67 assessment in moderately differentiated breast cancer. A retrospective analysis of the SAKK 28/12 study.

BACKGROUND: Proliferative activity (Ki-67 Labelling Index) in breast cancer increasingly serves as an additional tool in the decision for or against adjuvant chemotherapy in midrange hormone receptor positive breast cancer. Ki-67 Index has been previously shown to suffer from high inter-observer variability especially in midrange (G2) breast carcinomas. In this study we conducted a systematic approach using different Ki-67 assessments on large tissue sections in order to identify the method with the highest reliability and the lowest variability. MATERIALS AND METHODS: Five breast pathologists retrospectively analyzed proliferative activity of 50 G2 invasive breast carcinomas using large tissue sections by assessing Ki-67 immunohistochemistry. Ki-67-assessments were done on light microscopy and on digital images following these methods: 1) assessing five regions, 2) assessing only darkly stained nuclei and 3) considering only condensed proliferative areas ('hotspots'). An individual review (the first described assessment from 2008) was also performed. The assessments on light microscopy were done by estimating. All measurements were performed three times. Inter-observer and intra-observer reliabilities were calculated using the approach proposed by Eliasziw et al. Clinical cutoffs (14% and 20%) were tested using Fleiss' Kappa. RESULTS: There was a good intra-observer reliability in 5 of 7 methods (ICC: 0.76-0.89). The two highest inter-observer reliability was fair to moderate (ICC: 0.71 and 0.74) in 2 methods (region-analysis and individual-review) on light microscopy. Fleiss'-kappa-values (14% cut-off) were the highest (moderate) using the original recommendation on light-microscope (Kappa 0.58). Fleiss' kappa values (20% cut-off) were the highest (Kappa 0.48 each) in analyzing hotspots on light-microscopy and digital-analysis. No methodologies using digital-analysis were superior to the methods on light microscope. CONCLUSION: Our results show that all methods on light-microscopy for Ki-67 assessment in large tissue sections resulted in a good intra-observer reliability. Region analysis and individual review (the original recommendation) on light-microscopy yielded the highest inter-observer reliability. These results show slight improvement to previously published data on poor-reproducibility and thus might be a practical-pragmatic way for routine assessment of Ki-67 Index in G2 breast carcinomas.

MRI phenotype of breast cancer: Kinetic assessment for molecular subtypes.

PURPOSE: To evaluate the dynamic contrast-enhanced magnetic resonance imaging (MRI) kinetic characteristics of newly diagnosed breast cancer molecular subtypes. MATERIALS AND METHODS: Breast MRI examinations of 112 patients with newly diagnosed breast cancer were reviewed. Cases of newly diagnosed invasive ductal carcinoma were sorted by molecular subtype (28 TN, 11 HER2 +, 73 Lum A/B) and MRI field strength, with lesion segmentation and kinetic analyses performed on a dedicated workstation. For kinetic assessment, 50% and 100% thresholds were employed for display of medium and rapid uptake. Kinetic profiles in terms of percent volume for six kinetic types (medium-persistent, medium-plateau, medium-washout, fast-persistent, fast-plateau, fast-washout) relative to the whole volume of the lesion were obtained. Statistical analysis of the kinetic profiles was performed using Welch's t-test. RESULTS: Percent volume of HER2-positive lesions with >100% uptake at early phase on 3T strength MRI exams was significantly greater compared with luminal A/B (93.8 ± 0.92 vs. 77.3 ± 7.2; P < 0.01) and triple negative (93.8 ± 0.92 vs. 81.3 ± 8.2; P < 0.05) subtypes. The >50% early phase uptake for HER2+ lesions was also higher than Lum A/B (99.1 ± 0.73 vs. 93.6 ± 3.05; P < 0.01) at 3T. In the 1.5T subgroup the percent volume of HER2+ tumors with >50% and >100% early phase uptake trended higher than Lum A/B lesions without reaching significance. CONCLUSION: The percent volume of HER2-positive tumors demonstrating rapid early contrast uptake is significantly increased compared to other molecular subtypes.

Quality of pathology reporting is crucial for cancer care and registration: a baseline assessment for breast cancers diagnosed in Belgium in 2008.

OBJECTIVES: Given the crucial role of pathology reporting in the management of breast cancers, we aimed to investigate the quality and variability of breast cancer pathology reporting in Belgium. MATERIALS AND METHODS: Detailed information on non-molecular and molecular parameters was retrieved from the pathology protocols available at the Belgian Cancer Registry for 10,007 breast cancers diagnosed in Belgium in 2008. RESULTS: Substantial underreporting was shown for several clinically relevant non-molecular parameters, such as lymphovascular invasion. High-volume laboratories performed only slightly better than others, and analyses at the individual laboratory level showed clear inter-laboratory variability in reporting for all volume categories. Information on ER/PR and HER2 IHC was mentioned in respectively 91.7% and 90.8% of evaluative cases. HER2 ISH data were available for 78.5% of the cases judged to be 2+ for HER2 IHC. For cases with different specimens analysed, discordance between these specimens was highest for HER2, followed by PR. For HER2, results obtained from different laboratories were even less concordant. In addition, inter-laboratory differences were noted in the used ER/PR scoring systems, the proportion of ER-/PR+ cases, and the relation between histological grade and ER/PR positivity. Data on Ki67 were only available for 43.8% of the investigated cases, and showed inconsistent use of cut-off values. CONCLUSION: Breast pathology reporting in Belgium in 2008 was suboptimal and showed considerable inter-laboratory variability. Synoptic reporting has been proposed as a facilitator towards increased reporting quality and harmonization, but the lack of aligned informatics remains a major hurdle in its concrete implementation.

Incorporating microarray assessment of HER2 status in clinical practice supports individualised therapy in early-stage breast cancer.

Accurate determination of human epidermal growth factor receptor-2 (HER2) status is essential for optimal selection of breast cancer patients for gene targeted therapy. The analytical performance of microarray analysis using TargetPrint for assessment of HER2 status was evaluated in 138 breast tumours, including 41 fresh and 97 formalin-fixed paraffin embedded (FFPE) specimens. Reflex testing using immunohistochemistry/in situ hybridization (IHC/ISH) in four discordant cases confirmed the TargetPrint results, achieving 100% agreement regardless of whether fresh tissue or FFPE specimens were used. One equivocal IHC/ISH case was classified as HER2-positive based on the microarray result. The proven clinical utility in resolving equivocal and borderline cases justifies modification of the testing algorithm under these circumstances, to obtain a definitive positive or negative test result with the use of microarrays. Determination of HER2 status across three assay platforms facilitated improved quality assurance and led to a higher level of confidence on which to base treatment decisions.

Lymphatic vessel assessment by podoplanin (D2-40) immunohistochemistry in breast cancer.

CONTEXT: Lymph node metastasis has an important bearing on the staging of breast cancer. Lymph node metastasis occurs by hematogenous and lymphatic spread. The hematogenous and lymphatic spread can be quantified by the blood vessel and lymphatic vessel density in the intra-tumoral and peri-tumoral zone by specific markers for blood vessels and lymph vessels. AIMS: In this study, we are trying to study the localization of podoplanin in lymph vessels of invasive breast carcinoma, to quantify lymphangiogenesis in tissue sections of invasive breast carcinomas by podoplanin immunohistochemistry (IHC) by D2-40 antibody, and compare it with blood microvessel count using CD-31 antibody and correlating clinicopathologic parameters with the results of IHC. MATERIALS AND METHODS: IHC for biomarkers D2-40 and CD-31 were performed on sections from 30 mastectomy specimens to assess blood vessel and lymphatic vessel density in intra-tumoral and peri-tumoral zone. The data were analyzed using Statistical Package for Social Sciences (SPSS) version 15.0 statistical analysis software. RESULTS: The results showed that both lymph vessel density and blood vessel density increased with the increase in lymph node ratio. Lymph node ratio is the ratio of positive lymph nodes to the total number of lymph nodes removed. CONCLUSION: Taking into account our small sample size, we conclude that a further large-sized study should be carried out to further prove the role of lymphatics in tumor dissemination. New therapeutic options can be developed targeting the lymphatic channels to arrest the lymphatic spread of the breast cancer.

High agreement between whole slide imaging and optical microscopy for assessment of HER2 expression in breast cancer: whole slide imaging for the assessment of HER2 expression.

UNLABELLED: Whole slide imaging (WSI) technology has been used for training, teaching, researching, and remote consultation. Few studies compared HER2 expression using optical microscopy (OM) and WSI evaluations in breast carcinomas. However, no consensus has been achieved comparing both assessments. MATERIAL AND METHODS: Sections from tissue microarray containing 200 preselected invasive breast carcinomas were submitted to immunohistochemistry applying three anti-HER2 antibodies (HercepTest™, CB11, SP3) and in situ hybridization (DDISH). Slides were evaluated using OM and WSI (Pannoramic MIDI and Viewer, 3DHISTECH). Sensitivity and specificity were calculated comparing the anti-HER2 antibodies and DDISH. RESULTS: WSI and OM HER2 evaluations agreement was considered good (SP3, k=0.80) to very good (CB11 and HercepTest™, k=0.81). WSI evaluation led to higher sensitivity (ranging from 100 of SP3 and HercepTest™ to 97 of CB11) and lower specificity (ranging from 86.4 of SP3 to 89.4 of HercepTest™) compared to OM evaluation (sensitivity ranged from 92.1 of CB11 to 98 of SP3 and specificity ranged from 95.2 of SP3 and HercepTest™ to 97.1 of CB11 and SP3). CONCLUSION: High agreement was achieved between WSI and OM evaluations. All three antibodies were highly sensitive and specific using both evaluations. WSI can be considered a useful tool for HER2 immunohistochemical assessment.

Rapid point-of-care breath test for biomarkers of breast cancer and abnormal mammograms.

BACKGROUND: Previous studies have reported volatile organic compounds (VOCs) in breath as biomarkers of breast cancer and abnormal mammograms, apparently resulting from increased oxidative stress and cytochrome p450 induction. We evaluated a six-minute point-of-care breath test for VOC biomarkers in women screened for breast cancer at centers in the USA and the Netherlands. METHODS: 244 women had a screening mammogram (93/37 normal/abnormal) or a breast biopsy (cancer/no cancer 35/79). A mobile point-of-care system collected and concentrated breath and air VOCs for analysis with gas chromatography and surface acoustic wave detection. Chromatograms were segmented into a time series of alveolar gradients (breath minus room air). Segmental alveolar gradients were ranked as candidate biomarkers by C-statistic value (area under curve [AUC] of receiver operating characteristic [ROC] curve). Multivariate predictive algorithms were constructed employing significant biomarkers identified with multiple Monte Carlo simulations and cross validated with a leave-one-out (LOO) procedure. RESULTS: Performance of breath biomarker algorithms was determined in three groups: breast cancer on biopsy versus normal screening mammograms (81.8% sensitivity, 70.0% specificity, accuracy 79% (73% on LOO) [C-statistic value], negative predictive value 99.9%); normal versus abnormal screening mammograms (86.5% sensitivity, 66.7% specificity, accuracy 83%, 62% on LOO); and cancer versus no cancer on breast biopsy (75.8% sensitivity, 74.0% specificity, accuracy 78%, 67% on LOO). CONCLUSIONS: A pilot study of a six-minute point-of-care breath test for volatile biomarkers accurately identified women with breast cancer and with abnormal mammograms. Breath testing could potentially reduce the number of needless mammograms without loss of diagnostic sensitivity.

Assessment of maturation status of tumor-infiltrating dendritic cells in invasive ductal carcinoma of the breast: relation with vascular endothelial growth factor expression.

OBJECTIVE: Poor immunogenicity has been described in breast carcinoma although dendritic cells, the major antigen presenters, are known to infiltrate the tumor. Vascular endothelial growth factor has been proposed to reduce local immune response in tumors. We investigated the maturation status of dendritic cells in invasive ductal carcinoma of the breast in relation to vascular endothelial growth factor expression and clinicopathological parameters. MATERIAL AND METHOD: Fifty invasive ductal carcinomas of the breast were immunostained with CD1a (marker of immature dendritic cells); CD83 (marker of mature dendritic cells), vascular endothelial growth factor, estrogen receptor and progesterone receptor. RESULTS: Mature dendritic cells were detected in 36 cases (72%), and correlated with smaller tumor size, negative lymph nodes, positive steroid receptor status, and lower grade (P < 0.001). Immature dendritic cells were found in 100% of cases and correlated only with negative steroid receptor expression (estrogen receptor and progesterone receptor) (P=0.006 and 0.020 respectively). Vascular endothelial growth factor expression was detected in 44 cases (88%), and correlated directly with positive nodal metastases (P=0.014), correlated inversely with mature dendritic cell count (P=0.005); and did not correlate with immature dendritic cell count (P=0.104). CONCLUSION: Mature dendritic cell count correlates with good prognostic features in invasive ductal carcinoma of the breast, suggesting their role in initiating primary anti-tumor immune response. Vascular endothelial growth factor expression may play a role in inhibition of dendritic cell maturation sequence in the tumor microenvironment.

Impact of different tumour stroma assessment methods regarding podoplanin expression on clinical outcome in patients with invasive ductal breast carcinoma.

BACKGROUND: Podoplanin, a small mucin-type transmembrane protein has been shown in several studies to be expressed in cancer-associated fibroblasts (CAFs) and affect patient outcome. MATERIALS AND METHODS: We evaluated podoplanin expression in CAFs in a cohort of 257 patients with invasive ductal breast carcinomas (IDCs) using three different assessment scales based on the number of positive cells alone or in combination with the reaction intensity. RESULTS: Two of the utilized scales yielded prognostic information concerning patients' overall survival (OS), but scores were not independent prognostic factors in the multivariate analysis. On the contrary, two scales based on the combination of cell positivity and reaction intensity had no significant impact on patients' OS, but they were significantly correlated with a greater number of analysed clinicopathological parameters. CONCLUSION: In summary, podoplanin expression in CAFs may be considered a possible marker of poor prognosis in IDC, however, caution should be taken as the results varied regarding the utilized scales.