Denái R. Milton

Complete Loss of PTEN Protein Expression Correlates with Shorter Time to Brain Metastasis and Survival in Stage IIIB/C Melanoma Patients with BRAFV600 Mutations

Purpose: Loss of function of PTEN is a frequent event in melanoma, particularly in tumors with BRAFV600 mutations. The prevalence, pathologic features, and clinical outcomes associated with PTEN loss in patients with stage IIIB/C melanoma were interrogated to improve our understanding of the clinical significance of this molecular event. Experimental Design: Archival tissue from lymphadenectomy specimens among patients (n = 136) with stage IIIB or IIIC melanoma was assessed by DNA sequencing for activating BRAF and NRAS mutations, and by immunohistochemistry for the expression of PTEN protein. Associations of these molecular aberrations with demographics, tumor characteristics, and clinical outcomes were determined. Results: The prevalence of BRAFV600 mutations (40% overall), NRAS mutations (10%), and PTEN loss (25%) did not vary by pathologic substage. BRAF/NRAS mutation status did not correlate with distant disease-free survival (DDFS) or overall survival (OS). Complete loss of PTEN expression correlated with shorter OS but not DDFS. When stratified by specific sites of distant metastasis, PTEN loss was associated with significantly shorter time to melanoma brain metastasis (MBM), but not to liver, lung, or bone metastasis. Analysis of PTEN in mutationally defined subsets showed that PTEN loss was significantly associated with OS and time to MBM in patients with BRAFV600 mutations. Conclusions: Loss of PTEN protein expression correlates significantly with decreased OS and time to MBM in stage IIIB/C melanoma patients with BRAFV600 mutations. The findings add to evidence supporting a significant role for PTEN loss and the PI3K–AKT pathway in melanoma. Clin Cancer Res; 20(21); 5527–36. ©2014 AACR.

Inducible Nitric Oxide Synthase Drives mTOR Pathway Activation and Proliferation of Human Melanoma by Reversible Nitrosylation of TSC2

Melanoma is one of the cancers of fastest-rising incidence in the world. Inducible nitric oxide synthase (iNOS) is overexpressed in melanoma and other cancers, and previous data suggest that iNOS and nitric oxide (NO) drive survival and proliferation of human melanoma cells. However, specific mechanisms through which this occurs are poorly defined. One candidate is the PI3K-AKT-mTOR pathway, which plays a major role in proliferation, angiogenesis, and metastasis of melanoma and other cancers. We used the chick embryo chorioallantoic membrane (CAM) assay to test the hypothesis that melanoma growth is regulated by iNOS-dependent mTOR pathway activation. Both pharmacologic inhibition and siRNA-mediated gene silencing of iNOS suppressed melanoma proliferation and in vivo growth on the CAM in human melanoma models. This was associated with strong downregulation of mTOR pathway activation by Western blot analysis of p-mTOR, p70 ribosomal S6 kinase (p-P70S6K), p-S6RP, and p-4EBP1. iNOS expression and NO were associated with reversible nitrosylation of tuberous sclerosis complex (TSC) 2, and inhibited dimerization of TSC2 with its inhibitory partner TSC1, enhancing GTPase activity of its target Ras homolog enriched in brain (Rheb), a critical activator of mTOR signaling. Immunohistochemical analysis of tumor specimens from stage III melanoma patients showed a significant correlation between iNOS expression levels and expression of the mTOR pathway members. Exogenously supplied NO was also sufficient to reverse the mTOR pathway inhibition by the B-Raf inhibitor vemurafenib. In summary, covalent modification of TSC2 by iNOS-derived NO is associated with impaired TSC2/TSC1 dimerization, mTOR pathway activation, and proliferation of human melanoma. This model is consistent with the known association of iNOS overexpression and poor prognosis in melanoma and other cancers.

Complement-Binding Donor-Specific Anti-HLA Antibodies and Risk of Primary Graft Failure in Hematopoietic Stem Cell Transplantation

Detection of donor-specific anti-HLA antibodies (DSA) has been associated with graft rejection in all forms of transplantation. The mechanism by which DSA increase the risk of graft failure remains unclear. We hypothesized that complement-binding DSA are associated with engraftment failure in hematopoietic stem cell transplantation (HSCT) and analyzed 122 haploidentical transplant recipients tested prospectively for DSA. Retrospective analysis to detect C1q binding DSA (C1q+DSA) was performed on 22 allosensitized recipients. Twenty-two of 122 patients (18%) had DSA, 19 of which were women (86%). Seven patients with DSA (32%) rejected the graft. Median DSA level at transplant for patients who failed to engraft was 10,055 mean fluorescence intensity (MFI) versus 2065 MFI for those who engrafted (P = .007). Nine patients with DSA were C1q positive in the initial samples with median DSA levels of 15,279 MFI (range, 1554 to 28,615), compared with 7 C1q-negative patients with median DSA levels of 2471 MFI (range, 665 to 12,254) (P = .016). Of 9 patients who were C1q positive in the initial samples, 5 patients remained C1q positive at time of transplant (all with high DSA levels [median, 15,279; range, 6487 to 22,944]) and experienced engraftment failure, whereas 4 patients became C1q negative pretransplant and all engrafted the donor cells (P = .008). In conclusion, patients with high DSA levels (>5000 MFI) and complement-binding DSA antibodies (C1q positive) appear to be at much higher risk of primary graft failure. The presence of C1q+DSA should be assessed in allosensitized patients before HSCT. Reduction of C1q+DSA levels might prevent engraftment failure in HSCT.

Factors affecting 223Ra therapy: clinical experience after 532 cycles from a single institution

PurposeThe aim of this study was to identify baseline features that predict outcome in 223Ra therapy.MethodsWe retrospectively reviewed 110 patients with metastatic castration-resistant prostate cancer treated with 223Ra. End points were overall survival (OS), progression-free survival (PFS), bone event-free survival (BeFS), and bone marrow failure (BMF). The following parameters were evaluated prior to the first 223Ra cycle: serum levels of hemoglobin (Hb), prostate-specific antigen (PSA), alkaline phosphatase (ALP), Eastern Cooperative Oncology Group (ECOG) status, pain score, use of chemotherapy, and external beam radiation therapy (EBRT). During/after 223Ra we evaluated: the total number of radium cycles (RaTot), the PSA doubling time (PSADT), and the use of chemotherapy, EBRT, abiraterone, and enzalutamide.ResultsA significant reduction of ALP (p < 0.001) and pain score (p = 0.041) occurred throughout the 223 Ra cycles. The risk of progression was associated with declining ECOG status [hazard ratio (HR) = 3.79; p < 0.001] and decrease in PSADT (HR = 8.22; p < 0.001). RaTot, ALP, initial ECOG status, initial pain score, and use of abiraterone were associated with OS (p ≤ 0.008), PFS (p ≤ 0.003), and BeFS (p ≤ 0.020). RaTot, ALP, initial ECOG status, and initial pain score were significantly associated with BMF (p ≤ 0.001) as well as Hb (p < 0.001) and EBRT (p = 0.009). On multivariable analysis, only RaTot and abiraterone remained significantly associated with OS (p < 0.001; p = 0.033, respectively), PFS (p < 0.001; p = 0.041, respectively), and BeFS (p < 0.001; p = 0.019, respectively). Additionally, RaTot (p = 0.027) and EBRT (p = 0.013) remained significantly associated with BMF.ConclusionConcomitant use of abiraterone and 223Ra seems to have a beneficial effect, while the EBRT may increase the risk of BMF.

Progesterone Receptor and PTEN Expression Predict Survival in Patients With Low- and Intermediate-Grade Pancreatic Neuroendocrine Tumors

CONTEXT The PI3K-AKT-mTOR (phosphatidylinositol 3-kinase-AKT-mammalian target of rapamycin) pathway plays a crucial role in a subset of advanced pancreatic neuroendocrine tumors (PanNETs). In breast and endometrial carcinoma, activation of this pathway inhibits progesterone receptor (PR) expression. OBJECTIVE To determine whether combined low expression of PR and phosphatase and tensin homologue (PTEN), a negative regulator of the PI3K-AKT-mTOR pathway, is a prognostic factor. DESIGN A total of 160 resected PanNETs (89 low grade and 71 intermediate grade) were analyzed for PR and PTEN immunohistochemical positivity and staining was correlated with metastasis-free survival (MFS) and overall survival (OS). Progesterone receptor staining was scored as positive by using 1% or greater as cutoff. Weak/faint staining in greater than 90% of tumor cells was considered low PTEN positivity. RESULTS Most PanNETs (110 cases, 69%) were both PR and PTEN positive, 45 (28%) were either PR or PTEN positive, and only 5 (3%) had a PR-negative and PTEN-low profile. Combined PR-PTEN positivity was significantly associated with MFS in patients with stage I and II disease (P <.001) and OS in all patients (P < .001) and remained a significant predictor of survival after adjusting for other factors. Patients with PR-negative-PTEN-low PanNETs had the shortest median MFS and OS, compared to those with tumors that were either PR or PTEN positive and with tumors positive for both PR and PTEN (P ≤ .001). CONCLUSION Combined immunohistochemical assessment of PR and PTEN may help identify a small subset of PanNETs with more aggressive behavior and may aid in risk stratification.

Real-time magnetic resonance imaging-guided cryoablation of small renal tumors at 1.5 T

ObjectivesReal-time magnetic resonance imaging (MRI)–guided cryoablation has been investigated in open MRI systems with low magnetic fields (0.2–0.5 T). More advanced imaging techniques and faster imaging rates are possible at higher magnetic fields, which often require a closed-bore magnet design. However, there is very little experience with real-time interventions in closed-bore 1.5-T MRI units. Herein, we report our initial experience with real-time MRI-guided cryoablation of small renal tumors using a prototype balanced steady-state free precession imaging sequence in a closed-bore 1.5-T MRI system. Materials and MethodsFrom August 2008 to April 2012, 18 patients underwent MRI-guided cryoablation of small renal tumors. A 1.5-T cylindrical MRI scanner with a 125 cm × 70 cm bore and a prototype balanced steady-state free precession sequence (BEAT interactive real-time tip tracking) were used to guide the placement of 17-gauge cryoprobes in real time. Ice ball formation was monitored every 3 minutes in 1 or more imaging planes. Each ablation consisted of 2 freeze-thaw cycles. Contrast-enhanced MRI was performed after the second active thaw period. Follow-up consisted of clinical evaluation and renal protocol computed tomography (CT) or MRI performed at 1, 6, 12, 18, and 24 months and annually thereafter. ResultsDuring the study period, we successfully ablated 18 tumors in 18 patients in 18 sessions. The mean tumor size was 2.2 cm (median, 2 cm; range, 1.2–4.4 cm). The number of cryoprobes used per patient was determined based on tumor size. The mean number of cryoprobes used per patient was 3 (median, 3 cryoprobes; range, 2–4 cryoprobes). Fifty-six cryoprobes, 9 biopsy needles, and 2 hydrodissection needles were successfully placed under real-time MRI guidance using BEAT interactive real-time tip tracking sequence. Hydrodissection under MRI guidance was successfully performed in 4 patients. In each patient, contrast-enhanced MRI performed after the second active thaw period revealed a sharply defined avascular zone surrounding the targeted tumor, which confirmed complete ablation of the tumor with adequate margins. Although contrast media slowly accumulated in the targeted tumor in 9 patients immediately after the procedure, follow-up imaging studies performed at a mean of 16.7 months revealed no contrast enhancement within the ablation zone in these patients. Disease-specific, metastasis-free, and local recurrence-free survival rates were all 100%. ConclusionsReal-time placement and manipulation of cryoprobes during MRI-guided cryoablation of small renal tumors in a closed-bore, high–magnetic field scanner are feasible. Technical and clinical success rates are similar to those of patients who undergo CT-guided radiofrequency ablation or cryoablation of small renal tumors. Our findings suggest that MRI-guided ablation has several advantages over CT-guided ablation, including real-time guidance for probe placement, multiplanar imaging, exquisite soft tissue contrast, and lack of ionizing radiation.

Dual Roles of RNF2 in Melanoma Progression

UNLABELLED Epigenetic regulators have emerged as critical factors governing the biology of cancer. Here, in the context of melanoma, we show that RNF2 is prognostic, exhibiting progression-correlated expression in human melanocytic neoplasms. Through a series of complementary gain-of-function and loss-of-function studies in mouse and human systems, we establish that RNF2 is oncogenic and prometastatic. Mechanistically, RNF2-mediated invasive behavior is dependent on its ability to monoubiquitinate H2AK119 at the promoter of LTBP2, resulting in silencing of this negative regulator of TGFβ signaling. In contrast, RNF2s oncogenic activity does not require its catalytic activity nor does it derive from its canonical gene repression function. Instead, RNF2 drives proliferation through direct transcriptional upregulation of the cell-cycle regulator CCND2. We further show that MEK1-mediated phosphorylation of RNF2 promotes recruitment of activating histone modifiers UTX and p300 to a subset of poised promoters, which activates gene expression. In summary, RNF2 regulates distinct biologic processes in the genesis and progression of melanoma via different molecular mechanisms. SIGNIFICANCE The role of epigenetic regulators in cancer progression is being increasingly appreciated. We show novel roles for RNF2 in melanoma tumorigenesis and metastasis, albeit via different mechanisms. Our findings support the notion that epigenetic regulators, such as RNF2, directly and functionally control powerful gene networks that are vital in multiple cancer processes.

Absence of terminal deoxynucleotidyl transferase expression identifies a subset of high-risk adult T-lymphoblastic leukemia/lymphoma

Terminal deoxynucleotidyl transferase (TdT) can be downregulated in minimal residual disease of T-acute lymphoblastic leukemia/lymphoma (T-ALL/LBL) after chemotherapy. TdT-negative T-ALL/LBL cases are rare and have not been well characterized. We studied the clinicopathologic features of de novo T-ALL/LBL patients treated at our institution during 2003–2011, with an emphasis on immunophenotype and survival of TdT-negative versus TdT-positive cases. Absence of TdT expression was defined as <10% lymphoblasts positive. Seven (12%) TdT-negative cases were identified from a cohort of 59 de novo T-ALL/LBL. The TdT-negative and TdT-positive cases were similar with regard to gender, percentage of patients with a high leukocyte count (>100 × 109/l), central nervous system involvement, and an abnormal karyotype. However, patients with TdT-negative T-ALL/LBL had a significantly higher rate of disease progression and shorter overall survival. Although not statistically significant, TdT-negative T-ALL/LBL cases were associated with an older median age and higher percentage of ‘early T precursor’ (ETP) immunophenotype than TdT-positive cases. Absence of TdT expression identifies a subset of high-risk T-ALL/LBL that overlaps with, but is not identical to, the ETP leukemia, providing additional prognostic value.

Haploidentical Transplantation with Post-Transplantation Cyclophosphamide for High-Risk Acute Lymphoblastic Leukemia

Haploidentical transplantation performed with post-transplantation cyclophosphamide (PTCy)-based graft-versus-host disease (GVHD) prophylaxis has been associated with favorable outcomes for patients with acute myeloid leukemia and lymphomas. However, it remains unclear if such approach is effective for patients with acute lymphoblastic leukemia (ALL). We analyzed outcomes of 109 consecutively treated ALL patients 18 years of age and older at 5 institutions. The median age was 32 years and the median follow-up for survivors was 13 months. Thirty-two patients were in first complete remission (CR1), while the rest were beyond CR1. Neutrophil engraftment occurred in 95% of the patients. The cumulative incidences of grades II to IV and III and IV acute GVHD at day 100 after transplantation were 32% and 11%, respectively, whereas chronic GVHD, nonrelapse mortality, relapse rate, and disease-free survival (DFS) at 1 year after transplantation were 32%, 21%, 27%, and 51%, respectively. Patients in CR1 had 52% DFS at 3 years. These results suggest that haploidentical transplants performed with PTCy-based GVHD prophylaxis provide a very suitable alternative to HLA-matched transplantations for patients with ALL.

Prognostic factors of hepatosplenic T-cell lymphoma clinicopathologic study of 28 cases

Hepatosplenic T-cell lymphoma (HSTCL) is a rare type of lymphoma. Patients have a poor prognosis, and there is no standard of care. We evaluated 28 HSTCL patients to determine factors that may be associated with outcome. There were 19 men and 9 women with a median age of 32.5 years. Most patients had massive splenomegaly, and bone marrow showed sinusoidal involvement by lymphoma. The HSTCL cells expressed &ggr;&dgr; T-cell receptor (TCR) in 20 (74%), &agr;&bgr; TCR in 5 (19%), and neither in 2 (7%) patients (1 case not assessed). Conventional cytogenetics and/or fluorescence in situ hybridization analysis in 24 patients at diagnosis showed isochromosome 7q (i7q) in 10 (42%) and trisomy 8 in 8 (33%) patients. Median overall survival (OS) and event-free survival (EFS) were each 28.3 months. Serum bilirubin level ≥1.5 mg/dL, &agr;&bgr; TCR expression, and trisomy 8 each correlated significantly with shorter OS and EFS. Patients with HSTCL received a variety of chemotherapy regimens with no regimen better than any other. However, patients who underwent stem cell transplant showed longer survival (OS: hazard ratio 0.3, P=0.09; EFS: hazard ratio 0.2, P=0.034). In conclusion, although HSTCL patients have a poor prognosis overall, the data presented support the novel suggestions that HSTCL patients can be stratified into 2 prognostic groups, with an elevated serum bilirubin level, &agr;&bgr; TCR expression, and trisomy 8 identifying a poorer prognostic group. In addition, the outcomes of this patient cohort suggest that stem cell transplantation has value for the treatment of patients with HSTCL.