Sanjeeve Balasubramaniam

Vaccine-Stimulated, Adoptively Transferred CD8+ T Cells Traffic Indiscriminately and Ubiquitously while Mediating Specific Tumor Destruction

It has been suggested that antitumor T cells specifically traffic to the tumor site, where they effect tumor destruction. To test whether tumor-reactive CD8+ T cells specifically home to tumor, we assessed the trafficking of gp100-specific pmel-1 cells to large, vascularized tumors that express or do not express the target Ag. Activation of tumor-specific CD8+ pmel-1 T cells with IL-2 and vaccination with an altered peptide ligand caused regression of gp100-positive tumors (B16), but not gp100-negative tumors (methylcholanthrene 205), implanted on opposing flanks of the same mouse. Surprisingly, we found approximately equal and very large numbers of pmel-1 T cells (>25% of all lymphocytes) infiltrating both Ag-positive and Ag-negative tumors. We also found evidence of massive infiltration and proliferation of activated antitumor pmel-1 cells in a variety of peripheral tissues, including lymph nodes, liver, spleen, and lungs, but not peripheral blood. Most importantly, evidence for T cell function, as measured by production of IFN-γ, release of perforin, and activation of caspase-3 in target cells, was confined to Ag-expressing tumor. We thus conclude that CD8+ T cell-mediated destruction of tumor is the result of specific T cell triggering at the tumor site. The ability to induce ubiquitous homing and specific tumor destruction may be important in the case of noninflammatory metastatic tumor foci.

Targeting the Epigenome in Lung Cancer: Expanding Approaches to Epigenetic Therapy

Epigenetic aberrations offer dynamic and reversible targets for cancer therapy; increasingly, alteration via overexpression, mutation, or rearrangement is found in genes that control the epigenome. Such alterations suggest a fundamental role in carcinogenesis. Here, we consider three epigenetic mechanisms: DNA methylation, histone tail modification and non-coding, microRNA regulation. Evidence for each of these in lung cancer origin or progression has been gathered, along with evidence that epigenetic alterations might be useful in early detection. DNA hypermethylation of tumor suppressor promoters has been observed, along with global hypomethylation and hypoacetylation, suggesting an important role for tumor suppressor gene silencing. These features have been linked as prognostic markers with poor outcome in lung cancer. Several lines of evidence have also suggested a role for miRNA in carcinogenesis and in outcome. Cigarette smoke downregulates miR-487b, which targets both RAS and MYC; RAS is also a target of miR-let-7, again downregulated in lung cancer. Together the evidence implicates epigenetic aberration in lung cancer and suggests that targeting these aberrations should be carefully explored. To date, DNA methyltransferase and histone deacetylase inhibitors have had minimal clinical activity. Explanations include the possibility that the agents are not sufficiently potent to invoke epigenetic reversion to a more normal state; that insufficient time elapses in most clinical trials to observe true epigenetic reversion; and that doses often used may provoke off-target effects such as DNA damage that prevent epigenetic reversion. Combinations of epigenetic therapies may address those problems. When epigenetic agents are used in combination with chemotherapy or targeted therapy it is hoped that downstream biological effects will provoke synergistic cytotoxicity. This review evaluates the challenges of exploiting the epigenome in the treatment of lung cancer.

The VEGF inhibitor axitinib has limited effectiveness as a therapy for adrenocortical cancer

CONTEXT Adrenocortical carcinoma (ACC) is a rare malignancy with a poor prognosis in need of more effective treatment options. Published evidence indicates many ACCs express the vascular endothelial growth factor receptor (VEGFR), suggesting inhibiting vascular endothelial growth factor signaling could potentially impact tumor growth. OBJECTIVE The objective of the study was to determine the antitumor efficacy of axitinib (AG-013736), a potent, selective inhibitor of VEGFR1, -2, and -3. DESIGN This was a phase II, open-label trial using a two-stage design. PATIENTS Thirteen patients with metastatic ACC previously treated with at least one chemotherapy regimen with or without mitotane participated in the study. INTERVENTION Starting axitinib dose was 5 mg orally twice daily. Dose escalations were permitted if the administered dose was tolerable. RESULTS Thirteen patients were enrolled. Dose escalation was possible in seven patients, but the majority could not tolerate a dose higher than the starting 5 mg, twice-daily dose for prolonged periods of time. All patients experienced known grade 1/2 toxicities, and 10 of 13 patients had at least one grade 3/4 adverse event. No patient tumor could be scored as a Response Evaluation Criteria in Solid Tumors response, although the growth rate on therapy compared with that prior to starting axitinib was reduced in 4 of the 13 patients. The median progression-free survival was 5.48 months, and the median overall survival was longer than 13.7 months. CONCLUSION Axitinib has limited effectiveness in ACC. Together with 48 patients previously reported who received either sorafenib or sunitinib, a total of 61 ACC patients have now been treated with a VEGFR tyrosine kinase inhibitor without an objective Response Evaluation Criteria in Solid Tumors response. Future trials in ACC should look to other targets for possible active agents.

Association between Benign Thyroid and Endocrine Disorders and Subsequent Risk of Thyroid Cancer among 4.5 Million U.S. Male Veterans

CONTEXT Risk factors for thyroid cancer (TC) in males are poorly understood. OBJECTIVES, SETTING, AND PARTICIPANTS: Our aim was to evaluate the relationship between history of benign thyroid and endocrine disorders and risk of TC among 4.5 million male veterans admitted to U.S. Veterans Affairs hospitals between July 1, 1969, and September 30, 1996. DESIGN We conducted a retrospective cohort study based on hospital discharge records with 1053 cases of TC. MAIN OUTCOME MEASURES We estimated relative risks (RR) and computed 95% confidence intervals (CI) for TC using time-dependent Poisson regression models. To evaluate potential ascertainment bias and/or delayed diagnosis of TC, we also analyzed RR by time between diagnosis of benign disorder and TC (<5 or ≥ 5 yr). RESULTS RR for TC were significantly elevated with many disorders and were often higher less than 5 yr compared with 5 yr or more before TC diagnosis. RR (95% CI) less than 5 yr/at least 5 yr were 67.9 (42.4-108.8)/28.9 (9.2-90.2) for thyroid adenoma, 77.8 (64.5-93.1)/25.9 (17.9-38.0) for nontoxic nodular goiter, 23.9 (13.8-41.3)/12.9 (4.8-34.4) for thyroiditis, 8.8 (6.9-11.3)/6.0 (3.8-9.6) for hypothyroidism, 6.4 (4.4-9.4)/ 2.0 (0.8-4.8) for thyrotoxicosis, and 1.2 (1.0-1.4)/1.1 (0.9-1.5) for diabetes. For some disorders, RR also significantly varied by attained age and race with younger patients and Blacks having higher RR than older patients and Whites. CONCLUSIONS We found strong associations for a history of thyroid adenoma, nodular goiter, thyroiditis, or hypothyroidism with TC in males allowing for increased surveillance/delayed diagnosis and evidence that some of these associations are modified by age and race.

Effects of UGT1A1 genotype on the pharmacokinetics, pharmacodynamics, and toxicities of belinostat administered by 48-hour continuous infusion in patients with cancer.

The histone deacetylase inhibitor belinostat is eliminated through glucuronidation by UGT1A1. Polymorphisms that reduce UGT1A1 function could result in increased belinostat exposure and toxicities. We wanted to determine which single‐nucleotide polymorphisms alter belinostat exposure and toxicity. In a phase 1 trial (belinostat over 48 hours in combination with cisplatin and etoposide), belinostat (400, 500, 600, or 800 mg/m2/24 h, 48‐hour continuous infusion) was administered to patients with cancer in combination with cisplatin and etoposide (n = 25). Patients were genotyped for UGT1A1 variants associated with reduced function: UGT1A1*6, UGT1A1*28, and UGT1A1*60. End points were associations between UGT1A1 genotype and belinostat pharmacokinetics (PK), toxicities, and global protein lysine acetylation (AcK). Belinostat AUC was increased (P = .003), and t1/2 increased (P = .0009) in UGT1A1*28 and UGT1A1*60 carriers who received more than 400 mg/m2/24 h. The incidence of grades 3–4 thrombocytopenia (P = .0081) was associated with UGT1A1 polymorphisms. The US Food and Drug Administration–approved package insert recommends dose adjustment of belinostat for UGT1A1*28. However, our data suggest dose adjustment is also necessary for UGT1A1*60. UGT1A1 polymorphisms were associated with increased systemic belinostat exposure, increased AcK, and increased incidence of toxicities, particularly at doses > 400 mg/m2/24 h.

UGT1A1 genotype‐dependent dose adjustment of belinostat in patients with advanced cancers using population pharmacokinetic modeling and simulation

Belinostat is a second‐generation zinc‐binding histone deacetylase inhibitor that is approved for peripheral T‐cell lymphoma and is currently being studied in small cell lung cancer and other advanced carcinomas as a 48‐hour continuous intravenous infusion. Belinostat is predominantly metabolized by UGT1A1, which is polymorphic. Preliminary analyses revealed a difference in belinostat clearance based on UGT1A1 genotype. A 2‐compartment population pharmacokinetic (PK) model was developed and validated that incorporated the UGT1A1 genotype, albumin, and creatinine clearance on the clearance parameter; body weight was a significant covariate on volume. Simulated doses of 600 and 400 mg/m2/24 h given to patients considered extensive or impaired metabolizers, respectively, provided equivalent AUCs. This model and subsequent simulations supported additional PK/toxicity and pharmacogenomics/toxicity analyses to suggest a UGT1A1 genotype‐based dose adjustment to normalize belinostat exposure and allow for more tolerable therapy. In addition, global protein lysine acetylation was modeled with PK and demonstrated a reversible belinostat exposure/response relationship, consistent with previous reports.

A Phase I Study of DMS612, a Novel Bifunctional Alkylating Agent

Purpose: DMS612 is a dimethane sulfonate analog with bifunctional alkylating activity and preferential cytotoxicity to human renal cell carcinoma (RCC) in the NCI-60 cell panel. This first-in-human phase I study aimed to determine dose-limiting toxicity (DLT), maximum tolerated dose (MTD), pharmacokinetics (PK), and pharmacodynamics (PD) of DMS612 administered by 10-minute intravenous infusion on days 1, 8, and 15 of an every-28-day schedule. Experimental Design: Patients with advanced solid malignancies were eligible. Enrollment followed a 3+3 design. PKs of DMS612 and metabolites were assessed by mass spectroscopy and PD by γ-H2AX immunofluorescence. Results: A total of 31 patients, including those with colorectal (11), RCC (4), cervical (2), and urothelial (1) cancers, were enrolled. Six dose levels were studied, from 1.5 mg/m2 to 12 mg/m2. DLTs of grade 4 neutropenia and prolonged grade 3 thrombocytopenia were observed at 12 mg/m2. The MTD was determined to be 9 mg/m2 with a single DLT of grade 4 thrombocytopenia in 1 of 12 patients. Two patients had a confirmed partial response at the 9 mg/m2 dose level, in renal (1) and cervical (1) cancer. DMS612 was rapidly converted into active metabolites. γ-H2AX immunofluorescence revealed dose-dependent DNA damage in both peripheral blood lymphocytes and scalp hairs. Conclusions: The MTD of DMS12 on days 1, 8, and 15 every 28 days was 9 mg/m2. DMS612 appears to be an alkylating agent with unique tissue specificities. Dose-dependent PD signals and two partial responses at the MTD support further evaluation of DMS612 in phase II trials. Clin Cancer Res; 21(4); 721–9. ©2014 AACR.

Phase I/II Trial of Vandetanib and Bortezomib in Adults with Locally Advanced or Metastatic Medullary Thyroid Cancer

Lessons Learned. Vandetanib at a dose of 300 mg orally every day plus bortezomib 1.3 mg/m2 intravenously on days 1, 4, 8, and 11 could be administered safely. Assessing outcomes in 17 patients with medullary thyroid cancer, investigators considered the combination to be more difficult to administer than single‐agent vandetanib and that achieving better outcomes was unlikely. Consequently, a planned phase II study was terminated early. Background. The proto‐oncogene RET (REarranged during Transfection) has a critical role in the pathogenesis of medullary thyroid cancer (MTC). Vandetanib (V), a multitargeted tyrosine kinase inhibitor approved for the treatment of MTC, is thought to inhibit RET in MTC. Supported by preclinical studies demonstrating that bortezomib (B) administration lowered RET mRNA and protein levels, we conducted a phase I study in advanced solid tumors of vandetanib in combination with bortezomib. The goal was to establish an RP2D (recommended phase II dose) for the combination of vandetanib plus bortezomib, a regimen envisioned as a dual strategy for targeting RET in MTC. Methods. Patients with advanced solid tumors were treated with escalating doses of bortezomib or vandetanib to assess the safety and tolerability of daily oral vandetanib and intravenous (IV) bortezomib administered on days 1, 4, 8, and 11 of a 28‐day cycle. Intrapatient dose escalation was allowed. Results. Twenty‐two patients were enrolled and received escalating mg/m2 bortezomib and mg vandetanib (number of patients) at initial doses of 1 and 100 (3), 1.3 and 100 (6), 1.3 and 200 (6), and 1.3 and 300 (7), respectively. Patients received a median of four cycles of bortezomib/vandetanib (range: 1–10), with 13 patients escalating to 1.3/200 and 10 to 1.3/300. G3 toxicities occurring in more than one patient included hypertension (24%), fatigue (19%), thrombocytopenia (10%), diarrhea (10%), and arthralgia (10%). There were no drug‐related G4/5 toxicities. There was one dose‐limiting toxicity, G3 thrombocytopenia, at bortezomib/vandetanib doses of 1.3/200 in cycle 2 that resolved without intervention. Four patients with a diagnosis of MTC (27%) had a partial response (PR). Conclusion. The MTD of the combination was established as bortezomib, 1.3 mg/m2 IV days 1, 4, 8, and 11 with vandetanib 300 mg p.o. daily. RECIST responses were observed in patients with a diagnosis of MTC.

Abstract B43: ANG1005, a novel brain-penetrant drug conjugate, in CNS metastases from breast cancer: FLT-PET imaging as a predictor of response

Background: The novel drug conjugate ANG1005 consists of 3 molecules of paclitaxel covalently linked to Angiopep-2. After binding to the low-density lipoprotein receptor-related protein, ANG1005 crosses the blood brain barrier (BBB) by endocytosis. A multi-center Phase II study, with the primary endpoint of intracranial response in patients with breast cancer brain metastases is in progress. At the NCI, a biomarker substudy is evaluating 18F-FLT (39-Fluoro-39 deoxythymidine)-PET for response assessment. Methods: Patients with measurable brain metastases from breast cancer received ANG1005 at a dose of 550mg/m2 IV once every 21 days. Before and after cycle 1, all patients on study underwent imaging with 18F-FLT, a thymidine analog and novel imaging agent; retention of 18F-FLT correlating with DNA synthesis. In order to detect brain metastases and assess response to treatment, we compared FLT PET images with MRI-gadolinium contrast scans, obtaining both dynamic and static images. We determined the percentage (%) change after treatment with ANG1005; a decrease in ≥20% was considered significant. Results: Ten patients were enrolled on study. A total of 32 target and 20 non target CNS lesions in 10 patients were imaged and analyzed by MRI and FLT-PET. At 30 minutes, SUVmax ranged from 0.8 to 6.3 at baseline (mean 2.64), and the SUV80%, (mean of top 20% SUV units) ranged from 0.7 to 5.12. The median% change from baseline to post one cycle of ANG1005 was -19.9% for SUVmax (range +85.4 to -67.7, and was median -20.2% for SUV80% (range 95.7 to -69%). Two patients had confirmed partial responses lasting 6 and 18 cycles, respectively. Six patients had stable disease and received a median of 6 cycles. Tumor reductions, as determined by MRI per CNS RECIST version 1.1 ranged from -5% to -60% in lesion size, compared to baseline. Both FLT-PET parameters correlated with the% change in MRI measurements in the target lesions (R2 = 0.64, P = 0.0002). Conclusion: The development of CNS-directed therapies designed to cross the BBB, such as the paclitaxel conjugate ANG1005, is a research priority. As contrast-enhanced MRI detection of brain metastases is representative of gadolinium leakage through the BBB, as opposed to actual tumor volume, better approaches are needed to evaluate drug efficacy. Pilot evaluations of FLT-PET imaging in this setting suggest that it is a promising tool that may serve as a complementary assessment method for breast cancer brain metastases going forward. Citation Format: Ciara C. O9Sullivan, Maria Lindenberg, Christine Bryla, Nicole Davarpanah, Cody Peer, Nicholas Patronas, Laleh Amiri-Kordestani, Sanjeeve Balasubramaniam, Tito Fojo, William D. Figg, Peter Choyke. ANG1005, a novel brain-penetrant drug conjugate, in CNS metastases from breast cancer: FLT-PET imaging as a predictor of response. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B43.

Abstract 5480: Effects of UGT1A1 genotype on pharmacokinetics and toxicities of belinostat administered by continuous infusion in two clinical trials

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background: Belinostat (B), a histone deacetylase inhibitor recently approved for peripheral T-cell lymphoma, is extensively glucuronidated by UGT1A1. Genotypes with reduced UGT1A1 function could lead to higher B exposure and toxicities (tox). Methods: In a Phase I (BPE) and Phase I/II trial (BPAC), B (400-1000 mg/m2/24 h) was administered as a 48 h continuous infusion in combination with either cisplatin (P) and etoposide (E), or P, doxorubicin (A) and cyclophosphamide (C), respectively. Pts with small cell lung cancer and other cancers of neuroendrocrine origin (n = 25 in BPE) or advanced or unresectable thymic epithelial tumors (n = 26 in BPAC) underwent genotyping for UGT1A1 variants associated with reduced function: UGT1A1*6, *28 and *60. In both trials PK of B was analyzed and during Cycle 1 clinical outcomes were monitored. In 9 pts the Drug Metabolizing Enzymes and Transporter (DMET) genotyping array was carried out to study relationships between drug metabolizing enzymes and transporters with B PK or tox. Results: Pts carrying 1 or 2 UGT1A1*60 variants (n = 18) in BPE, had significantly higher B AUCs than WT pts (n = 5): median 11.4 vs 8.3 ng/mL*h/mg, P = 0.043. Consistent with greater exposure, pts carrying at least 1 variant UGT1A1*60 allele (n = 18) were at significantly higher risk for developing Gr 3-4 thrombocytopenia vs. WT pts (n = 5): OR (95%CI) = 21.2 (1.01-445.30), P = 0.014. A trend towards an increased risk of Gr 3-4 thrombocytopenia (P = 0.038), Gr 3-4 neutropenia (P = 0.045) and QTc prolongation (P = 0.030) was observed for UGT1A1*28. For Gr 3-4 neutropenia a marginal trend was also seen in *60 carriers (P = 0.088). In the BPAC trial UGT1A1*28 and UGT1A1*60 status did not significantly affect B PK (AUC, Cmax and CL, P>0.05) nor the incidence of most tox (P>0.05). Only Gr 3-4 thrombocytopenia occurred more frequently in *28 carriers (n = 11) than in WTs (n = 15): OR (95%CI) = 18.6 (0.88-392.7), P = 0.022. The DMET array revealed significant associations between SNPs in SLC15A1 and CYP24A1 and B PK (P<0.01). SNPs in SLCO1B3, SLCO1B1 and ABCB4 were significantly associated with the incidence of lymphocytopenia (P<0.01). Significant trends were observed between SNPs in UGT1A1, CYP2C19, CHST7, ABCB7 and ATP7A and the incidence of neutropenia. A SNP in XDH significantly trended with the incidence of thrombocytopenia (P<0.01). Conclusion: This is the first report of UGT1A1 polymorphisms effect on the PK and tox of B in combination with PE or PAC. In the BPE trial UGT1A1 status was associated with the extent of systemic exposure to B and the incidence of hematological tox. These associations were less profound in the BPAC trial and possible explanations are still under investigation. The DMET results indicate that besides UGT1A1 other drug transporters or enzymes could affect B PK and tox. Based on these findings, a population PK model accounting for genotype has been developed to titrate B dose based on UGT1A1 genotype. Citation Format: Andrew K.L. Goey, Tristan M. Sissung, Cody J. Peer, Sheryl Ehrlich, Christina Bryla, Arlene W. Berman, Sanjeeve Balasubramaniam, Arun Rajan, Giuseppe Giaccone, Susan E. Bates, William D. Figg. Effects of UGT1A1 genotype on pharmacokinetics and toxicities of belinostat administered by continuous infusion in two clinical trials. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5480. doi:10.1158/1538-7445.AM2015-5480