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Neuroblastomas harbor ALK aberrations clinically resistant to crizotinib yet sensitive pre-clinically to the third-generation ALK inhibitor lorlatinib. We conducted a first-in-child study evaluating lorlatinib with and without chemotherapy in children and adults with relapsed or refractory ALK-driven neuroblastoma. The trial is ongoing, and we report here on three cohorts that have met pre-specified primary endpoints: lorlatinib as a single agent in children (12 months to 123I-metaiodobenzylguanidine (MIBG) response. Lorlatinib was evaluated at 45–115 mg/m2/dose in children and 100–150 mg in adults. Common adverse events (AEs) were hypertriglyceridemia (90%), hypercholesterolemia (79%) and weight gain (87%). Neurobehavioral AEs occurred mainly in adults and resolved with dose hold/reduction. The RP2D of lorlatinib with and without chemotherapy in children was 115 mg/m2. The single-agent adult RP2D was 150 mg. The single-agent response rate (complete/partial/minor) for NCT03107988.

Legends to supplementary figures - PDF file 87K, Legends to supplementary figures 1 to 4

Supplementary Figure 6 from The Activity of Zoledronic Acid on Neuroblastoma Bone Metastasis Involves Inhibition of Osteoclasts and Tumor Cell Survival and Proliferation

Supplementary Figure 2 from Interleukin-6 in the Bone Marrow Microenvironment Promotes the Growth and Survival of Neuroblastoma Cells

Supplementary Figure 3 - PDF file 455K, A. CHLA-255 cells were cultures alone or in the presence of sIL-6R (25ng/mL) or IL-6 (10 ng/mL) before being treated with etoposide as indicated in Figure 3. After 24 hours, cells were examined for Annexin V expression by Flow cytometry. The data represents the mean percent of Annexin V positive cells from triplicate samples. B. CHLA-255 and human monocytes were co-cultured as described in figure 6E. Cells were then separated by flow cytometry using anti-GD2 antibody (CHLA-255) and an anti-CD14 (monocytes) and examined for nuclear pSTAT3 by flow cytometry. C. Increased expression of in bone marrow samples from patients with metastatic neuroblastoma. Paraffin-embedded sections of bone marrow biopsies stained by immunohistochemistry of patients without (Left panel) and with (Right panel) tumor cells (scale bar = 50 �m). Increased expression of pSTAT3 and survivin (top) and Bcl-xL (bottom) in bone marrow samples from patients with metastatic neuroblastoma. Left panel, Representative paraffin-embedded sections of bone marrow biopsies from patients, stained by immunohistochemistry for pSTAT3 and survivin of Bcl-xL as indicated in Materials and Methods (scale bar = 50 �m). Right panel, The graph represents the mean number (�SD) of pSTAT3 and survivin positive cells in five 40x fields examined in bone marrow without (n=5) and with (n=5) tumor cells

Supplementary Figure 1 - PDF file 231K, Inhibition of STAT3 activation in CHLA-255 cells by anti-IL-6R blocking antibody. A, Expression of pSTAT3 and STAT3 in CHLA-255 cells was examined by Western blot analysis as in Fig. 1A. When indicated, cells were treated with tocilizumab (2�g/mL) for 12 hours before treatment with IL-6 with or without sIL-6R. The data are representative of 2 separate experiments showing similar results. B, Nuclear extracts from CHLA-255 treated with IL-6 and sIL-6R in the presence and absence of tocilizumab as above described were examined for STAT3 DNA-binding activity by EMSA as indicated in Materials and Methods. Lane 8 is same as lane 5, except for the addition of a �supershifting� anti-STAT3 antibody also added to lane 2 (DU145 control). Top band (labeled STAT3) represents STAT3/STAT3 homodimers bound to DNA. C, CHLA-255 transfected with a STAT3 Firefly luciferase vector and a Renilla luciferase control vector were treated with IL-6 (10ng/mL) alone or in combination with sIL-6R (25ng/mL) as shown in Fig. 1 in the absence or presence of tocilizumab (4�g/mL). After 24 hours, the activity of the STAT3 reporter was examined by Dual Luciferase assay as indicated in Materials and Methods. The data represent the mean (�SD) ratio Firefly/Renilla luciferase activity from five samples for each experimental condition

Metastasis to the bone is seen in 56% of patients with neuroblastoma and contributes to morbidity and mortality. Using a murine model of bone invasion, we have reported previously that neuroblastoma cells invade the bone by activating osteoclasts. Here, we investigated the antitumoral and antiosteolytic activities of zoledronic acid, a bisphosphonate inhibitor of osteoclasts, in combination with cytotoxic chemotherapy in our model. We first show that zoledronic acid given at the same time (early prevention) or 2 weeks after tumor cell injection (late prevention) significantly prevented the formation of severe osteolytic lesions. It also prevented formation of these lesions when given 4 weeks after tumor cell injection (intervention) when combined with chemotherapy including cyclophosphamide and topotecan. The combination of zoledronic acid + cyclophosphamide/topotecan also significantly improved survival (P < 0.001). In mice treated with zoledronic acid, we observed a marked inhibition of osteoclasts inside the bone associated with a decrease in tumor cell proliferation and increase in tumor cell apoptosis. In vitro, zoledronic acid inhibited neuroblastoma cell proliferation and induced apoptosis, and these effects were significantly enhanced by the addition of 4-hydroxyperoxycyclophosphamide (4-HC). The proapoptotic effect of zoledronic acid and zoledronic acid in combination with 4-HC on tumor cells was associated with an increase in caspase-3 activity and a decrease in phosphorylated Bcl-2, Bcl-2, and Bcl-XL expression. Zoledronic acid inhibited the association of Ras with the plasma membrane and activation of c-Raf, Akt, and extracellular signal-regulated kinase 1/2. The data indicate that zoledronic acid, in addition to inhibiting osteoclasts, is active against tumor cells and suggest that zoledronic acid in combination with cytotoxic chemotherapy may be effective in children with neuroblastoma that has metastasized to the bone. [Cancer Res 2007;67(19):9346–55]

Neuroblastoma, the second most common solid tumor in children, frequently metastasizes to the bone marrow and the bone. Neuroblastoma cells present in the bone marrow stimulate the expression of interleukin-6 (IL-6) by bone marrow stromal cells (BMSC) to activate osteoclasts. Here we have examined whether stromal-derived IL-6 also has a paracrine effect on neuroblastoma cells. An analysis of the expression of IL-6 and its receptor, IL-6R, in 11 neuroblastoma cell lines indicated the expression of IL-6 in 4 cell lines and of IL-6R in 9 cell lines. Treatment of IL-6R–positive cells with recombinant human IL-6 resulted in signal transducer and activator of transcription-3 and extracellular signal–regulated kinase-1/2 activation. Culturing IL-6R–positive neuroblastoma cells in the presence of BMSC or recombinant human IL-6 increased proliferation and protected tumor cells from etoposide-induced apoptosis, whereas it had no effect on IL-6R–negative tumor cells. In vivo, neuroblastoma tumors grew faster in the presence of a paracrine source of IL-6. IL-6 induced the expression of cyclooxygenase-2 in neuroblastoma cells with concomitant release of prostaglandin-E2, which increased the expression of IL-6 by BMSC. Supporting a role for stromal-derived IL-6 in patients with neuroblastoma bone metastasis, we observed elevated levels of IL-6 in the serum and bone marrow of 16 patients with neuroblastoma bone metastasis and in BMSC derived from these patients. Altogether, the data indicate that stromal-derived IL-6 contributes to the formation of a bone marrow microenvironment favorable to the progression of metastatic neuroblastoma. [Cancer Res 2009;69(1):329–37]

Supplementary Figure 4 from Interleukin-6 in the Bone Marrow Microenvironment Promotes the Growth and Survival of Neuroblastoma Cells

Supplementary Figure 4 - PDF file 91K, A. Representative data of the analysis on one fresh patient bone marrow sample by Flow Cytometry showing the expression of pSTAT3 in various sub-populations of bone marrow cells as indicated in Materials and Methods. B. Left panel: Percentage of the 6 sub-populations of cells identified as shown in A. The data represents the mean percentage (�SD) of all mononuclear cells isolated from 8 individual patient samples. Right panel: The data represents the mean percentage of pSTAT3 positivity in each sub-population of bone marrow cells identified in the 8 samples as shown in the left panel

Drug resistance is a major cause of treatment failure in cancer. Here, we have evaluated the role of STAT3 in environment-mediated drug resistance (EMDR) in human neuroblastoma. We determined that STAT3 was not constitutively active in most neuroblastoma cell lines but was rapidly activated upon treatment with interleukin (IL)-6 alone and in combination with the soluble IL-6 receptor (sIL-6R). Treatment of neuroblastoma cells with IL-6 protected them from drug-induced apoptosis in a STAT3-dependent manner because the protective effect of IL-6 was abrogated in the presence of a STAT3 inhibitor and upon STAT3 knockdown. STAT3 was necessary for the upregulation of several survival factors such as survivin (BIRC5) and Bcl-xL (BCL2L1) when cells were exposed to IL-6. Importantly, IL-6–mediated STAT3 activation was enhanced by sIL-6R produced by human monocytes, pointing to an important function of monocytes in promoting IL-6–mediated EMDR. Our data also point to the presence of reciprocal activation of STAT3 between tumor cells and bone marrow stromal cells including not only monocytes but also regulatory T cells (Treg) and nonmyeloid stromal cells. Thus, the data identify an IL-6/sIL-6R/STAT3 interactive pathway between neuroblastoma cells and their microenvironment that contributes to drug resistance. Cancer Res; 73(13); 3852–64. ©2013 AACR.

Supplementary Figures 1-5 from The Activity of Zoledronic Acid on Neuroblastoma Bone Metastasis Involves Inhibition of Osteoclasts and Tumor Cell Survival and Proliferation

Supplementary Table 1 from The Activity of Zoledronic Acid on Neuroblastoma Bone Metastasis Involves Inhibition of Osteoclasts and Tumor Cell Survival and Proliferation

9014 Background: Osimertinib (Osi) is standard of care in 1st line (1L) EGFR mut NSCLC and TKI resistant T790Mpos NSCLC but acquired resistance emerges; outcomes are less robust in T790Mneg, C797Xpos and EGFR exon 20 insertion (ex20ins) disease. We examined Osi with the EGFR monoclonal antibody Necitumumab (Neci) in select settings of EGFR TKI resistance. Methods: Pts were accrued to 5 expansion cohorts (ExC) at recommended phase 2 dose (RP2D) of Osi 80 mg daily and Neci 800 mg D1 + D8 of q21d cycle. ExC (18 pts/cohort): A) T790Mneg progressive disease (PD) on 1st/2nd gen TKI as last therapy, B) T790Mneg PD on 1st/2nd gen TKI and PD on 3rd gen TKI, C) T790Mpos PD on 1st/2nd gen TKI and PD on 3rd gen TKI, D) EGFR ex20ins PD on chemotherapy, E) PD on 1L osi. In ExC A-C, T790M was confirmed centrally (tissue) by ddPCR. Additional correlative studies include: tissue NGS (> 400 gene panel), EGFR FISH, plasma for PK and serial EGFR ctDNA by ddPCR. Adverse events were graded (Gr) by CTCAEv5; ORR, PFS by RECIST 1.1. Primary pre-specified efficacy endpoint ≥3/18 pts responding per cohort. Results: 101 patients accrued (100 evaluable). Efficacy is summarized in the Table. Drug related Gr 3 AEs were seen in 38% of pts, mainly rash (21%). ORR among all pts was 19% (95% CI 12-28%) that varied across cohorts (Table). In ExC A-C, 69% pts had detectable EGFR activating mutations in ctDNA, with decline in mutant allele frequency (AF) on treatment in 80% and ctDNA clearance in 33%. Conclusions: Osi/Neci is feasible and tolerable at the RP2D. EGFR ctDNA was detectable at baseline in the majority of pts with decrease in AF on treatment. Osi/Neci was active in select settings of EGFR-TKI resistance, meeting its prespecified efficacy endpoint in T790Mneg PD on 1st/2nd gen TKI as last therapy (ExC A), EGFR ex20ins post-chemo (ExC D) and PD on 1L osimertinib (ExC E). mPFS in the EGFR ex20ins cohort was within the range of current EGFR Exon 20 ins agents in development. EGFR monoclonal antibodies with osimertinib warrant further study in settings of de novo and acquired EGFR dependent resistance to EGFR-TKI. Clinical trial information: NCT02496663. [Table: see text]

4529 Background: Immune checkpoint blockade has revolutionized mRCC Tx, but primary and acquired resistance continues to result in poor patient outcomes. OX40 (CD-134) mediates IO resistance. Co-stimulatory OX40 (CD-134) activates exhausted T-cells. OX40 activation in dendritic cells increases the proliferation, effector function, and survival of T cells. PFOX is an agonist for OX40. We hypothesized that PFOX + the VEGFR inhibitor Axi would improve outcomes vs. Axi in patients (pts) with mRCC after IO Tx. Methods: Pts with predominantly clear cell mRCC were stratified for MSKCC risk groups then randomized 1:1 to Axi 5mg po bid plus PFOX 0.3mg/kg iv (Arm 1) or placebo (PL) iv (Arm 2) on Day 1 of a 2-week cycle. The primary endpoint was progression free survival (PFS); secondary endpoints included overall survival (OS), objective response rate (ORR), duration of response (DOR) per RECIST v1.1, and safety/tolerability. A prespecified interim analysis (IA) tested PFS at a 1-sided P < 0.02 when ≥ 33 events were observed. Results: Between February 2018 and October 2021 a total of 59 pts were randomly assigned and treated with Axi+PFOX (N = 29) or Axi+PL (N = 30). Pt and disease characteristics are summarized in the table. As of October 2021, 38 PFS events had occurred, 19 on each arm. The IA rejected the hypothesis of added efficacy for PFOX with a p of 0.0089. Subsequently the study was closed to new accrual. At a median follow up of 13.4 mo, median PFS was 13.1 (6-15.8) months (mo) for Arm 1 and 8.5 (5.5-11) mo for Arm 2 (HR = 0.85 [95% CI: 0.45-1.60] p = 0.61). After adjusting by MSKCC risk group and prior lines of Tx, HR = 0.74 [95% CI: 0.38-1.46] p = 0.39. Median OS was not reached (adjusted HR = 0.71 [95% CI: 0.24-2.12] p = 0.54). ORR Arm 1: 31% PR, 52% SD, 14% PD, and Arm 2: 37% PR, 50% SD, 13% PD. Median DOR 9.1 (3.3-23.9) mo for Arm 1, and 7.5 (1.8-32.7) mo. for Arm 2. Rates of any grade Tx related adverse events (TRAEs; 93% vs 100%), including grade 3 or 4 TRAEs (66% vs 47%), in Arm 1 and Arm 2, respectively. 4 pts discontinued Tx due to TRAE, 3 in Arm 1 (1 grade 3 hypertension, 1 grade 2 stroke, 1 grade 3 bullous dermatitis) and 1 in Arm 2 (grade 4 ALT elevation). The most common TRAEs were diarrhea 52%, hypertension 52%, fatigue 41%, nausea 41% for Arm 1 and hypertension 67%, diarrhea 53%, fatigue 50% for Arm 2. Conclusions: In IO-pretreated mRCC pts, Axi + PFOX did not improve outcomes compared to Axi alone. Clinical trial information: NCT03092856. [Table: see text]

10026 Background: 131I-metaiodobenzylguanidine (131I-MIBG) has demonstrated efficacy as a single agent for patients with neuroblastoma. Recent trials have focused on 131I-MIBG combination strategies, though little is known about the impact of combination agents on markers of radiation exposure. Methods: NANT11-01 (NCT02035137) was a multicenter, open label, randomized phase II clinical trial that evaluated 131I-MIBG therapy alone (Arm A) or in combination with vincristine/irinotecan (Arm B) or vorinostat (Arm C) for patients with resistant/relapsed neuroblastoma. We collected blood samples at baseline, 72 hours, 96 hours, and 15 days after 131I-MIBG infusion and determined levels of plasma FLT3 ligand, serum amylase, and gene expression for selected RNA transcripts (apoptosis, DNA damage response and cell cycle related). We evaluated marker association with treatment arm, clinical response using NANT response criteria, toxicity, and whole-body radiation dose. Results: The cohort included 99 patients who had at least one biomarker available for analysis (32 Arm A; 35 Arm B; 32 Arm C). We observed both positive and negative significant modulation in most biomarkers between baseline, 72 hours, and 96 hours following 131I-MIBG. Patients in Arm C had the lowest degree of modulation in FLT3 ligand. Elevated baseline levels of FLT3 ligand were significantly associated with improved Curie response but not overall response. Lower baseline BCL2 levels were associated with higher overall and Curie response. Patients with increased FLT3 ligand at 96 hours after 131I-MIBG therapy were significantly more likely to have grade 4 thrombocytopenia.Peripheral blood gene expression of the BCL2 family of apoptotic markers (BCL2/L1 and BAX) was significantly associated with grade 4 hematological toxicity. Whole-body radiation dose and PRKDC fold change at 72 hours were significantly correlated. No other individual biomarkers were correlated with whole body radiation dose at 72 or 96 hours post 131I-MIBG. Conclusions: Peripheral blood biomarkers relevant to radiation exposure demonstrate significant modulation over time after 131I-MIBG treatment. Biomarkers related to hematopoietic damage and apoptosis were associated with hematological toxicity. Patients treated with vorinostat and 31I-MIBG had differential modulation of FLT3-ligand Further use of these biomarkers may improve our ability to care for patients treated with 131I-MIBG.

10038 Background: 131I-metaiodobenzylguanidine (MIBG) is one of the most active salvage therapies for patients with relapsed or refractory (R/R) high-risk neuroblastoma (HRNB). Preclinical neuroblastoma studies show cooperative effects when radiation is combined with anti-GD2 monoclonal antibody (mAb). We hypothesized that MIBG would synergize with the anti-GD2 mAb dinutuximab to provide improved anti-tumor responses. The primary aims of Part A of this study were to determine the maximum tolerated dose (MTD) and/or recommended Phase II dose (RP2D) of MIBG administered with dinutuximab in children with R/R HRNB and to define and describe the toxicities. Methods: Patients 1-29 years of age with R/R HRNB who had MIBG uptake in ≥ 1 site were eligible. Prior anti-GD2 mAb therapy was allowed provided it was not administered with MIBG and not permanently discontinued due to toxicity. One prior MIBG therapy was allowed. MIBG was administered on day 1 at one of three dose levels (DLs): 12, 15, and 18 mCi/kg (DL1-DL3, respectively) with an expansion cohort at the RP2D. Doses were escalated using a rolling six design starting at DL1. The primary endpoint was dose-limiting toxicity (DLT) during course 1. Dinutuximab (17.5 mg/m2/dose) was administered intravenously on days 8-11 and 29-32 and GM-CSF (250 mcg/m2/dose) subcutaneously on days 8-17 and 29-38. Autologous peripheral blood stem cells were infused to all patients on day 15 (+/- 2 days). A maximum of 2 courses per patient were allowed. Response rate was defined as the proportion of patients with a complete or partial response. Results: Thirty-one patients were enrolled. Fourteen were evaluable for dose escalation (4 on DL1, 4 on DL2, and 6 on DL3); 5 evaluable patients were treated in the DL3 expansion. The median age was 7.4 years (range: 3.1 – 22.0) and 20 (65%) were male. Twenty-seven (87%) patients had previously received a median of 8.5 cycles of chemoimmunotherapy (range: 2 – 21). Eight patients previously progressed while receiving anti-GD2 mAb including 7 in DL3. Five (16%) patients had previously received MIBG. No patient at any dose level experienced DLT. Common grade 3/4 treatment-related toxicities were expected hematologic toxicities attributable to MIBG and non-hematologic toxicities attributable to dinutuximab or GM-CSF. Among 26 response-evaluable patients, the centrally-confirmed response rate was 31% across all dose levels: 2/6 (33%) in DL1, 3/5 (60%) in DL2, and 3/15 (20%) in DL3. There were 3 minor responses, 1 in DL2 and 2 in DL3. Conclusions: The RP2D of MIBG in combination with standard doses of dinutuximab and GM-CSF is 18 mCi/kg. This radioimmunotherapy regimen is well-tolerated without additive toxicity. Preliminary efficacy data are encouraging in this heavily pre-treated patient population. A phase 2 trial of this regimen is planned in patients with R/R HRNB. Clinical trial information: NCT03332667.

e22003 Background: 131I-metaiodobenzylguanidine (MIBG) remains one of the most active agents for neuroblastoma. The clinical and biologic predictors of response to MIBG therapy have not been systematically characterized in a recent era trial. Methods: Patients 1-30 years were enrolled on the randomized phase 2 trial of MIBG vs. MIBG/vincristine/irinotecan vs. MIBG/vorinostat for relapsed/refractory neuroblastoma (NANT2011-01, NCT02035137) between 2014-2019. Data were compared between those who had an objective response (partial response or better using NANT response criteria) to MIBG after the first cycle and those who did not according to clinical features (age at study enrollment; sex; response to prior therapy; prior receipt of MIBG therapy; bone marrow involvement at study enrollment; and measurable disease status at study enrollment) and MYCN status. Univariate analyses were performed using odds ratio and Fisher exact tests. Multivariate logistic regression analysis was used to identify predictors of response to MIBG therapy while controlling for key confounders. Results: 105 response-evaluable patients were included in the analytic cohort. Across the 3 study arms, 20% (21/105) had an objective response to the treatment. Only measurable disease status was statistically significantly associated with response to therapy, with higher rates of response among patients without measurable disease at study enrollment (30% vs. 13%, p = 0.049). Response to prior therapy, sex, MYCN status, and measurable disease status showed univariate odds ratios of 2 or greater (Table). Only measurable disease status remained statistically significant in the multivariate analysis (p = 0.043, Table). Conclusions: Patients without measurable disease have a higher rate of objective responses to MIBG therapy. Understanding these differences based upon predictors of response may help to inform stratification methods for future MIBG clinical trials.[Table: see text]

Until the advent of T cell check point inhibitors standard second-line therapy for patients with metastatic urothelial cancer (mUC) was undefined. Histone deacetylase inhibitors (HDACi) have anti-cancer activity in a variety of tumor models including modulation of apoptosis in bladder cancer cell lines. We evaluated the efficacy and toxicity of the HDACi vorinostat in patients with mUC failing first-line platinum-based therapy either in the adjuvant/neoadjuvant setting or for recurrent/advanced disease.Vorinostat was given orally 200 mg twice daily continuously until progression or unacceptable toxicity. The primary end point was RECIST response rate (RR); a RR 20% was deemed interesting in a 2-stage design requiring one response in the first 12 patients to proceed to 2nd stage for a total of 37 subjects. CT or MRI scan imaging occurred every 6 weeks.Fourteen patients were accrued characterized by: median age 66 years (43-84); Caucasian (79%); males (86%); and Karnofsky performance status ≥90 (50%). Accrual was terminated in the first stage as no responses were observed. Best response was stable disease (3 patients). Progression was observed in 8 patients. Two patients came off therapy prior to re-imaging and a 3rd patient died while on treatment and was not assessed for response. Median number of cycles was 2 (range 1-11). Median disease-free survival and overall survival times were 1.1 (0.8, 2.1)3.2 (2.1, 14.5) months, respectively. Toxicities were predominantly cytopenias and thrombocytopenic bleeding. Two pts. had grade 5 toxicity unlikely related to treatment. Two pts. had grade 4 and 6 had grade 3 toxicities observed. Two patients with stable disease remained on therapy for 6+ cycles.Vorinostat on this dose-schedule had limited efficacy and significant toxicity resulting in a unfavorable risk:benefit ratio in patients with mUC. NCT00363883.

Objective For nearly 20 years, the putative prognostic value of P53 immunohistochemistry in bladder cancer has been controversially discussed, and key questions are still unanswered. The aim of this article was to elucidate the different findings using the new concept of a combined analysis of raw data from previously published material. Materials and methods Twenty-six of 38 study centers approached contributed patient data sets according to the protocol requirements; 3,421 patients with bladder cancer from 25 centers are included in the further analysis. The entire study group (mean age: 66.5 years) comprised 2,697 males (78.8 %) and 719 females. For 2,298 patients (68%) with non-muscle-invasive tumors Ta (1314), TIS (37), and T1 (947) (38.9, 1.1, and 28%, respectively) a median survival time of 130 months was calculated. The remaining 1,082 patients (32%) had advanced tumors (>T2) and a median survival time of 26 months. Of the 1,241 patients who have died, 744 patients died from bladder cancer and another 497 patients from intercurrent disease. Results With regard to gender, age, and tumor stage, the patients included reflected a normal bladder cancer population. Statistical analysis revealed a highly significant correlation between P53 positivity vs. tumor grade and tumor stage. Uni- and multivariate analyses showed that P53 positivity was significantly correlated with tumor progression (as defined by the different centers) in T1 disease ( P P Conclusions P53 immunohistochemistry appears to be predictive in high grade bladder cancer, however, the magnitude of this association varies among tumor stages. The results of this trial demonstrate the relevance of sufficient study size, provide a basis to define suitable patient populations, and allow an estimation of an adequate size of study cohorts for prospective trials. It also demonstrates the importance of common recommendations for evaluation and reporting of marker studies. Furthermore, this initiative demonstrates the strong will of a large number of investigators to contribute to combined efforts in order to establish pathways for standardization of marker development and clinical use.

To examine interactions among clinical factors and pathologic biomarkers in predicting the outcome of patients with diffuse large B-cell lymphoma (DLBCL) treated with rituximab-based immunochemotherapy.In 85 patients treated at a single institution, clinicopathologic variables were analyzed, including the International Prognostic Index (IPI); germinal/nongerminal center phenotype; MYC, p53, BCL2, Ki-67, and Epstein-Barr virus (EBV) expression; and MYC translocation status.In univariate analysis, overall survival (OS) was worse for patients with high IPI scores, nongerminal center phenotype, high MYC and p53 expression by immunohistochemistry, and EBV positivity. In multivariable analysis, p53 expression was the strongest prognostic factor (P.05) independent of IPI and cell of origin. A significant positive association between p53 and MYC expression was found. Moreover, coexpression of p53/MYC had an enhanced negative effect on OS independent of BCL2 expression.Immunohistochemical assessment of p53, particularly in combination with MYC, could be useful in identifying a high-risk subgroup of DLBCL.

Zoledronic acid, a bisphosphonate, delays progression of bone metastases in adult malignancies. Bone is a common metastatic site of advanced neuroblastoma. We previously reported efficacy of zoledronic acid in a murine model of neuroblastoma bone invasion prompting this Phase I trial of zoledronic acid with cyclophosphamide in children with neuroblastoma and bone metastases. The primary objective was to determine recommended dosing of zoledronic acid for future trials.Escalating doses of intravenous zoledronic acid were given every 28 days with oral metronomic cyclophosphamide (25 mg/m(2)/day). Toxicity, response, zoledronic acid pharmacokinetics, bone turnover markers, serum IL-6, and sIL-6R were evaluated.Twenty-one patients, median age 7.5 (range 0.8-25.6) years were treated with 2 mg/m(2) (n = 4), 3 mg/m(2) (n = 3), or 4 mg/m(2) (n = 14) zoledronic acid. Fourteen patients were evaluable for dose escalation. A median of one (range 1-18) courses was given. Two dose limiting toxicities (grade 3 hypophosphatemia) occurred at 4 mg/m(2) zoledronic acid. Other grades 3-4 toxicities included hypocalcemia (n = 2), elevated transaminases (n = 1), neutropenia (n = 2), anemia (n = 1), lymphopenia (n = 1), and hypokalemia (n = 1). Osteosclerosis contributed to fractures in one patient after 18 courses. Responses in evaluable patients included 1 partial response, 9 stable disease (median 4.5 courses, range 3-18), and 10 progressions. Zoledronic acid pharmacokinetics were similar to adults. Markers of osteoclast activity and serum IL-6 levels decreased with therapy.Zoledronic acid with metronomic cyclophosphamide is well tolerated with clinical and biologic responses in recurrent/refractory neuroblastoma. The recommended dose of zoledronic acid is 4 mg/m(2) every 28 days.

Previous reports have identified risk factors for urethral recurrence following radical cystectomy for transitional cell carcinoma (TCC). However, reports of the clinical presentation, treatment and outcome in these patients are lacking. We report our experience with the diagnosis, management and outcome of urethral TCC after radical cystectomy for bladder cancer.A database of 1,054 patients who underwent radical cystectomy and urinary diversion for TCC from 1971 to 1997 was retrospectively reviewed. All patients with urethral TCC after surgery were identified.Urethral TCC was diagnosed in 47 men a median of 18.5 months (range 2 to 116) after cystectomy with 20 (42%) diagnosed within 1 year. Symptomatic recurrence developed in 24 of 42 evaluable patients (57%), 21 had bloody urethral discharge and 7 had pain or a palpable mass. A total of 13 patients (31%) were asymptomatic with abnormal cytology. The remaining 5 patients underwent prophylactic urethrectomy based on cystectomy pathology. Overall 41 patients underwent urethrectomy, which was total in 36 and distal with perineal urethrostomy in 5, including later conversion to total urethrectomy in 2. Overall at a median followup of 26 months (range 3 to 275) since diagnosis 36 of 47 patients (76%) were dead, including 25 of metastatic disease. Only 10 patients (21%) remained disease-free. Median overall survival in patients with urethral TCC after radical cystectomy was only 28 months after the diagnosis of urethral TCC. Urethral stage (superficial vs invasive disease) at diagnosis was the most import predictor of overall survival in this cohort of patients.Most patients with urethral recurrence present with symptoms. However, screening cytology alone still detects a significant proportion. The median survival of patients with urethral TCC after radical cystectomy is only 28 months after diagnosis. Urethral stage (superficial vs invasive disease) at diagnosis is the most import predictor of overall survival in this cohort of patients.

e15153 Background: The prognosis for patients with castration-resistant prostate cancer (CRPC) remains suboptimal and targeted therapies should be explored. One potential target is the receptor for luteinizing hormone-releasing hormone (LHRH), which is highly expressed on prostate cancer cells. AEZS-108 is an LHRH-cytotoxic hybrid whose rational design covalently couples an LHRH agonist and the cytotoxic doxorubicin. AEZS-108 exploits the presence of LHRH receptors to target delivery of the cytotoxic. We report the phase I trial of AEZS-108 in men with taxane-resistant CRPC. We also report correlative studies of a novel circulating tumor cell (CTC) capture device that will provide enumeration of CTCs and results from AEZS-108 internalization studies that exploit the auto-fluorescence of doxorubicin in captured CTCs. Methods: This is a single-arm, dose-escalation phase I study in men with CRPC to confirm the dose established in a completed phase I trial in females. Eligibility criteria included adequate organ function and progression of disease despite prior therapy with an LHRH agonist and at least one taxane-based regimen. Patients were required to discontinue LHRH agonists to avoid receptor competition. Due to potential cardiotoxicity, patients with an ejection fraction < 50% or prior exposure to doxorubicin or mitoxantrone were excluded. Pituitary function was closely monitored. Patients received AEZS-108 every 21 days for up to 6 cycles until progression or unacceptable toxicity. The primary endpoint was safety. Results: Enrollment began in November 2010 and is ongoing. Currently, 13 patients have been enrolled. The first two planned dose levels had no dose-limiting toxicities observed. Two patients on the third dose level experienced a dose limiting toxicity. The second dose level has been reopened for expansion. There have been no cardiac or pituitary toxicities. At the time of submission, a decrease in PSA was noted in 6 of the 13 patients. Final results detailing safety, response and the suggested dose for the phase II portion will be reported. All correlative studies will also be reported. Conclusions: In general, AEZS-108 has been well tolerated. The phase II portion of the study will begin once the MTD is established.

60 Background: The prognosis for patients with castration-resistant prostate cancer (CRPC) remains suboptimal and targeted therapies should be explored. One potential target is the receptor for luteinizing hormone-releasing hormone (LHRH-R), which is highly expressed on prostate cancer cells. AEZS-108 (AN-152) is an LHRH-cytotoxic hybrid whose rational design covalently couples an LHRH agonist and the cytotoxic doxorubicin. AEZS-108 exploits the presence of these receptors to target delivery of the cytotoxic. We report the phase I trial of AEZS-108 in men with taxane-resistant CRPC. We also report correlative studies of a novel circulating tumor cell (CTC) capture device that will provide both enumeration of CTCs and LHRH-R expression on captured CTCs as well as results from AEZS-108 internalization studies that exploit the auto-fluorescence of doxorubicin in captured CTCs. Methods: This is a single-arm, dose-escalation phase I study in men with CRPC to confirm the dose established in a completed phase I trial in females. Eligibility criteria included adequate organ function and progression of disease despite prior therapy with an LHRH agonist and at least one taxane-based regimen. Patients were required to discontinue LHRH agonists to avoid receptor competition. Patients received AEZS-108 every 21 days until progression or unacceptable toxicity for up to 6 cycles. The primary endpoint was safety. Results: Enrollment began in November 2010 and completed in September 2011. Twelve patients were accrued onto 3 dose levels. No DLTs have been noted. At the time of submission, a decrease in PSA was noted in 5 of the 10 evaluable patients. The grade 3 or 4 toxicities were primarily hematologic. Final reports detailing toxicity, RECIST response and PSA response will be reported. All correlative studies will also be reported. Conclusions: AEZS-108 is well tolerated and has demonstrated early signs of antitumor activity in men with CRPC. We will report the recommended dose for the planned phase II study.