Comparison of Prostate Biopsy with or without Prebiopsy Multiparametric Magnetic Resonance Imaging for Prostate Cancer Detection: An Observational Cohort Study.

PURPOSE: We hypothesized that 1) introducing prebiopsy multiparametric magnetic resonance imaging would increase the diagnostic yield of transrectal prostate biopsy and 2) this would inform recommendations regarding systematic transrectal prostate biopsy in the setting of negative prebiopsy multiparametric magnetic resonance imaging. MATERIALS AND METHODS: A total of 997 biopsy naïve patients underwent transrectal prostate biopsy alone to June 2016 (cohort 1) and thereafter 792 underwent transrectal prostate biopsy following prebiopsy multiparametric magnetic resonance imaging (cohort 2). Patients with lesions on prebiopsy multiparametric magnetic resonance imaging underwent cognitive targeted plus systematic transrectal prostate biopsy. Patients without lesions underwent systematic transrectal prostate biopsy. RESULTS: Cohort 2 comprised younger men (age 68 vs 69 years, p = 0.01) with lower prostate specific antigen (7.6 vs 7.9 ng/ml, p = 0.024) and smaller prostate volume (56.1 vs 62 cc, p = 0.006). In cohort 2 vs cohort 1 there was no increase in overall prostate cancer detection (57.6% vs 56.7%, p = 0.701), the Gleason Grade Group or the number of positive cores (each p >0.05). Increased multifocal prostatic intraepithelial neoplasia, maximum prostate cancer core length (5 mm or greater vs less than 5 mm) and radical surgery/high intensity focused ultrasound (each p <0.05) were observed in cohort 2. For Gleason Grade Group 2-5 prostate cancer negative prebiopsy multiparametric magnetic resonance imaging had 88.1% sensitivity, 59.8% specificity, 67.8% positive predictive value and 84% negative predictive value. For negative prebiopsy multiparametric magnetic resonance images a prostate specific antigen density cutoff of 0.15 ng/ml2 or greater increased clinically significant prostate cancer detection only if the latter was defined as Gleason Grade Group 3-5 disease and/or tumor length 6 mm or greater. CONCLUSIONS: Introducing prebiopsy multiparametric magnetic resonance imaging in our clinical setting increased the diagnostic yield of prostate cancer per biopsy core. Not performing a systematic transrectal prostate biopsy when prebiopsy multiparametric magnetic resonance imaging was negative would have led to under detection of 15.1% of Gleason Grade Group 2 or greater prostate cancer cases (approximately 1 in 6).

Does the introduction of prostate multiparametric magnetic resonance imaging into the active surveillance protocol for localized prostate cancer improve patient re-classification?

OBJECTIVES: To determine whether replacement of protocol-driven repeat prostate biopsy (PB) with multiparametric magnetic resonance imaging (mpMRI) ± repeat targeted prostate biopsy (TB) when evaluating men on active surveillance (AS) for low-volume, low- to intermediate-risk prostate cancer (PCa) altered the likelihood of or time to treatment, or reduced the number of repeat biopsies required to trigger treatment. PATIENTS AND METHODS: A total of 445 patients underwent AS in the period 2010-2016 at our institution, with a median (interquartile range [IQR]) follow-up of 2.4 (1.2-3.7) years. Up to 2014, patients followed a 'pre-2014' AS protocol, which incorporated PB, and subsequently, according to the 2014 National Institute for Health and Care Excellence (NICE) guidelines, patients followed a '2014-present' AS protocol that included mpMRI. We identified four groups of patients within the cohort: 'no mpMRI and no PB'; 'PB alone'; 'mpMRI ± TB'; and 'PB and mpMRI ± TB'. Kaplan-Meier plots and log-rank tests were used to compare groups. RESULTS: Of 445 patients, 132 (30%) discontinued AS and underwent treatment intervention, with a median (IQR) time to treatment of 1.55 (0.71-2.4) years. The commonest trigger for treatment was PCa upgrading after mpMRI and TB (43/132 patients, 29%). No significant difference was observed in the time at which patients receiving a PB alone or receiving mpMRI ± TB discontinued AS to undergo treatment (median 1.9 vs 1.33 years; P = 0.747). Considering only those patients who underwent repeat biopsy, a greater proportion of patients receiving TB after mpMRI discontinued AS compared with those receiving PB alone (29/66 [44%] vs 32/87 [37%]; P = 0.003). On average, a single set of repeat biopsies was needed to trigger treatment regardless of whether this was a PB or TB. CONCLUSIONS: Replacing a systematic PB with mpMRI ±TB as part of an AS protocol increased the likelihood of re-classifying patients on AS and identifying men with clinically significant disease requiring treatment. mpMRI ±TB as part of AS thereby represents a significant advance in the oncological safety of the AS protocol.

Reducing Mortality in the Ageing Patient: Treatment of the Primary Tumour Is Not Necessary.

Robot-assisted radical prostatectomy for older men remains an unproven intervention in terms of both cancer control and functional and voiding outcomes. We highlight some of the evidence against radical surgery for older men while acknowledging that this is the direction of travel.

Establishing nurse-led active surveillance for men with localised prostate cancer: development and formative evaluation of a model of care in the ProtecT trial.

OBJECTIVES: To develop a nurse-led, urologist-supported model of care for men managed by active surveillance or active monitoring (AS/AM) for localised prostate cancer and provide a formative evaluation of its acceptability to patients, clinicians and nurses. Nurse-led care, comprising an explicit nurse-led protocol with support from urologists, was developed as part of the AM arm of the Prostate testing for cancer and Treatment (ProtecT) trial. DESIGN: Interviews and questionnaire surveys of clinicians, nurses and patients assessed acceptability. SETTING: Nurse-led clinics were established in 9 centres in the ProtecT trial and compared with 3 non-ProtecT urology centres elsewhere in UK. PARTICIPANTS: Within ProtecT, 22 men receiving AM nurse-led care were interviewed about experiences of care; 11 urologists and 23 research nurses delivering ProtecT trial care completed a questionnaire about its acceptability; 20 men managed in urology clinics elsewhere in the UK were interviewed about models of AS/AM care; 12 urologists and three specialist nurses working in these clinics were also interviewed about management of AS/AM. RESULTS: Nurse-led care was commended by ProtecT trial participants, who valued the flexibility, accessibility and continuity of the service and felt confident about the quality of care. ProtecT consultant urologists and nurses also rated it highly, identifying continuity of care and resource savings as key attributes. Clinicians and patients outside the ProtecT trial believed that nurse-led care could relieve pressure on urology clinics without compromising patient care. CONCLUSIONS: The ProtecT AM nurse-led model of care was acceptable to men with localised prostate cancer and clinical specialists in urology. The protocol is available for implementation; we aim to evaluate its impact on routine clinical practice. TRIAL REGISTRATION NUMBERS: NCT02044172; ISRCTN20141297.

Variation between specialist uropatholgists in reporting extraprostatic extension after radical prostatectomy.

AIMS: Extraprostatic extension of prostate cancer in radical prostatectomy specimens significantly affects patient management. We evaluated the degree of interobserver variation between uropathologists at a tertiary referral teaching hospital in assessing the extraprostatic extension of prostate cancer in radical prostatectomy specimens. METHODS: Histopathological data from a consecutive series of 293 radical prostatectomy specimens (January 2007-December 2012) were reviewed. A subset of 50 consecutive radical prostatectomy cases originally staged as tumours confined to the prostate (pT2) or tumours extending into periprostatic tissue (pT3a) during this period were reviewed by four specialist uropathologists. RESULTS: Five consultant histopathologists reported these specimens with significant differences in the reported stage (p=0.0164) between pathologists. Double-blind review by 4 uropathologists of 50 consecutive radical prostatectomy cases showed a lack of consensus in 16/50 (32%) cases (κ score 0.58, moderate agreement). A consensus meeting was held, but consensus could still not be reached in 9/16 cases. CONCLUSIONS: Our findings highlight variability in the reporting of pT stage in radical prostatectomy specimens even by specialist uropathologists. Assessment of extraprostatic extension has important implications for patient management and there is a need for more precise guidance.

Contemporary practice and technique-related outcomes for radical prostatectomy in the UK: a report of national outcomes.

OBJECTIVE: To determine current radical prostatectomy (RP) practice in the UK and compare surgical outcomes between techniques. PATIENTS AND METHODS: All RPs performed between 1 January 2011 and 31 December 2011 in the UK with data entered into the British Association of Urological Surgeons (BAUS) database, were identified for analysis. Overall surgical outcomes were assessed and subgroup analyses of these outcomes, based on operative technique [open RP (ORP), laparoscopic RP (LRP) and robot-assisted laparoscopic RP (RALP)], were made. Continuous variables were compared using the Mann-Whitney U-test and categorical variables using the Pearson chi-squared test. Univariate and multivariate binary regression analyses were performed to assess the effect of patient, surgeon and technique-related variables on surgical outcomes. RESULTS: During the study period 2163 RPs were performed by 115 consultants with a median (range) of 11 (1-154) cases/consultant. Most RPs were performed laparoscopically (ORP 25.8%, LRP 54.6%, RALP 19.6%) and those performing minimally invasive techniques are more likely to have a higher annual case volume with <1% ORP, 39% LRP and 62% RALP being performed by consultants with an annual caseload of >50 cases/year. Most patients were classified as having intermediate- or high-risk disease preoperatively (1596 patients, 82.5%) and this increased to 97.2% (1649) on postoperative risk stratification. The overall intraoperative complication rate was 14.2% and was significantly greater for LRP (17.8%) vs ORP (8.2%) and RALP (12.4%), (P < 0.001). In all, 71% of patients had an estimated blood loss (EBL) of <500 mL, although there were significantly more patients undergoing ORP with >500, > 1000 and >2000 mL EBL compared with the other techniques (P < 0.001). The postoperative complication rate was 10.7% overall, with a significantly greater postoperative complication rate in the LRP group (LRP 14.6%, ORP 8.8% and RALP 10.3% respectively, P = 0.007). Positive surgical margin (PSM) rates were 17.5% for pT2 disease and 42.3% for pT3 disease. The PSM rate was significantly lower in the RALP patients compared with the ORP patients for those with pT2 disease (P = 0.025), while there was no difference between ORP and LRP (ORP 21.7%, LRP 18.1% and RALP 13.0%). There was no significant difference in the PSM rate in pT3 disease between surgical techniques. CONCLUSION: Most RPs in the UK are performed using minimally invasive techniques, which offer reduced blood loss and transfusion rates compared with ORP. The operation time, complication rate, PSM rates, and association with higher volume practice support RALP as the minimally invasive technique of choice, which could have implications for regions without access to such services. The disparity in outcomes between this national study and high-volume single centres, most probably reflects the low median national case volume, and combined with the positive effect of high case volume on multivariate analysis of surgical outcomes and PSM rates, strengthens the argument for centralisation of services.

PSA Velocity and Doubling Time in Diagnosis and Prognosis of Prostate Cancer.

Cancer is a growth process and it is natural that we should be concerned with how the routinely used marker of prostate cancer tumour burden - PSA - changes over time. Such change is measured by PSA velocity or PSA doubling time, described in general as "PSA kinetics". However, it turns out that calculation of PSA velocity and doubling time is far from straightforward. More than 20 different methods have been proposed, and many of these give quite divergent results. There is clear evidence that PSA kinetics are critical for understanding prognosis in advanced or relapsed prostate cancer. However, PSA kinetics have no value for men with an untreated prostate: neither PSA velocity nor doubling time have any role in diagnosing prostate cancer or providing a prognosis for men before treatment.

Depletion of the type 1 IGF receptor delays repair of radiation-induced DNA double strand breaks.

BACKGROUND AND PURPOSE: IGF-1R depletion sensitizes prostate cancer cells to ionizing radiation and DNA-damaging cytotoxic drugs. This study investigated the hypothesis that IGF-1R regulates DNA double strand break (DSB) repair. METHODS: We tested effects of IGF-1R siRNA transfection on the repair of radiation-induced DSBs by immunoblotting and immunofluorescence for γH2AX, and pulsed-field gel electrophoresis. Homologous recombination (HR) was quantified by reporter assays, and cell cycle distribution by flow cytometry. RESULTS: We confirmed that IGF-1R depletion sensitized DU145 and PC3 prostate cancer cells to ionizing radiation. DU145 control transfectants resolved radiation-induced DSBs within 24 h, while IGF-1R depleted cells contained 30-40% unrepaired breaks at 24 h. IGF-1R depletion induced significant reduction in DSB repair by HR, although the magnitude of the repair defect suggests additional contributory factors. Radiation-induced G2-M arrest was attenuated by IGF-1R depletion, potentially suppressing cell cycle-dependent processes required for HR. In contrast, IGF-1R depletion induced only minor radiosensitization in LNCaP cells, and did not influence repair. Cell cycle profiles were similar to DU145, so were unlikely to account for differences in repair responses. CONCLUSIONS: These data indicate a role for IGF-1R in DSB repair, at least in part via HR, and support use of IGF-1R inhibitors with DNA damaging cancer treatments.

Prostate cancer: to screen or not to screen.

This paper evaluates the use of prostate-specific antigen (PSA) as a screening tool for prostate cancer. A current and contentious issue in both public and medical spheres, we are still lacking clear evidence and guidelines. Here, the Wilson and Jungner screening criteria are used as a framework to suggest that PSA-testing is not yet a proven tool for population screening. Additionally, the conflicting results of two recent randomised controlled trials are compared. The European Randomised trial of Screening for Prostate Cancer (ERSPC) found that PSA screening reduced prostate cancer-related deaths by 20% (adjusted p=0.04). Meanwhile the North American Prostate, Lung, Colon and Ovarian cancer trial (PLCO) found no significant impact of screening on mortality. The reasons for these differing outcomes are discussed in greater detail under the categories of methodology, study size, screening interval, cause of death and tumour demographics. The authors of this article conclude that PSA screening, at best, has a moderate impact on prostate cancer mortality. PSA-screening does, however, pose a high risk of over-diagnosis and over-treatment with its associated morbidity. Furthermore, economic and quality of life evaluations are lacking at present. Data are awaited from the UK Department of Health - funded ProtecT study,as well as longer-term outcomes of the ERSPC.

Cáncer de próstata: ¿hacer o no hacer cribado? / Prostate cancer: to screen or not to screen

Este artículo valora el uso del PSA (Antígeno prostático específico) como instrumento de cribado en cáncer de próstata. Siendo un tema de actualidad y polémico, tanto en la esfera médica como en la pública, todavía carecemos de evidencia clara y guías de actuación. Aquí, los criterios de cribado de Wilson y Jungner se utilizan como marco para sugerir que la prueba de PSA no es todavía un instrumento probado de cribado poblacional.Además, se comparan los resultados en conflicto de dos estudios aleatorizados controlados. El ERSPC (European Randomised trial of Screening for Prostate Cancer) encontró que el cribado con PSA reducía un 20% la mortalidad relacionada con cáncer de próstata (ajustada, p=0,04). Mientras que el ensayo PLCO (North American Prostate, Lung, Colon and Ovarian cancer Trial) no encuentra impacto significativo del cribado en la mortalidad. Las razones de estas diferencias de resultados se discuten en gran detalle bajo las categorías de metodología, tamaño del estudio, intervalo de cribado, causa de la muerte y datos demográficos del tumor. Los autores de este artículo concluyen que el cribado con PSA tiene, como mucho, un impacto moderado en la mortalidad por cáncer de próstata. Sin embargo, el cribado con PSA representa un problema de sobrediagnóstico y sobretratamiento con su morbilidad asociada. Más aun, faltan evaluaciones económicas y de calidad de vida en el presente. Se esperan los datos del estudio Protect –T financiado por el Departamento de Salud del Reino Unido, así como los resultados a más largo plazo del ERSPC(AU)

This paper evaluates the use of prostate-specific antigen (PSA) as a screening tool for prostate cancer. A current and contentious issue in both public and medical spheres, we are still lacking clear evidence and guidelines. Here, the Wilson and Jungner screening criteria are used as a framework to suggest that PSA-testing is not yet a proven tool for population screening. Additionally, the conflicting results of two recent randomised controlled trials are compared. The European Randomised trial of Screening for Prostate Cancer (ERSPC) found that PSA screening reduced prostate cancer-related deaths by 20% (adjusted p=0.04). Meanwhile the North American Prostate, Lung, Colon and Ovarian cancer trial (PLCO) found no significant impact of screening on mortality. The reasons for these differing outcomes are discussed in greater detail under the categories of methodology, study size, screening interval, cause of death and tumour demographics. The authors of this article conclude that PSA screening, at best, has a moderate impact on prostate cancer mortality. PSA-screening does, however, pose a high risk of over-diagnosis and over-treatment with its associated morbidity. Furthermore, economic and quality of life evaluations are lacking at present. Data are awaited from the UK Department of Health- funded ProtecT study,as well as longer-term outcomes of the ERSPC(AU)

Serial analysis of resected prostate cancer suggests up-regulation of type 1 IGF receptor with disease progression.

OBJECTIVE: • To compare immunostaining protocols using different antibodies for the type 1 insulin-like growth factor receptor (IGF-1R) in channel transurethal resection of the prostate (chTURP) chips, and to investigate how IGF-1R expression varies with time in serial prostate cancer specimens from individual patients. METHODS: • We studied IGF-1R expression in 44 prostate cancer specimens from 18 patients who had undergone serial chTURP at least 3 months apart. • Retrospective analysis of the hospital notes was undertaken to obtain clinical information, including age, Gleason score, prostate-specific antigen (PSA) level, hormone treatment and metastatic disease status at the time of each operation. • After an optimization process using three commercially-available IGF-1R antibodies, we used two antibodies for semiquantititve immunostaining of serial chTURP chips. RESULTS: • Santa Cruz antibody sc713 gave positive staining in IGF-1R null R- cells, and was not used further. Antibodies from Cell Signaling Technology (Beverly, MA, USA) (CS) and NeoMarkers Inc. (Fremont, CA, USA) (NM) did not stain R- cells and, in prostate tissue, showed staining of the glandular epithelium, with negligible stromal staining. All 44 chTURP samples contained identifiable malignant tissue and, of these, 73% and 64% scored moderately or strongly (score 3 or 4) with the CS and NM antibodies respectively. • There was significant correlation of IGF-1R scores of malignant tissue between the two antibodies (P < 0.001). By contrast, staining of benign glands showed poor correlation between antibodies: CS gave significantly weaker staining than malignant epithelium in the same sections (P < 0.001), whereas NM showed poor discrimination between malignant and benign glands. IGF-1R staining scores generated by the CS antibody were used to analyze the clinical data. • Most patients (six of seven) with falling IGF-1R staining scores were responding to androgen deprivation therapy (confirmed by PSA response) between operations. Conversely, in seven of eight patients who had progression to androgen-independence between procedures, IGF-1R levels increased or remained high. Finally, seven of 11 patients who developed radiologically confirmed metastases between procedures showed stable or increasing IGF-1R staining scores. CONCLUSION: • The present study is the first to assess changes in IGF-1R expression in serial prostate cancer samples. The results obtained indicate that IGF-1R expression usually remains high throughout the course of histologically-proven disease progression in serial specimens, suggesting that the IGF-1R remains a valid treatment target for advanced prostate cancer.

Does the presence of robotic surgery affect demographics in patients choosing to undergo radical prostatectomy? A multi-center contemporary analysis.

We report on differences in patient demographics in those men choosing to undergo radical prostatectomy in a UK center where there is no influence of robotic surgery and in those choosing radical prostatectomy in a US center where there is a strong robotic influence. Demographic and pathologic data were prospectively recorded in parallel for 78 consecutive men undergoing robot-assisted radical prostatectomy in a tertiary care academic US center and 69 consecutive men concurrently undergoing open radical prostatectomy in a similar UK center. Although average patient age was significantly younger in the US cohort (58.8 years, range 43.1-77.6 vs. 62.2 years, range 51.7-70.5; P = 0.002), the US cohort encompassed a wider age range and older patients than the UK cohort. Average preoperative prostate-specific antigen (PSA) was significantly lower in the US group (6.0, range 2.0-6.0 vs. 8.60, range 4.6-12.6; P < 0.01). Biopsy Gleason score, clinical stage, final pathology Gleason score, pathologic staging and positive margin rate were not significantly different between the two groups. Blood loss and transfusion rate were significantly lower in the US group. 16.7% of men in the US cohort had overall positive surgical margins compared to 29% in the UK group (P = 0.07). This data confirms our belief that patient age ranges are different in a setting influenced by robotic surgery. Although pathologic parameters were similar, the age distribution of robotic surgery patients was much wider, suggesting robotics attracts men previously reluctant to undergo surgery in the open setting or to pursue active surveillance protocols. Larger studies are needed to verify this finding.

Mutations in the AXIN1 gene in advanced prostate cancer.

BACKGROUND: The Wnt signalling pathway directs aspects of embryogenesis and is thought to contribute to maintenance of certain stem cell populations. Disruption of the pathway has been observed in many different tumour types. In bowel, stomach, and endometrial cancer, this is usually due to mutation of genes encoding Wnt pathway components APC or beta-catenin. Such mutations are rare in hepatocellular carcinomas and medulloblastomas with Wnt pathway dysfunction, and there, mutation in genes for other Wnt molecules, such as Axin, is more frequently found. OBJECTIVE: Although evidence of abnormal activation of the Wnt pathway in prostate cancer has been demonstrated by several groups, APC and beta-catenin mutations are infrequent. We sought mutations in genes encoding Wnt pathway participants in a panel of prostate cancer clinical specimens and cell lines. DESIGN, SETTING, AND PARTICIPANTS: DNA was obtained from 49 advanced prostate cancer specimens using laser microdissection followed by whole genome amplification and 8 prostate cancer cell lines. MEASUREMENTS: The DNA samples were screened for mutations in the genes encoding APC, beta-catenin, and Axin. The subcellular distribution of beta-catenin expression was assessed in the clinical specimens using immunohistochemistry. RESULTS AND LIMITATIONS: Abnormal patterns of beta-catenin expression, suggesting Wnt pathway dysregulation, were observed in 71% of specimens. One APC mutation, two beta-catenin gene mutations, and 7 DNA sequence variations in the Axin gene were detected. Four different Axin polymorphisms were also found in the cell lines. The study does not provide definite evidence that the observed sequence changes alter protein function, promoting neoplasia, but the potential functional relevance of these variants is discussed. CONCLUSIONS: These data contribute to our understanding of the role of Wnt dysregulation in prostatic tumourigenesis and support the current interest in the pathway as a therapeutic target. Of particular interest, we identified three new potentially functionally relevant AXIN1 mutations.

A double-blind, randomized, controlled trial of topical glyceryl trinitrate for transrectal ultrasound guided prostate biopsy.

PURPOSE: We evaluated the efficacy and tolerance of topical 0.2% glyceryl trinitrate (GTN) paste vs placebo to decrease pain associated with transrectal ultrasound guided prostate biopsy. MATERIALS AND METHODS: Between October 2003 and April 2004, 134 consecutive patients referred for a first prostate biopsy were randomized to receive topical 0.2% GTN paste or placebo 30 minutes prior to biopsy. Participants completed a 10 point visual analog pain score following the procedure. RESULTS: Mean patient age was 68.5 years in the GTN group and 68.8 in the placebo group. There was a significant decrease in mean pain score in the GTN group compared with placebo (3.7 vs 4.8, p <0.05). Six patients (10%) in the GTN group complained of headache. CONCLUSIONS: Topical GTN paste is an effective and well tolerated method of decreasing pain associated with transrectal ultrasound guided prostate biopsy. It is safe and easy to use, and it should be offered to patients undergoing this procedure.

Silencing of the IGF1R gene enhances sensitivity to DNA-damaging agents in both PTEN wild-type and mutant human prostate cancer.

The type 1 insulin-like growth factor receptor (IGF1R) is overexpressed in prostate cancer, and mediates proliferation, motility, and survival. Many prostate cancers harbor inactivating PTEN mutations, enhancing Akt phosphorylation. This activates the principal antiapoptotic pathway downstream of the IGF1R, calling into question the value of IGF1R targeting in this tumor. The aim of the current study was to assess the effect of IGF1R gene silencing in prostate cancer cells that lack functional PTEN protein. In human DU145, LNCaP and PC3 prostate cancer cells, transfection with IGF1R small interfering RNA induced significant enhancement of apoptosis and inhibition of survival, not only in PTEN wild-type DU145 but also in PTEN mutant LNCaP and PC3. This was attributed to attenuation of IGF signaling via Akt, ERKs and p38. In both DU145 and PC3, IGF1R knockdown led to enhancement of sensitivity to mitoxantrone, etoposide, nitrogen mustard and ionizing radiation. There was no sensitization to paclitaxel or 5-fluorouracil, which do not damage DNA, suggesting that chemosensitization results from impairment of the DNA damage response, in addition to removal of apoptosis protection. These results support the concept of IGF1R targeting in prostate cancer, and indicate that PTEN loss does not render tumor cells refractory to this strategy.