A phase I dose-escalation study of enzalutamide in combination with the AKT inhibitor AZD5363 (capivasertib) in patients with metastatic castration-resistant prostate cancer.

BACKGROUND: Activation of the PI3K/AKT/mTOR pathway through loss of phosphatase and tensin homolog (PTEN) occurs in approximately 50% of patients with metastatic castration-resistant prostate cancer (mCRPC). Recent evidence suggests that combined inhibition of the androgen receptor (AR) and AKT may be beneficial in mCRPC with PTEN loss. PATIENTS AND METHODS: mCRPC patients who previously failed abiraterone and/or enzalutamide, received escalating doses of AZD5363 (capivasertib) starting at 320 mg twice daily (b.i.d.) given 4 days on and 3 days off, in combination with enzalutamide 160 mg daily. The co-primary endpoints were safety/tolerability and determining the maximum tolerated dose and recommended phase II dose; pharmacokinetics, antitumour activity, and exploratory biomarker analysis were also evaluated. RESULTS: Sixteen patients were enrolled, 15 received study treatment and 13 were assessable for dose-limiting toxicities (DLTs). Patients were treated at 320, 400, and 480 mg b.i.d. dose levels of capivasertib. The recommended phase II dose identified for capivasertib was 400 mg b.i.d. with 1/6 patients experiencing a DLT (maculopapular rash) at this level. The most common grade ≥3 adverse events were hyperglycemia (26.7%) and rash (20%). Concomitant administration of enzalutamide significantly decreased plasma exposure of capivasertib, though this did not appear to impact pharmacodynamics. Three patients met the criteria for response (defined as prostate-specific antigen decline ≥50%, circulating tumour cell conversion, and/or radiological response). Responses were seen in patients with PTEN loss or activating mutations in AKT, low or absent AR-V7 expression, as well as those with an increase in phosphorylated extracellular signal-regulated kinase (pERK) in post-exposure samples. CONCLUSIONS: The combination of capivasertib and enzalutamide is tolerable and has antitumour activity, with all responding patients harbouring aberrations in the PI3K/AKT/mTOR pathway. CLINICAL TRIAL NUMBER: NCT02525068.

Circulating tumour cell increase as a biomarker of disease progression in metastatic castration-resistant prostate cancer patients with low baseline CTC counts.

Background: The development of treatment response and surrogate biomarkers for advanced prostate cancer care is an unmet clinical need. Patients with baseline circulating tumour cell (BLCTCs) counts <5/7.5 mL represent a good prognosis subgroup but are non-evaluable for response assessment (decrease in CTCs). The aim of the study is to determine the value of any increase in CTCs (CTC progression) as an indicator of progression in prostate cancer patients with low pre-treatment CTCs (<5). Patients and methods: We carried out a post hoc analysis of patients with BLCTCs < 5 treated in the COU-AA-301 (abiraterone or placebo + prednisone) and IMMC-38 (chemotherapy) trials. The association of CTC progression (increase in CTCs at 4, 8 or 12 weeks) with overall survival (OS) was evaluated in multi-variable Cox regression models. Performance of survival models with and without CTC progression was evaluated by calculating ROC curve area under the curves (AUCs) and weighted c-indices. Results: Overall, 511 patients with CTCs < 5 (421 in COU-AA-301 and 90 in IMMC-38) were selected; 212 (41.7%) had CTC progression at 4, 8 or 12 weeks after treatment initiation. CTC progression was associated with significantly worse OS [27.1 versus 15.1 m; hazard ratio (HR) 3.4 (95% confidence interval [CI] 2.5-4.5; P < 0.001)], independent of baseline CTCs and established clinical variables. Adding CTC progression to the OS model significantly improved ROC AUC (0.77 versus 0.66; P < 0.001). Models including CTC progression had superior ROC AUC (0.77 versus 0.69; P < 0.001) and weighted c-index [0.750 versus 0.705; delta c-index: 0.045 (95% CI 0.019-0.071)] values than those including CTC conversion (increase to CTCs ≥ 5). In COU-AA-301, the impact of CTC progression was independent of treatment arm. Conclusions: Increasing CTCs during the first 12 weeks of treatment are independently associated with worse OS from advanced prostate cancer in patients with baseline CTCs < 5 treated with abiraterone or chemotherapy and improve models with established prognostic variables. These findings must be prospectively validated.

CHD1 loss sensitizes prostate cancer to DNA damaging therapy by promoting error-prone double-strand break repair.

BACKGROUND: Deletion of the chromatin remodeler chromodomain helicase DNA-binding protein 1 (CHD1) is a common genomic alteration found in human prostate cancers (PCas). CHD1 loss represents a distinct PCa subtype characterized by SPOP mutation and higher genomic instability. However, the role of CHD1 in PCa development in vivo and its clinical utility remain unclear. PATIENTS AND METHODS: To study the role of CHD1 in PCa development and its loss in clinical management, we generated a genetically engineered mouse model with prostate-specific deletion of murine Chd1 as well as isogenic CHD1 wild-type and homozygous deleted human benign and PCa lines. We also developed patient-derived organoid cultures and screened patients with metastatic PCa for CHD1 loss. RESULTS: We demonstrate that CHD1 loss sensitizes cells to DNA damage and causes a synthetic lethal response to DNA damaging therapy in vitro, in vivo, ex vivo, in patient-derived organoid cultures and in a patient with metastatic PCa. Mechanistically, CHD1 regulates 53BP1 stability and CHD1 loss leads to decreased error-free homologous recombination (HR) repair, which is compensated by increased error-prone non-homologous end joining (NHEJ) repair for DNA double-strand break (DSB) repair. CONCLUSIONS: Our study provides the first in vivo and in patient evidence supporting the role of CHD1 in DSB repair and in response to DNA damaging therapy. We uncover mechanistic insights that CHD1 modulates the choice between HR and NHEJ DSB repair and suggest that CHD1 loss may contribute to the genomic instability seen in this subset of PCas.

Androgen receptor expression in circulating tumour cells from castration-resistant prostate cancer patients treated with novel endocrine agents.

BACKGROUND: Abiraterone and enzalutamide are novel endocrine treatments that abrogate androgen receptor (AR) signalling in castration-resistant prostate cancer (CRPC). Here, we developed a circulating tumour cells (CTCs)-based assay to evaluate AR expression in real-time in CRPC and investigated nuclear AR expression in CTCs in patients treated with enzalutamide and abiraterone. METHODS: CTCs were captured and characterised using the CellSearch system. An automated algorithm to identify CTCs and quantify AR expression was employed. The primary aim was to evaluate the association between CTC AR expression and prior treatment with abiraterone or enzalutamide. RESULTS: AR expression in CTCs was evaluated in 94 samples from 48 metastatic CRPC patients. We observed large intra-patient heterogeneity of AR expression in CTCs. Prior exposure to abiraterone or enzalutamide was not associated with a change in CTCs AR expression (median intensity and distribution of AR-positive classes). In support of this, we also confirmed maintained nuclear AR expression in tissue samples collected after progression on abiraterone. AR staining also identified additional AR-positive CD45-negative circulating cells that were CK-negative/weak and therefore missed using standard protocols. The number of these events correlated with traditional CTCs and was associated with worse outcome on univariate analysis. CONCLUSIONS: We developed a non-invasive method to monitor AR nuclear expression in CTCs. Our studies confirm nuclear AR expression in CRPC patients progressing on novel endocrine treatments. Owing to the significant heterogeneity of AR expression in CTCs, studies in larger cohorts of patients are required to identify associations with outcome.

Circulating allergen-reactive T cells from patients with atopic dermatitis and allergic contact dermatitis express the skin-selective homing receptor, the cutaneous lymphocyte-associated antigen.

The cutaneous lymphocyte-associated antigen (CLA) is the major T cell ligand for the vascular adhesion molecule E-selectin, and it has been proposed to be involved in the selective targeting of memory T cells reactive with skin-associated Ag to cutaneous inflammatory sites. To further investigate the relation of CLA and cutaneous T cell responses, we analyzed the CLA phenotype of circulating memory T cells in patients with allergic contact dermatitis and atopic dermatitis (AD) alone vs in patients manifesting bronchopulmonary atopy (asthma with or without AD) and nonallergic individuals. Significant T cell proliferative responses to Ni, a contact allergen, and to the house dust mite (HDM), an allergen to which sensitization is often observed in AD and/or asthma, was noted only in allergic and atopic individuals, respectively. When the minor circulating CLA+CD3+CD45RO+ subset was separated from the major CLA-CD3+CD45RO+ subpopulation in Ni-sensitive subjects, the Ni-dependent memory T cell response was largely confined to the CLA+ subset. A similar restriction of the T cell proliferative response to the CLA+ memory subset was observed for HDM in patients with AD alone. In HDM-sensitive patients with asthma with or without AD, however, the CLA- subset exhibited a strong antigen-dependent proliferation, in contrast to patients with AD alone, whose CLA- subset proliferated very weakly to HDM. In asthma with or without AD, the HDM-dependent proliferation slightly predominated in the CLA- when compared to the CLA+ subset. The functional linkage between CLA expression and disease-associated T cell effector function in AD was also demonstrated by the finding that the circulating CLA+ T cell subset in AD patients, but not nonatopic controls, selectively showed both evidence of prior activation (human histocompatibility antigen-DR expression) and spontaneous production of interleukin 4 but not interferon-gamma. Taken together, these observations demonstrate the correlation of CLA expression on circulating memory T cells and disease-associated memory T cell responses in cutaneous hypersensitivity, and they suggest the existence of mechanisms capable of sorting particular T cell Ag specificities and lymphokine patterns into homing receptor-defined memory subsets.

Molecular characterisation of ERG, ETV1 and PTEN gene loci identifies patients at low and high risk of death from prostate cancer.

BACKGROUND: The discovery of ERG/ETV1 gene rearrangements and PTEN gene loss warrants investigation in a mechanism-based prognostic classification of prostate cancer (PCa). The study objective was to evaluate the potential clinical significance and natural history of different disease categories by combining ERG/ETV1 gene rearrangements and PTEN gene loss status. METHODS: We utilised fluorescence in situ hybridisation (FISH) assays to detect PTEN gene loss and ERG/ETV1 gene rearrangements in 308 conservatively managed PCa patients with survival outcome data. RESULTS: ERG/ETV1 gene rearrangements alone and PTEN gene loss alone both failed to show a link to survival in multivariate analyses. However, there was a strong interaction between ERG/ETV1 gene rearrangements and PTEN gene loss (P<0.001). The largest subgroup of patients (54%), lacking both PTEN gene loss and ERG/ETV1 gene rearrangements comprised a 'good prognosis' population exhibiting favourable cancer-specific survival (85.5% alive at 11 years). The presence of PTEN gene loss in the absence of ERG/ETV1 gene rearrangements identified a patient population (6%) with poorer cancer-specific survival that was highly significant (HR=4.87, P<0.001 in multivariate analysis, 13.7% survival at 11 years) when compared with the 'good prognosis' group. ERG/ETV1 gene rearrangements and PTEN gene loss status should now prospectively be incorporated into a predictive model to establish whether predictive performance is improved. CONCLUSIONS: Our data suggest that FISH studies of PTEN gene loss and ERG/ETV1 gene rearrangements could be pursued for patient stratification, selection and hypothesis-generating subgroup analyses in future PCa clinical trials and potentially in patient management.

Diversity of TMPRSS2-ERG fusion transcripts in the human prostate.

TMPRSS2-ERG gene fusions have recently been reported to be present in a high proportion of human prostate cancers. In the current study, we show that great diversity exists in the precise structure of TMPRSS2-ERG hybrid transcripts found in human prostates. Fourteen distinct hybrid transcripts are characterized, each containing different combinations of sequences from the TMPRSS2 and ERG genes. The transcripts include two that are predicted to encode a normal full-length ERG protein, six that encode N-terminal truncated ERG proteins and one that encodes a TMPRSS2-ERG fusion protein. Interestingly, distinct patterns of hybrid transcripts were found in samples taken from separate regions of individual cancer-containing prostates, suggesting that TMPRSS2-ERG gene fusions may be arising independently in different regions of a single prostate.

Heterogeneity and clinical significance of ETV1 translocations in human prostate cancer.

A fluorescence in situ hybridisation (FISH) assay has been used to screen for ETV1 gene rearrangements in a cohort of 429 prostate cancers from patients who had been diagnosed by trans-urethral resection of the prostate. The presence of ETV1 gene alterations (found in 23 cases, 5.4%) was correlated with higher Gleason Score (P=0.001), PSA level at diagnosis (P=<0.0001) and clinical stage (P=0.017) but was not linked to poorer survival. We found that the six previously characterised translocation partners of ETV1 only accounted for 34% of ETV1 re-arrangements (eight out of 23) in this series, with fusion to the androgen-repressed gene C15orf21 representing the commonest event (four out of 23). In 5'-RACE experiments on RNA extracted from formalin-fixed tissue we identified the androgen-upregulated gene ACSL3 as a new 5'-translocation partner of ETV1. These studies report a novel fusion partner for ETV1 and highlight the considerable heterogeneity of ETV1 gene rearrangements in human prostate cancer.

Analysis of DNA methylation in single circulating tumor cells.

Direct analysis of circulating tumor cells (CTCs) can inform on molecular mechanisms underlying systemic spread. Here we investigated promoter methylation of three genes regulating epithelial-to-mesenchymal transition (EMT), a key mechanism enabling epithelial tumor cells to disseminate and metastasize. For this, we developed a single-cell protocol based on agarose-embedded bisulfite treatment, which allows investigating DNA methylation of multiple loci via a multiplex PCR (multiplexed-scAEBS). We established our assay for the simultaneous analysis of three EMT-associated genes miR-200c/141, miR-200b/a/429 and CDH1 in single cells. The assay was validated in solitary cells of GM14667, MDA-MB-231 and MCF-7 cell lines, achieving a DNA amplification efficiency of 70% with methylation patterns identical to the respective bulk DNA. Then we applied multiplexed-scAEBS to 159 single CTCs from 11 patients with metastatic breast and six with metastatic castration-resistant prostate cancer, isolated via CellSearch (EpCAMpos/CKpos/CD45neg/DAPIpos) and subsequent FACS sorting. In contrast to CD45pos white blood cells isolated and processed by the identical approach, we observed in the isolated CTCs methylation patterns resembling more those of epithelial-like cells. Methylation at the promoter of microRNA-200 family was significantly higher in prostate CTCs. Data from our single-cell analysis revealed an epigenetic heterogeneity among CTCs and indicates tumor-specific active epigenetic regulation of EMT-associated genes during blood-borne dissemination.

[ESWL centers and determination of urinary calculus etiology. Results of a survey of 150 ESWL centers]. / ESWL-Zentren und Harnsteinabklärung. Umfrageergebnisse an 150 ESWL-Standorten.

Questionnaires were mailed anonymously to 150 German shock wave centers. Twenty questions addressed the following areas of interest: Facilities of the extracorporeal shock wave lithotripsy (ESWL) center (technical, personnel, laboratory, etc.) Cooperation at ESWL center with referring urologists Laboratory facilities versus actual metabolic work-up. The return rate was 114 of 150 (76%). Surprisingly, at 58% of the centers the average number of treatments is less than two per day. In 30% of the centers only chemical stone analysis is done! The final conclusion was that ESWL has largely replaced the causal metabolic work-up and subsequent metaphylaxis as a symptomatic measure against urolithiasis.

[PSA volume quotient: an additional parameter in diagnosis of locally confined prostate cancer]. / PSA-Volumenquotient: Ein zusätzlicher Parameter in der Diagnostik des lokal begrenzten Prostatakarzinoms.

The sensitivity and specificity of prostate-specific antigen density (PSAD), a quotient of serum PSA and prostate volume, in the detection of localized prostate cancer was analysed in a prospective study. A total of 235 patients were examined, 145 without prostate cancer and 90 patients before radical prostatectomy for localized prostate cancer. PSAD was determined by dividing the serum level of PSA by the volume of the entire prostate (estimated by transrectal ultrasound) and multiplying by 100. Using a PSAD of 15, the specificity achieved in our collective was the same as with an absolute PSA value of 4 ng/ml (88.9-90%). On the other hand, with the PSAD of 15 we also found the same sensitivity as with an ab-solute PSA of 10 ng/ml (75.2-76.5%).

[Ileal neobladder with anastomosis to the female urethra]. / Die Ileumneoblase mit Anschluss an die weibliche Harnröhre.

Orthotopic reconstruction to the native urethra has revolutionized urinary diversion, allowing patients to void per the urethra. This form of urinary diversion was initially performed solely in male patients after cystectomy. More recently, however, with a better understanding of the female continence mechanism, including the urethral/vaginal support mechanism, and the ability to select appropriate female candidates properly for this type of surgery, orthotopic reconstruction has become a viable option in women. Since November 1986, 24 women aged 53 years (range 17-76) have undergone orthotopic reconstruction using the ileal neobladder. Indications for cystectomy included transitional cell carcinoma of the bladder (8), fibrotic radiated bladder (4), interstitial cystitis (5), tuberculotic bladder (2), urge incontinence (2), neurogenic fibrotic bladder (2), and fibrotic bladder of unknown etiology (1). Nineteen patients are available with a median follow-up of 48 months (range 3 to 109 months). There were no perioperative deaths, with few early and late complications. Two women previously irradiated developed a neovesicovaginal fistula and had to be diverted by an ileal loop. Three patients from the far East are no longer available for follow-up. Ten years of experience with 24 patients have led to a nerve- and urethral-support-sparing cystectomy technique with the ileal neobladder anastomosed to the proximal urethra. However, even then, retention in 20% of the patients rather than the expected incontinence is the critical issue. Incontinence has never been a problem. The advent of orthotopic lower urinary reconstruction in women is a major achievement in the evolution of urinary diversion. With our increasing understanding of the continence mechanism in women and with increasing evidence that the female urethra can be safely preserved after cystectomy, orthotopic lower urinary tract reconstruction by the ileal neobladder can now be offered safely not only to males, but also to female patients undergoing cystectomy, and the functional results are superb.

[Venous tumor invasion by renal cell carcinoma. Surgical technique, complications and survival rate]. / Venöse Tumorinvasion beim Nierenzellkarzinom. Operative Technik, Komplikationen und Uberlebensrate.

Renal cell carcinoma invades the vena cava in 4-10% of cases. Another 10% of patients present with invasion of the renal vein. The surgical approach, complications and long-term outcome of 95 patients were investigated. Intraoperative complications occurred in 1 of 73 patients with involvement of the renal vein and 5 of 22 patients with vena cava thrombus. One patient in each group died due to pulmonary emboli in the perioperative period. Minor renal insufficiency occurred in 39 (54.2%) and 11 (50%) of the patients respectively. The rates of minor complications such as wound infections, haematoma and pneumonia were similar in the two groups. The mean intra-operative blood loss of 780 ml in patients undergoing tumour nephrectomy was significantly lower than the blood loss of 1485 ml in patients with tumour thrombus extension into the vena cava. The 5-year survival rate of patients with V1 tumours (71%) is comparable to that of patients without venous involvement. Tumour extension into the vena cava (V2) influences the 5-year survival rate significantly, decreasing it to 56.7%. In conclusion, long-term survival can be achieved for patients with renal cell carcinoma and venous involvement, though for patients with lymph node metastases or distant metastases only short-term palliation can be achieved. However, the potential benefits should be carefully weighed against the possible complications, the surgical morbidity and the resources expended in vena cava resection.

[Is radical prostatectomy a suitable model for determination of PSA half-life?]. / Ist die radikale Prostatektomie ein geeignetes Modell zur Bestimmung der PSA-Halbwertszeit?

A review of the literature relating to PSA half-life reveals great variability in absolute values and pharmacokinetic models. A critical view is needed, however, since some authors suggest that the PSA half-life has implications for diagnosis and prognosis after radical prostatectomy. The aim of our study, therefore, was to characterize the value of PSA half-life determination after radical prostatectomy. Serial serum PSA detections were performed in 16 patients with localized prostatic cancer who had undergone radical prostatectomy. Serum PSA was detected on days 0, 1, 2, 3, 6, 9, 12, 15, 18, after radical prostatectomy. In all patients elimination of PSA from serum followed a biphasic logarithmic decay pattern indicating a two-compartment model of first order elimination kinetics (t1 = 1.01 +/- 0.06 days, t2 = 3.42 +/- 0.23 days; P < 0.00001). In this two-compartment model 56.3 +/- 4.8% of the preoperative PSA serum concentration was cleared by the first compartment. To find a biological correlative for the first compartment a mathematical model was developed to approximate the effect of operative blood and plasma loss on PSA serum concentration. In this model changes of hematocrit were used to estimate blood and plasma loss. These calculations showed that 50.12 +/- 3.04% of the preoperative PSA serum concentration was excreted by operative blood loss. This value was not significantly different from the clearance rate calculated for the first compartment. It is, therefore, concluded that the determination of PSA half-life after radical prostatectomy without correction of the operation-related PSA loss is only of limited value.

[Can examination of spontaneous urine samples adequately replace 24-hour-urine samples for determining excretory rate of various lithogenic and inhibitory substances in metabolic evaluation of kidney calculi patients?]. / Ist die Untersuchung von Spontanurinproben ein ausreichender Ersatz für die Bestimmung der Exkretionsraten unterschiedlicher lithogener und inhibitorischer Substanzen im 24-h-Urin bei der metabolischen Abklärung von Steinpatienten?

The gold standard for metabolic evaluation of stone-forming patients is the 24-h urine specimen. Recently, some authors have suggested that for routine metabolic evaluation spot urine samples are as valuable as the 24-h urine specimen. The purpose of our study, was to determine the value of the spot urine sample in comparison with the 24-h urine specimens. Eighty-eight healthy volunteers on different diets were investigated (32 vegetarians, 12 body-builders without protein concentrates, 28 body-builders on protein concentrates, and 16 subjects on a regular European diet). Using 24-h specimens, excretion rates of oxalate, calcium, sodium and potassium were determined. The concentration ratio of these electrolytes to creatinine was calculated for spot urine samples. A highly significant correlation between the excretion rates and the results of the spot urine samples was found for all parameters. However, the correlations showed considerable variations. On the other hand, we were able to show that creatinine excretion is highly dependent on daily protein intake, body weight and glomerular filtration rate. This leads to a considerable inter- and intraindividual variation in creatinine excretion. This variation of the creatinine excretion is the major cause for the variation in the results of spot urine samples. It is concluded that spot urine samples are an inadequate substitute for the 24-h urine specimen and that the 24-h urine specimen is still the basis for metabolic evaluation in stone patients.

High-density tissue microarrays from prostate needle biopsies.

BACKGROUND: Formalin-fixed prostate biopsies are frequently the only tissue collected at the time of prostate cancer diagnosis. There is therefore a requirement for techniques that allow the use of these prostate biopsy specimens in a high-throughput analysis of immunohistochemical and fluorescence-in-situ-hybridisation-detected biomarkers. METHODS: The authors have previously described methods that allow tissue microarray (TMA) construction from prostate biopsies. Here, we describe significant technical innovations that provide an easier and more robust system of biopsy-TMA construction. RESULTS AND DISCUSSION: The TMAs produced are of a high density (up to 104 cores each, 8 × 13) and allow a multiplex analysis of biomarkers in the context of clinical trials.

The identification of chromosomal translocation, t(4;6)(q22;q15), in prostate cancer.

Our previous work identified a chromosomal translocation t(4;6) in prostate cancer cell lines and primary tumors. Using probes located on 4q22 and 6q15, the breakpoints identified in LNCaP cells, we performed fluorescence in situ hybridization analysis to detect this translocation in a large series of clinical localized prostate cancer samples treated conservatively. We found that t(4;6)(q22;q15) occurred in 78 of 667 cases (11.7%). The t(4;6)(q22;q15) was not independently associated with patient outcome. However, it occurs more frequently in high clinical T stage, high tumor volume specimens and in those with high baseline PSA (P=0.001, 0.001 and 0.01, respectively). The t(4;6)(q22;q15) occurred more frequently in samples with two or more TMPRSS2:ERG fusion genes caused by internal deletion than in samples without these genomic alterations, but this correlation is not statistically significant (P=0.0628). The potential role of this translocation in the development of human prostate cancer is discussed.

Hormone-sensitive prostate cancer: a case of ETS gene fusion heterogeneity.

Fusion of the hormone-regulated gene TMPRSS2 with ERG occurs in 50-70% of prostate cancers; fusions of ETV1 with one of several partners occur in approximately 10% of prostate cancers. These two translocations are mutually exclusive. The presence of subclasses of these chromosomal rearrangements may indicate worse prognosis, with the subclass 2+Edel, which has duplication of TMPRSS2:ERG fusion sequences, indicating particularly poor survival. However as this case shows, significant heterogeneity can exist with ERG and ETV1 rearrangements occurring in both prostate intra-epithelial neoplasia and cancer in the same prostatectomy specimen and with adjacent cancer areas containing a single copy, duplication and even triplication of the rearranged locus. As the majority of ETS gene fusions are hormone regulated, they could explain the pathogenesis underlying exquisitely hormone-sensitive prostate cancer. This is exemplified by the case presented here of a patient diagnosed in 1991 who remains asymptomatic and chemotherapy-naïve after having long-lasting tumour responses to multiple lines of systemic hormonal treatments.

An improved method for constructing tissue microarrays from prostate needle biopsy specimens.

BACKGROUND: Prostate cancer diagnosis is routinely made by the histopathological examination of formalin fixed needle biopsy specimens. Frequently this is the only cancer tissue available from the patient for the analysis of diagnostic and prognostic biomarkers. There is, therefore, an urgent need for methods that allow the high-throughput analysis of these biopsy samples using immunohistochemical (IHC) markers and fluorescence in situ hybridisation (FISH) analysis based markers. METHODS: A method that allows the construction of tissue microarrays (TMAs) from diagnostic prostate needle biopsy cores has previously been reported. However, the technique only allows the production of low-density biopsy TMAs with a maximum of 20 cores per TMA. Here two methods are presented that allow the rapid and uniform production of biopsy TMAs containing between 54 and 72 biopsy cores. IHC and FISH techniques were used to detect biomarker status. RESULTS: Biopsy TMAs were constructed from prostate needle biopsy specimens taken from 102 patients entered into an active surveillance trial and 201 patients in a radiotherapy trial. The detection rate for cancer in slices of these biopsy TMAs was 66% and 79% respectively. Slices of a biopsy TMA prepared from biopsies from active surveillance patients were used to detect multiple IHC markers and to score TMPRSS2-ERG fusion status in a FISH-based assay. CONCLUSIONS: The construction of biopsy TMAs provides an effective method for the multiplex analysis of IHC and FISH markers and for their assessment as prognostic biomarkers in the context of clinical trials.

Complex patterns of ETS gene alteration arise during cancer development in the human prostate.

An ERG gene 'break-apart' fluorescence in situ hybridization (FISH) assay has been used to screen whole-mount prostatectomy specimens for rearrangements at the ERG locus. In cancers containing ERG alterations the observed pattern of changes was often complex. Different categories of ERG gene alteration were found either together in a single cancerous region or within separate foci of cancer in the same prostate slice. In some cases the juxtaposition of particular patterns of ERG alterations suggested possible mechanisms of tumour progression. Prostates harbouring ERG alterations commonly also contained cancer that lacked rearrangements of the ERG gene. A single trans-urethral resection of the prostate specimen examined harboured both ERG and ETV1 gene rearrangements demonstrating that the observed complexity may, at least in part, be explained by multiple ETS gene alterations arising independently in a single prostate. In a search for possible precursor lesions clonal ERG rearrangements were found both in high grade prostatic intraepithelial neoplasia (PIN) and in atypical in situ epithelial lesions consistent with the diagnosis of low grade PIN. Our observations support the view that ERG gene alterations represent an initiating event that promotes clonal expansion initially to form regions of epithelial atypia. The complex patterns of ERG alteration found in prostatectomy specimens have important implications for the design of experiments investigating the clinical significance and mechanism of development of individual prostate cancers.