Bruce Malkowicz

A screen of the complete protein kinase gene family identifies diverse patterns of somatic mutations in human breast cancer

We examined the coding sequence of 518 protein kinases, ∼1.3 Mb of DNA per sample, in 25 breast cancers. In many tumors, we detected no somatic mutations. But a few had numerous somatic mutations with distinctive patterns indicative of either a mutator phenotype or a past exposure.

ENDORECTAL MAGNETIC RESONANCE IMAGING AS A PREDICTOR OF BIOCHEMICAL OUTCOME AFTER RADICAL PROSTATECTOMY IN MEN WITH CLINICALLY LOCALIZED PROSTATE CANCER

PURPOSE Endorectal magnetic resonance imaging (MRI) of the prostate is sometimes performed before radical prostatectomy but to our knowledge its role for predicting outcome after radical prostatectomy is not yet established. We evaluated the clinical usefulness of endorectal MRI for predicting time to prostate specific antigen (PSA) failure after radical prostatectomy in 1,025 consecutive men with clinically localized or PSA detected prostate cancer. Our analysis controlled for PSA level, biopsy Gleason score, clinical T stage and percent of positive biopsies. MATERIALS AND METHODS Using Cox regression analysis we prospectively assessed time to PSA failure to determine the role of endorectal MRI in predicting PSA outcome after radical prostatectomy at our institution, where an expert prostate magnetic resonance radiologist is available. The main outcome measure was actuarial freedom from PSA failure. RESULTS Endorectal MRI did not add clinically meaningful information in 834 of our 1,025 cases (81%) after accounting for the prognostic value of PSA, biopsy Gleason score, clinical T stage and percent of positive biopsies. However, this modality provided a clinically and statistically relevant stratification of 5-year PSA outcome in the remaining 191 patients at intermediate risk based on established prognostic factors. Specifically when endorectal MRI was interpreted as indicating extracapsular versus organ confined disease the relative risk of PSA failure was 3.6 (95% confidence interval 2.0 to 6.3), and 5-year actuarial freedom from PSA failure was 33% versus 72% (p <0.0001). CONCLUSIONS Despite expert radiological interpretation endorectal MRI had potential clinical value in less than 20% of the cases in our study after accounting for established prognostic factors. While further study of the value of this modality for predicting clinical outcome after radical prostatectomy should be performed in this select cohort, routine use of endorectal MRI cannot be justified based on these data.

Detrusor overactivity is associated with downregulation of large-conductance calcium- and voltage-activated potassium channel protein

Large-conductance voltage- and calcium-activated potassium (BK) channels have been shown to play a role in detrusor overactivity (DO). The goal of this study was to determine whether bladder outlet obstruction-induced DO is associated with downregulation of BK channels and whether BK channels affect myosin light chain 20 (MLC(20)) phosphorylation in detrusor smooth muscle (DSM). Partial bladder outlet obstruction (PBOO) was surgically induced in male New Zealand White rabbits. The rabbit PBOO model shows decreased voided volumes and increased voiding frequency. DSM from PBOO rabbits also show enhanced spontaneous contractions compared with control. Both BK channel alpha- and beta-subunits were significantly decreased in DSM from PBOO rabbits. Immunostaining shows BKbeta mainly expressed in DSM, and its expression is much less in PBOO DSM compared with control DSM. Furthermore, a translational study was performed to see whether the finding discovered in the animal model can be translated to human patients. The urodynamic study demonstrates several overactive DSM contractions during the urine-filling stage in benign prostatic hyperplasia (BPH) patients with DO, while DSM is very quiet in BPH patients without DO. DSM biopsies revealed significantly less BK channel expression at both mRNA and protein levels. The degree of downregulation of the BK beta-subunit was greater than that of the BK alpha-subunit, and the downregulation of BK was only associated with DO, not BPH. Finally, the small interference (si) RNA-mediated downregulation of the BK beta-subunit was employed to study the effect of BK depletion on MLC(20) phosphorylation. siRNA-mediated BK channel reduction was associated with an increased MLC(20) phosphorylation level in cultured DSM cells. In summary, PBOO-induced DO is associated with downregulation of BK channel expression in the rabbit model, and this finding can be translated to human BPH patients with DO. Furthermore, downregulation of the BK channel may contribute to DO by increasing the basal level of MLC(20) phosphorylation.

Radiofrequency Ablation of Small Renal Cell Carcinomas Using Multitined Expandable Electrodes: Preliminary Experience

PURPOSE Radiofrequency ablation is a minimally invasive, nephron-sparing option for renal cell carcinoma (RCC) in poor surgical candidates. We report our contemporary experience with RCC radiofrequency ablation using multitined expandable electrodes along with an aggressive treatment strategy to displace adjacent viscera away from probe tines. Involution of the treatment zone was assessed over time. MATERIALS AND METHODS Over a 36-month period, a quality-assurance database identified 22 patients with 26 sporadic RCC who underwent 43 ablations during 27 radiofrequency ablation sessions. The mean age of the cohort was 71 years (range, 47-89 y). Mean RCC diameter was 2.2 cm (range, 1-4 cm). Twenty-six of radiofrequency ablation sessions were performed using multitined expandable electrodes. All ablations used CT guidance with moderate sedation. Adjunctive techniques used during ablation were recorded, as were instances in which ablation mandated penetration of tines beyond the kidney margin. Post-treatment ablation zones were measured from CT/MR images to evaluate serial involution and treatment response. RESULTS Technical success in targeting and ablation was 100%. Follow-up periods ranged from 1 to 31 months (mean, 11.2). During this period, one patient presented with marginal local recurrence and underwent repeat radiofrequency ablation. Adjunctive techniques in four patients included water injection for displacement of the tail of the pancreas (n = 1) or descending colon (n = 3). Deliberate penetration of tines beyond the margins of the kidney was performed in 41% of cases; no hemorrhage occurred in these cases. No major complications occurred. Minor complications occurred in 17% of patients, including asymptomatic pneumothorax, perirenal hematomas, subcutaneous hematoma, and subcutaneous abscess. After 6 months, mean involution of the ablation zone was 15% from baseline volume per year. CONCLUSION Multitined expandable radiofrequency electrodes produce a high rate of local control for small RCCs with a low complication rate, even when tine penetration of the kidney is required for an adequate tumor treatment margin. Adjacent organs can be protected with adjunctive percutaneous maneuvers.

Using PSA, biopsy Gleason score, clinical stage, and the percentage of positive biopsies to identify optimal candidates for prostate-only radiation therapy.

PURPOSE An identification of prostate cancer patients most likely to benefit from prostate-only radiation was made based upon the pretreatment prostate-specific antigen (PSA), biopsy Gleason score, clinical stage, percentage of positive biopsies, and the 5-year postoperative PSA outcome. METHODS Between 1989 and 2000, 2099 patients underwent radical prostatectomy for clinically localized prostate cancer. The primary end points were pathologic evidence of seminal vesicle invasion 2(SVI), extracapsular extension (ECE) with or without positive surgical margins, and the 5-year postoperative PSA outcome. RESULTS Pretreatment PSA, biopsy Gleason score, and clinical stage were used to assign patients to low-, intermediate-, and high-risk groups. These risk groups were stratified by the percentage of positive biopsies and the primary pathologic and biochemical outcomes examined. The rates of SVI, ECE with positive margin, and no biochemical evidence of disease (bNED) for low-risk patients with < or =50% positive biopsies were 2%, 7%, and 93%, respectively. Patients with >50% positive biopsies had higher rates of SVI and ECE (5% and 11%, respectively) and 52% bNED (p < 0.0001). For intermediate-risk patients with < or =17% positive biopsies, the rates of SVI, ECE with positive margin, and bNED were 3%, 9%, and 90%, respectively. As the percentage of positive biopsies increased above 17% in intermediate-risk patients, there was a statistically significant increase in SVI and ECE and a significant decrease in bNED. CONCLUSIONS Low-risk patients with < or =50% positive biopsies and intermediate-risk patients with < or =17% positive biopsies had a very low risk of SVI and ECE with positive surgical margins. Given that the presence of SVI and ECE with positive surgical margins was uncommon (<10%) with a > or =90% PSA failure-free survival after radical prostatectomy, these patients may be optimal candidates for radiation therapy directed at the prostate only (prostate gland + 1.5-cm margin).

The presence of lymphovascular invasion in radical cystectomy specimens from patients with urothelial carcinoma portends a poor clinical prognosis.

To assess the prognostic significance of lymphovascular invasion (LVI) on clinical outcomes in patients with transitional cell carcinoma of the bladder treated with radical cystectomy (RC).

Transurethral Biopsy of the Prostate for Persistently Elevated or Increasing Prostate Specific Antigen Following Multiple Negative Transrectal Biopsies

PURPOSE Treatment of the patient with persistently elevated prostate specific antigen (PSA) levels after pathologically negative transrectal or manually directed prostate needle biopsy is unclear. We retrospectively evaluated the use of transurethral biopsy of the prostate as an adjunctive study for the diagnosis of prostate cancer in these patients. MATERIALS AND METHODS From January 1993 through February 1996, 71 patients underwent transurethral biopsy in conjunction with repeat prostatic needle biopsy for a persistently elevated PSA (greater than 4 ng./ml.) after previously negative needle biopsy. All patients had at least 1 previous ultrasound guided sextant prostatic needle biopsy (mean 1.85, range 1 to 7) with or without manually directed biopsies. Following negative prostatic needle biopsy these patients subsequently underwent a minimum of a 4-quadrant transurethral sampling of the prostatic fossa followed by repeat sextant prostatic needle biopsy. A subset of patients underwent sampling of the anterior prostatic tissue or transition zone using transrectal ultrasound guided prostatic needle biopsy at transurethral biopsy. RESULTS Of the 71 patients with elevated PSA (mean 16.2 ng./ml., range 4.2 to 171) 17 (24%) had prostate cancer on the repeat prostatic needle biopsy. Both patients who had prostate cancer on the transurethral biopsy specimens also had prostate cancer on the repeat prostatic needle biopsy specimens. A total of 68 patients had benign prostatic tissue and 1 had high grade prostatic intraepithelial neoplasia on transurethral biopsy specimens. Of 19 patients with high grade prostatic intraepithelial neoplasia on the initial prostatic needle biopsy, transurethral biopsy specimens revealed no prostate cancer or prostatic intraepithelial neoplasia. Repeat prostatic needle biopsy in these patients with high grade prostatic intraepithelial neoplasia revealed prostate cancer in 6 and high grade prostatic intraepithelial neoplasia in 4. CONCLUSIONS In patients with persistently elevated or increasing serum PSA after a previously negative prostatic needle biopsy, transurethral biopsy is not a useful adjunct in diagnosing prostate cancer. In this high risk group of patients transurethral biopsy adds little or no diagnostic value to prostatic needle biopsy even in those with high grade prostatic intraepithelial neoplasia.

African American men with low-grade prostate cancer have increased disease recurrence after prostatectomy compared with Caucasian men.

PURPOSE To explore whether disparities in outcomes exist between African American (AA) and Caucasian (CS) men with low-grade prostate cancer and similar cancer of the prostate risk assessment-postsurgery (CAPRA-S) features following prostatectomy (RP). METHODS The overall cohort consisted of 1,265 men (234 AA and 1,031 CS) who met the National comprehensive cancer network criteria for low- to intermediate-risk prostate cancer and underwent RP between 1990 and 2012. We first evaluated whether clinical factors were associated with adverse pathologic outcomes and freedom from biochemical failure (FFbF) using the entire cohort. Next, we studied a subset of 705 men (112 AA and 593 CS) who had pathologic Gleason score≤6 (low-grade disease). Using this cohort, we determined whether race affected FFbF in men with RP-proven low-grade disease and similar CAPRA-S scores. RESULTS With a median follow-up time of 27 months, the overall 7-year FFbF rate was 86% vs. 79% in CS and AA men, respectively (P = 0.035). There was no significant difference in one or more adverse pathologic features between CS vs. AA men (27% vs. 31%; P = 0.35) or CAPRA-S score (P = 0.28). In the subset analysis of patients with low-grade disease, AA race was associated with worse FFbF outcomes (P = 0.002). Furthermore, AA race was a significant predictor of FFbF in men with low-grade disease (hazard ratio = 2.01, 95% CI: 1.08-3.72; P = 0.029). CONCLUSIONS AA race is a predictor of worse FFbF outcomes in men with low-grade disease after RP. These results suggest that a subset of AA men with low-grade disease may benefit from more aggressive treatment.

GATA-6 and NF-κB activate CPI-17 gene transcription and regulate Ca2+ sensitization of smooth muscle contraction.

ABSTRACT Protein kinase C (PKC)-potentiated inhibitory protein of 17 kDa (CPI-17) inhibits myosin light chain phosphatase, altering the levels of myosin light chain phosphorylation and Ca2+ sensitivity in smooth muscle. In this study, we characterized the CPI-17 promoter and identified binding sites for GATA-6 and nuclear factor kappa B (NF-κB). GATA-6 and NF-κB upregulated CPI-17 expression in cultured human and mouse bladder smooth muscle (BSM) cells in an additive manner. CPI-17 expression was decreased upon GATA-6 silencing in cultured BSM cells and in BSM from NF-κB knockout (KO) mice. Moreover, force maintenance by BSM strips from KO mice was decreased compared with the force maintenance of BSM strips from wild-type mice. GATA-6 and NF-κB overexpression was associated with CPI-17 overexpression in BSM from men with benign prostatic hyperplasia (BPH)-induced bladder hypertrophy and in a mouse model of bladder outlet obstruction. Thus, aberrant expression of NF-κB and GATA-6 deregulates CPI-17 expression and the contractile function of smooth muscle. Our data provide insight into how GATA-6 and NF-κB mediate CPI-17 transcription, PKC-mediated signaling, and BSM remodeling associated with lower urinary tract symptoms in patients with BPH.

LIMD2 Is a Small LIM-Only Protein Overexpressed in Metastatic Lesions That Regulates Cell Motility and Tumor Progression by Directly Binding to and Activating the Integrin-Linked Kinase

Proteins that communicate signals from the cytoskeleton to the nucleus are prime targets for effectors of metastasis as they often transduce signals regulating adhesion, motility, and invasiveness. LIM domain proteins shuttle between the cytoplasm and the nucleus, and bind to partners in both compartments, often coupling changes in gene expression to extracellular cues. In this work, we characterize LIMD2, a mechanistically undefined LIM-only protein originally found to be overexpressed in metastatic lesions but absent in the matched primary tumor. LIMD2 levels in fresh and archival tumors positively correlate with cell motility, metastatic potential, and grade, including bladder, melanoma, breast, and thyroid tumors. LIMD2 directly contributes to these cellular phenotypes as shown by overexpression, knockdown, and reconstitution experiments in cell culture models. The solution structure of LIMD2 that was determined using nuclear magnetic resonance revealed a classic LIM-domain structure that was highly related to LIM1 of PINCH1, a core component of the integrin-linked kinase-parvin-pinch complex. Structural and biochemical analyses revealed that LIMD2 bound directly to the kinase domain of integrin-linked kinase (ILK) near the active site and strongly activated ILK kinase activity. Cells that were null for ILK failed to respond to the induction of invasion by LIMD2. This strongly suggests that LIMD2 potentiates its biologic effects through direct interactions with ILK, a signal transduction pathway firmly linked to cell motility and invasion. In summary, LIMD2 is a new component of the signal transduction cascade that links integrin-mediated signaling to cell motility/metastatic behavior and may be a promising target for controlling tumor spread.