Patient-Derived Xenografts and Organoids Recapitulate Castration-Resistant Prostate Cancer with Sustained Androgen Receptor Signaling.

Castration-resistant prostate cancer (CRPC) remains an incurable and lethal malignancy. The development of new CRPC treatment strategies is strongly impeded by the scarcity of representative, scalable and transferable preclinical models of advanced, androgen receptor (AR)-driven CRPC. Here, we present contemporary patient-derived xenografts (PDXs) and matching PDX-derived organoids (PDXOs) from CRPC patients who had undergone multiple lines of treatment. These models were comprehensively profiled at the morphologic, genomic (n = 8) and transcriptomic levels (n = 81). All are high-grade adenocarcinomas that exhibit copy number alterations and transcriptomic features representative of CRPC patient cohorts. We identified losses of PTEN and RB1, MYC amplifications, as well as genomic alterations in TP53 and in members of clinically actionable pathways such as AR, PI3K and DNA repair pathways. Importantly, the clinically observed continued reliance of CRPC tumors on AR signaling is preserved across the entire set of models, with AR amplification identified in four PDXs. We demonstrate that PDXs and PDXOs faithfully reflect donor tumors and mimic matching patient drug responses. In particular, our models predicted patient responses to subsequent treatments and captured sensitivities to previously received therapies. Collectively, these PDX-PDXO pairs constitute a reliable new resource for in-depth studies of treatment-induced, AR-driven resistance mechanisms. Moreover, PDXOs can be leveraged for large-scale tumor-specific drug response profiling critical for accelerating therapeutic advances in CRPC.

Comedonecrosis Gleason pattern 5 is associated with worse clinical outcome in operated prostate cancer patients.

Individual growth patterns and cribriform architecture are increasingly considered in risk stratification and clinical decision-making in men with prostate cancer. Our objective was to establish the prognostic value of individual Gleason 5 patterns in a radical prostatectomy (RP) cohort. We reviewed 1064 RPs and recorded Grade Group (GG), pT-stage, surgical margin status, Gleason 4 and 5 growth patterns as well as intraductal carcinoma. The clinical endpoints were biochemical recurrence and post-operative distant metastasis. Gleason pattern 5 was present in 339 (31.9%) RPs, of which 47 (4.4%) presented as primary, 166 (15.6%) as secondary, and 126 (11.8%) as tertiary pattern. Single cells/cords were present in 321 (94.7%) tumors with Gleason pattern 5, solid fields in 90 (26.5%), and comedonecrosis in invasive carcinoma in 32 (9.4%) tumors. Solid fields demonstrated either a small nested morphology (n = 50, 14.7%) or medium to large solid fields (n = 61, 18.0%). Cribriform architecture was present in 568 (53.4%) RPs. Medium to large solid fields and comedonecrosis coincided with cribriform architecture in all specimens, and were not observed in cribriform-negative cases. In multivariable analysis adjusted for Prostate-Specific Antigen, pT-stage, GG, surgical margin status and lymph node metastases, cribriform architecture (Hazard Ratio (HR) 9.9; 95% Confidence Interval (CI) 3.9-25.5, P < 0.001) and comedonecrosis (HR 2.1, 95% CI 1.2-3.7, P = 0.01) were independent predictors for metastasis-free survival, while single cells/cords (HR 1.2; 95% CI 0.7-1.8, P = 0.55) and medium to large solid fields (HR 1.6, 95% CI 0.9-2.7, P = 0.09) were not. In conclusion, comedonecrosis in invasive carcinoma is an independent prognostic Gleason 5 pattern for metastasis-free survival after RP. These data support the current recommendations to routinely include cribriform pattern in pathology reports and indicate that comedonecrosis should also be commented on.

Cribriform architecture in radical prostatectomies predicts oncological outcome in Gleason score 8 prostate cancer patients.

The Gleason score is an important parameter for clinical outcome in prostate cancer patients. Gleason score 8 is a heterogeneous disease including Gleason score 3 + 5, 4 + 4, and 5 + 3 tumors, and encompasses a broad range of tumor growth patterns. Our objective was to characterize individual growth patterns and identify prognostic parameters in Gleason score 8 prostate cancer patients. We reviewed 1064 radical prostatectomy specimens, recorded individual Gleason 4 and 5 growth patterns as well as presence of intraductal carcinoma, and evaluated biochemical recurrence- and metastasis-free survival. Gleason score 8 disease was identified in 140 (13%) patients, of whom 76 (54%) had Gleason score 3 + 5, 46 (33%) 4 + 4, and 18 (13%) 5 + 3 disease. Invasive cribriform and/or intraductal carcinoma (n = 87, 62%) was observed more frequently in Gleason score 4 + 4 (93%) than 3 + 5 (47%; P < 0.001) and 5 + 3 (44%; P < 0.001) patients. Gleason pattern 5 was present in 110 (79%) men: as single cells and/or cords in 99 (90%) and solid fields in 32 (29%) cases. Solid field pattern 5 coexisted with cribriform architecture (23/32, 72%) more frequently than nonsolid pattern 5 cases (36/78, 46%, P = 0.02). In multivariable analysis including age, prostate-specific antigen, pT-stage, surgical margin status, and lymph node metastases, presence of cribriform architecture was an independent parameter for biochemical recurrence-free (hazard ratio (HR) 2.0, 95% confidence interval (CI) 1.0-3.7; P = 0.04) and metastasis-free (HR 3.5, 95% CI 1.0-12.3; P = 0.05) survival. In conclusion, invasive cribriform and/or intraductal carcinoma occurs more frequently in Gleason score 4 + 4 prostate cancer patients than in Gleason score 3 + 5 and 5 + 3, and is an independent parameter for biochemical recurrence and metastasis. Therefore, cribriform architecture has added value in risk stratification of Gleason score 8 prostate cancer patients.

Combined transmission, dark field and fluorescence microscopy for intact, 3D tissue analysis of biopsies.

SIGNIFICANCE: Currently, tissue biopsies are sectioned into 3- to 5-µm-thick slices that are used for conventional pathology analysis. Previous work by confocal microscopy and light-sheet microscopy has shown that analyzing biopsies intact in three-dimensions (3D) is possible and may lead to a better understanding of cancer growth patterns. Although accurate, these methods require fluorescent staining of the tissue, in addition to tissue clearing. If the 3D biopsy analysis could be done sufficiently swiftly, this approach may be used for on-site assessment of the adequacy of a biopsy taken. AIM: We aim to show that, by transmission microscopy of optically cleared tissue punches, the tissue architecture can be determined without the need for fluorescent staining. APPROACH: Transmission microscopy is used by combining bright field microscopy with dark field and epifluorescent microscopy to compare samples that have also been analyzed by fluorescent confocal microscopy. RESULTS: With increasing distance to the focal plane, the higher-frequency part of the spatial frequency spectrum of transmitted light is attenuated increasingly. This property is exploited for tissue segmentation, detecting whether tissue is present at a certain position in the focal plane image. Using this approach, we show that a 3D rendering of the internal cavity or tubules structure of punch biopsies, which are up to 1-mm thick, can be acquired in ≈1 min scan time per imaging modality. The images of the overall tissue architecture that are obtained are similar to those from the confocal microscopy benchmark, without requiring fluorescent staining. CONCLUSIONS: Images of the overall tissue architecture can be obtained from transmission microcopy; they are similar to those from the confocal microscopy benchmark without requiring fluorescent staining. Tissue clearing is still needed. The total scan time of the present method is significantly shorter at a fraction of the device costs.

Prostate cancer growth patterns beyond the Gleason score: entering a new era of comprehensive tumour grading.

The Gleason grading system is one of the most important factors in clinical decision-making for prostate cancer patients, and is entirely based on the classification of tumour growth patterns. In recent years it has become clear that some individual growth patterns themselves have independent prognostic value, and could be used for better personalised risk stratification. In this review we summarise recent literature on the clinicopathological value and molecular characteristics of individual prostate cancer growth patterns, and show how these, most particularly cribriform architecture, could alter treatment decisions for prostate cancer patients.

Clinicopathological characteristics of glomeruloid architecture in prostate cancer.

Glomeruloid architecture is the least common Gleason 4 growth pattern in prostate adenocarcinoma. Its clinicopathological features and relation with cribriform architecture, which has been recognized as an adverse feature, remains to be established. Our objective was to investigate clinicopathological features of glomeruloid architecture in radical prostatectomies. We reviewed 1064 radical prostatectomy specimens and recorded Grade Group, pT-stage, margin status, Gleason pattern percentages, and growth patterns. Simple and complex glomerulations were distinguished by gland size and intraluminal cribriform protrusions. Clinical endpoint was biochemical recurrence-free survival. Glomerulations were identified in 365 (34%) specimens. In 472 Grade Group 2 patients, 210 (44%) had simple and 92 (19%) complex glomerulations. Complex glomerulations coincided with cribriform architecture more often than simple glomerulations (67% versus 52%; P = 0.01). Men with simple glomerulations had significantly lower prostate specific antigen (PSA) levels (9.7 versus 12.1 ng/ml; P = 0.03), percentage Gleason pattern 4 (19% versus 25%; P = 0.001), extra-prostatic extension (34% versus 50%; P = 0.01), and positive surgical margins (25% versus 39%; P = 0.04) than those with cribriform architecture. Extra-prostatic extension (37%) and positive surgical margins (30%) in men with complex glomerulations resembled those with simple glomeruloid rather than those with cribriform architecture. In multivariate Cox regression analysis adjusted for PSA, pT-stage, margin status, and lymph node metastases, cribriform architecture had independent predictive value for biochemical recurrence-free survival (hazard ratio (HR)) 1.9; 95% confidence interval (CI) 1.2-2.9; P = 0.004), while simple (HR 0.8; 95% CI 0.5-1.2; P = 0.26) and complex (HR 0.9; 95% CI 0.5-1.6; P = 0.67) glomerulations did not. Both simple and complex glomeruloid architecture are associated with better outcome than cribriform architecture in Grade Group 2 prostate cancer patients. Therefore, glomeruloid pattern and particularly complex glomerulations should not be classified as a cribriform growth pattern variant in radical prostatectomy specimens.

Comparison of Tumor Volume Parameters on Prostate Cancer Biopsies.

CONTEXT.­: Prostate biopsy reports require an indication of prostate cancer volume. No consensus exists on the methodology of tumor volume reporting. OBJECTIVE.­: To compare the prognostic value of different biopsy prostate cancer volume parameters. DESIGN.­: Prostate biopsies of the European Randomized Study of Screening for Prostate Cancer were reviewed (n = 1031). Tumor volume was quantified in 6 ways: average estimated tumor percentage, measured total tumor length, average calculated tumor percentage, greatest tumor length, greatest tumor percentage, and average tumor percentage of all biopsies. Their prognostic value was determined by using either logistic regression for extraprostatic expansion (EPE) and surgical margin status after radical prostatectomy (RP), or Cox regression for biochemical recurrence-free survival (BCRFS) and disease-specific survival (DSS) after RP (n = 406) and radiation therapy (RT) (n = 508). RESULTS.­: All tumor volume parameters were significantly mutually correlated (R2 > 0.500, P < .001). None were predictive for EPE, surgical margin, or BCRFS after RP in multivariable analysis, including age, prostate-specific antigen, number of positive biopsies, and grade group. In contrast, all tumor volume parameters were significant predictors for BCRFS (all P < .05) and DSS (all P < .05) after RT, except greatest tumor length. In multivariable analysis including only all tumor volume parameters as covariates, calculated tumor length was the only predictor for EPE after RP (P = .02) and DSS after RT (P = .02). CONCLUSIONS.­: All tumor volume parameters had comparable prognostic value and could be used in clinical practice. If tumor volume quantification is a threshold for treatment decision, calculated tumor length seems preferential, slightly outperforming the other parameters.

Clinical outcome comparison of Grade Group 1 and Grade Group 2 prostate cancer with and without cribriform architecture at the time of radical prostatectomy.

AIMS: Invasive cribriform and intraductal carcinoma are associated with aggressive disease in Grade Group 2 (GG2) prostate cancer patients. However, the characteristics and clinical outcome of patients with GG2 prostate cancer without cribriform architecture (GG2-) as compared with those with Grade Group 1 (GG1) prostate cancer are unknown. The aim of this study was to investigate the clinical and pathological characteristics of GG1 and GG2- prostate cancer in radical prostatectomy specimens. METHODS AND RESULTS: We reviewed 835 radical prostatectomy specimens for Grade Group, pT stage, surgical margin status, and the presence of cribriform architecture. Biochemical recurrence-free survival and metastasis were used as clinical outcomes. GG1 prostate cancer was seen in 207 patients, and GG2 prostate cancer was seen in 420 patients, of whom 228 (54%) showed cribriform architecture (GG2+) and 192 (46%) did not. GG2- patients had higher prostate-specific antigen levels (9.4 ng/ml versus 7.0 ng/ml; P < 0.001), more often had extraprostatic extension (36% versus 11%; P < 0.001) and had more positive surgical margins (27% versus 17%; P = 0.01) than GG1 patients. GG2- patients had shorter biochemical recurrence-free survival (hazard ratio 2.7, 95% confidence interval 1.4-4.9; P = 0.002) than GG1 patients. Lymph node and distant metastasis were observed neither in GG2- nor in GG1 patients, but occurred in 22 of 228 (10%) GG2+ patients. CONCLUSION: In conclusion, patients with GG2- prostate cancer at radical prostatectomy have more advanced disease and shorter biochemical recurrence-free survival than those with GG1 prostate cancer, but both groups have a very low risk of developing metastasis.

Improved Prostate Cancer Biopsy Grading by Incorporation of Invasive Cribriform and Intraductal Carcinoma in the 2014 Grade Groups.

BACKGROUND: Grade groups (GGs) are an important parameter for therapeutic decision making in prostate cancer (Pca) patients. Invasive cribriform and/or intraductal carcinoma (CR/IDC) has an independent prognostic value for disease outcome, but are not included in the GG limiting their clinical use. OBJECTIVE: To perform a proof-of-principle study incorporating CR/IDC in the current GG. DESIGN, SETTING, AND PARTICIPANTS: All prostate biopsies of 1031 men with screen-detected Pca between 1993 and 2000 were reviewed for the current GG (ranging from 1 to 5) and CR/IDC. The cribriform grade (cGrade) was equal to the GG if CR/IDC was present and GG minus 1 if not. GG1 was cGrade1 if intraductal carcinoma was absent. INTERVENTION: Biopsy review for GG and CR/IDC. A total of 406 patients had received radical prostatectomy (RP), 508 radiotherapy (RT), 108 surveillance, and eight hormonal therapy, and the treatment was unknown for one patient. Outcome measurements and statistical analysis disease-specific survival (DSS), metastasis-free survival (MFS), and biochemical recurrence-free survival (BCRFS) after 15.1 yr (interquartile range 10.9-19.7 yr) follow-up were compared using Harrell's C-statistic. RESULTS AND LIMITATIONS: The biopsy GGs were 486 GG1, 310 GG2, 104 GG3, 64 GG4, and 67 GG5; cGrade distributions were 738 cGrade1, 102 cGrade2, 91 cGrade3, 58 cGrade4, and 42 cGrade5. The cGrade had a better discriminative value than the GG for DSS (C-index 0.79; 95% confidence interval 0.74-0.83 vs 0.76; 0.71-0.82) and MFS (0.79; 0.74-0.84 vs 0.77; 0.72-0.82). The discriminative value for BCRFS after RP and RT was similar for both models. Different diagnostic, such as use of sextant biopsies, and therapeutic strategies in the 1990s are the limitations of this study. CONCLUSIONS: The cGrade is a simple Pca grade modification with better discriminative values for DSS and MFS than the GG, particularly impacting decision making in men with current GG2 Pca. PATIENT SUMMARY: Microscopic grading is an important factor for decision making in prostate cancer (Pca) patients. We show that a simple grade modification better predicts Pca outcome and might improve treatment choices.

Prostate Carcinoma Grade and Length But Not Cribriform Architecture at Positive Surgical Margins Are Predictive for Biochemical Recurrence After Radical Prostatectomy.

Postoperative biochemical recurrence occurs in up to 40% of prostate carcinoma patients treated with radical prostatectomy. Primary tumor grade and cribriform architecture are important parameters for clinical outcome; however, their relevance at positive surgical margins has not been completely elucidated yet. We reviewed 835 radical prostatectomy specimens and recorded pT-stage, surgical margin status, Grade Group, and cribriform architecture of the primary tumor and at positive surgical margins. Clinicopathologic parameters and biochemical recurrence-free survival (BCRFS) were used as endpoints. Positive surgical margins were present in 284 (34%) patients, with a median cumulative length of 5.0 mm. In 46%, the Grade Group at the margin was equal to the primary tumor grade, while being lower in 42% and higher in 12%. In multivariable analysis, Grade Group at the margin outperformed the Grade Group of the primary tumor in predicting BCRFS. Among primary Grade Group 2 patients, 56% had Grade Group 1 disease at the margin. Multivariable analysis identified cumulative length, Grade Group at the margin, and lymph node metastasis as independent predictors for BCRFS, while percentage Gleason pattern 4, tertiary Gleason pattern 5 of the primary tumor, and cribriform architecture at the margin were not. In conclusion, the Grade Group at the positive surgical margin was dissimilar to the primary tumor grade in 54% and better predicted BCRFS than the primary tumor grade. Cumulative length and tumor grade at the margin were independent predictors for BCRFS, whereas cribriform architecture at the margin was not.

APOBEC3B Gene Expression in Ductal Carcinoma In Situ and Synchronous Invasive Breast Cancer.

The underlying mechanism of the progression of ductal carcinoma in situ (DCIS), a non-obligate precursor of invasive breast cancer (IBC), has yet to be elucidated. In IBC, Apolipoprotein B mRNA Editing Enzyme, Catalytic Polypeptide-Like 3B (APOBEC3B) is upregulated in a substantial proportion of cases and is associated with higher mutational load and poor prognosis. However, APOBEC3B expression has never been studied in DCIS. We performed mRNA expression analysis of APOBEC3B in synchronous DCIS and IBC and surrounding normal cells. RNA was obtained from 53 patients. The tumors were categorized based on estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (Her2) and phosphoinositide-3-kinase, catalytic, alpha polypeptide (PIK3CA) mutation status. APOBEC3B mRNA levels were measured by RT-qPCR. The expression levels of paired DCIS and adjacent IBC were compared, including subgroup analyses. The normal cells expressed the lowest levels of APOBEC3B. No differences in expression were found between DCIS and IBC. Subgroup analysis showed that APOBEC3B was the highest in the ER subgroups of DCIS and IBC. While there was no difference in APOBEC3B between wild-type versus mutated PIK3CA DCIS, APOBEC3B was higher in wild-type versus PIK3CA-mutated IBC. In summary, our data show that APOBEC3B is already upregulated in DCIS. This suggests that APOBEC3B could already play a role in early carcinogenesis. Since APOBEC3B is a gain-of-function mutagenic enzyme, patients could benefit from the therapeutic targeting of APOBEC3B in the early non-invasive stage of breast cancer.

Concordance of cribriform architecture in matched prostate cancer biopsy and radical prostatectomy specimens.

AIMS: Invasive cribriform and/or intraductal carcinoma have been identified as independent adverse parameters for prostate cancer outcome. Little is known on biopsy undersampling of cribriform architecture. Our aim was to determine the extent of cribriform architecture undersampling and to find predictive factors for identifying false cribriform-negative cases. METHODS AND RESULTS: We reviewed 186 matched prostate biopsies and radical prostatectomy specimens. Of 97 biopsy grade group 2 (Gleason score 3 + 4 = 7) patients, 22 (23%) had true cribriform-negative (TN), 39 (40%) false-negative (FN) and 36 (37%) true-positive (TP) biopsies. Patients with FN biopsies had higher, although not statistically significant (P = 0.06), median PSA levels than patients with TP biopsies (12 versus 8 ng/ml). A PI-RADS 5 lesion was present in nine of 16 (54%) FN and three of 11 (27%) TN biopsies (P = 0.05). Positive biopsy rate (P = 0.47), percentage Gleason pattern 4 (P = 0.55) and glomeruloid architecture (P = 1.0) were not different. Logistic regression identified PSA as an independent predictor (odds ratio = 3.5; 95% confidence interval = 1.2-9.4, P = 0.02) for cribriform architecture on radical prostatectomy, but not PI-RADS score. The FN rate for large cribriform architecture at radical prostatectomy was 27%, which was lower than for any cribriform architecture (P = 0.01). During follow-up (median 27 months), biochemical recurrence-free survival of patients with TP biopsies was significantly shorter than that of those with FN biopsies (P = 0.03). CONCLUSION: In conclusion, 40% of grade group 2 prostate cancer biopsies were FN for cribriform architecture. These patients had higher PSA levels and more frequent PI-RADS score 5 lesions than men with TN biopsies.

Differential tissue expression of extracellular vesicle-derived proteins in prostate cancer.

BACKGROUND: Proteomic profiling of extracellular vesicles (EVs) from prostate cancer (PCa) and normal prostate cell lines, led to the identification of new candidate PCa markers. These proteins included the nuclear exportin proteins XPO1 (also known as CRM1), the EV-associated PDCD6IP (also known as ALIX), and the previously published fatty acid synthase FASN. In this study, we investigated differences in expression of XPO1 and PDCD6IP on well-characterized prostate cancer cohorts using mass spectrometry and tissue microarray (TMA) immunohistochemistry to determine their diagnostic and prognostic value. METHODS: Protein fractions from 67 tissue samples (n = 33 normal adjacent prostate [NAP] and n = 34 PCa) were analyzed by mass spectrometry (nano-LC-MS-MS). Label-free quantification of EVs was performed to identify differentially expressed proteins between PCa and NAP. Prognostic evaluation of the candidate markers was performed with a TMA, containing 481 radical prostatectomy samples. Samples were stained for the candidate markers and correlated with patient information and clinicopathological outcome. RESULTS: XPO1 was higher expressed in PCa compared to NAP in the MS data analysis (P > 0.0001). PDCD6IP was not significantly higher expressed (P = 0.0501). High cytoplasmic XPO1 staining in the TMA immunohistochemistry, correlated in a multivariable model with high Gleason scores (P = 0.002) and PCa-related death (P = 0.009). CONCLUSION: High expression of cytoplasmic XPO1 shows correlation with prostate cancer and has added clinical value in tissue samples. Furthermore, as an extracellular vesicles-associated protein, it might be a novel relevant liquid biomarker.

Three-dimensional architecture of common benign and precancerous prostate epithelial lesions.

AIMS: Many glandular lesions can mimic prostate cancer microscopically, including atrophic glands, adenosis and prostatic intraepithelial neoplasia. While the characteristic histopathological and immunohistochemical features of these lesions have been well established, little is known about their three-dimensional architecture. Our objective was to evaluate the three-dimensional organisation of common prostate epithelial lesions. METHODS AND RESULTS: 500 µm-thick punches (n = 42) were taken from radical prostatectomy specimens, and stained with antibodies targeting keratin 8-18 and keratin 5 for identification of luminal and basal cells, respectively. Tissue samples were optically cleared in benzyl alcohol:benzyl benzoate and imaged using a confocal laser scanning microscope. The three-dimensional architecture of peripheral and transition zone glands was acinar, composed of interconnecting and blind-ending saccular tubules. In simple atrophy, partial atrophy and post-atrophic hyperplasia, the acinar structure was attenuated with branching blind-ending tubules from parental tubular structures. Three-dimensional imaging revealed a novel variant of prostate atrophy characterised by large Golgi-like atrophic spaces parallel to the prostate surface, which were represented by thin, elongated tubular structures on haematoxylin and eosin (H&E) slides. Conversely, adenosis lacked acinar organisation, so that it closely mimicked low-grade prostate cancer. High-grade prostatic intraepithelial neoplasia displayed prominent papillary intraluminal protrusions but retained an acinar organisation, whereas intraductal carcinoma predominantly consisted of cribriform proliferations with either spheroid, ellipsoid or complex interconnecting lumens. CONCLUSIONS: While various prostate epithelial lesions might mimic malignancy on H&E slides, their three-dimensional architecture is acinar and clearly different from the tubular structure of prostate cancer, with adenosis as an exception.

PIK3CA mutations in ductal carcinoma in situ and adjacent invasive breast cancer.

PIK3CA is one of the most frequently mutated genes in invasive breast cancer (IBC). These mutations are generally associated with hyper-activation of the phosphatidylinositol 3-kinase signaling pathway, which involves increased phosphorylation of AKT (p-AKT). This pathway is negatively regulated by the tumor suppressor PTEN. Data are limited regarding the variant allele frequency (VAF) of PIK3CA, PTEN and p-AKT expression during various stages of breast carcinogenesis. Therefore, the aim of this study was to gain insight into PIK3CA VAF and associated PTEN and p-AKT expression during the progression from ductal carcinoma in situ (DCIS) to IBC. We isolated DNA from DCIS tissue, synchronous IBC and metastasis when present. These samples were pre-screened for PIK3CA hotspot mutations using the SNaPshot assay and, if positive, validated and quantified by digital PCR. PTEN and p-AKT expression was evaluated by immunohistochemistry using the Histo-score (H-score). Differences in PIK3CA VAF, PTEN and p-AKT H-scores between DCIS and IBC were analyzed. PIK3CA mutations were detected in 17 out of 73 DCIS samples, 16 out of 73 IBC samples and 3 out of 23 lymph node metastasis. We detected a significantly higher VAF of PIK3CA in the DCIS component compared to the adjacent IBC component (P = 0.007). The expression of PTEN was significantly higher in DCIS compared to the IBC component in cases with a wild-type (WT) PIK3CA status (P = 0.007), while it remained similar in both components when PIK3CA was mutated. There was no difference in p-AKT expression between DCIS and the IBC component. In conclusion, our data suggest that PIK3CA mutations could be essential specifically in early stages of breast carcinogenesis. In addition, these mutations do not co-occur with PTEN expression during DCIS progression to IBC in the majority of patients. These results may contribute to further unraveling the process of breast carcinogenesis, and this could aid in the development of patient-specific treatment.

Three-dimensional analysis reveals two major architectural subgroups of prostate cancer growth patterns.

The Gleason score is one of the most important parameters for therapeutic decision-making in prostate cancer patients. Gleason growth patterns are defined by their histological features on 4- to 5-µm cross sections, and little is known about their three-dimensional architecture. Our objective was to characterize the three-dimensional architecture of prostate cancer growth patterns. Intact tissue punches (n = 46) of representative Gleason growth patterns from radical prostatectomy specimens were fluorescently stained with antibodies targeting Keratin 8/18 and Keratin 5 for the detection of luminal and basal epithelial cells, respectively. Punches were optically cleared in benzyl alcohol-benzyl benzoate and imaged using a confocal laser scanning microscope up to a depth of 500 µm. Gleason pattern 3, poorly formed pattern 4, and cords pattern 5 all formed a continuum of interconnecting tubules in which the diameter of the structures and the lumen size decreased with higher grades. In fused pattern 4, the interconnections between the tubules were markedly closer together. In these patterns, all tumor cells were in direct contact with the surrounding stroma. In contrast, cribriform Gleason pattern 4 and solid pattern 5 demonstrated a three-dimensional continuum of contiguous tumor cells, in which the vast majority of cells had no contact with the surrounding stroma. Transitions between cribriform pattern 4 and solid pattern 5 were seen. There was a decrease in the number and size of intercellular lumens from cribriform to solid growth pattern. Glomeruloid pattern 4 formed an intermediate structure consisting of a tubular network with intraluminal epithelial protrusions close to the tubule splitting points. In conclusion, three-dimensional microscopy revealed two major architectural subgroups of prostate cancer growth patterns: (1) a tubular interconnecting network including Gleason pattern 3, poorly formed and fused Gleason pattern 4, and cords Gleason pattern 5, and (2) serpentine contiguous epithelial proliferations including cribriform Gleason pattern 4 and solid Gleason pattern 5.

Large cribriform growth pattern identifies ISUP grade 2 prostate cancer at high risk for recurrence and metastasis.

Invasive cribriform and intraductal carcinoma are associated with adverse clinical outcome in patients with Gleason score 7 prostate cancer. It is yet unclear whether invasive cribriform and intraductal carcinoma of the prostate both have independent prognostic value, or whether field size of invasive cribriform carcinoma has impact on disease outcome. Our objective was to determine the prognostic impact of intraductal and invasive cribriform prostate cancer histological subtypes in radical prostatectomies. We reviewed 420 prostatectomy specimens with ISUP grade 2 prostate cancer, assessed the percentages of Gleason grade 4 and tertiary 5, and performed immunohistochemistry for basal cells to discriminate intraductal from invasive cribriform growth. Small and large invasive cribriform fields were distinguished based on a diameter of at least twice the size of adjacent pre-existent normal glands. Clinicopathological parameters and biochemical recurrence-free survival were used as endpoints. Cribriform architecture was observed in 228 (54.3%) men, 103 (24.5%) of whom had intraductal, 194 (46.2%) small invasive, and 34 (8.1%) large invasive cribriform growth. Large invasive cribriform architecture was associated with older age (P < 0.001), higher percentage Gleason grade 4 (P = 0.001), extraprostatic expansion (P < 0.001), and more frequent lymph node metastases (P = 0.002), when compared with small invasive cribriform and/or intraductal carcinoma. Univariate analysis identified PSA, pT-stage, surgical margin status, and intraductal and invasive cribriform growth as significant predictors for biochemical recurrence-free survival. In multivariable Cox regression analysis, pT-stage (hazard ratio = 1.64, 95% CI: 1.02-2.63, P = 0.04), positive surgical margins (hazard ratio = 3.28, 95% CI: 2.06-5.23, P < 0.001), and large cribriform growth (hazard ratio = 4.36, 95% CI: 2.08-9.17, P < 0.001) were independent predictors for biochemical recurrence-free survival, while intraductal carcinoma, small cribriform growth, and percentage of Gleason grade 4 were not. In conclusion, large cribriform fields represent an aggressive subpattern of invasive cribriform prostate cancer and are an independent predictive factor for biochemical recurrence-free survival in ISUP grade 2 prostate cancer patients.

Tissue proteomics outlines AGR2 AND LOX5 as markers for biochemical recurrence of prostate cancer.

Although many patients are cured from prostate cancer (PCa) by surgery only, there are still patients who will experience rising prostate-specific antigen (PSA) levels after surgery, a condition known as biochemical recurrence (BCR). Novel protein prognostic markers in PCa tissue might enable finding better treatment for those patients experiencing BCR with a high chance of metastasis. In this study, we aimed to identify altered proteins in prostate cancer tissue, and to evaluate their potential role as prognostic markers. We used two proteomics strategies to analyse 34 prostate tumours (PCa) and 33 normal adjacent prostate (NAP) tissues. An independent cohort of 481 samples was used to evaluate the expression of three proteins: AGR2, FASN and LOX5 as prognostic markers of the disease. Tissue microarray immunohistochemical staining indicated that a low percentage of positive tumour cells for AGR2 (HR (95% CI) = 0.61 (0.43-0.93)), and a low percentage of positive tumour cells for LOX5 expression (HR (95% CI) = 2.53 (1.23-5.22)) are predictors of BCR after RP. In contrast, FASN expression had no prognostic value for PCa.

Epithelial-Mesenchymal Transition in Human Prostate Cancer Demonstrates Enhanced Immune Evasion Marked by IDO1 Expression.

Cancer invasion and metastasis are driven by epithelial-mesenchymal transition (EMT), yet the exact mechanisms that account for EMT in clinical prostate cancer are not fully understood. Expression of N-cadherin is considered a hallmark of EMT in clinical prostate cancer. In this study, we determined the molecular mechanisms associated with N-cadherin expression in patients with prostate cancer. We performed laser capture microdissection of matched N-cadherin-positive and -negative prostate cancer areas from patient samples (n = 8), followed by RNA sequencing. N-cadherin expression was significantly associated with an immune-regulatory signature including profound upregulation of indoleamine 2,3-dioxygenase (IDO1; log2-fold change = 5.1; P = 2.98E-04). Fluorescent immunostainings of patient samples confirmed expression of IDO1 protein and also its metabolite kynurenine in primarily N-cadherin-positive areas. N-cadherin-positive areas also exhibited a local decrease of intraepithelial cytotoxic (CD8+) T cells and an increase of immunosuppressive regulatory T cells (CD4+/FOXP3+). In conclusion, EMT in clinical prostate cancer is accompanied by upregulated expression of IDO1 and an increased number of regulatory T cells. These data indicate that EMT, which is an important step in tumor progression, can be protected from effective immune control in patients with prostate cancer.Significance: These findings demonstrate EMT is linked to an immunosuppressive environment in clinical prostate cancer, suggesting that patients with prostate cancer can potentially benefit from combinatorial drug therapy. Cancer Res; 78(16); 4671-9. ©2018 AACR.

Characteristics and outcome of prostate cancer patients with overall biopsy Gleason score 3 + 4 = 7 and highest Gleason score 3 + 4 = 7 or > 3 + 4 = 7.

AIM: Prostate cancer heterogeneity and multifocality might result in different Gleason scores (GS) at individual biopsy cores. According to World Health Organisation/International Society of Urologic Pathology (WHO/ISUP) guidelines, the GS in each biopsy core should be recorded with optional reporting of overall GS for the entire case. We aimed to compare the clinicopathological characteristics and outcome of men with overall biopsy GS 3 + 4 = 7 with highest GS 3 + 4 = 7 (HI = OV) to those with highest GS > 3 + 4 = 7 (HI > OV). METHODS AND RESULTS: Prostate cancer biopsies from the European Randomised Study of Screening for Prostate Cancer (ERSPC) were revised according to WHO/ISUP 2014 guidelines (n = 1031). In total, 370 patients had overall GS 3 + 4 = 7, 60 of whom (16%) had had at least one biopsy core with GS 4 + 3 = 7 or 4 + 4 = 8. Men with higher GS than 3 + 4 (HI > OV) in any of the cores had higher age, prostate-specific antigen (PSA) level, number of positive biopsies, percentage tumour involvement, percentage Gleason grade 4 and cribriform or intraductal growth (all P < 0.05) than those with GS 3 + 4 = 7 at highest (HI = OV). In multivariable Cox regression analysis, including PSA, percentage positive biopsies and percentage tumour involvement, biochemical recurrence-free survival after radical prostatectomy (P = 0.52) or radiotherapy (P = 0.35) was not statistically different between both groups. CONCLUSION: Among patients with overall GS 3 + 4 = 7, those with highest GS > 3 + 4 = 7 had worse clinicopathological features, but clinical outcome was not statistically significant. Therefore, the use of overall GS instead of highest GS for clinical decision-making is justified, potentially preventing overtreatment in prostate cancer patients.