Autoantibodies, antigen-autoantibody complexes and antigens complement CA125 for early detection of ovarian cancer.

BACKGROUND: Multiple antigens, autoantibodies (AAb), and antigen-autoantibody (Ag-AAb) complexes were compared for their ability to complement CA125 for early detection of ovarian cancer. METHODS: Twenty six biomarkers were measured in a single panel of sera from women with early stage (I-II) ovarian cancers (n = 64), late stage (III-IV) ovarian cancers (186), benign pelvic masses (200) and from healthy controls (502), and then split randomly (50:50) into a training set to identify the most promising classifier and a validation set to compare its performance to CA125 alone. RESULTS: Eight biomarkers detected ≥ 8% of early stage cases at 98% specificity. A four-biomarker panel including CA125, HE4, HE4 Ag-AAb and osteopontin detected 75% of early stage cancers in the validation set from among healthy controls compared to 62% with CA125 alone (p = 0.003) at 98% specificity. The same panel increased sensitivity for distinguishing early-stage ovarian cancers from benign pelvic masses by 25% (p = 0.0004) at 95% specificity. From 21 autoantibody candidates, 3 AAb (anti-p53, anti-CTAG1 and annt-Il-8) detected 22% of early stage ovarian cancers, potentially lengthening lead time prior to diagnosis. CONCLUSION: A four biomarker panel achieved greater sensitivity at the same specificity for early detection of ovarian cancer than CA125 alone.

B cell profiles, antibody repertoire and reactivity reveal dysregulated responses with autoimmune features in melanoma.

B cells are known to contribute to the anti-tumor immune response, especially in immunogenic tumors such as melanoma, yet humoral immunity has not been characterized in these cancers to detail. Here we show comprehensive phenotyping in samples of circulating and tumor-resident B cells as well as serum antibodies in melanoma patients. Memory B cells are enriched in tumors compared to blood in paired samples and feature distinct antibody repertoires, linked to specific isotypes. Tumor-associated B cells undergo clonal expansion, class switch recombination, somatic hypermutation and receptor revision. Compared with blood, tumor-associated B cells produce antibodies with proportionally higher levels of unproductive sequences and distinct complementarity determining region 3 properties. The observed features are signs of affinity maturation and polyreactivity and suggest an active and aberrant autoimmune-like reaction in the tumor microenvironment. Consistent with this, tumor-derived antibodies are polyreactive and characterized by autoantigen recognition. Serum antibodies show reactivity to antigens attributed to autoimmune diseases and cancer, and their levels are higher in patients with active disease compared to post-resection state. Our findings thus reveal B cell lineage dysregulation with distinct antibody repertoire and specificity, alongside clonally-expanded tumor-infiltrating B cells with autoimmune-like features, shaping the humoral immune response in melanoma.

Mucin 13 (MUC13) as a candidate biomarker for ovarian cancer detection: potential to complement CA125 in detecting non-serous subtypes.

OBJECTIVES: Ovarian cancer is the most lethal gynecological malignancy in developed countries. One of the key associations with the high mortality rate is diagnosis at late stages. This clinical limitation is primarily due to a lack of distinct symptoms and detection at the early stages. The ovarian cancer biomarker, CA125, is mainly effective for identifying serous ovarian carcinomas, leaving a gap in non-serous ovarian cancer detection. Mucin 13 (MUC13) is a transmembrane, glycosylated protein with aberrant expression in malignancies, including ovarian cancer. We explored the potential of MUC13 to complement CA125 as an ovarian cancer biomarker, by evaluating its ability to discriminate serous and non-serous subtypes of ovarian cancer at FIGO stages I-IV from benign conditions. METHODS: We used our newly developed, high sensitivity ELISA to measure MUC13 protein in a large, well-defined cohort of 389 serum samples from patients with ovarian cancer and benign conditions. RESULTS: MUC13 and CA125 serum levels were elevated in malignant compared to benign cases (p<0.0001). Receiver-operating characteristic (ROC) curve analysis showed similar area under the curve (AUC) of 0.74 (MUC13) and 0.76 (CA125). MUC13 concentrations were significantly higher in mucinous adenocarcinomas compared to benign controls (p=0.0005), with AUC of 0.80. MUC13 and CA125 showed significant elevation in early-stage cases (stage I-II) in relation to benign controls (p=0.0012 and p=0.014, respectively). CONCLUSIONS: We report the novel role of MUC13 as a serum ovarian cancer biomarker, where it could complement CA125 for detecting some subtypes of non-serous ovarian carcinoma and early-stage disease.

Quantitation of cardiac troponin I in cancer patients treated with immune checkpoint inhibitors: a case-control study.

OBJECTIVES: Immune checkpoint inhibitors (ICIs) cause a variety of toxicities, including immune-related adverse events (irAEs), but there are no biomarkers to predict their development. Guidelines recommend measuring circulating cardiac troponin I (cTnI) during ICI therapy to detect related cardiotoxicities. Moreover, elevated cTnI has also been associated with worse outcomes in non-cardiac patients, including cancer. Thus here, we investigated whether cTnI levels were higher in patients with irAEs. METHODS: The study consisted of three groups; 21 cancer patients undergoing ICI immunotherapies who presented with irAEs, four patients without irAEs, and 20 healthy controls. Patient samples were assessed at baseline (n=25), during ICI treatment (n=25, median=6 weeks of treatment) and at toxicity (n=6, median=13 weeks of treatment). In addition to blood high sensitivity cardiac troponin I (hs-cTnI), anti-thyroglobulin (TG) and anti-thyroid peroxidase (TPO) antibodies were also quantitated to detect thyroid dysfunction, constituting the second leading toxicity (23.8%) after pneumonitis (28.6%). RESULTS: Four patients with irAEs (n=4/21; 19%) and one without irAEs (n=1/4; 25%) showed higher hs-cTnI levels at any time-point; the remaining had physiological levels. None of these patients developed cardiotoxicity. Concurrent elevated levels of anti-thyroid antibodies and hs-cTnI were detected in one patient with thyroid dysfunction (n=1/5, 20%). However, these antibodies were also elevated in three patients (n=3/16, 19%) with non-thyroid irAEs and in up to 40% of healthy controls. CONCLUSIONS: hs-cTnI was not elevated in patients with irAEs, but larger studies are needed to confirm these observations.

Kallikrein-Related Peptidase 6 (KLK6) as a Contributor toward an Aggressive Cancer Cell Phenotype: A Potential Role in Colon Cancer Peritoneal Metastasis.

Kallikrein-related peptidases (KLKs) are implicated in many cancer-related processes. KLK6, one of the 15 KLK family members, is a promising biomarker for diagnosis of many cancers and has been associated with poor prognosis of colorectal cancer (CRC) patients. Herein, we evaluated the expression and cellular functions of KLK6 in colon cancer-derived cell lines and in clinical samples from CRC patients. We showed that, although many KLKs transcripts are upregulated in colon cancer-derived cell lines, KLK6, KLK10, and KLK11 are the most highly secreted proteins. KLK6 induced calcium flux in HT29 cells by activation and internalization of protease-activated receptor 2 (PAR2). Furthermore, KLK6 induced extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylation. KLK6 suppression in HCT-116 colon cancer cells decreased the colony formation, increased cell adhesion to extracellular matrix proteins, and reduced spheroid formation and compaction. Immunohistochemistry (IHC) analysis demonstrated ectopic expression of KLK6 in human colon adenocarcinomas but not in normal epithelia. Importantly, high levels of KLK6 protein were detected in the ascites of CRC patients with peritoneal metastasis, but not in benign ascites. These data indicate that KLK6 overexpression is associated with aggressive CRC, and may be applied to differentiate between benign and malignant ascites.

Transcriptome profiling and proteomic validation reveals targets of the androgen receptor signaling in the BT-474 breast cancer cell line.

BACKGROUND: Accumulating evidence suggests that the androgen receptor (AR) and its endogenous ligands influence disease progression in breast cancer (BCa). However, AR-mediated changes in BCa differ among the various BCa subtypes according to their hormone receptor profile [i.e., presence/absence of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2, (HER2)]. Thus, we explored the androgen-regulated transcriptomic changes in the ER+PR+HER2+ BCa cell line, BT-474, and compared them with PR-mediated changes. METHODS: We performed RNA sequencing analysis in treated BT-474 cells with dihydrotestosterone (DHT) and progesterone. Validation of the top ten differentially androgen-regulated genes and a number of other genes found in enriched signaling pathways was performed by qRT-PCR in BT-474 and other BCa cell lines. In addition, a parallel reaction monitoring targeted proteomic approach was developed to verify selected transcripts at the protein level. RESULTS: In total 19,450 transcripts were detected, of which 224 were differentially regulated after DHT treatment. The increased expression of two well-known androgen-regulated genes, KLK2 (p < 0.05) and KLK3 (p < 0.001), confirmed the successful androgen stimulation in BT-474 cells. The transcription factor, ZBTB16, was the most highly upregulated gene, with ~ 1000-fold change (p < 0.001). Pathway enrichment analysis revealed downregulation of the DNA replication processes (p < 0.05) and upregulation of the androgen signaling and fatty acid metabolism pathways (p < 0.05). Changes related to progesterone treatment showed opposite effects in gene expression than DHT treatment. Similar expression profiles were observed among other BCa cell lines expressing high levels of AR (ZR75.1 and MBA-MB-453). The parallel reaction monitoring targeted proteomic analysis further confirmed that altered protein expression (KLK3, ALOX15B) in the supernatant and cell lysate of DHT-treated BT-474 cells, compared to control cells. DISCUSSION: Our findings suggest that AR modulates the metabolism of BT-474 cells by affecting the expression of a large number of genes and proteins. Based on further pathway analysis, we suggest that androgen receptor acts as a tumor suppressor in the BT-474 cells.

Comparison of two multiplexed technologies for profiling >1,000 serum proteins that may associate with tumor burden.

Background: In this pilot study, we perform a preliminary comparison of two targeted multiplex proteomics technologies for discerning serum protein concentration changes that may correlate to tumor burden in ovarian cancer (OC) patients. Methods: Using the proximity extension assay (PEA) and Quantibody® Kiloplex Array (QKA), we measured >1,000 proteins in the pre-surgical and post-surgical serum from nine OC patients (N=18 samples). We expect that proteins that have decreased significantly in the post-surgical serum concentration may correlate to tumor burden in each patient. Duplicate sera from two healthy individuals were used as controls (N=4 samples). We employed in-house ELISAs to measure five proteins with large serum concentration changes in pre- and post-surgical sera, from four of the original nine patients and the two original controls. Results: Both platforms showed a weak correlation with clinical cancer antigen 125 (CA125) data. The two multiplexed platforms showed a significant correlation with each other for >400 overlapping proteins. PEA uncovered 15 proteins, while QKA revealed 11 proteins, with more than a two-fold post-surgical decrease in at least six of the nine patients. Validation using single enzyme-linked immunosorbent assays (ELISAs) showed at least a two-fold post-surgical decrease in serum concentration of the same patients, as indicated by the two multiplex assays. Conclusion: Both methods identified proteins that had significantly decreased in post-surgical serum concentration, as well as recognizing proteins that had been implicated in OC patients. Our findings from a limited sample size suggest that novel targeted proteomics platforms are promising tools for identifying candidate serological tumor-related proteins.  However further studies are essential for the improvement of accuracy and avoidance of false results.

Investigating a novel multiplex proteomics technology for detection of changes in serum protein concentrations that may correlate to tumor burden.

Background: To account for cancer heterogeneity, we previously introduced the concept of "personalized" tumor markers, which are biomarkers that are informative in subsets of patients or even a single patient. Recent developments in various multiplex protein technologies create excitement for the discovery of markers of tumor burden in individual patients, but the reliability of the technologies remains to be tested for this purpose. Here, we sought to explore the potential of a novel proteomics platform, which utilizes a multiplexed antibody microarray, to detect changes in serum protein concentration that may correlate to tumor burden in pancreatic cancer. Methods: We applied the Quantibody® Human Kiloplex Array to simultaneously measure 1,000 proteins in sera obtained pre- and post-surgically from five pancreatic cancer patients. We expected that proteins which decreased post-surgery may correlate to tumor burden. Sera from two healthy individuals, split into two aliquots each, were used as controls. To validate the multiplexed results, we used single-target ELISA assays to measure the proteins with the largest serum concentration changes after surgery in sera collected pre- and post-surgically from the previous five patients and 10 additional patients. Results: The multiplexed array revealed nine proteins with more than two-fold post-surgical decrease in at least two of five patients. However, validation using single ELISAs showed that only two proteins tested displayed more than two-fold post-surgical decrease in one of the five original patients. In the independent cohort, six of the proteins tested showed at least a two-fold decrease post-surgery in at least one patient. Conclusions: Our study found that the Quantibody® Human Kiloplex Array results could not be reliably replicated with individual ELISA assays and most hits would likely represent false positives if applied to biomarker discovery. These findings suggest that data from novel, high-throughput proteomic platforms need stringent validation to avoid false discoveries.

Predicting response and toxicity to PD-1 inhibition using serum autoantibodies identified from immuno-mass spectrometry.

Background: Validated biomarkers are needed to identify patients at increased risk of immune-related adverse events (irAEs) to immune checkpoint blockade (ICB). Antibodies directed against endogenous antigens can change after exposure to ICB. Methods: Patients with different solid tumors stratified into cohorts received pembrolizumab every 3 weeks in a Phase II trial (INSPIRE study). Blood samples were collected prior to first pembrolizumab exposure (baseline) and approximately 7 weeks (pre-cycle 3) into treatment. In a discovery analysis, autoantibody target immuno-mass spectrometry was performed in baseline and pre-cycle 3 pooled sera of 24 INSPIRE patients based on clinical benefit (CBR) and irAEs. Results: Thyroglobulin (Tg) and thyroid peroxidase (TPO) were identified as the candidate autoantibody targets. In the overall cohort of 78 patients, the frequency of CBR and irAEs from pembrolizumab was 31% and 24%, respectively. Patients with an anti-Tg titer increase ≥1.5x from baseline to pre-cycle 3 were more likely to have irAEs relative to patients without this increase in unadjusted, cohort adjusted, and multivariable models (OR=17.4, 95% CI 1.8-173.8, p=0.015). Similarly, patients with an anti-TPO titer ≥ 1.5x from baseline to pre-cycle 3 were more likely to have irAEs relative to patients without the increase in unadjusted and cohort adjusted (OR=6.1, 95% CI 1.1-32.7, p=0.035) models. Further, the cohort adjusted analysis showed patients with anti-Tg titer greater than median (10.0 IU/mL) at pre-cycle 3 were more likely to have irAEs (OR=4.7, 95% CI 1.2-17.8, p=0.024). Patients with pre-cycle 3 anti-TPO titers greater than median (10.0 IU/mL) had a significant difference in overall survival (23.8 vs 11.5 months; HR=1.8, 95% CI 1.0-3.2, p=0.05). Conclusions: Patient increase ≥1.5x of anti-Tg and anti-TPO titers from baseline to pre-cycle 3 were associated with irAEs from pembrolizumab, and patients with elevated pre-cycle 3 anti-TPO titers had an improvement in overall survival.

Utility of a Fifth-Generation Ultrasensitive Prostate-Specific Antigen Assay for Monitoring Prostate Cancer Patients after Radical Prostatectomy with 3 Years of Follow-Up.

BACKGROUND: We investigated an ultrasensitive prostate-specific antigen (uPSA) immunoassay (MesoScale; lower limit of detection (LLD) of 0.0035 pg/mL) to monitor patients with prostate cancer (PCa) following radical prostatectomy (RP) and to examine whether changes in PSA in the conventionally undetectable range (<1 pg/mL) can predict biochemical relapse (BCR). METHODS: We measured uPSA in serial serum samples (N = 100) collected from 20 RP cases with a third-generation ELISA (LLD of 1 pg/mL) and the fifth-generation MesoScale assay. We analyzed the PSA nadir changes to classify patients into BCR or non-BCR groups, observed the trends in PSA kinetics, and associated BCR status with clinicohistopathological features. RESULTS: The ELISA could quantify PSA in only 38% of the RP samples, detecting BCR in 7 of 20 patients with PCa. The MesoScale assay quantified PSA in all samples, showing 8 of 20 patients with BCR. However, there was no significant difference between the median time to BCR detection based on ELISA (1016 days) compared with MesoScale data (949 days). Gleason scores were higher in the BCR groups compared with non-BCR. There was no significant difference for other clinicohistopathological parameters. CONCLUSIONS: The uPSA MesoScale technology could track miniscule changes in serum PSA in the range of 0.003-1 pg/mL in all RP cases. However, PSA kinetics and nadir at concentrations <2 pg/mL fluctuated, and increases below this range could not reliably suggest signs of BCR. Instead, ultrasensitive fifth-generation PSA assays may hold clinical potential for measuring the low concentrations of PSA in women for various medical contexts.

Serum PD-1 Is Elevated after Pembrolizumab Treatment but Has No Predictive Value.

Immune-checkpoint blockade (ICB) uses antibody targeting of specific inhibitory receptors and ligands. The major limitations of ICB, such as high cost, limited success rate, and immune-related adverse events (irAE), highlight the need for predictive biomarkers. We analyzed pre-immunotherapy and post-immunotherapy serum samples of 24 patients treated with pembrolizumab for changes in PD-1 and over 1,000 additional protein markers using a multiplex proximity extension assay (PEA) to identify potential predictive biomarkers of response and/or toxicity. Candidates were selected based on the criteria that at least 2 patients within any of 3 patient groups (responders without irAEs, responders with irAEs, or nonresponders with irAEs) had either a ≥4-fold increase or 4-fold decrease in expression post-immunotherapy. Female and male control samples were used as technical duplicates. A patient group with no response and no irAEs was used to exclude candidates. Following treatment with pembrolizumab, there was a relative increase of PD-1 in the serum of all patients, compared with controls (average 4.4-fold). We identified 7 additional serum proteins that met our candidate selection criteria. These candidate markers did not have any significant association with response or toxicity to pembrolizumab. Overall, we show that serum PD-1 increases post-therapy with pembrolizumab treatment but has no predictive value for response or toxicity in this small set of patients.

Liquid biopsy of cerebrospinal fluid identifies neuronal pentraxin receptor (NPTXR) as a biomarker of progression of Alzheimer's disease.

Background Alzheimer's disease (AD) is the most prevalent form of dementia. Currently, the most studied biomarkers of AD are cerebrospinal fluid (CSF) amyloid ß 1-42, total tau and phosphorylated tau. However, misdiagnosis can exceed 20%. Recently, we found that CSF amyloid ß precursor-like protein-1 (APLP1) and neuronal pentraxin receptor (NPTXR) are promising biomarkers of AD. The aim of the present study is to validate CSF APLP1 and NPTXR as biomarkers of AD severity. Methods APLP1 and NPTXR concentrations were measured in the CSF of patients with mild cognitive impairment (MCI) (n = 14), mild AD (n = 21), moderate AD (n = 43) and severe AD (n = 30) using enzyme-linked immunosorbent assays (ELISAs). Results CSF APLP1 and NPTXR were not associated with age or sex. CSF APLP1 was not different between any of the AD severity groups (p = 0.31). CSF NPTXR was significantly different between MCI and mild AD (p = 0.006), mild and moderate AD (p = 0.016), but not between moderate and severe AD (p = 0.36). NPTXR concentration progressively declined from MCI to mild, to moderate and to severe AD patients (p < 0.0001, Kruskal-Wallis test). CSF NPTXR positively correlated with the Mini-Mental Status Examination (MMSE) score (p < 0.001). Conclusions NPTXR concentration in CSF is a promising biomarker of AD severity and could inform treatment success and disease progression in clinical settings.

Expression profile of human tissue kallikrein 15 provides preliminary insights into its roles in the prostate and testis.

BACKGROUND: Human tissue kallikrein 15 (KLK15) is the latest member of the kallikrein-related peptidase family. Little is known about the pathophysiological roles of KLK15. Previous studies implied a role of KLK15 in prostate cancer. METHODS: In the present study, we examined KLK15 protein expression using a new immunoassay (ELISA) and immunohistochemistry (IHC). RESULTS: Highest KLK15 levels were detected in the testis and seminal fluid, whereas lower levels were observed in prostate and other tissues. Immunohistochemical analysis of testis suggests that KLK15 is strongly expressed in mature spermatids, but not in immature germ cells. KLK15 displayed predominantly nuclear localization in the basal cell layer of the prostatic epithelium. We also measured KLK15 in supernatants of various cell lines. Highest KLK15 levels were primarily detected in prostate cancer cell lines and KLK15 expression was hormone-independent, in contrast to KLK3. CONCLUSIONS: Collectively, our data provide insights into the localization and possible role of KLK15 in human physiology.

Exploring the potential of mucin 13 (MUC13) as a biomarker for carcinomas and other diseases.

BACKGROUND: Mucin 13 (MUC13) is a cell surface glycoprotein aberrantly expressed in a variety of epithelial carcinomas. Thus far, the role of MUC13 in various diseases remains elusive. To the best of our knowledge, this is the first study to examine the potential of MUC13 as a serum biomarker in a variety of carcinomas and other conditions. METHODS: We developed a recombinant MUC13 protein, mouse monoclonal antibodies and enzyme immunoassay (ELISA) for MUC13. We used this assay to measure MUC13 levels in the supernatants of cancer cell lines and a large cohort of serum samples from healthy and diseased individuals. RESULTS: MUC13 is secreted from cancer cell lines, with highest levels found in ovarian cancer cell lines. MUC13 levels in human sera were significantly increased in patients with renal failure and 20%-30% of patients with ovarian, liver, lung and other cancers. MUC13 was also elevated in 70% of patients with active cutaneous melanoma, but not uveal melanoma. Furthermore, we identified significant MUC13 elevations in the serum of patients with vasculitis (ANCA-positive) autoantibodies, but not in those with inflammatory bowel disease. CONCLUSIONS: Serum MUC13 is frequently elevated not only in a variety of malignant cases but also in some benign pathologies, thus appearing to be a non-specific disease biomarker. Nonetheless, serum MUC13 is clearly highly elevated in some carcinoma patients, and its relationship with tumor progression in this context warrant further research. Future studies that examine the correlation between serum MUC13 levels to stage of cancer could elucidate prognostic potential.

Differentiating Psoriatic Arthritis From Psoriasis Without Psoriatic Arthritis Using Novel Serum Biomarkers.

OBJECTIVE: There is a high prevalence of undiagnosed psoriatic arthritis (PsA) in patients with psoriasis. Identifying soluble biomarkers for PsA will help in screening psoriasis patients for appropriate rheumatology referral. We therefore aimed to investigate whether serum levels of novel markers previously discovered by quantitative mass spectrometric analysis of synovial fluid and skin biopsies performs better than the C-reactive protein (CRP) level in differentiating PsA patients from those with psoriasis without PsA (PsC). METHODS: In this case-control study, serum samples were obtained from 100 subjects with PsA, 100 with PsC, and 100 healthy controls. Patients with PsA and PsC were group matched for age, sex, psoriasis duration, and Psoriasis Area and Severity Index and were not currently receiving biologic treatment. Using enzyme-linked immunosorbent assay, 4 high-priority markers (Mac-2-binding protein [M2BP], CD5-like protein [CD5L], myeloperoxidase [MPO], and integrin ß5 [ITGß5]), as well as previously established markers (matrix metalloproteinase 3 [MMP-3] and CRP level) were assayed. Data were analyzed using logistic regression. Receiver operating characteristic (ROC) curves were plotted. RESULTS: In comparisons to controls, CD5L, ITGß5, M2BP, MPO, MMP-3, and CRP level were independently associated with PsA, while only CD5L, M2BP, and MPO were independently associated with PsC alone. In comparisons to PsC, ITGß5, M2BP, and CRP level were independently associated with PsA. ROC analysis of this model shows an area under the curve (AUC) of 0.85 (95% confidence interval [95% CI] 0.80-0.90). The model that included CRP level alone had an AUC of 0.71 (95% CI 0.64-0.78). CONCLUSION: CD5L, ITGß5, M2BP, MPO, MMP-3, and CRP level are markers for PsA. The combination of ITGß5, M2BP, and CRP level differentiates PsA from PsC, and performs better than CRP level alone.

Study of kallikrein-related peptidase 6 (KLK6) and its complex with α1-antitrypsin in biological fluids.

BACKGROUND: Human kallikrein-related peptidase 6 (KLK6) is a member of the kallikrein family of serine proteases. KLK6 is synthesized as a preproenzyme, mainly in tissues of the central nervous system (CNS), and secreted as an inactive precursor. Serum KLK6 is a biomarker of unfavorable prognosis for ovarian cancer, but its sensitivity for early detection is relatively low. Differential glycosylation of KLK6 has been identified in ascites fluid obtained from ovarian cancer patients, suggesting the presence of unique KLK6 isoforms in biological samples. METHODS: In the present study, we applied a two-step enrichment approach for KLK6 in ovarian cancer ascites, followed by mice immunization and production of monoclonal antibodies. Immunoaffinity techniques coupled to mass spectrometric methods were employed for hybridoma screening and target antigen identification. RESULTS: We found that the main target of the newly-generated monoclonal antibodies target was the serine protease inhibitor α1-antitrypsin (A1AT). Additional experiments confirmed that A1AT is the main inhibitor of KLK6 in biological fluids. One new antibody (24ED138) was chosen to build a hybrid assay for the accurate quantification of the A1AT-KLK6 complex in biological samples. The aforementioned assay was evaluated with serum samples collected from patients with ovarian cancer (n=24) and normal donors (n=16) and showed slight improvement in sensitivity (~12%) compared to the standard in-house KLK6 assay. CONCLUSIONS: We conclude that KLK6 is present in biological fluids either as free form, or bound to A1AT, and the bound form performs better than total KLK6 as a biomarker of ovarian carcinoma.

Aberrant expression of kallikrein-related peptidase 7 is correlated with human melanoma aggressiveness by stimulating cell migration and invasion.

Members of the tissue kallikrein-related peptidase (KLK) family not only regulate several important physiological functions, but aberrant expression has also been associated with various malignancies. Clinically, KLKs have been suggested as promising biomarkers for diagnosis and prognosis in many types of cancer. As of yet, expression of KLKs and their role in skin cancers are, however, poorly addressed. Malignant melanoma is an aggressive disease associated with poor prognosis. Hence, diagnostic biomarkers to monitor melanoma progression are needed. Herein, we demonstrate that although mRNA of several KLKs are aberrantly expressed in melanoma cell lines, only the KLK7 protein is highly secreted in vitro. In line with these findings, ectopic expression of KLK7 in human melanomas and its absence in benign nevi were demonstrated by immunohistochemistry in vivo. Interestingly, overexpression of KLK7 induced a significant reduction in melanoma cell proliferation and colony formation. Moreover, KLK7 overexpression triggered an increase in cell motility and invasion associated with decreased expression of E-cadherin and an upregulation of MCAM/CD146. Our results demonstrate, for the first time, that aberrant KLK7 expression leads to a switch from proliferative to invasive phenotype, suggesting a potential role of KLK7 in melanoma progression. Thus, we hypothesize that KLK7 may represent a potential biomarker for melanoma progression.

Germline Mutations in the Kallikrein 6 Region and Predisposition for Aggressive Prostate Cancer.

Background: There is a need for markers that can specifically identify individuals at increased risk of harboring aggressive forms of prostate cancer (PCa). Methods: We surveyed the Kallikrein ( KLK ) region ( KLK 1-15) for single-nucleotide polymorphisms (SNPs) associated with aggressive PCa (Gleason Score ≥ 8) in 1858 PCa patients. Discovery cohorts (Swiss arm of the European Randomized Study of Screening for PCa, n = 379; Toronto, Canada, n = 540) and a validation cohort (Prostate, Lung, Colorectal and Ovarian [PLCO] screening trial, n = 939) were analyzed. Fine-mapping within the KLK region was carried out by genotyping and imputation in the discovery cohort, whereas PLCO data were provided through database of Genotypes and Phenotypes ( dbGaP ). The influence of SNPs of interest on biochemical-free survival was evaluated in a cohort of localized PCa from the International Cancer Genome Consortium (ICGC; n = 130) analyzed with next-generation sequencing. Single- and multi-SNP association studies, as well as haplotype analyses, were performed. All statistical tests were two-sided. Results: Several SNPs in very strong linkage disequilibrium in the KLK 6 region and located within the same haplotype (rs113640578, rs79324425, rs11666929, rs28384475, rs3810287), identified individuals at increased risk of aggressive PCa in both discovery (odds ratio [OR] = 3.51-3.64, 95% confidence interval [CI] = 2.01 to 6.36, P = 1.0x10 -5 -8.4x10 -6 ) and validation (OR = 1.89-1.96, 95% CI = 0.99 to 3.71, P = .04-.05) cohorts. The overall test of haplotype association was highly statistically significant in each cohort ( P = 3.5x10 -4 and .006, respectively) and in the three data sets combined ( P = 2.3x10 -5 ). These germline SNPs independently predicted relapse in the ICGC cohort (hazard ratio = 3.15, 95% CI = 1.57 to 6.34, P = .001). Conclusions: Our fine-mapping study has identified novel loci in the KLK 6 region strongly associated with aggressive PCa.

Human tissue Kallikreins: Blood levels and response to radiotherapy in intermediate risk prostate cancer.

OBJECTIVES: Kallikreins are serine proteases over expressed in many malignancies. In this study, we measure changes in serum kallikrein (KLKs) levels during intensity-modulated radiotherapy (IMRT) in prostate cancer patients, and find potential correlations between serum kallikrein level and normal tissues toxicity during radiation. METHODS: Forty-nine patients with prostate cancer were recruited as follows: group 1, definitive standard fractionation IMRT (78Gy in 39 fractions, n=15); group 2, definitive hypofractionated IMRT (60Gy in 20 fractions, n=15); and group 3, IMRT postprostatectomy (66Gy in 33 fractions, n=19). Patients treated with definitive radiation therapy were intermediate risk. Blood samples were collected at baseline and quarterly during IMRT and at each follow-up visit. Acute toxicity was graded weekly during radiotherapy using CTC-AE v4.0 criteria. Multiplexed immunoassays were used to quantify total, free, and intact Prostate Specific Antigen (PSA), as well as Kallikreins 2, 4, 6, and 11. RESULTS: The serum kallikreins, PSA (total, free and intact), KLK2, 6, and 11 change significantly after definitive radiotherapy. KLK2 and intact PSA decrease as fast as two weeks after initiation of radiation, while the first significant decrease in total and free PSA is noted only at the completion of radiation. KLK6 and KLK11 surge temporarily during radiation therapy and decrease below baseline levels at 8weeks and 12months, respectively after completion of radiation. KLK4 levels did not change with radiation. There was no correlation between GU or GI toxicities and serum kallikreins. CONCLUSIONS: PSA, KLK2, 6, and 11, change significantly after definitive prostate radiotherapy, though KLK2 and PSA decrease by the end of the radiation course while KLK6 and KLK11 decrease significantly starting at 2 and 12months, respectively, after radiation. There was no correlation between GU or GI toxicities and serum kallikreins.

Novel immunoassays for detection of CUZD1 autoantibodies in serum of patients with inflammatory bowel diseases.

BACKGROUND: Pancreatic autoantibodies (PABs) are detected in patients with inflammatory bowel disease (IBD). Their prevalence is higher in Crohn's disease (CrD) than in ulcerative colitis (UC). Glycoprotein 2 (GP2) and, more recently, CUB and zona pellucida-like domain-containing protein 1 (CUZD1) have been identified as target autoantigens of PAB. The clinical utility of CUZD1 autoantibodies has only recently been assessed by indirect immunofluorescence (IIF) assays. In this study, we developed and validated novel immunoassays for the detection of CUZD1 autoantibodies. METHODS: Recombinant CUZD1 protein was utilized as a solid-phase antigen for the development of two immunoassays for the detection of IgG and IgA CUZD1 autoantibodies. Serum samples from 100 patients with CrD, 100 patients with UC, 129 patients assessed for various autoimmune diseases (vADs) and 50 control individuals were analyzed. RESULTS: Two immunofluorometric assays for the detection of IgG and IgA CUZD1-specific antibodies were developed. CUZD1 autoantibodies were detected in 12.5% (25/200) IBD patients, including 16% of patients with CrD and in 9% of patients with UC (CrD vs. UC, p<0.05), compared with 3.1% (4/129) patients suspected of having vADs (CrD vs. ADs, p<0.05; UC vs. ADs, p=0.08). CUZD1 autoantibody positivity was not found to be related to disease location, age of disease onset or disease phenotype. CONCLUSIONS: This is the first study to describe novel IgA and IgG CUZD1 autoantibody enzyme-linked immunosorbent assay. These immunoassays agree well with standard IIF techniques and can be utilized in multicenter studies to investigate the diagnostic and clinical utility of CUZD1 autoantibodies.