The use of Lutetium-177 PSMA radioligand therapy with high dose rate brachytherapy for locally recurrent prostate cancer after previous definitive radiation therapy: a randomized, single-institution, phase I/II study (ROADSTER).

BACKGROUND: Isolated local failure (ILF) can occur in patients who initially receive definitive radiation therapy for prostate cancer. Salvage therapy for ILF includes high dose rate (HDR) brachytherapy. Prostate Specific Membrane Antigen (PSMA) Positron Emission Tomography (PET) can accurately detect ILF and can exclude extraprostatic disease. Lutetium-177 PSMA Radioligand Therapy (RLT) is a novel treatment for prostate cancer that can target prostate cancer accurately, while sparing radiation dose to normal tissues. METHODS: ROADSTER is a phase I/II randomized, single-institution study. Patients with an ILF of prostate cancer after definitive initial radiation therapy are eligible. The ILF will be confirmed with biopsy, magnetic resonance imaging (MRI) and PSMA PET. Patients will be randomized between HDR brachytherapy in two fractions (a standard of care salvage treatment at our institution) (cohort 1) or one treatment of intravenous Lutetium-177 PSMA RLT, followed by one fraction of HDR brachytherapy (cohort 2). The primary endpoints for the phase I portion of the study (n = 12) will be feasibility, defined as 10 or more patients completing the study protocol within 24 months of study activation; and safety, defined as zero or one patients in cohort 2 experiencing grade 3 or higher toxicity in the first 6 months post-treatment. If feasibility and safety are achieved, the study will expand to a phase II study (n = 30 total) where preliminary efficacy data will be evaluated. Secondary endpoints include changes in prostate specific antigen levels, acute toxicity, changes in quality of life, and changes in translational biomarkers. Translational endpoints will include interrogation of blood, urine, and tissue for markers of DNA damage and immune activation with each treatment. DISCUSSION: ROADSTER explores a novel salvage therapy for ILF after primary radiotherapy with combined Lutetium-177 PSMA RLT and HDR brachytherapy. The randomized phase I/II design will provide a contemporaneous patient population treated with HDR alone to facilitate assessment of feasibility, tolerability, and biologic effects of this novel therapy. TRIAL REGISTRATION: NCT05230251 (ClinicalTrials.gov).

Systematically higher Ki67 scores on core biopsy samples compared to corresponding resection specimen in breast cancer: a multi-operator and multi-institutional study.

Ki67 has potential clinical importance in breast cancer but has yet to see broad acceptance due to inter-laboratory variability. Here we tested an open source and calibrated automated digital image analysis (DIA) platform to: (i) investigate the comparability of Ki67 measurement across corresponding core biopsy and resection specimen cases, and (ii) assess section to section differences in Ki67 scoring. Two sets of 60 previously stained slides containing 30 core-cut biopsy and 30 corresponding resection specimens from 30 estrogen receptor-positive breast cancer patients were sent to 17 participating labs for automated assessment of average Ki67 expression. The blocks were centrally cut and immunohistochemically (IHC) stained for Ki67 (MIB-1 antibody). The QuPath platform was used to evaluate tumoral Ki67 expression. Calibration of the DIA method was performed as in published studies. A guideline for building an automated Ki67 scoring algorithm was sent to participating labs. Very high correlation and no systematic error (p = 0.08) was found between consecutive Ki67 IHC sections. Ki67 scores were higher for core biopsy slides compared to paired whole sections from resections (p ≤ 0.001; median difference: 5.31%). The systematic discrepancy between core biopsy and corresponding whole sections was likely due to pre-analytical factors (tissue handling, fixation). Therefore, Ki67 IHC should be tested on core biopsy samples to best reflect the biological status of the tumor.

An international multicenter study to evaluate reproducibility of automated scoring for assessment of Ki67 in breast cancer.

The nuclear proliferation biomarker Ki67 has potential prognostic, predictive, and monitoring roles in breast cancer. Unacceptable between-laboratory variability has limited its clinical value. The International Ki67 in Breast Cancer Working Group investigated whether Ki67 immunohistochemistry can be analytically validated and standardized across laboratories using automated machine-based scoring. Sets of pre-stained core-cut biopsy sections of 30 breast tumors were circulated to 14 laboratories for scanning and automated assessment of the average and maximum percentage of tumor cells positive for Ki67. Seven unique scanners and 10 software platforms were involved in this study. Pre-specified analyses included evaluation of reproducibility between all laboratories (primary) as well as among those using scanners from a single vendor (secondary). The primary reproducibility metric was intraclass correlation coefficient between laboratories, with success considered to be intraclass correlation coefficient >0.80. Intraclass correlation coefficient for automated average scores across 16 operators was 0.83 (95% credible interval: 0.73-0.91) and intraclass correlation coefficient for maximum scores across 10 operators was 0.63 (95% credible interval: 0.44-0.80). For the laboratories using scanners from a single vendor (8 score sets), intraclass correlation coefficient for average automated scores was 0.89 (95% credible interval: 0.81-0.96), which was similar to the intraclass correlation coefficient of 0.87 (95% credible interval: 0.81-0.93) achieved using these same slides in a prior visual-reading reproducibility study. Automated machine assessment of average Ki67 has the potential to achieve between-laboratory reproducibility similar to that for a rigorously standardized pathologist-based visual assessment of Ki67. The observed intraclass correlation coefficient was worse for maximum compared to average scoring methods, suggesting that maximum score methods may be suboptimal for consistent measurement of proliferation. Automated average scoring methods show promise for assessment of Ki67 scoring, but requires further standardization and subsequent clinical validation.

Tumoral BRD4 expression in lymph node-negative breast cancer: association with T-bet+ tumor-infiltrating lymphocytes and disease-free survival.

BACKGROUND: We previously observed that T-bet+ tumor-infiltrating T lymphocytes (T-bet+ TILs) in primary breast tumors were associated with adverse clinicopathological features, yet favorable clinical outcome. We identified BRD4 (Bromodomain-Containing Protein 4), a member of the  Bromodomain and Extra Terminal domain (BET) family, as a gene that distinguished T-bet+/high and T-bet-/low tumors. In clinical studies, BET inhibitors have been shown to suppress inflammation in various cancers, suggesting a potential link between BRD4 and immune infiltration in cancer. Hence, we examined the BRD4 expression and clinicopathological features of breast cancer. METHODS: The cohort consisted of a prospectively ascertained consecutive series of women with axillary node-negative breast cancer with long follow-up. Gene expression microarray data were used to detect mRNAs differentially expressed between T-bet+/high (n = 6) and T-bet-/low (n = 41) tumors. Tissue microarrays (TMAs) constructed from tumors of 612 women were used to quantify expression of BRD4 by immunohistochemistry, which was analyzed for its association with T-bet+ TILs, Jagged1, clinicopathological features, and disease-free survival. RESULTS: Microarray analysis indicated that BRD4 mRNA expression was up to 44-fold higher in T-bet+/high tumors compared to T-bet-/low tumors (p = 5.38E-05). Immunohistochemical expression of BRD4 in cancer cells was also shown to be associated with T-bet+ TILs (p = 0.0415) as well as with Jagged1 mRNA and protein expression (p = 0.0171, 0.0010 respectively). BRD4 expression correlated with larger tumor size (p = 0.0049), pre-menopausal status (p = 0.0018), and high Ki-67 proliferative index (p = 0.0009). Women with high tumoral BRD4 expression in the absence of T-bet+ TILs exhibited a significantly poorer outcome (log rank test p = 0.0165) relative to other subgroups. CONCLUSIONS: The association of BRD4 expression with T-bet+ TILs, and T-bet+ TIL-dependent disease-free survival suggests a potential link between BRD4-mediated tumor development and tumor immune surveillance, possibly through BRD4's regulation of Jagged1 signaling pathways. Further understanding BRD4's role in different immune contexts may help to identify an appropriate subset of breast cancer patients who may benefit from BET inhibitors without the risk of diminishing the anti-tumoral immune activity.

Single cell-derived clonal analysis of human glioblastoma links functional and genomic heterogeneity.

Glioblastoma (GBM) is a cancer comprised of morphologically, genetically, and phenotypically diverse cells. However, an understanding of the functional significance of intratumoral heterogeneity is lacking. We devised a method to isolate and functionally profile tumorigenic clones from patient glioblastoma samples. Individual clones demonstrated unique proliferation and differentiation abilities. Importantly, naïve patient tumors included clones that were temozolomide resistant, indicating that resistance to conventional GBM therapy can preexist in untreated tumors at a clonal level. Further, candidate therapies for resistant clones were detected with clone-specific drug screening. Genomic analyses revealed genes and pathways that associate with specific functional behavior of single clones. Our results suggest that functional clonal profiling used to identify tumorigenic and drug-resistant tumor clones will lead to the discovery of new GBM clone-specific treatment strategies.

Loss of heterozygosity impacts MHC expression on the immune microenvironment in CDK12-mutated prostate cancer.

BACKGROUND: In prostate cancer (PCa), well-established biomarkers such as MSI status, TMB high, and PDL1 expression serve as reliable indicators for favorable responses to immunotherapy. Recent studies have suggested a potential association between CDK12 mutations and immunotherapy response; however, the precise mechanisms through which CDK12 mutation may influence immune response remain unclear. A plausible explanation for immune evasion in this subset of CDK12-mutated PCa may be reduced MHC expression. RESULTS: Using genomic data of CDK12-mutated PCa from 48 primary and 10 metastatic public domain samples and a retrospective cohort of 53 low-intermediate risk primary PCa, we investigated how variation in the expression of the MHC genes affected associated downstream pathways. We classified the patients based on gene expression quartiles of MHC-related genes and categorized the tumors into "High" and "Low" expression levels. CDK12-mutated tumors with higher MHC-expressed pathways were associated with the immune system and elevated PD-L1, IDO1, and TIM3 expression. Consistent with an inflamed tumor microenvironment (TME) phenotype, digital cytometric analyses identified increased CD8 + T cells, B cells, γδ T cells, and M1 Macrophages in this group. In contrast, CDK12-mutated tumors with lower MHC expression exhibited features consistent with an immune cold TME phenotype and immunoediting. Significantly, low MHC expression was also associated with chromosome 6 loss of heterozygosity (LOH) affecting the entire HLA gene cluster. These LOH events were observed in both major clonal and minor subclonal populations of tumor cells. In our retrospective study of 53 primary PCa cases from this Institute, we found a 4% (2/53) prevalence of CDK12 mutations, with the confirmation of this defect in one tumor through Sanger sequencing. In keeping with our analysis of public domain data this tumor exhibited low MHC expression at the RNA level. More extensive studies will be required to determine whether reduced HLA expression is generally associated with primary tumors or is a specific feature of CDK12 mutated PCa. CONCLUSIONS: These data show that analysis of CDK12 alteration, in the context of MHC expression levels, and LOH status may offer improved predictive value for outcomes in this potentially actionable genomic subgroup of PCa. In addition, these findings highlight the need to explore novel therapeutic strategies to enhance MHC expression in CDK12-defective PCa to improve immunotherapy responses.

Investigating the Role of SNAI1 and ZEB1 Expression in Prostate Cancer Progression and Immune Modulation of the Tumor Microenvironment.

Prostate cancer (PCa) is an immunologically cold tumor and the molecular processes that underlie this behavior are poorly understood. In this study, we investigated a primary cohort of intermediate-risk PCa (n = 51) using two NanoString profiling panels designed to study cancer progression and immune response. We identified differentially expressed genes (DEGs) and pathways associated with biochemical recurrence (BCR) and clinical risk. Confirmatory analysis was performed using the TCGA-PRAD cohort. Noteworthy DEGs included collagens such as COL1A1, COL1A2, and COL3A1. Changes in the distribution of collagens may influence the immune activity in the tumor microenvironment (TME). In addition, immune-related DEGs such as THY1, IRF5, and HLA-DRA were also identified. Enrichment analysis highlighted pathways such as those associated with angiogenesis, TGF-beta, UV response, and EMT. Among the 39 significant DEGs, 11 (28%) were identified as EMT target genes for ZEB1 using the Harmonizome database. Elevated ZEB1 expression correlated with reduced BCR risk. Immune landscape analysis revealed that ZEB1 was associated with increased immunosuppressive cell types in the TME, such as naïve B cells and M2 macrophages. Increased expression of both ZEB1 and SNAI1 was associated with elevated immune checkpoint expression. In the future, modulation of EMT could be beneficial for overcoming immunotherapy resistance in a cold tumor, such as PCa.

Validation of the Prognostic Performance of Breast Cancer Index in Hormone Receptor-Positive Postmenopausal Breast Cancer Patients in the TEAM Trial.

PURPOSE: Patients with early-stage hormone receptor-positive (HR+) breast cancer face a prolonged risk of recurrence even after adjuvant endocrine therapy. The Breast Cancer Index (BCI) is significantly prognostic for overall (0-10 years) and late (5-10 years) distant recurrence (DR) risk in N0 and N1 patients. Here, BCI prognostic performance was evaluated in HR+ postmenopausal women from the Tamoxifen and Exemestane Adjuvant Multinational (TEAM) trial. EXPERIMENTAL DESIGN: 3,544 patients were included in the analysis (N = 1,519 N0, N = 2,025 N+). BCI risk groups were calculated using pre-specified cutoff points. Kaplan-Meier analyses and log-rank tests were used to assess the prognostic significance of BCI risk groups based on DR. Hazard ratios (HR) and confidence intervals (CI) were calculated using Cox models with and without clinical covariates. RESULTS: For overall 10-year DR, BCI was significantly prognostic in Ni0 (N = 1,196) and N1 (N = 1,234) patients who did not receive prior chemotherapy (P < 0.001). In patients who were DR-free for 5 years, 10-year late DR rates for low- and high-risk groups were 5.4% and 9.3% (N0 cohort, N = 1,285) and 4.8% and 12.2% (N1 cohort, N = 1,625) with multivariate HRs of 2.25 (95% CI, 1.30-3.88; P = 0.004) and 2.67 (95% CI, 1.53-4.63; P < 0.001), respectively. Late DR performance was substantially improved using previously optimized cutoff points, identifying BCI low-risk groups with even lower 10-year late DR rates of 3.8% and 2.7% in N0 and N1 patients, respectively. CONCLUSIONS: The TEAM trial represents the largest prognostic validation study for BCI to date and provides a more representative assessment of late DR risk to guide individualized treatment decision-making for HR+ patients with early-stage breast cancer.

Adjunctive Statistical Standardization of Adjuvant Estrogen Receptor and Progesterone Receptor in Canadian Cancer Trials Group MA.27 Postmenopausal Breast Cancer Trial of Exemestane Versus Anastrozole.

PURPOSE: ASCO/College of American Pathologists guidelines recommend reporting estrogen receptor (ER) and progesterone receptor (PgR) as positive with (1%-100%) staining. Statistically standardized quantitated positivity could indicate differential associations of positivity with breast cancer outcomes. METHODS: MA.27 (ClinicalTrials.gov identifier: NCT00066573) was a phase III adjuvant trial of exemestane versus anastrozole in postmenopausal women with early-stage breast cancer. Immunochemistry ER and PgR HSCORE and % positivity (%+) were centrally assessed by machine image quantitation and statistically standardized to mean 0 and standard deviation (SD) 1 after Box-Cox variance stabilization transformations of square for ER; for PgR, (1) natural logarithm (0.1 added to 0 HSCOREs and 0%+) and (2) square root. Our primary end point was MA.27 distant disease-free survival (DDFS) at a median 4.1-year follow-up, and secondary end point was event-free survival (EFS). Univariate survival with cut points at SDs about a mean of 0 (≤-1; (-1, 0]; (0, 1]; >1) was described with Kaplan-Meier plots and examined with Wilcoxon (Peto-Prentice) test statistic. Adjusted Cox multivariable regressions had two-sided Wald tests and nominal significance P < .05. RESULTS: Of 7,576 women accrued, 3,048 women's tumors had machine-quantitated image analysis results: 2,900 (95%) for ER, 2,726 (89%) for PgR, and 2,582 (85% of 3,048) with both ER and PgR. Higher statistically standardized ER and PgR HSCORE and %+ were associated with better univariate DDFS and EFS (P < .001). In multivariable assessments, ER HSCORE and %+ were not significantly associated (P = .52-.88) with DDFS in models with PgR, whereas higher PgR HSCORE and %+ were significantly associated with better DDFS (P = .001) in models with ER. CONCLUSION: Adjunctive statistical standardization differentiated quantitated levels of ER and PgR. Patients with higher ER- and PgR-standardized units had superior DDFS compared with those with HSCOREs and %+ ≤-1.

Copy number and expression alterations of miRNAs in the ovarian cancer cell line OVCAR-3: impact on kallikrein 6 protein expression.

BACKGROUND: Kallikrein-related peptidase 6 (KLK6), a member of the serine protease family of kallikrein (KLK) genes, is dysregulated in ovarian carcinomas (OCa) and its overexpression is associated with poor prognosis. Regulation of its expression is poorly understood and is likely to be influenced by multiple mechanisms. The KLK locus is subject to copy number changes and heterogeneity in serous OCas. These copy number imbalances generally correlate with KLK6 protein expression; however, this is not always the case. In this study we explored the role of miRNAs in the posttranscriptional control of KLK6 expression and the contributions of copy numbers, not only of the KLK locus, but also of the miRNAs predicted to regulate it. METHODS AND RESULTS: By miRNA profiling of the KLK6-overexpressing OCa cell line, OVCAR-3, we identified overexpressed and underexpressed miRNAs. Publically available miRNA databases identified the human miRNA lethal 7 (hsa-let-7) family members as putative regulating miRNAs, from which hsa-let-7a was chosen for functional analysis. The transient transfection of hsa-let-7a to OVCAR-3 resulted in a decrease of KLK6 secreted protein. Moreover, such transfection was also able to weakly affect the expression of another member of the KLK gene family, KLK10 (kallikrein-related peptidase 10). Cytogenomic analysis, including array comparative genomic hybridization, fluorescence in situ hybridization, and spectral karyotyping revealed the overall net copy number losses of hsa-let-7a and other miRNAs predicted to target KLK6. CONCLUSIONS: The hsa-let-7 family member hsa-let-7a is a modulator of KLK6 protein expression that is independent of the KLK6 copy number status.

Emerging clinical importance of the cancer biomarkers kallikrein-related peptidases (KLK) in female and male reproductive organ malignancies.

BACKGROUND: Tumor tissue-associated KLKs (kallikrein-related peptidases) are clinically important biomarkers that may allow prognosis of the cancer disease and/or prediction of response/failure of cancer patients to cancer-directed drugs. Regarding the female/male reproductive tract, remarkably, all of the fifteen KLKs are expressed in the normal prostate, breast, cervix uteri, and the testis, whereas the uterus/endometrium and the ovary are expressing a limited number of KLKs only. CONCLUSIONS: Most of the information regarding elevated expression of KLKs in tumor-affected organs is available for ovarian cancer; depicting them as valuable biomarkers in the cancerous phenotype. In contrast, for breast cancer, a series of KLKs was found to be downregulated. However, in breast cancer, KLK4 is elevated which is also true for ovarian and prostate cancer. In such cases, selective synthetic KLK inhibitors that aim at blocking the proteolytic activities of certain KLKs may serve as future candidate therapeutic drugs to interfere with tumor progression and metastasis.

Identification of tumor-agnostic biomarkers for predicting prostate cancer progression and biochemical recurrence.

The diverse clinical outcomes of prostate cancer have led to the development of gene signature assays predicting disease progression. Improved prostate cancer progression biomarkers are needed as current RNA biomarker tests have varying success for intermediate prostate cancer. Interest grows in universal gene signatures for invasive carcinoma progression. Early breast and prostate cancers share characteristics, including hormone dependence and BRCA1/2 mutations. Given the similarities in the pathobiology of breast and prostate cancer, we utilized the NanoString BC360 panel, comprising the validated PAM50 classifier and pathway-specific signatures associated with general tumor progression as well as breast cancer-specific classifiers. This retrospective cohort of primary prostate cancers (n=53) was stratified according to biochemical recurrence (BCR) status and the CAPRA-S to identify genes related to high-risk disease. Two public cohort (TCGA-PRAD and GSE54460) were used to validate the results. Expression profiling of our cohort uncovered associations between PIP and INHBA with BCR and high CAPRA-S score, as well as associations between VCAN, SFRP2, and THBS4 and BCR. Despite low levels of the ESR1 gene compared to AR, we found strong expression of the ER signaling signature, suggesting that BCR may be driven by ER-mediated pathways. Kaplan-Meier and univariate Cox proportional hazards regression analysis indicated the expression of ESR1, PGR, VCAN, and SFRP2 could predict the occurrence of relapse events. This is in keeping with the pathways represented by these genes which contribute to angiogenesis and the epithelial-mesenchymal transition. It is likely that VCAN works by activating the stroma and remodeling the tumor microenvironment. Additionally, SFRP2 overexpression has been associated with increased tumor size and reduced survival rates in breast cancer and among prostate cancer patients who experienced BCR. ESR1 influences disease progression by activating stroma, stimulating stem/progenitor prostate cancer, and inducing TGF-ß. Estrogen signaling may therefore serve as a surrogate to AR signaling during progression and in hormone-refractory disease, particularly in prostate cancer patients with stromal-rich tumors. Collectively, the use of agnostic biomarkers developed for breast cancer stratification has facilitated a precise clinical classification of patients undergoing radical prostatectomy and highlighted the therapeutic potential of targeting estrogen signaling in prostate cancer.

Stromal cell-associated expression of kallikrein-related peptidase 6 (KLK6) indicates poor prognosis of ovarian cancer patients.

Several members of the human kallikrein-related peptidase family, including KLK6, are up-regulated in ovarian cancer. High KLK6 mRNA or protein expression, measured by quantitative polymerase chain reaction and enzyme-linked immunoassay, respectively, was previously found to be associated with a shortened overall and progression-free survival (OS and PFS, respectively). In the present study, we aimed at analyzing KLK6 protein expression in ovarian cancer tissue by immunohistochemistry. Using a newly developed monospecific polyclonal antibody, KLK6 immunoexpression was initially evaluated in normal tissues. We observed strong staining in the brain and moderate staining in the kidney, liver, and ovary, whereas the pancreas and the skeletal muscle were unreactive, which is in line with previously published results. Next, both tumor cell- and stromal cell-associated KLK6 immunoexpression were analyzed in tumor tissue specimens of 118 ovarian cancer patients. In multivariate Cox regression analysis, only stromal cell-associated expression, besides the established clinical parameters FIGO stage and residual tumor mass, was found to be statistically significant for OS and PFS [high vs. low KLK6 expression; hazard ratio (HR), 1.92; p=0.017; HR, 1.80; p=0.042, respectively]. These results indicate that KLK6 expressed by stromal cells may considerably contribute to the aggressiveness of ovarian cancer.

Upregulation of IFNÉ£-mediated chemokines dominate the immune transcriptome of muscle-invasive urothelial carcinoma.

Tumor inflammation is prognostically significant in high-grade muscle-invasive bladder cancer (MIBC). However, the underlying mechanisms remain elusive. To identify inflammation-associated immune gene expression patterns, we performed transcriptomic profiling of 40 MIBC archival tumors using the NanoString nCounter Human v.1.1 PanCancer Panel. Findings were validated using the TCGA MIBC dataset. Unsupervised and supervised clustering identified a distinctive immune-related gene expression profile for inflammation, characterized by significant upregulation of 149 genes, particularly chemokines, a subset of which also had potential prognostic utility. Some of the most enriched biological processes were lymphocyte activation and proliferation, leukocyte adhesion and migration, antigen processing and presentation and cellular response to IFN-γ. Upregulation of numerous IFN-γ-inducible chemokines, class II MHC molecules and immune checkpoint genes was detected as part of the complex immune response to MIBC. Further, B-cell markers linked to tertiary lymphoid structures were upregulated, which in turn is predictive of tumor response to immunotherapy and favorable outcome. Our findings of both an overall activated immune profile and immunosuppressive microenvironment provide novel insights into the complex immune milieu of MIBC with inflammation and supports its clinical significance for predicting prognosis and immunotherapeutic responsiveness, which warrants further investigation. This may open novel opportunities to identify mechanisms for developing new immunotherapeutic strategies.

Discordance between Immunohistochemistry and Erb-B2 Receptor Tyrosine Kinase 2 mRNA to Determine Human Epidermal Growth Factor Receptor 2 Low Status for Breast Cancer.

Novel human epidermal growth factor receptor 2 (HER2)-directed antibody-drug conjugates have demonstrated efficacy in HER2-low expressing breast cancers, which are currently defined as those with immunohistochemistry (IHC) scores of 1+ or 2+ with a negative in situ hybridization assay. However, current HER2 testing methods are designed to identify HER2-amplified tumors with high expression levels. The true definition of HER2-low expressing breast cancers remains controversial. Using quantitative molecular analysis of breast cancers based on RNA expression, the dynamic range of HER2 expression exceeds that detected by in situ IHC approaches. Erb-B2 receptor tyrosine kinase 2 (ERBB2) mRNA expression levels across IHC groups using patient samples derived from the Tamoxifen Exemestane Adjuvant Multicenter Trial were investigated. The standardized mean differences in ERBB2 mRNA scores in log base 2 are 0.47 (95% CI, 0.36-0.57), 0.58 (95% CI, 0.26-0.70), and 0.32 (95% CI, -0.12 to 0.75) when comparing IHC 0+ without staining versus IHC 0+ with some staining, IHC 0+ with some staining versus IHC 1+, and IHC 1+ versus IHC 2+/fluorescence in situ hybridization-negative, respectively. The results showed immunohistochemical methods have a comparatively limited dynamic range for measuring HER2 protein expression. The range of expression based on RNA abundance suggests a molecular method defining HER2-low cancers may better serve the treatment decision needs of this group. Indeed, the validity of RNA abundance to identify HER2-low cancers and predict treatment response needs to be further evaluated by prospective clinical trials.

Imaging Biomarkers in Prostate Stereotactic Body Radiotherapy: A Review and Clinical Trial Protocol.

Advances in imaging have changed prostate radiotherapy through improved biochemical control from focal boost and improved detection of recurrence. These advances are reviewed in the context of prostate stereotactic body radiation therapy (SBRT) and the ARGOS/CLIMBER trial protocol. ARGOS/CLIMBER will evaluate 1) the safety and feasibility of SBRT with focal boost guided by multiparametric MRI (mpMRI) and 18F-PSMA-1007 PET and 2) imaging and laboratory biomarkers for response to SBRT. To date, response to prostate SBRT is most commonly evaluated using the Phoenix Criteria for biochemical failure. The drawbacks of this approach include lack of lesion identification, a high false-positive rate, and delay in identifying treatment failure. Patients in ARGOS/CLIMBER will receive dynamic 18F-PSMA-1007 PET and mpMRI prior to SBRT for treatment planning and at 6 and 24 months after SBRT to assess response. Imaging findings will be correlated with prostate-specific antigen (PSA) and biopsy results, with the goal of early, non-invasive, and accurate identification of treatment failure.

The physiology and pathobiology of human kallikrein-related peptidase 6 (KLK6).

The human kallikrein-related peptidase 6 (KLK6) gene belongs to the 15-member kallikrein (KLK) gene family mapping to chromosome 19q13.3-13.4. Encoding for an enzyme with trypsin-like properties, KLK6 can degrade components of the extracellular matrix. The successful utilisation of another KLK member (KLK3/PSA) for prostate cancer diagnosis has led many to evaluate KLK6 as a potential biomarker for other cancer and diseased states. The observed dysregulated expression in cancers, neurodegenerative diseases and skin conditions has led to the discovery that KLK6 participates in other cellular pathways including inflammation, receptor activation and regulation of apoptosis. Moreover, the improvements in high-throughput genomics have not only enabled the identification of sequence polymorphisms, but of transcript variants, whose functional significances have yet to be realised. This comprehensive review will summarise the current findings of KLK6 pathophysiology and discuss its potential as a viable biomarker.

Immune gene expression profiles in high-grade urothelial carcinoma of the bladder: a NanoString study.

AIMS: The advent of immune checkpoint inhibitor therapy has proven beneficial in a subset of high-grade urothelial carcinomas (HGUC) of the bladder. Although treatment selection is currently largely determined by programmed death-ligand 1 (PD-L1) status, multiple factors in the immune system may modulate the host immune response to HGUC and immunotherapy. In this pilot study, we used a transcriptomic approach to identify the immune milieu associated with PD-L1 expression to enhance our understanding of the HGUC immune evasion network. METHODS: The immune transcriptome of 40 HGUC cystectomy cases was profiled using the NanoString nCounter Human V.1.1 PanCancer Panel. All cases were assessed for associated PD-L1 status (SP263) using whole tissue sections. PD-L1 status was determined as high or low using 25% tumour and/or immune cell staining. RESULTS: The most significantly differentially expressed gene was PD-L1 messenger RNA (CD274), which strongly correlated with protein expression (r=0.720, p<0.001). The sensitivity, specificity, positive and negative predictive values of CD274 for PD-L1 expression were 85%, 96%, 92% and 93%, respectively. The PD-L1 associated gene signature also included complement components C1QA and CD46 and NOD2 (innate immune system), proinflammatory cytokines CXCL14, CXCL16, CCL3, CCL3L1 and OSM along with the immune response mediator SMAD3, among others. Pathway analysis determined enrichment of these genes in interleukin-10 production, lymphocyte chemotaxis and aberrant IFNγ, NF-κB and ERK signalling networks. CONCLUSIONS: We report key genes and pathways in the immune transcriptome and their association with PD-L1 status, which may be involved in immune evasion of HGUC and warrants further investigation.

Comparative survival analysis of multiparametric tests-when molecular tests disagree-A TEAM Pathology study.

Multiparametric assays for risk stratification are widely used in the management of both node negative and node positive hormone receptor positive invasive breast cancer. Recent data from multiple sources suggests that different tests may provide different risk estimates at the individual patient level. The TEAM pathology study consists of 3284 postmenopausal ER+ve breast cancers treated with endocrine therapy Using genes comprising the following multi-parametric tests OncotypeDx®, Prosigna™ and MammaPrint® signatures were trained to recapitulate true assay results. Patients were then classified into risk groups and survival assessed. Whilst likelihood χ2 ratios suggested limited value for combining tests, Kaplan-Meier and LogRank tests within risk groups suggested combinations of tests provided statistically significant stratification of potential clinical value. Paradoxically whilst Prosigna-trained results stratified Oncotype-trained subgroups across low and intermediate risk categories, only intermediate risk Prosigna-trained cases were further stratified by Oncotype-trained results. Both Oncotype-trained and Prosigna-trained results further stratified MammaPrint-trained low risk cases, and MammaPrint-trained results also stratified Oncotype-trained low and intermediate risk groups but not Prosigna-trained results. Comparisons between existing multiparametric tests are challenging, and evidence on discordance between tests in risk stratification presents further dilemmas. Detailed analysis of the TEAM pathology study suggests a complex inter-relationship between test results in the same patient cohorts which requires careful evaluation regarding test utility. Further prognostic improvement appears both desirable and achievable.

Multisite verification of the accuracy of a multi-gene next generation sequencing panel for detection of mutations and copy number alterations in solid tumours.

Molecular variants including single nucleotide variants (SNVs), copy number variants (CNVs) and fusions can be detected in the clinical setting using deep targeted sequencing. These assays support low limits of detection using little genomic input material. They are gaining in popularity in clinical laboratories, where sample volumes are limited, and low variant allele fractions may be present. However, data on reproducibility between laboratories is limited. Using a ring study, we evaluated the performance of 7 Ontario laboratories using targeted sequencing panels. All laboratories analysed a series of control and clinical samples for SNVs/CNVs and gene fusions. High concordance was observed across laboratories for measured CNVs and SNVs. Over 97% of SNV calls in clinical samples were detected by all laboratories. Whilst only a single CNV was detected in the clinical samples tested, all laboratories were able to reproducibly report both the variant and copy number. Concordance for information derived from RNA was lower than observed for DNA, due largely to decreased quality metrics associated with the RNA components of the assay, suggesting that the RNA portions of comprehensive NGS assays may be more vulnerable to variations in approach and workflow. Overall the results of this study support the use of the OFA for targeted sequencing for testing of clinical samples and suggest specific internal quality metrics that can be reliable indicators of assay failure. While we believe this evidence can be interpreted to support deep targeted sequencing in general, additional studies should be performed to confirm this.