Tumour heterogeneity in primary prostate cancer is a well-established phenomenon. However, how the subclonal diversity of tumours changes during metastasis and progression to lethality is poorly ...understood. Here we reveal the precise direction of metastatic spread across four lethal prostate cancer patients using whole-genome and ultra-deep targeted sequencing of longitudinally collected primary and metastatic tumours. We find one case of metastatic spread to the surgical bed causing local recurrence, and another case of cross-metastatic site seeding combining with dynamic remoulding of subclonal mixtures in response to therapy. By ultra-deep sequencing end-stage blood, we detect both metastatic and primary tumour clones, even years after removal of the prostate. Analysis of mutations associated with metastasis reveals an enrichment of TP53 mutations, and additional sequencing of metastases from 19 patients demonstrates that acquisition of TP53 mutations is linked with the expansion of subclones with metastatic potential which we can detect in the blood.
The Illumina HumanMethylation450 BeadChip (HM450K) measures the DNA methylation of 485,512 CpGs in the human genome. The technology relies on hybridization of genomic fragments to probes on the chip. ...However, certain genomic factors may compromise the ability to measure methylation using the array such as single nucleotide polymorphisms (SNPs), small insertions and deletions (INDELs), repetitive DNA, and regions with reduced genomic complexity. Currently, there is no clear method or pipeline for determining which of the probes on the HM450K bead array should be retained for subsequent analysis in light of these issues.
We comprehensively assessed the effects of SNPs, INDELs, repeats and bisulfite induced reduced genomic complexity by comparing HM450K bead array results with whole genome bisulfite sequencing. We determined which CpG probes provided accurate or noisy signals. From this, we derived a set of high-quality probes that provide unadulterated measurements of DNA methylation.
Our method significantly reduces the risk of false discoveries when using the HM450K bead array, while maximising the power of the array to detect methylation status genome-wide. Additionally, we demonstrate the utility of our method through extraction of biologically relevant epigenetic changes in prostate cancer.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Study Type – Diagnostic (exploratory cohort)
Level of Evidence 2b
What's known on the subject? and What does the study add?
Due to sampling error, the Gleason score of clinically localized prostate ...cancer is frequently underestimated at the time of initial biopsy. Given that this may lead to inappropriate surveillance of patients with high‐risk disease, there is considerable interest in identifying predictors of significant undergrading. Recently PSAD has been proposed to be an accurate predictor of subsequent upgrading in patients diagnosed with Gleason 6 disease on biopsy.
We examined the predictive characteristics of PSAD in patients with low‐ and intermediate‐risk disease on biopsy subsequently treated with radical prostatectomy. We found that although PSAD was a significant predictor of upgrade of biopsy Gleason 6 and 3 + 4 = 7 tumours, it failed to predict upgrading in patients with Gleason 7 tumours taken as a whole. When we explored reasons for this discrepancy, we found that the amount of PSA produced per unit tumour volume decreased with increasing Gleason score, thereby diminishing the predictive value of PSAD.
OBJECTIVES
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To analyse the performance of PSA density (PSAD) as a predictor of Gleason score upgrade in a large cohort stratified by Gleason score.
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We and others have shown that an upgrade in Gleason score between initial prostate biopsy and final radical prostatectomy (RP) pathology is a significant risk factor for recurrence after local therapy.
PATIENTS AND METHODS
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Patients undergoing RP with matching biopsy information were identified from two prospective databases.
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Patients were analysed according to the concordance between biopsy and final pathology Gleason score in three paired groups: 6/>6, 3 + 4/>3 + 4, 7/>7.
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Receiver‐operating characteristic (ROC) curves were generated stratified by Gleason score, and the area under the curve (AUC) calculated.
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Logistic regression models were fitted to identify significant predictors of tumour upgrade.
RESULTS
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From 1516 patients, 435 (29%) had an upgrade in Gleason score. ROC analysis showed a decline in AUC with increasing biopsy Gleason score, from 0.64 for biopsy Gleason score 6, to 0.57 for Gleason score 7.
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In logistic regression models containing pretreatment variables, e.g. clinical stage and number of positive cores, for Gleason score 6 and 3 + 4, PSAD was the strongest predictor of subsequent tumour upgrade (odds ratio OR 1.46, 95% confidence interval 95% CI 1.18–1.83, P= 0.001 and OR 1.37, 95% CI 1.14–1.67, P= 0.002, respectively).
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Surprisingly, in tumours upgraded from Gleason score 7 to >7, PSAD was not predictive even on univariable analysis, whereas clinical stage and number of positive cores were significant independent predictors. To explore the relationship between serum PSA and Gleason score, tumour volume was calculated in 669 patients.
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There was a strong association between Gleason score and tumour volume, with the median volume of Gleason score 7 and Gleason score >7 tumours being approximately twice and four‐times that of Gleason score 6 tumours, respectively (P < 0.001).
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In contrast, the median serum PSA level per millilitre tumour volume decreased significantly with increasing grade, from 5.4 ng/mL for Gleason score 6 to 2.1 ng/mL for >7 (P < 0.001).
CONCLUSIONS
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There is a strong correlation between Gleason score and tumour volume in well/intermediate differentiated tumours, and as they produce relatively high amounts of PSA per unit volume of cancer, high PSAD is the strongest single predictor of tumour undergrading.
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However, as higher grade tumours produce less PSA per unit volume, PSAD loses its predictive ability, and other clinical markers of tumour volume such as palpable disease and numbers of positive cores become more predictive.
The purpose of this study was to determine if microRNA profiling of urine and plasma at radical prostatectomy can distinguish potentially lethal from indolent prostate cancer.
A panel of microRNAs ...was profiled in the plasma of 70 patients and the urine of 33 patients collected prior to radical prostatectomy. Expression of microRNAs was correlated to the clinical endpoints at a follow-up time of 3.9 years to identify microRNAs that may predict clinical response after radical prostatectomy. A machine learning approach was applied to test the predictive ability of all microRNAs profiled in urine, plasma, and a combination of both, and global performance assessed using the area under the receiver operator characteristic curve (AUC). Validation of urinary expression of miRNAs was performed on a further independent cohort of 36 patients.
The best predictor in plasma using eight miRs yielded only moderate predictive performance (AUC = 0.62). The best predictor of high-risk disease was achieved using miR-16, miR-21 and miR-222 measured in urine (AUC = 0.75). This combination of three microRNAs in urine was a better predictor of high-risk disease than any individual microRNA. Using a different methodology we found that this set of miRNAs was unable to predict high-volume, high-grade disease.
Our initial findings suggested that plasma and urinary profiling of microRNAs at radical prostatectomy may allow prognostication of prostate cancer behaviour. However we found that the microRNA expression signature failed to validate in an independent cohort of patients using a different platform for PCR. This highlights the need for independent validation patient cohorts and suggests that urinary microRNA signatures at radical prostatectomy may not be a robust way to predict the course of clinical disease after definitive treatment for prostate cancer.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
It has been recognized for almost a decade that concentrations of signaling androgens sufficient to activate the androgen receptor are present in castration-resistant prostate cancer tissue. The ...source of these androgens is highly controversial, with three competing models proposed. We, therefore, wished to determine the androgenic potential of human benign and malignant (hormone-naïve and treated) prostate tissue when incubated with various precursors and examine concomitant changes in enzyme expression.
Freshly harvested prostate tissue benign, hormone-naïve, and hormone-refractory prostate cancer (HRPC) was incubated in excess concentrations of cholesterol, progesterone, DHEA, androstenedione, or testosterone for 96 hours, and steroid concentrations in the conditioned media measured by gas chromatography-mass spectroscopy. Changes in the expression of androgen synthetic and/or degradative enzymes were determined by expression microarray and qPCR. Significant changes were confirmed in an independent dataset.
Of the precursor molecules tested, only incubation with androstenedione gave rise to significant concentrations of signaling androgens. Although this was observed in all tissue types, it occurred to a significantly greater degree in hormone-refractory compared with hormone-naïve cancer. Consistent with this, gene set enrichment analysis of the expression microarray data revealed significant upregulation of 17HSD17B activity, with overexpression of the canonical enzyme AKR1C3 confirmed by qPCR in the same samples and in a publicly available expression dataset. Importantly, we found no evidence to support a significant contribution from either the "backdoor" or "5-α dione" pathway.
Reduction of androstenedione to testosterone by the canonical HSD17B AKR1C3 is the predominant source of signaling androgens in HRPC.
Study Type – Diagnostic (case series)
Level of Evidence 4
What's known on the subject? and What does the study add?
The Gleason score of prostate cancer is frequently underestimated at the time of ...diagnostic biopsy, although the contribution of sampling error to its incidence is unknown.
We show that under‐graded tumours are significantly smaller that tumours concordant for the higher grade, indicating that incomplete tumour sampling plays a significant role in Gleason score assignment error.
OBJECTIVE
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To determine the influence of tumour and prostate gland volumes on the underestimation of prostate cancer Gleason score in diagnostic core biopsies.
PATIENTS AND METHODS
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Patients undergoing radical prostatectomy with matched diagnostic biopsies were identified from a prospectively recorded database.
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Tumour volumes were measured in serial whole‐mount sections with image analysis software as part of routine histological assessment.
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Differences in various metrics of tumour and prostate volume between upgraded tumours and tumours concordant for the lower or higher grade were analysed.
RESULTS
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In all, 684 consecutive patients with Gleason score 6 or 7 prostate cancer on diagnostic biopsy were identified.
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Of 298 patients diagnosed with Gleason 6 tumour on biopsy, 201 (67.4%) were upgraded to Gleason 7 or higher on final pathology. Similarly, of 262 patients diagnosed with Gleason 3 + 4 = 7 prostate cancer on initial biopsy, 60 (22.9%) were upgraded to Gleason score 4 + 3 = 7 or higher.
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Tumours upgraded from Gleason 6 to 7 had a significantly lower index tumour volume (1.73 vs 2 mL, P= 0.029), higher calculated prostate volume (41.6 vs 39 mL, P= 0.017) and lower relative percentage of tumour to benign glandular tissue (4.3% vs 5.9%, P= 0.001) than tumours concordant for the higher grade.
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Similarly, tumours that were Gleason score 3 + 4 on biopsy and upgraded on final pathology to 4 + 3 were significantly smaller as measured by both total tumour volume (2.3 vs 3.3 mL, P= 0.005) and index tumour volume (2.2 vs 3, P= 0.027) and occupied a smaller percentage of the gland volume (6.3% vs 8.9%, P= 0.017) compared with tumours concordant for the higher grade.
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On multivariate analysis, lower prostate weight (hazard ratio 0.97, 95% confidence interval 0.96–0.99, P < 0.001) and larger total tumour volume (hazard ratio 1.87, 95% confidence interval 1.4–2.6, P < 0.001) independently predicted an upgrade in Gleason score from 6 to 7. In tumours upgraded from biopsy Gleason 3 + 4, only higher index tumour volume (hazard ratio 3.1, 95% confidence interval 1.01–9.3, P= 0.048) was a significant predictor of upgrading on multivariate analysis.
CONCLUSIONS
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Under‐graded tumours are significantly smaller than tumours concordant for the higher grade, indicating that incomplete tumour sampling plays a significant role in Gleason score assignment error.
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Surrogate measures of tumour volume may predict those at greatest risk of Gleason score upgrade.
Chromosomal rearrangements involving the mixed-lineage leukemia (MLL) gene occur in primary and treatment-related leukemias and confer a poor prognosis. Studies based primarily on mouse models have ...substantially advanced our understanding of MLL leukemia pathogenesis, but often use supraphysiological oncogene expression with uncertain implications for human leukemia. Genome editing using site-specific nucleases provides a powerful new technology for gene modification to potentially model human disease, however, this approach has not been used to re-create acute leukemia in human cells of origin comparable to disease observed in patients. We applied transcription activator-like effector nuclease–mediated genome editing to generate endogenous MLL-AF9 and MLL-ENL oncogenes through insertional mutagenesis in primary human hematopoietic stem and progenitor cells (HSPCs) derived from human umbilical cord blood. Engineered HSPCs displayed altered in vitro growth potentials and induced acute leukemias following transplantation in immunocompromised mice at a mean latency of 16 weeks. The leukemias displayed phenotypic and morphologic similarities with patient leukemia blasts including a subset with mixed phenotype, a distinctive feature seen in clinical disease. The leukemic blasts expressed an MLL-associated transcriptional program with elevated levels of crucial MLL target genes, displayed heightened sensitivity to DOT1L inhibition, and demonstrated increased oncogenic potential ex vivo and in secondary transplant assays. Thus, genome editing to create endogenous MLL oncogenes in primary human HSPCs faithfully models acute MLL-rearranged leukemia and provides an experimental platform for prospective studies of leukemia initiation and stem cell biology in a genetic subtype of poor prognosis leukemia.
•Genome editing of primary human HSPCs generates MLL leukemias that model clinical disease features and molecular pathogenesis.
Recent publications have shown patients with defects in the DNA mismatch repair (MMR) pathway driven by either MSH2 or MSH6 loss experience a significant increase in the incidence of prostate cancer. ...Moreover, this increased incidence of prostate cancer is accompanied by rapid disease progression and poor clinical outcomes.
We show that androgen-receptor activation, a key driver of prostate carcinogenesis, can disrupt the MSH2 gene in prostate cancer. We screened tumours from two cohorts (recurrent/non-recurrent) of prostate cancer patients to confirm the loss of MSH2 protein expression and identified decreased MSH2 expression in recurrent cases. Stratifying the independent TCGA prostate cancer cohort for MSH2/6 expression revealed that patients with lower levels of MSH2/6 had significant worse outcomes, in contrast, endometrial and colorectal cancer patients with lower MSH2/6 levels. MMRd endometrial and colorectal tumours showed the expected increase in mutational burden, microsatellite instability and enhanced immune cell mobilisation but this was not evident in prostate tumours.
We have shown that loss or reduced levels of MSH2/MSH6 protein in prostate cancer is associated with poor outcome. However, our data indicate that this is not associated with a statistically significant increase in mutational burden, microsatellite instability or immune cell mobilisation in a cohort of primary prostate cancers.
Study Type – Prognosis (retrospective cohort)
Level of Evidence 2b
OBJECTIVE
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To determine the effect of an upgrade in Gleason score between initial prostate biopsy and final prostatectomy specimen ...on the risk of postoperative biochemical recurrence.
PATIENTS AND METHODS
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A total of 1629 patients with paired biopsy and radical prostatectomy histology were identified from two prospectively recorded prostate cancer databases.
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Information on key clinical and pathological characteristics as well as prostate‐specific antigen follow‐up was recorded.
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Patients who experienced an upgrade in their Gleason score were compared with corresponding patients with concordant tumours of the lower and higher grade.
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Kaplan–Meier curves and multivariate models were generated to examine the impact of Gleason score upgrade on the risk of postoperative biochemical recurrence.
RESULTS
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Overall, 466 patients (28.6%) experienced an upgrade in their Gleason score post radical prostatectomy, in 88.4% of cases involving a change in a single Gleason score point.
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Patients upgraded from Gleason 6 (3 + 3) to Gleason 7 (3 + 4) had pathological characteristics that were very similar to Gleason 7 (3 + 4) concordant tumours, with an identical risk of biochemical recurrence. In contrast, patients upgraded from Gleason score 6 (3 + 3) to Gleason 7 (4 + 3) had tumours with pathological characteristics intermediate between the two concordant groups, which was mirrored by their risk of biochemical recurrence.
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Patients with Gleason 7 tumours who experienced a change in the predominant pattern from 3 + 4 to 4 + 3 had tumours that resembled Gleason 7 (4 + 3) concordant tumours, with a similar risk of biochemical recurrence. In contrast, patients upgraded from Gleason 7 to Gleason >7 had tumours with intermediate pathological characteristics, and a risk of biochemical recurrence that was significantly different to concordant tumours of the lower and higher grade.
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In multivariate models, a change in Gleason score was an independent predictor of biochemical recurrence in the preoperative setting only.
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Although a difference in Gleason score was an independent predictor of recurrence in concordant tumours in models based on postoperative variables, an upgrade in Gleason score in discordant tumours was not, with differences in co‐segregated adverse pathological characteristics being more predictive.
CONCLUSIONS
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Patients experiencing an upgrade in their Gleason score between biopsy and final specimen exhibit significantly more aggressive pathological features than corresponding concordant tumours, and a higher risk of biochemical recurrence post radical prostatectomy.
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As Gleason score can be more accurately assessed preoperatively than other prognostic tumour features, continued effort is required to identify those most at risk of upgrading, and to refine biopsy strategies to reduce sampling error.
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