Kallikreins Research Articles

Kallikrein 5 overexpression is associated with poor prognosis in uterine cervical cancer.

ObjectiveKallikrein 5 (KLK5), which is frequently observed in normal cervico-vaginal fluid, is known to be related to prognosis in several solid tumors. We investigated the prognostic significance of KLK5 in uterine cervical cancer using tumor tissue microarray and immunohistochemistry staining.MethodsWe analyzed samples of 165 patients with uterine cervical cancer who received definitive radiation therapy between 2004 and 2012. We divided patients into two groups stratified by their KLK5 activity by immunohistochemistry staining: negative/weak (0–1+) (n=120 patients) and moderate/strong (2–3+) group (n=45 patients). Patient and tumor characteristics, patterns of failure, and survival outcomes were compared. Univariable and multivariable analyses were performed to identify prognostic factors.ResultsPatients with KLK5 2–3+ were younger (median: 52 vs. 60 years) and had frequent paraaortic lymph node involvement (40.0% vs. 18.3%) than those with KLK5 0–1+. With a median follow-up of 60.8 (interquartile range, 47.5–77.9) months, patients with KLK5 2–3+ had inferior 5-year locoregional recurrence-free survival and distant metastasis-free survival of 61.7% (vs. 77.5% in KLK5 0–1+ group) and 59.4% (vs. 72.8% in the KLK5 0–1+ group), respectively (all p<0.05). KLK5 2–3+ expression retained its significance after adjusting for other well-known prognostic factors of tumor size and stage in multivariable analysis.ConclusionsKLK5 overexpression is associated with the aggressiveness of cervical cancer and may underlie the diminished response to conventional treatments. Therefore, KLK5 could be a reliable prognostic factor in cervical cancer.

Hyaluronan Oligosaccharides Improve Rosacea-Like Phenotype through Anti-Inflammatory and Epidermal Barrier-Improving Effects.

BackgroundRosacea is a common skin disease associated with increased expression of cathelicidin, kallikrein 5 (KLK5), toll-like receptor (TLR) 2, and abnormal barrier function. Recently, it was reported that hyaluronan (HA) could influence immune function via various receptors and HA oligosaccharides (oligo-HAs) could suppress TLR-dependent cytokine expression.ObjectiveWe investigated if oligo-HAs could influence on inflammation and epidermal barrier induced by LL-37, which had a major role in rosacea.MethodsWe cultured normal human keratinocytes and treated them with LL-37 and oligo-HAs or the LL-37 alone. A rosacea-like BALB/c mouse model injected with LL-37 was used to determine the role of oligo-HAs in rosacea in vivo.ResultsInterleukin-8 (IL-8) and tumor necrosis factor (TNF)-α release was suppressed when keratinocytes were co-treated with oligo-HAs and LL-37 compared with keratinocytes treated with LL-37 only. Treatment with oligo-HAs resulted in decreased transepidermal water loss as well as improved redness. Decreased inflammatory cell infiltration, IL-17A and KLK5 expression and increased CD44 and filaggrin expression were also noted.ConclusionOur findings suggest that oligo-HA improves rosacea-like phenotype through anti-inflammatory and epidermal barrier improving effect.

Kallikrein 7 Promotes Atopic Dermatitis-Associated Itch Independently of Skin Inflammation

Atopic dermatitis (AD) is a highly prevalent, itchy inflammatory skin disorder that is thought to arise from a combination of skin barrier defect and immune dysregulation. Kallikreins (KLK), a family of serine proteases with a diverse array of homeostatic functions, including skin desquamation and innate immunity, are hypothesized to contribute to AD pathogenesis. However, their precise role in AD has not been clearly defined. In this study, RNA sequencing analyses identified KLK7 as the most abundant and differentially expressed KLK in both human AD and murine AD-like skin. Further, in mice, Klk7 expression was localized to the epidermis in both steady state and inflammation. Unexpectedly, KLK7 was dispensable for the development of AD-associated skin inflammation. Instead, KLK7 was selectively required for AD-associated chronic itch. Even without the alleviation of skin inflammation, KLK7-deficient mice exhibited significantly attenuated scratching, compared with littermate controls, after AD-like disease induction. Collectively, our findings indicate that KLK7 promotes AD-associated itch independently from skin inflammation and reveal a previously unrecognized epidermal-neural mechanism of AD associated itch.

Compound K improves skin barrier function by increasing SPINK5 expression

BackgroundThe skin acts as a barrier to protect organisms against harmful exogenous agents. Compound K (CK) is an active metabolite of ginsenoside Rb1, Rb2 and Rc, and researchers have focused on its skin protective efficacy. In this study, we hypothesized that increased expression of the serine protease inhibitor Kazal type-5 (SPINK5) may improve skin barrier function. MethodsWe screened several ginsenosides to increase SPINK5 gene promoter activity using a transactivation assay and found that CK can increase SPINK5 expression. To investigate the protective effect of CK on the skin barrier, RT-PCR and Western blotting were performed to investigate the expression levels of SPINK5, kallikrein 5 (KLK5), KLK7 and PAR2 in UVB-irradiated HaCaT cells. Measurement of transepidermal water loss (TEWL) and histological changes associated with the skin barrier were performed in a UVB-irradiated mouse model and a 1-chloro-2,4-dinitrobenzene (DNCB)-induced atopic dermatitis–like model. ResultsCK treatment increased the expression of SPINK5 and decreased the expression of its downstream genes, such as KLKs and PAR2. In the UVB-irradiated mouse model and the DNCB-induced atopic dermatitis model, CK restored increased TEWL and decreased hydration and epidermal hyperplasia. In addition, CK normalized the reduced SPINK5 expression caused by UVB or DNCB, thereby restoring the expression of the proteins involved in desquamation to a level similar to normal. ConclusionsOur data showed that CK contributes to improving skin-barrier function in UVB-irradiated and DNCB-induced atopic dermatitis–like models through SPINK5. These results suggest that therapeutic attempts with CK might be useful in treating barrier-disrupted diseases.

Design and development of a series of borocycles as selective, covalent kallikrein 5 inhibitors.

The connection between Netherton syndrome and overactivation of epidermal/dermal proteases, particularly Kallikrein 5 (KLK5) has been well established and it is expected that a KLK5 inhibitor would improve the dermal barrier and also reduce the pain and itch that afflict Netherton syndrome patients. One of the challenges of covalent protease inhibitors has been achieving selectivity over closely related targets. In this paper we describe the use of structural insight to design and develop a selective and highly potent reversibly covalent KLK5 inhibitor from an initial weakly binding fragment.

Inhibitory Effects of Ceramide From the Seeds of Brassica napus L. on the Atopic Function via the Regulation of Human Kallikrein 5 and 7 Protease

The stratum corneum tryptic enzyme kallikrein 5 (KLK5) is a serine protease that is involved in the cell renewal and maintenance of the skin barrier functions. The excessive activation of KLK5 causes an exacerbation of dermatoses, such as rosacea and atopic dermatitis. Brassica napus play a well-known role in the treatment of canola oil through their anti-oxidative and DNA protective properties. We aimed to investigate whether the bioactive ceramide modulate the KLK5 protease. The ceramides were evaluated using an enzymatic assay to measure the anti-KLK5 activity. Our study revealed that the ceramides modulate the KLK5 and 7 protease activity. Ceramides may affect the skin barrier and atopic function via the regulation of proteases.

426 Type I interferon-driven pathogenic angiogenesis initiated by antimicrobial killing of B.oleronius during flare ups of rosacea

Rosacea is a common inflammatory skin disorder characterised by recurrent flare-ups and neoangiogenesis, eventually culminating in persistent erythema. Cathelicidin antimicrobial peptides and their upstream activating protease Kallikrein 5 (KLK5) have been implicated in rosacea, yet the pathogenic mechanisms that lead to flare-ups remain elusive. In gene expression analyses, we find selective overexpression of type I interferon (IFN) specifically during acute flare-ups of disease. Using an established in vivo model of rosacea, we find that IFN expression in situ, critically dependent on plasmacytoid dendritic cells (pDCs), drives a predominant TH17/22 profile, similar to the signature found in our patient cohort. Transgenic mice overexpressing KLK5 in the skin recapitulate the cathelicidin-pDC-IFN-TH17/22 axis, in support of a consistent overarching pathway. Finally, we find that IFN cooperates with IL22 to promote survival and proliferation of vascular endothelial cells, and in the sprouting of dermal microvasculature in vivo. Furthermore, B.oleronius, a bacterium that is linked to severity in rosacea, is exquisitely sensitive to killing by cathelicidins leading to potent activation of pDCs to produce IFN in vitro and in vivo. Taken together, proteolytic activation of cathelicidins, along with skin microbes lead to rapid pDC activation and IFN production during flare-ups of rosacea. The latter plays a critical role in inducing a predominant TH17/22 profile promoting pathogenic angiogenesis. Our study identifies a pathogenic KLK5-cathelicidin-pDC-IFN-IL22 axis in rosacea and mechanistically links B.oleronius to the induction of characteristic rosacea skin lesions, providing novel actionable targets for treatment along this pathway.

Kallikrein 12 Regulates Innate Resistance of Murine Macrophages against Mycobacterium bovis Infection by Modulating Autophagy and Apoptosis.

Mycobacterium bovis (M. bovis) is a member of the Mycobacterium tuberculosis (Mtb) complex causing bovine tuberculosis (TB) and imposing a high zoonotic threat to human health. Kallikreins (KLKs) belong to a subgroup of secreted serine proteases. As their role is established in various physiological and pathological processes, it is likely that KLKs expression may mediate a host immune response against the M. bovis infection. In the current study, we report in vivo and in vitro upregulation of KLK12 in the M. bovis infection. To define the role of KLK12 in immune response regulation of murine macrophages, we produced KLK12 knockdown bone marrow derived macrophages (BMDMs) by using siRNA transfection. Interestingly, the knockdown of KLK12 resulted in a significant downregulation of autophagy and apoptosis in M. bovis infected BMDMs. Furthermore, we demonstrated that this KLK12 mediated regulation of autophagy and apoptosis involves mTOR/AMPK/TSC2 and BAX/Bcl-2/Cytochrome c/Caspase 3 pathways, respectively. Similarly, inflammatory cytokines IL-1β, IL-6, IL-12 and TNF-α were significantly downregulated in KLK12 knockdown macrophages but the difference in IL-10 and IFN-β expression was non-significant. Taken together, these findings suggest that upregulation of KLK12 in M. bovis infected murine macrophages plays a substantial role in the protective immune response regulation by modulating autophagy, apoptosis and pro-inflammatory pathways. To our knowledge, this is the first report on expression and the role of KLK12 in the M. bovis infection and the data may contribute to a new paradigm for diagnosis and treatment of bovine TB.

Mechanisms of itching in mycosis fungoides: grade of itching correlates with eosinophil infiltration and kallikrein 5 expression.

Mycosis fungoides (MF) is the most common variant of cutaneous T-cell lymphomas (CTCL). Itching can be a major symptom for patients with CTCL, however, itching associated with MF is not relieved by conventional therapy using anti-histamines, suggesting that histamine is not the main pruritogen. Therefore, the underlying mechanisms of itching in MF patients remain unclear. To investigate the clinical and histopathological features associated with MF-related itching. Skin sections from MF patients and healthy subjects were used for pathophysiological analysis and evaluation of protease activity. These results were compared with the degree of itching. Of the MF patients, 40% did not report itching and 60% reported itching (moderate itching: 40%; strong itching: 20%). The number of eosinophils, but not mast cells, that infiltrated into skin was increased in the group with strong itching. In the skin of patients, both serine protease activity and immunoreactivity to kallikrein 5 (KLK5), a known itch mediator, increased relative to the grade of itching. These results suggest that KLK5 and eosinophil infiltration may be involved in itching in patients with MF.

Evaluation of a crystallographic surrogate for kallikrein 5 in the discovery of novel inhibitors for Netherton syndrome.

The inhibition of kallikrein 5 (KLK5) has been identified as a potential strategy for treatment of the genetic skin disorder Netherton syndrome, in which loss-of-function mutations in the SPINK5 gene lead to down-regulation of the endogenous inhibitor LEKTI-1 and profound skin-barrier defects with severe allergic manifestations. To aid in the development of a medicine for this target, an X-ray crystallographic system was developed to facilitate fragment-guided chemistry and knowledge-based drug-discovery approaches. Here, the development of a surrogate crystallographic system in place of KLK5, which proved to be challenging to crystallize, is described. The biochemical robustness of the crystallographic surrogate and the suitability of the system for the study of small nonpeptidic fragments and lead-like molecules are demonstrated.

Kallikrein 5 inhibitors identified through structure based drug design in search for a treatment for Netherton Syndrome

Netherton syndrome (NS) is a rare and debilitating severe autosomal recessive genetic skin disease with high mortality rates particularly in neonates. NS is caused by loss-of-function SPINK5 mutations leading to unregulated kallikrein 5 (KLK5) and kallikrein 7 (KLK7) activity. Furthermore, KLK5 inhibition has been proposed as a potential therapeutic treatment for NS. Identification of potent and selective KLK5 inhibitors would enable further exploration of the disease biology and could ultimately lead to a treatment for NS. This publication describes how fragmentation of known trypsin-like serine protease (TLSP) inhibitors resulted in the identification of a series of phenolic amidine-based KLK5 inhibitors 1. X-ray crystallography was used to find alternatives to the phenol interaction leading to identification of carbonyl analogues such as lactam 13 and benzimidazole 15. These reversible inhibitors, with selectivity over KLK1 (10–100 fold), provided novel starting points for the guided growth towards suitable tool molecules for the exploration of KLK5 biology.

Crystal structures of the complex of a kallikrein inhibitor from Bauhinia bauhinioides with trypsin and modeling of kallikrein complexes.

Structures of a recombinant Kunitz-type serine protease inhibitor from Bauhinia bauhinioides (BbKI) complexed with bovine trypsin were determined in two crystal forms. The crystal structure with the L55R mutant of BbKI was determined in space group P64 at 1.94 Å resolution and that with native BbKI in the monoclinic space group P21 at 3.95 Å resolution. The asymmetric unit of the latter crystals contained 44 independent complexes, thus representing one of the largest numbers of independent objects deposited in the Protein Data Bank. Additionally, the structure of the complex with native BbKI was determined at 2.0 Å resolution from P64 crystals isomorphous to those of the mutant. Since BbKI has previously been found to be a potent inhibitor of the trypsin-like plasma kallikrein, it was also tested against several tissue kallikreins. It was found that BbKI is a potent inhibitor of human tissue kallikrein 4 (KLK4) and the chymotrypsin-like human tissue kallikrein 7 (KLK7). Structures of BbKI complexed with the catalytic domain of human plasma kallikrein were modeled, as well as those with KLK4 and KLK7, and the structures were analyzed in order to identify the interactions that are responsible for inhibitory potency.

Depsipeptides Featuring a Neutral P1 Are Potent Inhibitors of Kallikrein-Related Peptidase 6 with On-Target Cellular Activity.

Kallikrein-related peptidase 6 (KLK6) is a secreted serine protease that belongs to the family of tissue kallikreins (KLKs). Many KLKs are investigated as potential biomarkers for cancer as well as therapeutic drug targets for a number of pathologies. KLK6, in particular, has been implicated in neurodegenerative diseases and cancer, but target validation has been hampered by a lack of selective inhibitors. This work introduces a class of depsipeptidic KLK6 inhibitors, discovered via high-throughput screening, which were found to function as substrate mimics that transiently acylate the catalytic serine of KLK6. Detailed structure-activity relationship studies, aided by in silico modeling, uncovered strict structural requirements for potency, stability, and acyl-enzyme complex half-life. An optimized scaffold, DKFZ-251, demonstrated good selectivity for KLK6 compared to other KLKs, and on-target activity in a cellular assay. Moreover, DKFZ-633, an inhibitor-derived activity-based probe, could be used to pull down active endogenous KLK6.

Mining human cancer datasets for kallikrein expression in cancer: the ‘KLK-CANMAP’ Shiny web tool

The dysregulation of the serine-protease family kallikreins (KLKs), comprising 15 genes, has been reportedly associated with cancer. Their expression in several tissues and physiological fluids makes them potential candidates as biomarkers and therapeutic targets. There are several databases available to mine gene expression in cancer, which often include clinical and pathological data. However, these platforms present some limitations when comparing a specific set of genes and can generate considerable unwanted data. Here, several datasets that showed significant differential expression (p<0.01) in cancer vs. normal (n=118), metastasis vs. primary (n=15) and association with cancer survival (n=21) have been compiled in a user-friendly format from two open and/or publicly available databases Oncomine and OncoLnc for the 15 KLKs. The data have been included in a free web application tool: the KLK-CANMAP https://cancerbioinformatics.shinyapps.io/klk-canmap/. This tool integrates, analyses and visualises data and it was developed with the R Shiny framework. Using KLK-CANMAP box-plots, heatmaps and Kaplan-Meier graphs can be generated for the KLKs of interest. We believe this new cancer KLK focused web tool will benefit the KLK community by narrowing the data visualisation to only the genes of interest.

Discovery, Synthesis, Pharmacological Profiling, and Biological Characterization of Brintonamides A-E, Novel Dual Protease and GPCR Modulators from a Marine Cyanobacterium.

Five novel modified linear peptides named brintonamides A-E (1-5) were discovered from a marine cyanobacterial sample collected from Brinton Channel, Florida Keys. The total synthesis of 1-5 in addition to two other structurally related analogues (6 and 7) was achieved, which provided more material to allow rigorous biological evaluation and SAR studies. Compounds were subjected to cancer-focused phenotypic cell viability and migration assays and orthogonal target-based pharmacological screening platforms to identify their protease and GPCR modulatory activity profiles. The cancer related serine protease kallikrein 7 (KLK7) was inhibited to similar extents with an IC50 near 20 μM by both representative members 1 and 4, which differed in the presence or lack of the N-terminal unit. In contrast to the biochemical protease profiling study, clear SAR was observed in the functional GPCR screens, where five GPCRs in antagonist mode (CCR10, OXTR, SSTR3, TACR2) and agonist mode (CXCR7) were modulated by compounds 1-7 to varying extents. Chemokine receptor type 10 (CCR10) was potently modulated by brintonamide D (4) with an IC50 of 0.44 μM. We performed in silico modeling to understand the structural basis underlying the differences in the antagonistic activity among brintonamides toward CCR10. Because of the significance of KLK7 and CCR10 in cancer progression and metastasis, we demonstrated the ability of brintonamide D (4) at 10 μM to significantly target downstream cellular substrates of KLK7 (Dsg-2 and E-cad) in vitro and to inhibit CCL27-induced CCR10-mediated proliferation and the migration of highly invasive breast cancer cells.

Kallikreins Stepwise Scoring Reveals Three Subtypes of Papillary Thyroid Cancer with Prognostic Implications.

Papillary thyroid cancer (PTC) is the most common type of thyroid cancer. Unlike most cancers, its incidence has dramatically increased in the last decades mainly due to increased diagnosis of indolent PTCs. Adequate risk stratification is crucial to avoid the over-treatment of low-risk patients, as well as the under-treatment of high-risk patients, but the currently available markers are still insufficient. Kallikreins (KLKs) are emergent biomarkers in cancer, but their involvement in PTC is unknown. This study analyzed DNA methylation (HumanMethylation arrays) and gene expression (RNA-Seq) of KLKs, BRAF and RAS mutations, and clinical data from four published thyroid cancer data sets including normal and tumor tissues (n = 73, n = 475, n = 20, and n = 82) as discovery, training, and validation series. The C4.5 classification algorithm was used to generate a decision tree. Disease-free survival was estimated using Kaplan-Meier and Cox approaches. Specific analyses were performed using real-time polymerase chain reaction and immunohistochemistry. The entire KLK family was deregulated in PTC, displaying a specific epigenetic and transcriptional profile strongly associated with BRAFV600E or RAS mutations. Thus, a decision-tree algorithm was developed based on three KLKs with >80% sensitivity and >95% specificity, identifying BRAF- and RAS-mutated tumors. Notably, tumors lacking these mutations were classified as BRAF- or RAS-like. Most importantly, the KLK algorithm uncovered a novel PTC subtype showing favorable prognostic features. The KLK algorithm could lead to a new clinically applicable strategy with important implications for the risk stratification of PTC and the management of patients.

909 Kallikrein 6 (KLK6) mediated keratin 10 degradation is required for VZV propagation in skin

909 Kallikrein 6 (KLK6) mediated keratin 10 degradation is required for VZV propagation in skin

Effect of microRNA-143 on interleukin-13-induced expression of kallikrein 7 in human epidermal keratinocytes

Objective To evaluate the effect of microRNA-143 (miR-143) on interleukin (IL) -13-induced expression of kallikrein 7 (KLK7) in primary normal human epidermal keratinocytes (NHEKs) . Methods Some NHEKs at exponential growth phase were divided into 4 groups to be treated with recombinant human IL-13 at different concentrations of 0, 2, 10 and 50 μg/L respectively for 24 hours, and some NHEKs were treated with 50 μg/L IL-13 for 0, 6, 12, 24 and 48 hours separately. After the treatment, NHEKs were collected, and total RNA was extracted. Real-time fluorescence-based quantitative PCR was performed to determine the mRNA expression of KLK7. Some other NHEKs were divided into another 4 groups: NHEK group (blank control group) receiving no treatment, IL-13 group treated with 50 μg/L IL-13, miR-NC group transfected with miRNA mimics negative control followed by the treatment with 50 μg/L IL-13, and miR-143 group transfected with miR-143 mimics followed by the treatment with 50 μg/L IL-13. After 24-hour treatment with IL-13, real-time fluorescence-based quantitative PCR and Western blot analysis were conducted to determine the mRNA and protein expression of KLK7 respectively in the above groups. Results After 24-hour treatment with IL-13 at concentrations of 0, 2, 10 and 50 μg/L, the mRNA expression of KLK7 in NHEKs was 1.00 ± 0.12, 0.89 ± 0.04, 1.15 ± 0.09 and 1.70 ± 0.10 respectively, and significantly increased along with the increase of IL-13 concentrations (F= 92.48, P < 0.05) . After 0-, 6-, 12-, 24- and 48-hour treatment with 50 μg/L IL-13, the mRNA expression of KLK7 in NHEKs was 1.00 ± 0.05, 1.05 ± 0.12, 1.71 ± 0.20, 1.97 ± 0.19 and 2.48 ± 0.13 respectively, and significantly increased over time (F= 206.44, P < 0.05) . Compared with the miR-NC group, the miR-143 group showed significantly decreased mRNA and protein expression of KLK7 (t= 6.76, 4.23 respectively, both P < 0.05) . Conclusion In NHEKs, IL-13 can up-regulate the expression of KLK7, likely by the regulation of miR-143. Key words: Dermatitis, atopic; Keratinocytes; Interleukin-13; Kallikrein 7; MicroRNA-143

Kallikrein-related peptidases in lung diseases.

Human tissue kallikreins (KLKs) are 15 members of the serine protease family and are present in various healthy human tissues including airway tissues. Multiple studies have revealed their crucial role in the pathophysiology of a number of chronic, infectious and tumour lung diseases. KLK1, 3 and 14 are involved in asthma pathogenesis, and KLK1 could be also associated with the exacerbation of this inflammatory disease caused by rhinovirus. KLK5 was demonstrated as an influenza virus activating protease in humans, and KLK1 and 12 could also be involved in the activation and spread of these viruses. KLKs are associated with lung cancer, with up- or downregulation of expression depending on the KLK, cancer subtype, stage of tumour and also the microenvironment. Functional studies showed that KLK12 is a potent pro-angiogenic factor. Moreover, KLK6 promotes malignant-cell proliferation and KLK13 invasiveness. In contrast, KLK8 and KLK10 reduce proliferation and invasion of malignant cells. Considering the involvement of KLKs in various physiological and pathological processes, KLKs appear to be potential biomarkers and therapeutic targets for lung diseases.

Abstract PD7-12: Molecular subtyping of male breast cancer by the International male breast cancer program (IMBC): EORTC 10085/TBCRC 0-29/BIG 2-07/NABCG/BOOG 2009-04

Abstract Introduction. Male breast cancer (male BC) is a rare disease for which disease management is extrapolated from females. IMBC, an international consortium, which previously reported on clinico-pathological aspects, now reports on molecular subgroups revealed by RNA sequencing and their relation to patient outcome. Methods. Tumor samples from the retrospective MALE BC registry diagnosed between 1990-2010 and with pathology and outcome data (relapse-free- (RFS) and overall survival (OS)) were included (n=699). To allow the discovery of prognostic features, we selected, stratified for known risk factors (TN stage, grade, age at diagnose and adjuvant endocrine treatment), from the cohort 152 cases with poor (RFS &amp;lt;= 4 yrs) and good outcome (RFS &amp;gt; 7yrs) evenly distributed. Here, we report RNA sequencing results of the first 73 cases, 38 with poor and 35 with good outcome. RNA sequencing reads were used to generate gene expression values and to report transcripts carrying driver mutations. Unsupervised clustering identified subgroups and within subgroups differentially expressed genes were identified. The reported prognostic male BC subgroups M1 and M2 (Johansson BCR 2012(14):R31) were also annotated. All identified subgroups were related to outcome using logistic regression (p-value using Wald test). Results. Unsupervised clustering revealed 2 main subgroups of which group 1 was enriched for expression of ER target genes, WNT3 and genes from amplicons known for female BC, e.g. 19p13 (CCNE1), 8q24 (MYC), and 11q13 (CCND1). The biology of the smaller group 2 was less defined but TGFβ2 expression was high as were various kallikreins (KLK) including interestingly KLK3 (prostate specific antigen). Other known amplified regions [(8p11 (FGFR1), 20q13 (ZNF217) and 12q15 (MDM2)] and mutated transcripts [PIK3CA (H1047R/L/Q; E542K, E545K, N345K; 16% of cases), TP53 and SF3B1 (K700E) (2% of cases)] were identified. Profound tumor infiltrate gene expression was present in 5% of cases and one third of cases expressed proliferation markers. Except for TP53, none of these latter characteristics were unevenly distributed among the 2 main subgroups. ER and AR were highly correlated, particularly in group 1. The two main groups could be further subdivided. Group 1 comprised 3 subgroups of which subgroup 1a expressed TFF1/3 and NAT1, well-known ER targets, while subgroups 1b and 1c expressed other ER targets,respectively BEX1 and PITX1. HOXC cluster expression differentiated subgroup 1b from 1a and 1c. None of these intrinsic subgroups were, however, related to RFS. The previously reported M2 subgroup, which largely segregated with subclusters 1a and 1b, was associated with a better RFS than the M1 subgroup (OR=2.9; 95%CI 1.1-7.5; p-value=0.03). Conclusions. 1) Intrinsic subtypes of male BC were revealed and their subgrouping is defined by ER associated subsets of genes. 2) The association of the reported M2 subgroup of male BC with longer RFS was validated; 3) Currently identified biological characteristics of male BC may improve future treatments. The full report on 152 cases including a comparison to female BC will be presented at the conference. This research was funded by Breast Cancer Research Foundation Citation Format: Martens JWM, Sieuwerts A, Ponchet C, Smid M, de Weerd V, Slaets L, Piper T, van Deurzen CHM, Schroder CP, Stangle C, Kloosterman W, van Leeuwen-Stok E, Nilsson C, Vermeij J, Peeters S, Goulioti T, Nowaczyk M, Aebi S, Rubio IT, Kelly C, Bayani J, Porter P, Murray M, Hudis C, Middleton L, Korde L, Ruddy K, Winer E, Bogler O, van den Weyngaert D, dal Lago L, Fraser J, Benstead K, van Asperen C, Linderholm B, Hedenfalk I, Tryfonidis K, Giordano S, Bartlett J, Cardoso F. Molecular subtyping of male breast cancer by the International male breast cancer program (IMBC): EORTC 10085/TBCRC 0-29/BIG 2-07/NABCG/BOOG 2009-04 [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD7-12.