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Purpose: Although the murine orthologue of glycoprotein nonmetastatic B (GPNMB), Osteoactivin, promotes breast cancer metastasis in an in vivo mouse model, its importance in human breast cancer is unknown. We have examined the significance of GPNMB expression as a prognostic indicator of recurrence and assessed its potential as a novel therapeutic target in breast cancer.Experimental Design: The clinical significance of GPNMB expression in breast cancer was addressed by analyzing GPNMB levels in several published gene expression data sets and two independent tissue microarrays derived from human breast tumors. GPNMB-expressing human breast cancer cell lines were further used to validate a toxin-conjugated anti-GPNMB antibody as a novel therapeutic agent.Results: GPNMB expression correlates with shorter recurrence times and reduced overall survival of breast cancer patients. Epithelial-specific GPNMB staining is an independent prognostic indicator for breast cancer recurrence. GPNMB is highly expressed in basal and triple-negative breast cancers and is associated with increased risk of recurrence within this subtype. GPNMB expression confers a more migratory and invasive phenotype on breast cancer cells and sensitizes them to killing by CDX-011 (glembatumumab vedotin), a GPNMB-targeted antibody-drug conjugate.Conclusions: GPNMB expression is associated with the basal/triple-negative subtype and is a prognostic marker of poor outcome in patients with breast cancer. CDX-011 (glembatumumab vedotin) is a promising new targeted therapy for patients with metastatic triple-negative breast cancers, a patient population that currently lacks targeted-therapy options. Clin Cancer Res; 16(7); 2147–56. ©2010 AACR.

Supplementary Data from Glycoprotein Nonmetastatic B Is an Independent Prognostic Indicator of Recurrence and a Novel Therapeutic Target in Breast Cancer

Intraductal carcinoma of the prostate (IDC-P) is one of the most aggressive types of prostate cancer (PCa). IDC-P is identified in approximately 20% of PCa patients and is associated with recurrence, metastasis, and PCa-specific death. The main feature of this histological variant is the colonization of benign glands by PCa cells. Although IDC-P is a well-recognized independent parameter for metastasis, mechanisms by which IDC-P cells can spread and colonize other tissues are not fully known. In this review, we discuss the molecular portraits of IDC-P determined by immunohistochemistry and genomic approaches and highlight the areas in which more research is needed.

Significance Prostate cancer is the most common cancer among men. An accurate diagnosis of its severity at detection plays a major role in improving their survival. Recently, machine learning models using biomarkers identified from Raman micro-spectroscopy discriminated intraductal carcinoma of the prostate (IDC-P) from cancer tissue with a ≥85 % detection accuracy and differentiated high-grade prostatic intraepithelial neoplasia (HGPIN) from IDC-P with a ≥97.8 % accuracy. Aim To improve the classification performance of machine learning models identifying different types of prostate cancer tissue using a new dimensional reduction technique. Approach A radial basis function (RBF) kernel support vector machine (SVM) model was trained on Raman spectra of prostate tissue from a 272-patient cohort (Centre hospitalier de l'Universite de Montreal, CHUM) and tested on two independent cohorts of 76 patients [University Health Network (UHN)] and 135 patients (Centre hospitalier universitaire de Quebec-Universite Laval, CHUQc-UL). Two types of engineered features were used. Individual intensity features, i.e., Raman signal intensity measured at particular wavelengths and novel Raman spectra fitted peak features consisting of peak heights and widths. Results Combining engineered features improved classification performance for the three aforementioned classification tasks. The improvements for IDC-P/cancer classification for the UHN and CHUQc-UL testing sets in accuracy, sensitivity, specificity, and area under the curve (AUC) are (numbers in parenthesis are associated with the CHUQc-UL testing set): +4 % (+8 % ), +7 % (+9 % ), +2 % (6%), +9 (+9) with respect to the current best models. Discrimination between HGPIN and IDC-P was also improved in both testing cohorts: +2.2 % (+1.7 % ), +4.5 % (+3.6 % ), +0 % (+0 % ), +2.3 (+0). While no global improvements were obtained for the normal versus cancer classification task [+0 % (-2 % ), +0 % (-3 % ), +2 % (-2 % ), +4 (+3)], the AUC was improved in both testing sets. Conclusions Combining individual intensity features and novel Raman fitted peak features, improved the classification performance on two independent and multicenter testing sets in comparison to using only individual intensity features.

Reliable cytopathological diagnosis requires new methods and approaches for the rapid and accurate determination of all cell types. This is especially important when the number of cells is limited, such as in the cytological samples of fine-needle biopsy. Immunoplasmonic-multiplexed- labeling may be one of the emerging solutions to such problems. However, to be accepted and used by the practicing pathologists, new methods must be compatible and complementary with existing cytopathology approaches where counterstaining is central to the correct interpretation of immunolabeling. In addition, the optical detection and imaging setup for immunoplasmonic-multiplexed-labeling must be implemented on the same cytopathological microscope, not interfere with standard H&amp, E imaging, and operate as a second easy-to-use imaging method. In this article, we present multiplex imaging of four types of nanoplasmonic markers on two types of H&amp, E-stained cytological specimens (formalin-fixed paraffin embedded and non-embedded adherent cancer cells) using a specially designed adapter for SI dark-field microscopy. The obtained results confirm the effectiveness of the proposed optical method for quantitative and multiplex identification of various plasmonic NPs, and the possibility of using immunoplasmonic-multiplexed-labeling for cytopathological diagnostics.

This protocol describes the acquisition of Raman spectra on formalin-fixed paraffin-embedded biological tissue with the following characteristics: 4 µm tissue section placed on a glass slide (H&E-IHC stained-slide for tissue visualization) 4 µm tissue section placed on an aluminum slide (Raman micro-spectroscopy) rapid dewaxing Raman micro-spectroscopy: 5 accumulations of 10 seconds, 50x short working distance objective (N.A. 0.75) Of note: this protocol addresses the Raman micro-spectroscopy slide preparation but does not fully describe the H&E-IHC slide used for tissue visualization (see Grosset et al, Appl Immunohistochem Mol Morphol, 2019)

To test the agreement between high-grade PCa at RP and TMA, and the ability of TMA to predict BCR. Validation of concordance between tissue microarray (TMA) and radical prostatectomy (RP) high-grade prostate cancer (PCa) is crucial because latter determines the treated natural history of PCa. We hypothesized that TMA Gleason score is in agreement with RP pathology and capable of accurately predicting biochemical recurrence (BCR). Data were provided from a multi-institutional Canadian sample of 1333 TMA and RP specimens with complete clinicopathological data. First, rate of agreement between TMA and high-grade Gleason at RP or biopsy and RP was tested. Second, ability of RP, TMA and biopsy to predict BCR was compared. Multivariable (MVA) Cox regression models were fitted and BCR rates were illustrated with Kaplan-Meier plots. Agreement between RP and TMA and between RP and biopsy was 72.6% (95% CI:69.7-75.5) and 60.4% (95% CI:57.2-63.6), respectively. In MVA predicting BCR, the accuracy for RP, TMA and biopsy was 0.73, 0.72 and 0.68, respectively. TMA added discriminatory ability among exclusively low-grade Gleason RP patients (p = 0.02), but did not improve BCR discrimination in exclusive high-grade PCa RP patients (p = 0.8). TMA Gleason grade accurately reflects presence of high-grade Gleason in RP specimen, accurately predicts BCR rates after RP and improves prediction of BCR in low-grade Gleason patients at RP.

Purpose Raman spectroscopy is a promising cancer detection technique for surgical guidance applications. It can provide quantitative information relating to global tissue properties associated with structural, metabolic, immunological and genetic biochemical phenomena in terms of molecular species including amino acids, lipids, proteins, and nucleic acid (DNA). To date in vivo Raman spectroscopy systems mostly included probes and biopsy needles typically limited to single-point tissue interrogation over a scale between 100 and 500 microns. The development of wider field handheld systems could improve tumor localization for a range of open surgery applications including brain, ovarian and skin cancers. Methods Here we present a novel Raman spectroscopy implementation using a coherent imaging bundle of fibers to create a probe capable of reconstructing molecular images over mesoscopic fields of view. Detection is performed using linear scanning with a rotation mirror and an imaging spectrometer. Different slits widths were tested at the entrance of the spectrometer to optimize spatial and spectral resolution while preserving sufficient signal to noise ratios to detect the principal Raman tissue features. The non-biological samples Calcite and Polytetrafluoroethylene (PTFE) were used to characterize the performance of the system. The new wide-field probe was tested on ex vivo samples of calf brain and swine tissue. Raman spectral content of both tissue types were validated with data from the literature and compared with data acquired with a single-point Raman spectroscopy probe. The single-point probe was used as the gold standard against which the new instrument was benchmarked since it has already been thoroughly validated for biological tissue characterization. Result We have developed and characterized a practical non-contact handheld Raman imager providing tissue information at a spatial resolution of 115 microns over a field of view >14 mm2 and a spectral resolution of 6 cm−1 over the whole fingerprint region. Typical integration time to acquire an entire Raman image over swine tissue was set to approximately 100 s. Spectra acquired with both probes (single point and wide-field) showed good agreement, with a Pearson correlation factor > 0.85 over different tissue categories. Protein and lipid content of imaged tissue were manifested into the measured spectra which correlated well with previous findings in the literature. An example of quantitative molecular map is presented for swine tissue and calf brain based on the ratio of protein-to lipid-content showing clear delineations between white and gray matter as well as between adipose and muscle tissue. Conclusion We presented the development of a Raman imaging probe with a field of view of a few millimeters and a spatial resolution consistent with standard surgical imaging methods using an imaging bundle. Spectra acquired with the newly developed system on swine tissue and calf brain correlated well with an establish single-point probe and observed spectral features agreed with previous finding in the literature. The imaging probe has demonstrated its ability to reconstruct molecular images of soft tissues. The approach presented here has a lot of potential for the development of surgical Raman imaging probe to guide the surgeon during cancer surgery. This article is protected by copyright. All rights reserved.

Objective To test if Raman spectroscopy (RS) is an appropriate tool for the diagnosis and possibly grading of prostate cancer (PCa). Patients and methods Between 20 and 50 Raman spectra were acquired from 32 fresh and non-processed post-prostatectomy specimens using a macroscopic handheld RS probe. Each measured area was characterized and categorized according to histopathological criteria: tissue type (extraprostatic or prostatic); tissue malignancy (benign or malignant); cancer grade (Grade Groups [GGs] 1-5); and tissue glandular level. The data were analysed using machine-learning classification with neural network. Results The RS technique was able to distinguish prostate from extraprostatic tissue with a sensitivity of 82% and a specificity of 83% and benign from malignant tissue with a sensitivity of 87% and a specificity of 86%. In an exploratory fashion, RS differentiated benign from GG1 in 726/801 spectra (91%; sensitivity 80%, specificity 91%), from GG2 in 588/805 spectra (73%; sensitivity 76%, specificity 73%), from GG3 in 670/797 spectra (84%; sensitivity 86%, specificity 84%), from GG4 in 711/802 spectra (88%; sensitivity 77%, specificity 89%) and from GG5 in 729/818 spectra (89%; sensitivity 90%, specificity 89%). Conclusion Current diagnostic approaches of PCa using needle biopsies have suboptimal cancer detection rates and a significant risk of infection. Standard non-targeted random sampling results in false-negative biopsies in 15-30% of patients, which affects clinical management. RS, a non-destructive tissue interrogation technique providing vibrational molecular information, resolved the highly complex architecture of the prostate and detect cancer with high accuracy using a fibre optic probe to interrogate radical prostatectomy (RP) specimens from 32 patients (947 spectra). This proof-of-principle paves the way for the development of in vivo tumour targeting spectroscopy tools for informed biopsy collection to address the clinical need for accurate PCa diagnosis and possibly to improve surgical resection during RP as a complement to histopathological analysis.

Background Prostate cancer (PC), initially diagnosed on biopsies by pathologists, is the most frequent cancer in North American men. However, better tools are needed for pathologists to diagnose intraductal carcinoma of the prostate (IDC), an aggressive histopathological variant of PC for which therapeutic options are now available. Indeed, no technique or biomarker is clinically available to support the diagnosis of IDC. Raman spectroscopy (RS) provides a global molecular characterisation of the tissue by analysing how photons interact with the molecules present in the tissue. Indeed, we and other groups previously used RS to detect cancer from multiple organ types, machine learning classification models being employed to process the complex Raman data. Methods We used Raman micro-spectroscopy (RµS) to detect IDC on tissues from 483 first-line radical prostatectomies from three Canadian institutions. Following a rapid, standardized and low-cost protocol, we acquired an average of 7 Raman spectra per patient and generated classification models using machine learning technology. Importantly, models were trained with data from one institution before independent testing on the data from the other two institutions. Results The three institutions included 272, 76 and 135 patients. Median age at diagnosis ranged from 61-62 years-old, with median pre-operative PSA ranging from 6.6-7.4 µg/L. Most patient had ≤3 + 4 Gleason score (60-80% of the specimens) and pT3-stage incidence was 31-55%. IDC was identified in 6-18% of the patients of each cohort. Overall, we acquired an average of 7 Raman spectra per patient. In the training cohort (N = 272), RµS identified IDC with a sensitivity of 95%, a specificity of 94% and an accuracy of 94%. Results from the testing cohort were in a similar range, with sensitivities of 88 and 92%, specificities of 83 and 91% and accuracies of 85 and 91%. Conclusions As clinically available biomarkers of IDC have reported sensitivities/specificities of ∼80%, we here identified IDC with accuracies ≥85%. Since our classification model was trained on a cohort and independently tested on the other two, these are likely to be close to real life experience making clinical implementation a realistic outcome. Legal entity responsible for the study The authors. Funding Centre de Recherche du Centre Hospitalier de l’Universite de Montreal (CRCHUM, continuum program) IVADO, Institut de valorisation des donnees. Disclosure All authors have declared no conflicts of interest.

International audience; Because of their ability to induce local immunosuppression and to confer cancer cells with resistance to apoptosis, members of the galectin family are emerging as a new class of actionable targets in cancer. Unfortunately, we have yet to obtain a clear picture of the galectin signatures in cancer cells and the surrounding tumor microenvironment. The aim of this study was to provide the first detailed analysis of the galectin signature in molecular subtypes of breast cancer. Expression signatures of galectins were obtained at the mRNA and protein levels. A particular attention was paid to stromal versus epithelial staining and to subcellular compartmentalization. Analysis of the stromal signature showed that gal-1, -3, -9-positive stroma were preferentially found in triple-negative (TN) and HER2 subtypes. In cancer cells, gal-1, -3, -8, and -9 showed a dual expression pattern, being found either in the cytosol or in the cytosol and the nucleus. TN patients with gal-8-positive nuclei had significantly better disease-free survival (DFS), distant-disease-free survival (DDFS), and overall survival (OS). In contrast, high expression of nuclear gal-1 correlated with poor DDFS and OS. TNBC patients who were positive for both nuclear gal-1 and gal-8 had 5-year DFS and DDFS of 100%, suggesting a dominance of the gal-8 phenotype. Overall, the results indicate that specific galectin expression signatures contribute to the phenotypic heterogeneity of aggressive subtypes of breast cancer. Our data also suggest that galectins have clinical utility as indicators of disease progression and therapeutic targets in aggressive molecular subtypes of breast cancer.

Resistance to apoptosis induced by anti-cancer drugs is a major obstacle for the treatment of aggressive forms of breast cancer. Galectin-7 was recently shown to be specifically expressed in basal-like and HER-2 positive but not in luminal subtypes of human breast cancer. Galectin-7 also increases the metastatic behavior of breast cancer cells since overexpression of this protein in poorly metastatic breast cancer cells increases their ability to metastasize to the bone and to the lung. This pro-tumoral activity of galectin-7 in breast cancer is surprising since galectin-7 had been previously associated with activation of p53 and breast cancer cells often harbor a transcriptionally inactive form of p53. We have recently reconcile this apparent contradiction by showing that elevated levels of galectin-7 in breast cancer cells occurs via a gain-of-function mechanism of mutant p53. In p53-null breast cancer cells, we have shown that C/EBPβ-2 (also known as LAP2), the most transcriptionally active of the C/EBPβ isoforms, is responsible for the upregulation of galectin-7. How galectin-7 contributes to the progression of breast cancer remains unclear. Up to now, regulation of apoptosis by intracellular galectins has been largely attributed to their ability to translocate to mitochondria, possibly following their interaction with bcl-2. Here, we report that a mutant of galectin-7 that is unable to translocate to mitochondria induces resistance of human breast cancer cells to apoptosis induced by etoposide or by hypoxia-mimicking conditions. Surprisingly, this mutant and the wild-type form of galectin-7 bind equally well to bcl-2 in vitro and in vivo. Interestingly, both forms decreases the translocation of p53 to the nucleus and reduce the expression of p21 following treatment with doxorubicin. We also found that galectin-7 was released by breast cancer cells and that recombinant galectin-7 increased apoptosis of monocytes, T CD4+ and T CD8+ cells. This suggests that galectin-7 contributes to the establishment of an immunosuppressive tumor microenvironment by killing helper and effector T cells. Taken together, these results challenges the current paradigm that mitochondrial galectins are important for resistance to apoptosis and call for a greater focus on the role of galectin-7 in breast cancer. They also indicate that targeting both cytosolic and extracellular galectin-7 could improve the efficacy of anti-cancer drugs for the treatment of aggressive forms of breast cancer. Citation Format: Yves St-Pierre, Andrée-Anne Grosset, Marilyne Labrie, Donald Gagné, Maria-Claudia Vladoiu, Louis Gaboury, Nicolas Doucet. Galectin-7 increases resistance of breast cancer cells to drug-induced apoptosis and promotes tumor escape by killing T cells [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-07-16.

Hematoxylin and eosin (H&E) staining is a well-established technique in histopathology. However, immunohistochemistry (IHC) interpretation is done exclusively with hematoxylin counterstaining. Our goal was to investigate the potential of H&E as counterstaining (H&E-IHC) to allow for visualization of a marker while confirming the diagnosis on the same slide. The quality of immunostaining and the fast-technical performance were the main criteria to select the final protocol. We stained multiple diagnostic tissues with class I IHC tests with different subcellular localization markers (anti-CK7, CK20, synaptophysin, CD20, HMB45, and Ki-67) and with double-staining on prostate tissues with anti-high molecular weight keratins/p63 (DAB detection) and p504s (alkaline phosphatase detection). To validate the efficacy of the counterstaining, we stained tissue microarrays from the Canadian Immunohistochemistry Quality Control (cIQc) with class II IHC tests (ER, PR, HER2, and p53 markers). Interobserver and intraobserver concordance was assessed by κ statistics. Excellent agreement of H&E-IHC interpretation was observed in comparison with standard IHC from our laboratory (κ, 0.87 to 1.00), and with the cIQc reference values (κ, 0.81 to 1.00). Interobserver and intraobserver agreement was excellent (κ, 0.89 to 1.00 and 0.87 to 1.00, respectively). We therefore show for the first time the potential of using H&E counterstaining for IHC interpretation. We recommend the H&E-IHC protocol to enhance diagnostic precision for the clinical workflow and research studies.

Background To investigate the effect of intraductal carcinoma of the prostate (IDC-P) in radical prostatectomy (RP) specimens in the context of the site of recurrence, time to recurrence, and cancer-specific survival in two academic cohorts of locally, regionally, or distantly recurrent prostate cancer. Methods Our cohort included men enrolled into two academic tissue repositories from 1993 to 2011, who were treated with first-line RP who later experienced local recurrence, regional recurrence, or distant metastasis (together termed clinical recurrence, CR). RP material was reviewed to identify IDC-P and to update grading to current standards. The primary endpoint was the initial location of CR. Secondary endpoints included time to CR and cancer-specific survival. Pearson's chi-square, Welch's t-test, Mann-Whitney U test and Fisher's exact test were performed for univariate analyses. Multinomial logistic regression was used for multivariate analyses. Cancer-specific survival was analyzed with the generalized Wilcoxon test and Cox regression. Results Eighty-five patients with CR were included in the analysis. IDC-P was present in 78.5% of patients from Center 1 and 70.0% from Center 2 (P = 0.547). IDC-P was independently associated with distant metastasis at initial CR (multivariate odds ratio = 6.27, P = 0.015). IDC-P status did not affect time to recurrence; median survival without recurrence was at 53 months for IDC-P(+) and at 50 months for IDC-P(-) (P = 0.441). Distant metastases at the initial CR event had a 36% reduction of cancer-specific survival compared to local recurrences (P = 0.007). Additionally, prostatic-bed radiotherapy (adjuvant or salvage for biochemical recurrence before distant metastasis) was associated with a 25% reduction in cancer-specific mortality compared to no radiotherapy (P = 0.023). Similar reduction in cancer-specific mortality was observed in the subgroup of patients with distant metastasis and IDC-P when treated with radiotherapy (29%, P = 0.050). Conclusions In our cohort, presence of IDC-P was an independent factor for distant metastasis at initial CR, but did not have a significant impact on time to CR. Furthermore, metastatic patients showed statistically reduced cancer-specific mortality when treated with radiotherapy. This reduction in cancer-specific mortality was also identified in patients with IDC-P. Future large scale validation studies should take into account the presence of IDC-P and confirm its impact on disease progression.

Prostate cancer is the most frequent diagnosed cancers among men. When prostate cancer occurs, the cancer does not result in only one or few localized malignant tumor, but is generally spread within the whole prostate. In order to counteract the very high level of heterogeneities exhibited by prostate tissues, we developed a method for high-resolution co-registration of Raman spectroscopy with prostate cancer diagnosis. Raman spectra were acquired on fresh ex vivo prostate within 2 hours after radical prostatectomy using a multi-wavelength hand-held contact probe. After the measurements, the prostate was reintegrated to the usual pathological workflow: formalin fixated and paraffin embedded (FFPE), and prepared for microscope histopathological analyses. The precise reconstruction of the prostate slice with hematoxylin and eosin (H and E) tissue allows the spatial correlation of the measured area (0.2 mm2) with the correspondent histopathological information, for point-by-point diagnosis determination. The tissue was classified into groups (normal/cancer) and subgroups according to the percentage of benign glands, stroma or cancer. Different machine learning algorithms were tested to classify the spectra with increasing levels of categorization. Preliminary results showed that Raman spectroscopy is capable of detecting prostate cancer with an accuracy >90%. In addition, high percentages of stroma (vs. glands) have been correlated with spectral signature of collagen, which is the main constituent of extracellular matrix.

Background: There is an urgent need for pathologists to better define patients with high-risk prostate cancer. One of the promising tools is Raman micro-spectroscopy, also known as Raman microscopy, a nondestructive and label-free imaging technique based on light scattered after reflection. Our group has recently developed a rapid standardized protocol for the preparation of formalin-fixed, paraffin-embedded (FFPE) diagnostic tissues suitable for Raman microscopy. The objective of this study was to evaluate the potential of Raman microscopy to assess the prognosis of prostate cancer patients with FFPE tissues from radical prostatectomy. Methods: Patients treated by first-line radical prostatectomy between 1994 and 2004 at Centre hospitalier de l’Université de Montréal (CHUM) were included in this study. FFPE prostate cancer tissues from surgery were used for the construction of tissue microarrays (TMAs). To enable Raman microscopy, TMA sections of 4 µm were placed on low-cost aluminum slides. The rapid dewaxing protocol of the hospital was used (8 minutes), followed by 20 minutes of vacuum drying. All Raman spectra were acquired with the Renishaw inVia confocal Raman microscope equipped with a 785-nm line focus laser. After removing background contributions (e.g., autofluorescence) for each spectrum with Wire 4.4 software, a custom toolbox in MATLAB was used to predict biochemical recurrence. Chemometric methods and calculated ratios were used for the analysis of Raman microscopy images. Results: A total of 320 Raman spectra from 80 patients were analyzed from prostate cancer TMAs, representing 25 patients with biochemical recurrence within 18 months after radical prostatectomy and 55 without. Using a Support Vector Machine (SVM) technique and correlation feature selection for classification, our results with Raman microscopy identified biochemical recurrence with an accuracy of 83.7%, a sensitivity of 84.0% and a specificity of 83.6%. Raman peak assignment of features was used to investigate the molecular differences between these two patient groups. We found that the molecular constituents of RNA and phosphorylated proteins were more important in prostate cancer with biochemical recurrence. In contrast, Raman peaks of the phospholipid head of cell membranes, DNA, and collagen were more intense in prostate cancer without biochemical recurrence. For the visualization of these different molecular constituents of prostate cancer, we developed two methods of Raman microscopy imaging. The first method involved analysis of chemometric data (i.e., extraction of chemical information) to identify the whole tissue (phenylalanine), nuclei (DNA), and red blood cells (hemoglobin), followed by background removal. The images of our chemometric analysis created a virtual staining of hematoxylin and eosin (H&E). The second method involved testing several ratios of Raman peaks associated with proteins, lipids, DNA and RNA. Calculated ratios distinguished specific structures of the prostate tissue, such as the cancerous and normal glands, by different colors. Conclusions: This is the first study demonstrating the potential of Raman microscopy for the prediction of biochemical recurrence within 18 months following radical prostatectomy for prostate cancer. Raman microscopy imaging of tissues is a promising method for the recognition of specific structures, which could help pathologists in the accuracy of diagnosis. The accessibility of this technology to clinicians could be useful for patient follow-up and treatment strategies. Citation Format: Andrée-Anne Grosset, Catherine St-Pierre, Karl St-Arnaud, Kelly Aubertin, Michael Jermyn, Frédéric Leblond, Dominique Trudel. Raman microscopy to assess biochemical recurrence risk after radical prostatectomy [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A014.

For the first time, molecular information deduced from Raman spectroscopy measurements was spatially correlated with histopathologic analysis to detect and characterize prostate cancer (PCa) using Raman spectroscopy (RS). Ex vivo post-prostatectomy specimen were interrogated using a handheld RS probe. Up to 50 areas (of 500µm in diameter each) per prostate slice were correlated spatially with hematoxylin, phloxine, and saffron (HPS) stained images for detailed histopathologic characteristics, including percentage of benign glands and cancer grade group (International Society of Urological Pathology, ISUP): (1) each measured area was identified with a spot of Indian ink; (2) a photograph was taken at the end of the measurements to build a mask including the prostate slice contour and circles corresponding to the measured areas; (3) the whole prostate slice was reconstructed from the HPS scans guided by anatomic landmarks to insure spatial correspondence with the fresh prostate slice photograph. The Raman spectra were analyzed in terms of univariate statistics of specific Raman peaks corresponding to biomolecules such as DNA/RNA, amino acids, or lipids. In addition, the spectra were submitted to multivariate statistical classification using machine learning algorithms (artificial neural network). In order to test the validity of ex vivo conditions regarding intraoperative (i.e., in vivo) applications, we measured and compared different rat organs (including brain, prostate, ovarian, spleen, kidney, and liver) in in vivo and ex vivo conditions. Previous studies about the use of RS for PCa detection were done exclusively on cell lines or snap-frozen human tissue by considering the overall patient diagnosis. PCa tissues are highly heterogeneous morphologically: cancer glands are intermingled with benign glands and tumors are often multiclonal. Therefore, local histopathologic criteria must be considered. We designed a method that allows us to correlate spatially each Raman spectrum with precise diagnostic criteria and we were able to distinguish extraprostatic from prostatic tissue with an accuracy of 82% (sensibility of 82%, specificity of 83%), malignant from benign prostatic tissue with an accuracy over 86% (sensibility of 87%, specificity of 86%), and different cancer grade groups, for instance, with an accuracy of 84% for distinguishing ISUP grade group 1 from 5. When comparing in vivo with ex vivo rat tissues, preliminary results showed that the difference between average spectra is of the same magnitude than the variability within the same tissue type (same organ from different rats), therefore meaning that ex vivo RS data could be used to train classifier for in vivo diagnosis. Thanks to our robust methodology spatially correlating the optical measurements with local histopathologic characteristics, our work on ex vivo human prostates has proved that we have the capability to characterize the heterogeneity of prostate tissue in the context of PCa. In addition, we established that the use of ex vivo specimen to mimic intraoperative condition is valid. These are promising results, especially in the context of optical guided biopsy procedure to help collect targeted tissue to decrease the false-negative rate nowadays associated with the procedure (~30%). Citation Format: Kelly Aubertin, Mirela Birlea, Michael Pinto, Karl St-Arnaud, Vincent Quoc Trinh, Andrée-Anne Grosset, Frédéric Leblond, Dominique Trudel. Towards the use of Raman spectroscopy to guide prostate biopsy procedure [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B063.

Purpose: Although the murine orthologue of glycoprotein nonmetastatic B (GPNMB), Osteoactivin, promotes breast cancer metastasis in an in vivo mouse model, its importance in human breast cancer is unknown. We have examined the significance of GPNMB expression as a prognostic indicator of recurrence and assessed its potential as a novel therapeutic target in breast cancer. Experimental Design: The clinical significance of GPNMB expression in breast cancer was addressed by analyzing GPNMB levels in several published gene expression data sets and two independent tissue microarrays derived from human breast tumors. GPNMB-expressing human breast cancer cell lines were further used to validate a toxin-conjugated anti-GPNMB antibody as a novel therapeutic agent. Results: GPNMB expression correlates with shorter recurrence times and reduced overall survival of breast cancer patients. Epithelial-specific GPNMB staining is an independent prognostic indicator for breast cancer recurrence. GPNMB is highly expressed in basal and triple-negative breast cancers and is associated with increased risk of recurrence within this subtype. GPNMB expression confers a more migratory and invasive phenotype on breast cancer cells and sensitizes them to killing by CDX-011 (glembatumumab vedotin), a GPNMB-targeted antibody-drug conjugate. Conclusions: GPNMB expression is associated with the basal/triple-negative subtype and is a prognostic marker of poor outcome in patients with breast cancer. CDX-011 (glembatumumab vedotin) is a promising new targeted therapy for patients with metastatic triple-negative breast cancers, a patient population that currently lacks targeted-therapy options. Clin Cancer Res; 16(7); 2147–56. ©2010 AACR.

// Maria Claudia Vladoiu 1 , Marilyne Labrie 1 , Myriam Letourneau 1 , Philippe Egesborg 1 , Donald Gagne 1 , Etienne Billard 1 , Andree-Anne Grosset 1 , Nicolas Doucet 1 , David Chatenet 1 , Yves St-Pierre 1 1 INRS-Institut Armand-Frappier, Universite du Quebec, Laval, Quebec, H7V 1B7 Canada Correspondence to: Yves St-Pierre, e-mail: yves.st-pierre@iaf.inrs.ca Keywords: galectin, inhibitor, peptide, apoptosis, T-cells Received: May 13, 2015 Accepted: August 20, 2015 Published: October 01, 2015 ABSTRACT Galectins are small soluble lectins that bind α-galactosides via their carbohydrate recognition domain (CRD). Their ability to dimerize is critical for the crosslinking of glycoprotein receptors and subsequent cellular signaling. This is particularly important in their immunomodulatory role via the induction of T-cell apoptosis. Because galectins play a central role in many pathologies, including cancer, they represent valuable therapeutic targets. At present, most inhibitors have been directed towards the CRD, a challenging task in terms of specificity given the high structural homology of the CRD among galectins. Such inhibitors are not effective at targeting CRD-independent functions of galectins. Here, we report a new class of galectin inhibitors that specifically binds human galectin-7 (hGal-7), disrupts the formation of homodimers, and inhibits the pro-apoptotic activity of hGal-7 on Jurkat T cells. In addition to representing a new means to achieve specificity when targeting galectins, such inhibitors provide a promising alternative to more conventional galectin inhibitors that target the CRD with soluble glycans or other small molecular weight allosteric inhibitors.

International audience; The observation that galectin-7 (gal-7) is specifically expressed in mammary myoepithelial (basal) cells prompted us to investigate whether this protein is expressed in the basal cells of other tissues. Given that breast and prostate cancer have remarkable underlying biological similarities and given the important roles of basal cells in prostate cancer, we examined the expression patterns and role of gal-7 in human prostate cancer. Using tissue microarray, we found that although gal-7 is readily expressed in basal cells in normal prostate tissue, it is downregulated in prostate cancer (PCa) cells. De novo expression of gal-7 in prostate cancer cells increases their sensitivity to apoptosis in response to etoposide and cisplatin. The assessment of a carbohydrate-recognition domain (CRD)-defective mutant form of gal-7 (R7S) showed that the ability of this protein to modulate apoptosis was independent of its CRD activity. This activity was also independent of its ability to translocate to the mitochondrial and nuclear compartments. However, CRD activity was necessary to inhibit the invasive behaviors of prostate cancer cells. In vivo, gal-7 overexpression in PCa cells led to a modest yet significant reduction in tumor size, while its CRD-defective mutant form significantly increased tumor growth compared to controls. Taken together, these results suggest that although de novo expression of gal-7 may be an interesting means of increasing the tumorigenic phenotypes of PCa cells, alterations in the CRD activity of this protein drive a phenotypic switch in its role in PCa cells. This CRD-independent activity represents a paradigm shift in our understanding of the functions of galectin. The R74S model will be useful to distinguish CRD-dependent and CRD-independent functions of gal-7 in cancer progression.

80 Background: The CPCBN has assembled a TMA-based resource comprising the specimens of 1512 prostate cancer patients treated by radical prostatectomy. This richly annotated and multi-institutional resource is available to researchers who wish to access a large cohort to validate prognostic biomarkers (http://www.tfri.ca/en/research/translational-research/cpcbn.aspx). Over the last decade, our laboratory demonstrated with an independent cohort (Gannon PO, et al. Eur J Cancer. 2013 Jul;49(10):2441-8, Labouba I, et al. PLoS One. 2015 Jul 17;10(7):e0131024), the reproducible association of nuclear factor-kappa B (NF-kB) p65 with patient’s risk of biochemical recurrence. Here, we evaluated the CPCBN TMA-series for p65 expression. Methods: Two independent observers scored the frequency of p65 nuclear localisation on digital images obtained after automated immunohistochemistry analysis of p65. Over the available minimum 3 cores of tumour tissue per patient, an average percentage of positive nucleus frequency was used. Statistical analyses were performed using SPSS software. Results: High nuclear frequency of NF-kB p65 (cut-off at 3%) was associated with an increased risk for patients to experience a biochemical relapse (p < 0.001; Exp(B) = 1.599; 95%CI = 1.321-1.937), develop bone metastasis (p = 0.007; Exp(B) = 2.126; 95%CI = 1.234-3.663)and die from their disease (p = 0.001; Exp(B) = 3.117; 95%CI = 1.55-6.266). In multivariate analyses, p65 also remained independent from clinical parameters (PSA, Gleason score and pTNM): biochemical relapse (p = 0.005; Exp(B) = 1.331; 95%CI = 1.092-1.623), bone metastasis (p = 0.033; Exp(B) = 1.82; 95%CI = 1.04- 3.158), mortality (p = 0.007; Exp(B) = 2.626; 95%CI = 1.298-5.312) Conclusions: Using a large cohort of Canadian men, our study reiterates the previous study linking NF-kB p65 with prostate cancer progression and highlights the suitability of CPCBN TMAs for biomarker validation. Our results also reveal the role of p65 as a predictor of bone metastasis development and prostate cancer-specific mortality.

International audience; The identification of galectin-7 as a p53-induced gene and its ability to induce apoptosis in many cell types support the hypothesis that galectin-7 has strong antitumor activity. This has been well documented in colon cancer. However, in some cases, such as breast cancer and lymphoma, its high expression level correlates with aggressive subtypes of cancer, suggesting that galectin-7 may have a dual role in cancer progression. In fact, in breast cancer, overexpression of galectin-7 alone is sufficient to promote metastasis to the bone and lung. In the present work, we investigated the expression and function of galectin-7 in melanoma. An analysis of datasets obtained from whole-genome profiling of human melanoma tissues revealed that galectin-7 mRNA was detected in more than 90% of biopsies of patients with nevi while its expression was more rarely found in biopsies collected from patients with malignant melanoma. This frequency, however, was likely due to the presence of normal epidermis tissues in biopsies, as shown our studies at the protein level by immunohistochemical analysis. Using the experimental melanoma B16F1 cell line, we found that melanoma cells can express galectin-7 at the primary tumor site and in lung metastasis. Moreover, we found that overexpression of galectin-7 increased the resistance of melanoma cells to apoptosis while inducing de novo egr-1 expression. Overexpression of galectin-7, however, was insufficient to modulate the growth of tumors induced by the subcutaneous injection of B16F1 cells. It also failed to modulate the dissemination of B16F1 cells to the lung.

Prostate cancer (PCa) is the most frequently diagnosed cancer among men. PCa diagnosis is currently based on analyses of the morphological organization of tissue. The process from tissue collection to diagnosis is time consuming and can be too long for intraoperative diagnosis. Raman spectroscopy (RS) is a label-free and non-destructive laser-based technique that provides real-time molecular information about tissue. Histologically, cancer patterns are intermingled with entrapped benign glands. Because of the very high level of heterogeneity exhibited by prostate tissues, it is highly important to identify precisely the tissue type measured by RS. To this aim, we developed a method for high-resolution co-registration of RS with PCa diagnosis.Raman spectra are acquired on fresh ex vivo prostates within 2 hours after radical prostatectomy using a multi-wavelength hand-held contact Raman probe. The prostate is then reintegrated to the usual clinical pathological workflow for formalin fixation and paraffin embedding (FFPE) and preparation for optic microscope histopathological analyses. The precise reconstruction of the prostate slice with hematoxylin & eosin (H&E) tissue allows the spatial correlation of the measured areas (0.2 mm2) with the correspondent histopathological information, for point-by-point diagnosis. The tissue is then classified into groups (benign/malignant) and subgroups according to the percentage of benign glands, stroma or cancer grades.Different machine learning algorithms were tested to classify the spectra with different levels of categorization. Preliminary results showed that RS is capable of detecting PCa with an accuray of 89%. Within the benign tissue group, high stroma/glands rations have been correlated with the spectral signature of collagen, which is the main constituent of stroma. Within the cancer tissue group, high grade patterns were find to be different from low grade patterns, demonstrating that RS is able to distinguish aggressive from less aggressive cancers.

Introduction CT10 regulator of kinase (Crk) adaptor proteins (CrkI, CrkII and CrkL) play a role in integrating signals for migration and invasion of highly malignant breast cancer cell lines. This has important implications, as elevated CrkI/II protein levels were observed in a small cohort of breast cancer patients, which identified a potential role for Crk proteins in breast cancer progression. Numerous in vitro studies identified a role for Crk proteins in cell motility, but little is known about how Crk proteins contribute to breast cancer progression in vivo. Methods The clinical significance of Crk proteins in human breast cancer was assessed by analyzing published breast cancer datasets using a gene expression signature that was generated following CrkII over-expression and by examining Crk protein expression in tissue microarrays of breast tumors (n = 254). Stable knockdown of Crk (CrkI/CrkII/CrkL) proteins was accomplished using a short hairpin RNA (shRNA)-mediated approach in two basal breast cancer cell lines, MDA-231 1833TR and SUM1315, where the former have a high affinity to form bone metastases. Both in vitro assays (cell migration, invasion, soft agar growth) and in vivo experiments (intra-cardiac, tibial and mammary fat pad injections) were performed to assess the functional significance of Crk proteins in breast cancer. Results A gene signature derived following CrkII over-expression correlated significantly with basal breast cancers and with high grade and poor outcome in general. Moreover, elevated Crk immunostaining on tissue microarrays revealed a significant association with highly proliferative tumors within the basal subtype. RNAi-mediated knockdown of all three Crk proteins in metastatic basal breast cancer cells established a continued requirement for Crk in cell migration and invasion in vitro and metastatic growth in vivo. Furthermore, Crk ablation suppressed anchorage independent growth and in vivo orthotopic tumor growth. This was associated with diminished cell proliferation and was rescued by expression of non-shRNA targeted CrkI/II. Perturbations in tumor progression correlated with altered integrin signaling, including decreased cell spreading, diminished p130Cas phosphorylation, and Cdc42 activation. Conclusions These data highlight the physiological importance of Crk proteins in regulating growth of aggressive basal breast cancer cells and identify Crk-dependent signaling networks as promising therapeutic targets.

The galectins are a family of evolutionay-conserved carbohydrate-binding proteins. They are distributed widely in all living organisms and have been implicated in many essential functions including development, differentiation, cell-cell adhesion, cell-matrix interaction, growth regulation, apoptosis. Several members of the galectin family have also been shown to be involved in cancer progression and metastasis. In the case of galectin-7, several studies have reported alterations in its expression pattern during cancer progression. In a variety of tumors, its expression can range from being completely down-regulated to highly up-regulated. Accordingly, its precise role in this field is still debated. The evidence shows that galectin-7 may promote or inhibit cancer development. In this article, we review the data concerning expression and roles of galectin-7 in cancer and propose a comprehensive view of its contribution during cancer progression.

International audience; Galectins are members of a family of beta-galactosides-binding proteins that have recently emerged as novel modulators in different aspects of cancer. The expression of galectins in tumors and/or the tissue surrounding them has been well documented. Since galectin-7 expression has been associated with epithelial tissues and varies significantly in various types of cancer, we have investigated for the first time its role in breast cancer. Using two preclinical mouse models, high levels of galectin-7 expression in breast cancer cells drastically increased their ability to metastasize to lungs and bones. Significant increases in the number of pulmonary metastases and osteolytic lesions were induced by overexpression of galectin-7 compared with control cells. In human tissues, galectin-7 was specifically found in myoepithelial cells of normal human breast tissue, but not in luminal cells. Its expression was severely altered in breast carcinoma, many samples showing greater than 70% of galectin-7 positive cells. High expression levels of galectin-7 were restricted to high-grade breast carcinomas, including HER2 overexpressing and basal-like groups. In HER2 overexpressing cases, galectin-7 expression was associated with lymph node axillary metastasis. Taken together, our results indicate that galectin-7 may represent a potential target for both specific detection and therapeutic inhibition of metastatic breast cancer.

Resistance to apoptosis induced by anti-cancer drugs is a major obstacle for the treatment of aggressive forms of breast cancer. Galectin-7 was recently shown to be specifically expressed in basal-like and HER-2 positive but not in luminal subtypes of human breast cancer. Overexpression of galectin-7 in breast cancer cells also increases lung and bone metastases. However, the molecular mechanisms used by galectin-7 to increase the dissemination of breast cancer cells remain largely unknown. Although galectins are best known for their ability to bind cell surface receptors, there is increasing evidence that their intracellular forms have important functions in cancer cells. This is particularly true in the control of apoptosis. Up to now, regulation of apoptosis by intracellular galectins has been largely attributed to their ability to translocate to mitochondria, possibly following their interaction with bcl-2. Here, we report that a mutant of galectin-7 that is unable to translocate to mitochondria and nucleus induces resistance of human breast cancer cells to apoptosis induced by etoposide or by hypoxia-mimicking conditions. Surprisingly, this mutant and the wild-type form of galectin-7 bind equally well to bcl-2 in vitro and in vivo. Interestingly, both forms decreases the translocation of p53 to the nucleus and reduce the expression of p21 following treatment with doxorubicin. These results challenges the current paradigm that mitochondrial galectins are important for resistance to apoptosis and call for a greater focus on the role of cytosolic galectins in cancer cells. Our data also indicate that targeting cytosolic galectin-7 could improve the efficacy of anti-cancer drugs for the treatment of aggressive forms of breast cancer. Citation Format: Andrée-Anne Grosset, Donald Gagné, Marilyne Labrie, Maria Vladoiu, Louis Gaboury, Nicolas Doucet, Yves St-Pierre. Cytoplasmic galectin-7 has an antiapoptotic function by decreasing p53 nuclear translocation in breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1352. doi:10.1158/1538-7445.AM2014-1352

Ovarian cancer is the fifth leading cause of cancer-related deaths in the Western world, the second most common gynecological cancer and the leading cause of death from gynecological malignancies. Epithelial ovarian cancer (EOC) accounts for the majority of ovarian cancers. Here, we have investigated the role of galectin-7 in EOC. Galectin-7 is an epithelial cell marker which has been shown to play an important role in the progression of many types of cancer, including breast cancer and colon cancer. Galectins play an important role in several physiological processes, including cell migration, programmed cell death and regulation of the immune response. They are also involved in a number of pathological conditions, including cancer. In fact, galectins have been shown to create an immunosuppressive tumor microenvironment by killing helper and effector T cells. They can also bind to the surface of tumor cells to form cell surface lattice that stabilizes the expression of key growth factor receptors, thereby increasing cell growth, motility, and production of matrix metalloproteinases. Using tissue microarrays, we found that gal-7 expression increases in malignant tumors compared to normal tissues and benign tumors. Detection of galectin-7 was also more frequent in tissue samples with metastasis as compared to non-metastatic primary tumor samples. Western blot analysis showed that OVCAR-3 EOC cells secrete gal-7, while no expression was detected in COV-434, A2780 and SK-OV-3 cell lines. Treatment of EOC cells with recombinant gal-7 increased their invasive behavior through matrigel and their ability to express MMP-9, an extracellular protease which confers an invasive phenotype to cancer cells. Treatment of freshly isolated human PBMCs with gal-7 also showed gal-7 increased apoptosis of monocytes, T CD4+ and T CD8+ cells. Overall, these data provide impetus for further studies to determine the potential of gal-7 as a therapeutic target for the treatment of EOC. Citation Format: Marilyne Labrie, Maria Claudia Vladoiu, Andrée-Anne Grosset, Louis Gaboury, Yves St-Pierre. Galectin-7 increases the invasive behavior of ovarian cancer cells and promotes tumor escape. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 152. doi:10.1158/1538-7445.AM2014-152

Elevated levels of Crk proteins are observed in human cancers, including breast cancer, identifying a potential role for Crk in tumor progression. However, little is known about how Crk contributes to breast cancer progression in vivo. Crk proteins (CrkI, CrkII, CrkL) can regulate cell signaling downstream of integrins and various receptor tyrosine kinases through the formation of protein-protein complexes. We tested the hypothesis that Crk proteins are key signaling nodes for breast cancer tumorigenesis using shRNA-mediated knockdown of all 3 Crk proteins in highly aggressive human basal breast cancer cell lines. Crk knockdown cells show no differences in cell proliferation under 2D culture conditions, but show significantly decreased growth in soft agar under low serum conditions. This demonstrates that Crk loss imparts a renewed dependence on adhesion and growth factor signaling in highly aggressive breast tumor cell lines and highlights a requirement for Crk dependent signals for anchorage independent growth in vitro. Consistent with this, loss of Crk in basal breast cancer cells inhibited in vivo orthotopic tumor growth in nude mice. Using immunohistochemical staining of cells 3 days post-injection, Crk knockdown diminished proliferation in vivo, but did not alter cell apoptosis when compared to controls. This difference was reflected in the formation of smaller lesions by Crk knockdown cells by 8 days post-injection. Importantly, rescue of Crk protein expression restored in vivo tumor growth, demonstrating a specific requirement for Crk proteins in this process. To evaluate the significance of Crk in human breast cancer, immunohistochemistry was performed on two independent tissue microarrays of human breast tumors (n=209, n=234). Crk protein levels and the proliferative index were assessed by staining with antibodies to Crk and Ki67. Both Crk and Ki67 antigen positivity correlated strongly with high tumor grade (p=0.001, p=2.27e-10). Within both TMA datasets, CrkI/II (R2=0.3855, p=0.0004) and CrkL (R2=0.3845, p=0.0002) protein levels showed a strong positive correlation with cell proliferation within basal tumors, demonstrating a strong link between elevated Crk protein and an aggressive tumor phenotype. In addition to assessing Crk protein levels, a gene expression signature consisting of 151 genes was derived following Crk overexpression in a breast cancer cell line that initially had low levels of Crk. Notably, when this signature was applied to 5 breast cancer gene expression datasets (n=1469 breast cancers) there was a strong correlation with both the basal molecular subtype (p These findings demonstrate that Crk proteins are unexpectedly essential for growth of aggressive human breast cancer cell lines in vivo, and suggest a key role for Crk proteins in basal breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 918. doi:10.1158/1538-7445.AM2011-918

Purpose: While the murine orthologue of GPNMB (Glycoprotein non-metastatic B, DC-HIL, HGFIN), Osteoactivin, has been shown to promote breast cancer metastasis in an in vivo mouse model, its importance in human breast cancer is unknown. We have examined the significance of GPNMB expression as a prognostic indicator of recurrence and assessed its potential as a novel therapeutic target in breast cancer. Experimental Design: The clinical significance of GPNMB expression in breast cancer was addressed by analyzing GPNMB mRNA levels in several published gene expression datasets and by determining GPNMB protein expression in two independent tissue microarrays derived from human breast tumors. Human breast cancer cell lines endogenously or exogenously expressing GPNMB were further used to validate a toxin-conjugated anti-GPNMB antibody as a novel therapeutic agent. Results: GPNMB expression correlates with shorter times to recurrence and reduced overall survival of breast cancer patients. Epithelial-specific staining of GPNMB serves as an independent prognostic indicator for breast cancer recurrence. GPNMB is highly expressed in basal and triple-negative (TN) breast cancers and is associated with increased risk of recurrence within this subtype. GPNMB expression confers a more migratory and invasive phenotype on breast cancer cells. Treatment of mice harbouring endogenous GPNMB-expressing human breast cancer xenografts with CDX-011 (glembatumumab vedotin, CR011-vcMMAE), a GPNMB-targeted antibody-drug conjugate, induces and sustains tumor regression in vivo. Conclusions: GPNMB expression is associated with the basal/TN subtype and is a prognostic marker of poor outcome in patients with breast cancer. CDX-011 (glembatumumab vedotin) is a promising new targeted therapy for patients with metastatic TN breast cancers, a patient population that currently lacks targeted-therapy options. Citation Information: Clin Cancer Res 2010;16(7 Suppl):A8

Background Resistance to apoptosis induced by anti-cancer drugs is a major obstacle for the treatment of aggressive forms of breast cancer. Galectin-7 (gal-7) was recently shown to be specifically expressed in basal-like but not in luminal subtypes of human breast cancer. Methods We generated a mutant form of gal-7 (R74S). Arginine 74 is the structural equivalent of arginine 186 found in human galectin-3. Mutation R186S was previously shown to abolish the biological function of galectin-3. Results Mutation of arginine 74 induced only limited and local changes to the gal-7 fold. Recombinant forms of R74S and wtgal-7 were also equally effective at forming dimers in solution. Analysis of the thermodynamic parameters by isothermal titration calorimetry (ITC) indicated, however, that binding of lactose to gal-7 was inhibited by the R74S mutation. Using confocal microscopy and electron microscopy, we confirmed the expression of gal-7 in the cytosolic and nuclear compartments of breast cancer cells and the ability of gal-7 to translocate to mitochondria. The mutation at position 74, however, greatly reduced the expression of gal-7 in the nuclear and mitochondrial compartments. Interestingly, cells expressing mutated gal-7 were equally if not even more resistant to drug-induced apoptosis when compared to cells expressing wtgal-7. We also found that both wtgal-7 and R74S inhibited dox-induced PARP-1 cleavage and p53 protein expression. The inhibition of p53 correlated with a decrease in p21 protein expression and CDKN1A mRNA. Furthermore, analysis of nuclear and cytoplasmic fractions showed that both wild type and R74S mutant gal-7 inhibited p53 nuclear translocation, possibly by increasing degradation of cytosolic p53. Conclusions These findings pose a challenge to the paradigm that has guided the design of galectin-specific inhibitors for the treatment of cancer. This study suggests that targeting CRD-independent cytosolic gal-7 in breast cancer cells may be a valuable strategy for the treatment of this disease. Our study will thus complement efforts towards improving selectivity of targeted anticancer agents. Electronic supplementary material The online version of this article (doi:10.1186/1471-2407-14-801) contains supplementary material, which is available to authorized users.