Rapid Doubling Time Research Articles

Chronic Lymphocytic Leukemia: Proliferative and Apoptotic Profile on Lymph Node, Studied by Immunohistochemistry Including the Proliferation Centers

Abstract 2538Chronic lymphocytic leukemia (CLL) has a proliferation rate higher than previously recognized. Proliferation centers (PC) play an important role in the biology of CLL given the fact that they constitute its proliferative compartment. The complexity of the microenvironment of PC, as well as the molecular events taking place in the PC and their clinical significance remain to be elucidated.The aim was to identify the presence of PC in lymph node and other tissues, except the bone marrow; to analyze their proliferative status; to evaluate the expression of molecules implicated in the apoptotic process in PC, compared to their expression in the non-PC areas; and to correlate the aforementioned findings with the clinical and laboratory features in a series of CLL patients.Fifty patients, fulfilling the diagnostic criteria of CLL/SLL and in whom lymph node biopsy or other tissue material (44 lymph nodes, 5 spleens, 1 skin) were available, were enrolled in this study. Twenty five biopsies were performed at diagnosis while the rest prior to treatment initiation. All necessary clinical and biological data were recorded on each patient, including Binet stage, lymphocyte doubling time, bone marrow infiltration pattern, CD 38 expression, IgVH mutational status and FISH analysis for genetic abnormalities. PC were defined as pale areas containing large cells so called paraimmunoblasts and prolymphocytes, surrounded by a dark background of small lymphocytes. Immunohistochemical detection of the following molecules participating in the apoptotic process were studied in all tissue sections: Bcl-2, Fas, FasL, c-FLIP (in the PC and the non PC areas) as well as cleaved caspase 3 that was evaluated in the entire tumor area. ZAP-70 was also studied by immunohistochemistry while proliferation status was assessed by the Ki-67 immunostaining.The median age of our patients at diagnosis was 55 years (36–77). Twenty-nine (58%) had disease stage A, 17(34%) B and 4(8%) C, while 12% had B-symptoms, 26% elevated LDH levels and 25% presented with rapid lymphocyte doubling time. 39% needed treatment immediately after diagnosis.Proliferations centers were present and easily identified after staining with haematoxylin-eosin on lymph node and splenic sections with their numbers varying from case to case. Proliferation assessment revealed that median Ki67 proliferation index per PC was 10% (1–20%), while median Ki67 in the whole tissue section was 3% (1–8%) (p<0,0001).Apoptotic molecules evaluation disclosed that the expression of cleaved-caspase 3 was very low (median 0.0046%). Fas, FasL and cFLIP activity was expressed in various percentages in the PC and in the non-PC areas as shown in Table 1. Further on, in cases of overexpression of Fas and FasL, an analogous increase of cFLIP expression was not observed. Patients with elevated Ki67 proliferation index in PC area tended to express more Fas and FasL in the same areas. Strong homogenous Bcl-2 expression was observed both in the PC and in the non PC areas. Weaker Bcl-2 expression in PC compared to non PC areas was observed in 10 patients in whom a higher Fas (p=0.054) and FasL (p=0.005) was present. 48% of the cases were ZAP-70 positive. ZAP70 positivity was correlated with increased expression of cFLIP in PC (p=0.0216). The only statistically significant correlation between apoptotic molecule profile and clinical features was between an increased FasL expression in PC and B-symptoms (p: 0.0263). In univariate survival analysis, coexpression of Fas/FasL and cFLIP at increased levels in PC correlated with poor disease-free and overall survival (p=0.0031, p=0.0521 respectively).Table 1Fas, FasL and cFLIP expression in lymph node and splenic sections of CLL patientsMoleculePC median(%)Non-PC areas (median %)P valueFas50400,1581FasL30100,0107cFLIP50400,0726Proliferative centers in lymph node and splenic sections of CLL patients present particular proliferative and apoptotic profile. Inhibition of Fas mediated apoptosis in PC may not be attributable to cFLIP expression. Increased Fas,FasL and cFlip coexpression in PC was correlated with disease-free and overall survival. Disclosures:No relevant conflicts of interest to declare.

Clinical Utility of Molecular Surveillance for Cytomegalovirus After Antiviral Prophylaxis in High-Risk Solid Organ Transplant Recipients

Cytomegalovirus (CMV) disease after discontinuation of prophylaxis is a significant problem for CMV-seronegative recipients of CMV-seropositive organs (donor seropositive and recipient seronegative [D+/R-]). Virologic monitoring after prophylaxis has been proposed as a way to prevent late-onset disease. We reviewed the efficacy of this strategy. CMV D+/R- organ transplant recipients received 3 to 6 months of antiviral prophylaxis, and then viral loads were performed weekly for 8 weeks. Preemptive antiviral therapy was initiated at a predefined threshold. Seventy-one CMV D+/R- patients were assessed. Symptomatic CMV disease occurred in 29 of 71 (40.8%) patients during the first-year posttransplant. A significant portion of disease occurred only after the 8-week surveillance period (n=16). Viremia occurred in 19 of 71 (26.8%) patients during the 8-week surveillance. Preemptive therapy was successfully used in only 3 of 19 (15.8%) viremic patients with no further disease development. The remaining patients cleared low-level viremia spontaneously (n=3) or had CMV disease (n=13) either at the first detection of viremia or before preemptive therapy initiation because of rapid viral load doubling (median doubling time 1.1 days). CMV D+/R- patients had significant incidence of late-onset disease after prophylaxis. However, the use of a preemptive after prophylaxis strategy was of limited benefit in this group because of rapid viral doubling times and disease occurring after the surveillance period.

Poor Agreement of Prostate Specific Antigen Doubling Times Calculated Using Ultrasensitive Versus Standard Prostate Specific Antigen Values: Important Impact on Risk Assessment

In men with biochemical recurrence after radical prostatectomy, a rapid prostate specific antigen doubling time is associated with adverse outcomes, and is often used to guide the type and timing of salvage therapy. It is unknown whether prostate specific antigen doubling time calculated in the ultrasensitive range (prostate specific antigen less than 0.2 ng/ml) accurately reflects measures performed in the traditional range (prostate specific antigen greater than 0.2 ng/ml). We studied 394 men in a national disease registry of men with prostate cancer (CaPSURE™) who underwent radical prostatectomy, experienced biochemical failure, and had prostate specific antigen doubling time assessed using ultrasensitive and traditional prostate specific antigen values. Agreement between these measurements was assessed using Cohen's kappa score. Median ultrasensitive prostate specific antigen doubling time was 11.9 months (IQR 6-29) and median traditional prostate specific antigen doubling time was 240 months (IQR 18-240). Agreement between ultrasensitive and traditional prostate specific antigen doubling time was poor, with a weighted Cohen's kappa score of 0.04 (95% CI -0.02-0.10). Using a dichotomous prostate specific antigen doubling time cutoff of 9 months, there was a statistically significant difference between ultrasensitive and standard prostate specific antigen doubling time (exact McNemar p <0.01). Ultrasensitive prostate specific antigen doubling time was more or less rapid than traditional prostate specific antigen doubling time by more than 15 months in 244 (62%) and 35 (9%) patients, respectively. Agreement between prostate specific antigen doubling time calculated using ultrasensitive vs traditional prostate specific antigen values is poor. Ultrasensitive prostate specific antigen doubling time is often significantly more rapid than traditional prostate specific antigen doubling time, potentially overestimating the risk of clinical recurrence. Until the significance of ultrasensitive prostate specific antigen doubling time is better characterized, the decision to proceed with salvage therapy should not be based on prostate specific antigen doubling time calculated using ultrasensitive prostate specific antigen values.

Long-Term Risk of Clinical Progression After Biochemical Recurrence Following Radical Prostatectomy: The Impact of Time from Surgery to Recurrence

BackgroundThe natural history of biochemical recurrence (BCR) after radical retropubic prostatectomy (RRP) is variable and does not always translate into systemic progression or prostate cancer (PCa) death. ObjectiveTo evaluate long-term clinical outcomes of patients with BCR and to determine predictors of disease progression and mortality in these men. Design, setting, and participantsWe reviewed our institutional registry of 14 632 patients who underwent RRP between 1990 and 2006 to identify 2426 men with BCR (prostate-specific antigen [PSA] levels ≥0.4ng/ml) who did not receive neoadjuvant or adjuvant therapy. Median follow-up was 11.5 yr after RRP and 6.6 yr after BCR. InterventionRRP. MeasurementsPatients were grouped into quartiles according to time from RRP to BCR. Survival after BCR was estimated using the Kaplan-Meier method and compared using the log-rank test. Cox proportional hazard regression models were used to analyze clinicopathologic variables associated with systemic progression and death from PCa. Results and limitationsMedian systemic progression-free survival (PFS) and cancer-specific survival (CSS) had not been reached after 15 yr of follow-up after BCR. Cancer-specific mortality 10 yr after BCR was 9.9%, 9.3%, 7.8%, and 4.7% for patients who experienced BCR <1.2 yr, 1.2–3.1 yr, 3.1–5.9 yr, and >5.9 yr after RRP, respectively (p=0.10). On multivariate analysis, time from RRP to BCR was not significantly associated with the risk of systemic progression (p=0.50) or cancer-specific mortality (p=0.81). Older patient age, increased pathologic Gleason score, advanced tumor stage, and rapid PSA doubling time (DT) predicted systemic progression and death from PCa. Limitations included retrospective design, varied utilization of salvage therapies, and the inclusion of few patients with positive lymph nodes. ConclusionsOnly a minority of men experience systemic progression and death from PCa following BCR. The decision to institute secondary therapies must balance the risk of disease progression with the cost and morbidity of treatment, independent of time from RRP to BCR.

Immune Evasion by B-cell Lymphoma

Immune Evasion by B-cell Lymphoma

Autoimmune cytopenia in chronic lymphocytic leukemia: prevalence, clinical associations, and prognostic significance

AbstractWe analyzed prevalence, characteristics, clinical correlates, and prognostic significance of autoimmune cytopenia in patients with chronic lymphocytic leukemia. Seventy of 960 unselected patients (7%) had autoimmune cytopenia, of whom 19 were detected at diagnosis, 3 before diagnosis, and 48 during the course of the disease. Forty-nine patients had autoimmune hemolytic anemia, 20 had immune thrombocytopenic purpura, and 1 had both conditions. A clear association was observed between autoimmune cytopenia and poor prognostic variables (ie, high blood lymphocyte count, rapid blood lymphocyte doubling time, increased serum β-2 microglobulin level, and high expression of ζ-associated protein 70 and CD38). Nevertheless, the outcome of patients with autoimmune cytopenia as a whole was not significantly different from that of patients without this complication. Furthermore, no differences were observed according to time at which cytopenia was detected (ie, at diagnosis, during course of disease). Importantly, patients with advanced (Binet stage C) disease because of an autoimmune mechanism had a significantly better survival than patients in advanced stage related to a massive bone marrow infiltration (median survivals: 7.4 years vs 3.7 years; P = .02). These results emphasize the importance of determining the origin of cytopenia in patients with chronic lymphocytic leukemia for both treatment and prognostic purposes.

Adjuvant versus salvage radiation therapy for prostate cancer and the risk of death

To investigate whether salvage radiation therapy (RT) for prostate-specific antigen (PSA) failure can provide the same result as adjuvant RT, which decreases the risk of all-cause mortality (ACM) for men with positive margins (R1), or extra-capsular or seminal vesicle extension (pT3). We studied 1638 men at Duke University who underwent radical prostatectomy for unfavourable-risk prostate cancer and whose postoperative PSA was undetectable. Cox regression was used to evaluate whether salvage vs adjuvant RT in men with a rapid (<10 months) or slow (≥10 months) PSA doubling time (DT) was associated with the risk of ACM, adjusting for adverse features (pT3, R1, Gleason score 8-10), age, preoperative PSA level, comorbidity and hormonal therapy use. Despite fewer men with two or more adverse features (61 vs 82%; P=0.016), salvage for a rapid PSA DT vs adjuvant RT increased the risk of ACM [adjusted hazard ratio (AHR)=3.42; 95% confidence interval (CI)=1.27-9.20; P=0.015]. There was no difference (AHR=1.39; 95% CI=0.50-3.90; P=0.53) in the risk of ACM among men who received salvage for a slow PSA DT or adjuvant RT. Nearly all (90%) men with a slow PSA DT had Gleason score ≤7 and the majority (59%) had at most pT3 or R1 disease. Radiation therapy after PSA failure as compared with adjuvant RT was not associated with an increased risk of ACM in men with Gleason score ≤7 and pT3R0 or pT2R1 disease.

Small Cell Lung Cancer: Past, Present, and Future

Small cell lung cancer (SCLC) accounts for about 10% to 15% of all lung cancers. It is characterized by its rapid doubling time, high rate of dissemination, and increased sensitivity to chemotherapy and radiation. Although the incidence of SCLC has been steadily decreasing over time, it remains a serious public health problem given its aggressive clinical behavior and the lack of effective therapies. This review looks at the evolution of SCLC treatment and the standard treatments that are currently available, including platinum-based combination chemotherapy, hyperfractionated thoracic radiation, and prophylactic cranial irradiation. The development of novel therapies for SCLC has been lagging behind, but completed clinical trials and ongoing investigations are helping us define what will be the best therapeutic targets for this disease.

It's time to depolarise the unhelpful PSA‐testing debate and put into practice lessons from the two major international screening trials

TO THE EDITOR: When I looked at the cover of the 5 April issue of the MJA, I feared finding another article focused on discrediting the prostate-specific antigen (PSA) test. Instead, I congratulate the authors, and the Journal, for presenting one of the rare balanced articles on this topic. Denham and colleagues called for an end to taking sides in the debate over PSA testing, and focused instead on helpful guidance. The problem is not whether PSA helps us find prostate cancer, but that we lack clinical tools for deciding which patients would benefit from aggressive treatment. However, the authors point out that there are two important tools that can help with this decision: low free to total PSA ratios, and rapid PSA doubling time (< 3 years). The PSA test is now one of the most sensitive, precise and highly standardised immunoassays in the clinical laboratory. The difficulty does not lie with the measurement but with its application. The Royal College of Pathologists of Australasia, together with the Urological Society of Australia and New Zealand, recently produced a monograph that discussed the appropriate use of the PSA test. It emphasised using age-related cut-offs for PSA levels, the free to total PSA ratio, and the calculation of PSA doubling time as modern tools to achieve optimal benefit from the test. The Australian Medicare Benefits Schedule (MBS) was changed in May 2009 (following a suggestion from the Urological Society of Australia and New Zealand) to improve utilisation of free to total PSA ratios. The Box indicates the per capita request rates of PSA testing (MBS item number 66655) and free to total PSA ratios (MBS item number 66659) from May 2009 to February 2010. Consistent with Denham et al’s observation that there are regional differences in the attitudes to prostate cancer, there is a twofold variation in PSA requesting and a sevenfold difference in free to total PSA ratio requesting across the Australian states. Furthermore, the relationship between the two tests is, if anything, inverse, suggesting that increased use of PSA testing is less commonly followed up by modern tools such as free to total PSA ratio. As a chemical pathologist, the appropriate clinical use of the PSA test has been a careerlong concern of mine. Even though the discoverer of PSA has labelled the test a public health disaster, recent “case–controlled” studies using PSA in an outdated approach (without free to total PSA ratios or doubling times) have shown a marginal benefit for screening. The indiscriminate use of PSA testing can be helpful to some but disastrous for others. Modern PSA tools may significantly improve management beyond these marginal effects. As always, the value of medical investigations lies in how intelligently we use them.

Frequency of Screening Magnetic Resonance Imaging to Detect Occult Spinal Cord Compromise and to Prevent Neurological Deficit in Metastatic Castration-resistant Prostate Cancer

Aims Neurological deficit from malignant spinal cord compression (SCC) is a major complication of metastatic castration-resistant prostate cancer (CRPC). The aims of the present study were to determine the incidence of neurological deficit in metastatic prostate cancer patients and to determine the optimal frequency of screening magnetic resonance imaging (MRI) spine required to detect clinically occult radiological SCC (rSCC). Materials and methods A retrospective analysis of the clinical data of 130 consecutive patients with CRPC, with no functional neurological deficit, who had screening MRI spine from January 2001 to May 2005, was undertaken. Patients found to have rSCC received radiotherapy. All patients were followed-up to document the incidence of neurological deficit. Results Thirty-seven (28.4%) patients had rSCC on MRI. The proportion of patients free from neurological deficit at 3, 6, 12, 18 and 24 months was 94, 80, 59 and 43%, respectively, in patients who had rSCC on initial MRI and 97.5, 89, 75 and 63%, respectively, in patients who had no rSCC. A high prostate-specific antigen (PSA) level at initial MRI ( P = 0.035) and a short PSA doubling time < 3 months ( P = 0.009) significantly predicted for neurological deficit on univariate analysis, whereas back pain ( P = 0.059), although an important predictive factor, did not attain statistical significance. On multivariate analysis, only rapid PSA doubling time (<3 months) independently predicted for future neurological deficit ( P = 0.042). Conclusion MRI spine can be used to detect asymptomatic rSCC in patients with CRPC and serial estimations are required to maintain a low incidence of clinical SCC. If serial screening MRI spine is used to detect rSCC in 90% of patients before the development of neurological signs, the optimum frequency depends on the subset of patients studied. The results of our study suggest that the optimum frequency would be every 4–6 months for patients with previous SCC, rapid or high PSA or back pain and annually for asymptomatic patients.

Abstract CN02-02: CTCs as pharmacodynamic and /or predictive biomarkers

Abstract CTC analysis has potential utility in the development of novel therapeutics and particularly so for the management of those patients with cancer types for which obtaining tumor biopsies poses significant challenge, especially pre and serially post therapy. We have enumerated and begun to characterize CTCs from patients with lung cancer (both small cell lung cancer (SCLC) and non small cell lung cancer (NSCLC)) using two techniques; a) the Veridex CellSearch Platform wherein CTCs are identified based on EpCam and cytokeratin expression together with the absence of the white blood cell surface marker CD45, and b) the Metagenex ISET filter-based system that isolates CTCs on the basis of their size. SCLC accounts for ∼15% of all lung cancers, is highly aggressive with a rapid doubling time, high growth fraction and propensity to metastasise early. Primary SCLC biopsies are often of small volume, highly necrotic and thus present difficulties for biomarker analysis. As SCLC has neuro-endocrine characteristics, we examined initially, the ability of the CellSearch approach (with the epithelial cell marker EpCam as its primary discriminator) to detect SCLC CTCs. In chemo-naive SCLC, CTCs were detected in 86% patients (median 28, range 0–44896, n=50). Tumor stage correlated with CTC number (#) and was highest in patients with liver metastases. The median survival for patients with baseline CTC# &amp;gt;300/7.5ml blood was 134 days compared to 443 days when CTC# &amp;lt;2. We next asked whether the CTCs detected expressed the neuro-endocrine surface marker CD56 using the 4th immunofluorescence channel on the CellSearch platform. Expression of CD56 was heterogeneous but consistent with expression in primary tumors. CTC# was pharmacodynamic, decreasing after one cycle of etoposide/platinum chemotherapy mirroring radiological response. CTC# and % apoptotic CTCs (analyzed by nuclear morphology) are now being applied as pharmacodynamic biomarkers in early clinical trials of novel agents and in particular of pro-apoptotic agents. To test the hypothesis that CTCs could provide added value to novel drug development, we also assessed the feasibility of drug-target characterization in SCLC CTCs. Bcl-2, an antiapoptotic drug target, is amplified in ∼40% SCLC patients and was detectable in SCLC CTCs using fluorescence in situ-hybridization; potentially, Bcl-2 amplification will predict sensitivity to Bcl-2 targeted drugs. In a cohort of 93 chemo-naive stage III/IV NSCLC patients, 2 or more CTCs/7.5ml blood were detected using CellSearch in 6% patients (2/36) with stage IIIA/IIIB disease and 32% (18/57) patients with stage IV disease. A CTC# of &amp;gt;5 at baseline predicted for a worse progression free and overall survival compared to patients with &amp;lt;5 CTCs (p = &amp;lt; 0.0001). For the 20 patients with 2 of more CTCs at baseline, 55 % exhibited reduction in CTC# following a single cycle of standard platinum based chemotherapy, suggesting that CTC# may be useful in early prediction of response to novel therapeutics. Matched blood samples were taken for ISET analysis for 30 of this cohort of NSCLC patients. CTCs were detected in patients in whom no CTCs were identified using CellSearch. This may be due to loss of, or reduction in EpCam expression perhaps during eptithelial to mesenchymal transition and this now requires further investigation. After optimization of immunohistochemical assays using ISET filters, a panel of NSCLC markers including Thyroid Transcription Factor 1 (TTF1, a routinely used diagnostic marker for NSCLC) and EGFR are being used to confirm the identity of NSCLC CTCs distinct from peripheral blood mononuclear cells. This approach is being applied for detection of protein targets of novel therapies. Overall, our data suggest that CTC analysis is a powerful addition to the biomarker portfolio for drug development in lung cancer. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):CN02-02.

Targeted therapies in small cell lung cancer: a review.

Small cell lung cancer (SCLC) is an aggressive form of lung cancer that is characterized by a rapid doubling time, early onset of dissemination and high sensitivity to chemotherapy. Despite the potential for cure in patients with limited disease with concurrent chemoradiation and an initial good response to chemotherapy in extensive disease, there is a high chance of disease relapse with an overall poor median survival for both stages. With increasing translational research and a better understanding of the molecular basis of cancer, a number of molecular targets have been identified in various preclinical studies. This review summarizes potentially viable targets and new agents that have been developed and employed in recent, ongoing and future clinical trials to attempt to improve clinical outcomes in this disease.

Characterization and In Vivo Testing of Mesenchymal Stem Cells Derived From Human Embryonic Stem Cells.

Abstract 1451Poster Board I-474Mesenchymal stem cells (MSC, aka marrow stromal cells) present a promising tool for cell therapy, and have been shown to contribute to the recovery of tissues in myocardial infarction, stroke, meniscus injury, limb ischemia, and even neurodegenerative disorders. The percentage of engrafted MSC in these studies has been very low in comparison to the recipient tissue cells, suggesting that their efficacy relies upon actions other than differentiation. One theory of tissue repair and regeneration by adult MSC is that the injected stem cells home to the injured area, in particular to hypoxic, apoptotic, or inflamed areas, and release trophic factors that hasten endogenous repair. These secreted bioactive products can suppress the local immune system, enhance angiogenesis, inhibit fibrosis and apoptosis, and stimulate recruitment, retention, proliferation and differentiation of tissue-residing stem cells. Paracrine effects exerted by MSC are distinct from the classical model of direct differentiation of stem cells into the tissue to be regenerated. MSC can, however, directly contribute to the repair of bone, tendon and cartilage. In some cases, where the patient lacks expression of a critical gene product, genetic engineering of the MSC is desired. Using human embryonic stem cells (hESC), the integration site of a vector can be fully characterized and the clones with benign integration sites can be expanded. Homologous recombination is also now feasible for embryonic cells due to increased efficiencies, and clones with vectors targeted for gene correction can be expanded. For this reason we have generated MSC from the well-characterized human embryonic stem cell line H9. H9-derived MSC (H9-MSC) expressed CD105, CD90, CD73 and CD146, and lacked expression of CD45, CD34, CD14, CD31, and HLA-DR. H9-MSC also lacked expression of the hESC pluripotency markers SSEA-4 and Tra-1-81, which were expressed by the starting H9 line. Additionally, they lacked expression of SSEA-1, an early marker of hESC differentiation. Marrow-derived MSC showed a similar phenotype when analyzed concurrently with H9-MSC. Morphology was similar to adult MSC derived from marrow or adipose tissue. H9-MSC grew more robustly than MSC derived from marrow or adipose tissue, with a rapid doubling time and a failure to display contact inhibition. However, upon reaching maturity the cells did slow to the same rate as bone marrow-derived MSC, and then were finally subject to contact inhibition. Additionally, H9-MSC were injected with matrigel subcutaneously into the flank of immune deficient mice [NOD/SCID/IL2Rg-/- (NSG)] to assess their ability to form tumors due to possible growth abnormalities. No mice that received injections of H9-MSC formed teratoma or other tumors, whereas the parent H9 line robustly forms teratoma. H9-MSC could be robustly differentiated into bone, as shown by alizarin red staining of mineralized plaques after 21 days of culture in standard osteogenic differentiation medium containing 0.2 mM ascorbic acid, 0.1 m M dexamethasone, and 10 mM b-glycerophosphate with media changes every three days. H9-MSC morphology was noticeably different from that of H9-MSC undergoing differentiation as early as day 3 of the differentiation protocol. Finally, cells were tested for their capacity to respond to a hypoxic in vivo environment, using our standard hindlimb ischemia model in immune deficient [NOD/SCID/b2M-/-] mice. H9-MSC were found to have homed to the hypoxic muscle by 48 hours after injection into the bloodstream. In summary we have defined methods for differentiation of hESC into MSC, have defined their characteristics, and in vivo migratory properties. This system could be useful, following further safety studies, for production of large numbers of MSC from embryonic or induced pluripotent stem cells that have been corrected for gene defects by lentiviral vector integration with careful assessment of integration site, or by homologous integration, with subsequent expansion, characterization and banking of the line prior to differentiation into functional mesenchymal stem cells. DisclosuresNo relevant conflicts of interest to declare.

Why are pretreatment prostate‐specific antigen levels and biochemical recurrence poor predictors of prostate cancer survival?

The value of pretreatment (initial) prostate-specific antigen (iPSA) and biochemical recurrence (BR) as prognostic factors for survival remains unclear. The authors sought to determine why using randomized trial data with 7-year minimum follow-up. In the Trans-Tasman Radiation Oncology Group 96.01 trial, 802 men with T2b, T2c, T3, or T4 N0 prostate cancer (PC) were randomized to radiotherapy alone or with 3 or 6 months neoadjuvant androgen deprivation between 1996 and 2000. Cox modeling was used to identify outcome predictors at follow-up landmark points. Higher iPSA was found to be a potent predictor of BR-free survival (P < .01) but was not prognostic for prostate cancer-specific survival (PCSS) from randomization. Patients experiencing BR had unfavorable initial prognostic factors compared with patients who did not. After BR, these factors were not prognostic for PC death in models adjusted for time to BR (TTBR). In these models, TTBR predicted PCSS more satisfactorily than the occurrence of BR itself. Survival probability 5 years after BR exceeded 90% for men with TTBR >/=4 years; however, it dropped to 44% +/- 6% for men with TTBR <1 year. After BR, rapid PSA doubling time (DT), low iPSA, and short TTBR were identified as the most important predictors of inferior PCSS. When BR occurs, prognostic factors for survival change. Low iPSA, short TTBR, and rapid PSA DT take over at this point, providing reasons why iPSA and occurrence of BR alone predict PCSS unsatisfactorily.

Impact of age at diagnosis on survival of hormone-refractory prostate cancer (HRPC) patients

e16050 Background: Conflicting data exist for age as a determinant of overall survival (OS) in pts with HRPC. We hypothesize that young (&lt;55) HRPC pts represent a more aggressive biological phenotype and therefore have a decreased OS. Methods: A retrospective chart review was conducted on 334 consective HRPC pts treated between 1995–2005. Summary statistics for demographic and clinical factors were generated, and Kaplan-Meier (KM) OS curves were created. Bivariate Cox Proportional-Hazards regression was used to test the association of age at diagnosis while adjusting for a covariate, with significant covariates entered into multivariate models. Results: Overall median survivals in the age stratified categories (&lt;55, ≥55–65, ≥65–75, ≥75) were 5.5, 6.9, 7.9, and 4.3 yrs, with 5 yr survivals 51.9%, 67.4%, 67.0%, and 34.9%, respectively. KM curves showed divergence with an overall significant log-rank test (p &lt; 0.0001). Compared to pts ≥65–75, the hazard ratios (HR) for HRPC pts &lt;55 and ≥75 were 1.40 (95% CI 0.90–2.60) and 2.52 (95% CI 1.67–3.82), respectively. However, following multivariate analysis HRs for HRPC pts &lt;55 and ≥75 were 1.60 (95% CI 0.98–2.62) and 1.25 (95% CI 0.71–2.20). Pts &lt;55 and ≥75 presented with advanced stage at diagnosis and progressed to bone metastasis earlier. Pts ≥75 had decreased performance status, more comorbidities, higher PSA at diagnosis, shorter duration of hormone sensitive disease, and were less likely to receive chemotherapy than pts &lt;75. The percentage of rapid PSA doubling times was highest in the &lt;55 cohort. In multivariate analysis with age as a categorical variate, ECOG 3–4 (HR 2.65), time from diagnosis to both HRPC (HR 0.78) and bone metastasis (HR 0.80), and duration of response to androgen ablation (HR 0.86) remained highly predictive. Conclusions: Age at diagnosis influences OS in HRPC with a bimodal survival curve. Pts &lt;55 and ≥75 present with more aggressive disease, translating into reduced median and 5 yr survivals. Other covariates, especially ECOG status, likely account for the decreased OS in the ≥75 cohort. Conversely, pts &lt;55 had an adjusted HR of 1.60 (p = 0.06). Our study supports a growing body evidence that suggests a poor prognosis in younger men; correlating these differences at the molecular level could lead to better targeted therapies. No significant financial relationships to disclose.

Survival following early hormone therapy for men with rapid PSA doubling time within 2 years following radical prostatectomy

5065 Background: Early hormonal therapy has been used in the salvage setting for men with biochemical recurrence following radical prostatectomy (RP), but no studies to date have been able to evaluate whether such treatment prolongs survival. We examined the impact of salvage hormonal therapy on overall survival (OS) in a cohort with long-term follow-up, and attempted to identify the subgroup most likely to benefit. Methods: Retrospective analysis of a cohort of 488 men undergoing RP at Johns Hopkins Hospital from 1982–2004, who experienced biochemical recurrence and received no salvage therapy (n = 386) or salvage hormonal therapy (n = 102); no one received adjuvant therapy. Survival was defined from biochemical recurrence to death from all causes, and analyzed with proportional hazards models with time-dependent covariates. Results: With median follow-up of 6 years after recurrence and 9 years after RP, there were 143 deaths (29%), including 105 from prostate cancer. After adjusting for PSA doubling time (PSADT), RP Gleason score, and year of surgery, hormonal therapy did not significantly improve OS for all men, compared to no salvage therapy: hazard ratio (HR) = 0.72 (95% confidence interval (CI): 0.45–1.17), p = 0.187. However, when restricted to men with early recurrence, i.e. within 2 years of RP, and with a rapid PSADT&lt;6 months, hormonal therapy was associated with a large, significant improvement in OS: HR = 0.25 (95% CI: 0.08–0.71), p = 0.0095. This subgroup comprised 22% of the cohort. In contrast, there was no benefit of salvage hormonal therapy in men with early recurrence and PSADT&gt;6 months: HR = 1.96 (95% CI: 0.89–4.31), p = 0.093, nor those who recurred more than 2 years after RP, regardless of PSADT. Conclusions: This study suggests that early salvage hormonal therapy may significantly and substantially prolong overall survival in the subgroup of men who experience an early biochemical recurrence with a rapid PSADT. These results are consistent with early recurrences being indicative of metastatic disease, while later recurrences are more likely to represent local recurrence. If validated, these results may provide useful stratification criteria for clinical trials. No significant financial relationships to disclose.

Exposure to Agent Orange is a significant predictor of prostate‐specific antigen (PSA)‐based recurrence and a rapid PSA doubling time after radical prostatectomy

To investigate and report the clinicopathological characteristics and outcomes after radical prostatectomy (RP) in patients with prostate cancer and previous exposure to Agent Orange (AO), particularly in relationship to race. In 1495 veterans who had undergone RP the clinicopathological characteristics, biochemical progression rates, and prostate-specific antigen (PSA) doubling time (DT) after recurrence between AO-exposed and unexposed men were compared using logistic and linear regression and Cox proportional hazards analyses, and stratified by race. The 206 (14%) men with AO exposure were more likely to be black (P = 0.001), younger (P < 0.001), treated more recently (P < 0.001), have a higher body mass index (P = 0.001), have clinical stage T1 disease (P < 0.001), and have lower preoperative PSA levels (P = 0.001). After adjusting for several clinical characteristics, AO exposure was not significantly related to adverse pathological features but was significantly associated with biochemical progression risk (relative risk 1.55, 95% confidence interval 1.15-2.09, P = 0.004) and shorter PSADT (P < 0.001) after recurrence (8.2 vs 18.6 months). When stratified by race, these associations were present and similar in both races, with no significant interaction between race and AO exposure for predicting biochemical recurrence or mean adjusted PSADT (P interaction >0.20). Patients with AO exposure and treated with RP were more likely to be black, present with lower risk features, have an increased risk of biochemical progression, and shorter PSADT after recurrence. When stratified by race, the association between AO exposure and poor outcomes was present in both races. These findings suggest that among selected men who choose RP, AO exposure might be associated with more aggressive prostate cancer.

Dose escalation model for limited-stage small-cell lung cancer

Background and purpose To construct a model for the local dose–response relationship of limited-stage small-cell lung cancer (LS-SCLC) along with subsequent models for dose-per-fraction escalation. Materials and methods Reported doses and 2-year progression free survival ( PFS 2year ) rates from LS-SCLC studies were used to construct a dose–response model. Modeling incorporated effects of chemotherapy, incomplete repair of sublethal damage and variations in both kick-off time for rapid reproliferation and effective tumor cell doubling time. Response was modeled as a function of overall treatment duration in order to shed light on the optimum radiotherapy treatment duration. Subsequent models were constructed for more aggressive radiotherapy by means of dose-per-fraction escalation for once a day (QD) and twice a day (BID) fractionation schedules. Results Modeling response as a function of treatment duration resulted in an optimum treatment duration of 3 weeks for both fractionation schedules. Modeling for dose-per-fraction escalation resulted in an increase in the expected local PFS 2year for both fractionation schedules with possible gains in a BID schedule with lower expected late complication rates than a QD schedule yielding the same expected local PFS 2year . Conclusions This modeling suggests more aggressive treatment through escalation of dose-per-fraction, which may result in substantial gains in local PFS 2year . This points towards treatment durations of 3 weeks as being optimal with a BID schedule predicting possible gains in local PFS 2year at lower rates of expected late complications than a QD schedule.

CFAR, An Active Frontline Regimen for High-Risk Patients with CLL, Including Those with Del 17p.

High-risk patients (pts) with CLL can be identified by prognostic factors. Traditional prognostic factors that identify high-risk pts include Rai stage, absolute lymphocyte count (ALC), rapid lymphocyte doubling time, and serum beta-2 microglobulin (β2M). Del 17p is associated with loss of the tumor suppressor gene TP53. Patients with del 17p also tend to have mutations in TP53, thereby providing a second mechanism for complete TP53 knockout. Pts with del 17p have poor response to purine analogue-based treatments. β2M greater than twice the upper limit of normal (2XULN) was an independent high-risk feature for pts treated with the frontline fludarabine (F), cyclophosphamide (C), rituximab (R) regimen (FCR). Pts younger than 70 years with β2M >2XULN treated with FCR had a complete remission (CR) rate of 60% and median time to progression (TTP) of 70 months, compared to 84% and 86 months for pts <70 yrs old with β2M <2XULN. We evaluated the CFAR regimen (F 20mg/m2 d3–5; C 200mg/m2 d3–5; R 375–500mg/ m2 d2; and Alemtuzumab 30mg d1,3,5 of each 4-wk course) as frontline treatment in a high-risk group of pts with CLL. The trial completed planned enrollment of 60 pts; 48 are currently evaluable for response and follow-up. In this high-risk group, 28% of the 48 evaluable high-risk pts had del 17p prior to treatment by FISH analysis. Overall, the CR rate for the 48 pts was 69%, 54% CR for the 13 pts with del 17p. The overall response rates were 94% and 77% for the 48 pts and 13 pts with del 17p, respectively. CFAR was associated with more myelosuppression and fewer pts could receive all 6 intended courses compared with the historic high-risk group treated with FCR. However, a higher proportion of CFAR pts had no evidence of residual disease in the bone marrow by 2-color flow cytometry evaluation at response assessment. There is currently no difference in TTP or OS, comparing CFAR and FCR in this retrospective historical analysis with a short median follow-up time of 16 months for the CFAR group. There was no significant difference in incidence of infection during treatment with CFAR compared to FCR, with the exception of CMV reactivation. The CFAR regimen is active and tolerated in the highrisk group of pts with CLL, including those with del 17p. Follow-up continues for the pts treated on this trial in order to evaluate responses in all 60 enrolled pts and to evaluate time-to-event endpoints and compare with the historic FCR experience.

A Phase I Study of Immune Gene Therapy for Patients with CLL Using Intranodal Injection of ISF35, a Membrane-Stable CD40 Binding Protein.

ISF35 is a novel CD40-binding protein designed to maximize stable, high-level surface-expression of this potent immuno-stimulatory molecule on cells transduced to express this protein. In a recent clinical study, patients with chronic lymphocytic leukemia (CLL) received intravenous infusions of autologous leukemia cells transduced ex vivo to express ISF35 using a replication-defective adenovirus vector (Ad-ISF35). This treatment was well tolerated, did not have dose-limiting toxicity, and had apparent clinical activity. Moreover, injection of autologous, ISF35-expressing CLL cells induced upregulation of death receptors and pro-apoptotic proteins on bystander, non-infected CLL cells, resulting in acute cytoreductions in leukemia cell counts and reductions in the size of lymph nodes and spleen of the treated patients (Wierda et al. Blood . 2007; 110: a-2040). Because of this and preclinical studies demonstrating specific anti-lymphoma activity of Ad-ISF35 when directly injected into growing tumor nodules of experimental animals, we conducted a first-in-man phase I clinical study, evaluating the safety of direct intra-nodal injections of Ad-ISF35 in patients with CLL. Fifteen patients, ranging in age from 45 to 71 years (median age 55, 10 male and 5 female), with progressive CLL (Rai stage III and IV) and leukemia-cell doubling times of 0.9 to 22.5 months (median 3.3 months) participated in a dose-escalation study involving successive cohorts of 3 patients each. The patients in each cohort received a single injection of 1x1010, 3 x1010, 1x1011 or 3x1011 Ad-ISF35 viral particles into a pathologically enlarged axillary lymph node under ultrasound guidance. Intranodal injection with Ad-ISF35 was well tolerated. Adverse events included erythema, swelling and/or pain at the site of injection and “flu like symptoms”, which occurred primarily during the first 24 hours of treatment. At the two highest dosing cohorts, three of three patients in the 3x1011 cohort and two of six patients in the 1x1011 cohort developed Grade 3 and 4 asymptomatic and transient neutropenia and hypophosphatemia one to three weeks after the injection of Ad-ISF35. In all patients, the neutropenia and hypophosphatemia resolved following administration of filgastrim and oral phosphate. Although Ad-ISF35 was injected into only one axillary lymph node, we observed significant reductions in absolute numbers of CLL cells in the blood and reductions in the size of all lymph nodes and the spleen in 14 of 15 evaluable patients. The reduction was durable in 9 patients and the leukemia cell counts have remained below pre-treatment levels for up to four months after a single intra-nodal injection of Ad-ISF35, even in patients who had rapid CLL doubling times prior to therapy. Although we have no evidence for dissemination of Ad-ISF35 beyond the injected lymph node, we observed up-regulation of death receptors, immune co-stimulatory molecules, and pro-apoptotic proteins in the circulating, non-infected CLL cells of the treated patients, suggesting a bystander effect. In addition, we observed increased blood levels of interferon-gamma and interleukin-12 beginning 8 hours after injection. In summary, we found that direct intranodal injection of Ad-ISF35 into patients with CLL was well tolerated and had systemic biologic and clinical activity, suggesting that this approach might be effective in the treatment of patients with this disease and other B-cell lymphomas.