Samuel H. Whiting

Pancreatic ductal adenocarcinoma contains an effector and regulatory immune cell infiltrate that is altered by multimodal neoadjuvant treatment

Objective The immune response to pancreatic ductal adenocarcinoma (PDA) may play a role in defining its uniquely aggressive biology; therefore, we sought to clearly define the adaptive immune infiltrate in PDA. Design We used immunohistochemistry and flow cytometry to characterize the immune infiltrate in human PDA and compared our findings to the patients’ peripheral blood. Results In contrast to the myeloid cell predominant infiltrate seen in murine models, T cells comprised the majority of the hematopoietic cell component of the tumor stroma in human PDA. Most intratumoral CD8+ T cells exhibited an antigen-experienced effector memory cell phenotype and were capable of producing IFN-γ. CD4+ regulatory T cells (Treg) and IL-17 producing T helper cells were significantly more prevalent in tumor than in blood. Consistent with the association with reduced survival in previous studies, we observed higher frequencies of both myeloid cells and Treg in poorly differentiated tumors. The majority of intratumoral T cells expressed the co-inhibitory receptor programmed death-1 (PD-1), suggesting one potential mechanism through which PDA may evade antitumor immunity. Successful multimodal neoadjuvant therapy altered the immunoregulatory balance and was associated with reduced infiltration of both myeloid cells and Treg. Conclusion Our data show that human PDA contains a complex mixture of inflammatory and regulatory immune cells, and that neoadjuvant therapy attenuates the infiltration of intratumoral cells associated with immunosuppression and worsened survival.

Cleavage Specificities of Moloney Murine Leukemia Virus RNase H Implicated in the Second Strand Transfer During Reverse Transcription

Reverse transcription of a retroviral RNA genome requires two template jumps to generate the linear double-stranded DNA required for integration. The RNase H activity of reverse transcriptase has several roles during this process. We have examined RNase H cleavages that define the maximal 3′ and 5′ ends of Moloney murine leukemia virus minus strand DNA prior to the second template jump. In both the endogenous reaction and on model substrates in vitro, RNase H cleaves the genomic RNA template between the second and third ribonucleotides 5′ of the U5/PBS junction, but other minor cleavages between 1 and 10 nucleotides 5′ of this junction are also observed. Similar experiments examining the specificity of RNase H for tRNA primer removal revealed that cleavage generally leaves a ribo A residue at the 5′ end of minus strand DNA. These observations suggest that three bases are typically duplicated on the ends of the minus strands, leading to an intermediate following the second jump which contains unpaired nucleotides. Model substrates mimicking the structure of this intermediate demonstrate that reverse transcriptase has little difficulty in utilizing such a branched structure for the initiation of displacement synthesis.

New generation of ensemble-decision aliquot ranking based on simplified microfluidic components for large-capacity trapping of circulating tumor cells.

Ensemble-decision aliquot ranking (eDAR) is a sensitive and high-throughput method to analyze circulating tumor cells (CTCs) from peripheral blood. Here, we report the next generation of eDAR, where we designed and optimized a new hydrodynamic switching scheme for the active sorting step in eDAR, which provided fast cell sorting with an improved reproducibility and stability. The microfluidic chip was also simplified by incorporating a functional area for subsequent purification using microslits fabricated by standard lithography method. Using the reported second generation of eDAR, we were able to analyze 1 mL of whole-blood samples in 12.5 min, with a 95% recovery and a zero false positive rate (n = 15).

Abstract 5314: A proteomic signature predicts response to a therapeutic vaccine in pancreas cancer; analysis from the GI-4000-02 trial

Background: We have previously reported that adjuvant treatment with a therapeutic vaccine targeting the mutated Ras oncogene product generated mutation-specific T cell responses associated with a trend toward improved survival in patients with post-operative residual disease (R1 resections) but no improvement in the overall population 1 . Initial analysis of 90 pretreatment plasma samples using matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry (MS) showed the potential to predict improved RFS and OS for treatment with GI-4000/gemcitabine, but not placebo/gemcitabine. Methods: We have developed a novel technique, combining methods used in recent advances in learning theory (‘deep learning’) with newly-refined MS techniques that allow exploration deeper into the proteome to create diagnostic tests. Using 500,000 laser shot Deep MALDI spectra 2 more than 700 mass spectral features were identified. A subset of these was used to create many multivariate classifiers that were filtered for performance and combined using dropout regularization. This method allows the use of smaller training sets and so left a test set with which performance of the signature could be independently assessed. This new methodology was used to create a test (BDX-001) to identify patients likely to benefit from the addition of GI-4000 to gemcitabine. Results: Using BDX-001 for stratification, subjects who are BDX-001(+) demonstrated a 499 day advantage in median OS when treated with GI-4000/gemcitabine vs. placebo/gemcitabine. Additionally, these subjects demonstrated a 351 day improvement in median RFS. BDX-001 did not predict response for placebo/gemcitabine treated subjects. These results were obtained using only test set data, and although the small sample size prohibited statistical significance, it should give an unbiased test performance estimate to be validated independently. Conclusions: BDX-001 is a test developed using novel proteomic and learning theory methods that appears to predict treatment response to GI-4000 in resected pancreas cancer patients, potentially identifying patients with improved RFS and OS in the GI-4000/gemcitabine arm. We plan to prospectively validate BDX-001 as a companion diagnostic in a future study of GI-4000 in pancreas cancer. References 1. Richards et al, ESMO GI. Annals of Oncology, June 2012 23 (suppl 4) 2. Duncan et al, ASMS 2013, http://asms.inmerge.com/Proceedings/2013Proceedings.aspx. Citation Format: Donald A. Richards, Peter Muscarella, Tanios Bekaii-Saab, Lalan S. Wilfong, Vic Velanovich, Julian Raynov, Patrick J. Flynn, William E. Fisher, Samuel H. Whiting, Constana Timcheva, Tom Holmes, Claire Coeshott, Alicia Mattson, Heinrich Roder, Joanna Roder, Allen Cohn, Timothy C. Rodell. A proteomic signature predicts response to a therapeutic vaccine in pancreas cancer; analysis from the GI-4000-02 trial. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5314. doi:10.1158/1538-7445.AM2014-5314

Retroviral envelope protein fusions to secreted and membrane markers

We have analyzed a series of Moloney murine leukemia (M-MuLV) envelope (env) protein fusions to the marker proteins invertase and placental alkaline phosphatase (PLAP), expressed in Psi2 retrovirus packaging cells. The yeast invertase protein, fused at its third amino acid residue to the amino-terminal signal sequence and 17 residues of the mature M-MuLV env protein, retained its enzymatic activity and was secreted from mammalian cells. However, env protein fusions to the C-terminal portion of invertase were inactive. In contrast, some, but not all, env protein fusions at the C-terminal region of PLAP were enzymatically active: PLAP fusions containing long C-terminal portions of env localized to the rough endoplasmic reticulum (RER) and possessed low enzyme activity levels, while fusion constructs containing relatively short portions of the M-MuLV env gene localized to the Golgi and had higher activity levels. Those proteins that localized to the Golgi also were processed, in part, to forms of 67 to 68 kDa, the size of the mature PLAP protein. Since PLAP ordinarily is transferred to a phosphatidyl-inositol glycan tail (PIG-tail) in the Golgi and then transported to the plasma membrane, it appears that Golgi-localized PLAP-env fusions are processed imperfectly. PLAP itself, when expressed in Psi2 cells, accumulated at the plasma membrane and, unlike the PIG-tailed Thy-1 protein, was not incorporated into virus particles. Thus, the reported incorporation of the Thy-1 protein into M-MuLV virions does not appear to be a consequence of its glycoprotein tail.

Abstract PD3-13: Phase 1 study of CB-839, a first-in-class oral inhibitor of glutaminase, in combination with paclitaxel in patients with advanced triple negative breast cancer

Background: CB-839 is a first-in-class oral highly selective inhibitor of glutaminase (GLS), a mitochondrial enzyme that plays a key role in cancer cell metabolism. Triple negative breast cancer (TNBC) has high GLS expression and demonstrates high glutamine utilization and glutamine dependence in clinical and preclinical studies. CB-839 has monotherapy antitumor activity in vitro and in vivo in preclinical models of TNBC, and also demonstrates synergistic anti-cancer activity with the standard of care agent paclitaxel (Pac). In an ongoing Phase 1 study, CB-839 combined with Pac (Pac-CB) is being evaluated in patients (pts) with metastatic TNBC. We previously reported that Pac-CB was well-tolerated and active in heavily pre-treated TNBC pts, including those previously refractory to taxane treatment for metastatic disease. We report here updated results from this ongoing study. Methods: Pts with refractory metastatic TNBC (prior taxane therapy allowed) received escalating doses of CB-839 (400-800 mg BID) in combination with full dose Pac of 80 mg/m 2 Days 1, 8, 15 every 28 days. After demonstration of safety and tolerability, an expansion cohort was opened at the CB-839 recommended phase 2 dose of 800 mg PO BID. Results: As of the May 2017 data cut, 45 pts have enrolled across the dose escalation and expansion cohorts (7 pts at 400 mg, 12 at 600 mg, and 26 at 800 mg). 15 pts (33%) have received ≥5 prior lines of systemic therapy for metastatic disease (median 3 prior lines), and 39 pts (87%) have received prior taxane therapy. The Pac-CB regimen has been well tolerated with the most frequent treatment-related Grade ≥3 AEs being neutropenia (24%), anemia (7.6%), fatigue (2.6%), WBC decreased (2.6%), and peripheral neuropathy (2.6%). At CB-839 doses ≥600 mg BID (n=27 RECIST-evaluable), the ORR has been 22% and disease control rate (DCR=CR+PR+SD) 59%; in the subgroup of pts refractory to previous taxane therapy (n=19) the ORR has been 21% (4 pts) and DCR 47% (9 pts). The highest ORR in this study has been seen in pts of African ancestry (AA, n=11 RECIST evaluable) with an ORR of 36% (4 pts) and DCR 55% (6 pts), with all 4 AA responders being refractory to prior taxane treatment for advanced/metastatic disease. Conclusions: TNBC has elevated glutamine metabolism that requires mitochondrial GLS. In TNBC pts with heavy pretreatment and previous taxane exposure, the combination of CB-839 with Pac has demonstrated clinical activity including a 21% ORR in pts refractory to taxane. Notably, in patients of African ancestry, a population reported to have especially high glutamine utilization in TNBC tumors, the encouraging ORR of 36% suggests a potential genetic association with treatment response. The Pac-CB regimen has been well tolerated in late line TNBC pts, including a Gr 3 neuropathy signal of only 2.6%. A Phase 2 study, CX-839-007, has been initiated to further evaluate the activity and safety of Pac-CB in pts with TNBC, including defined cohorts of pts with 1 st line and 3 rd line+ metastatic disease in pts of African and non-African ancestry. Responses in relation to genetic background, molecular subtype of TNBC and glutamine biology will be studied. Citation Format: Kalinsky K, Harding JJ, DeMichele A, Infante JR, Gogineni K, Owonikoko TK, Isakoff S, Iliopoulos O, Patel MR, Munster P, Telli ML, Jenkins Y, Fiji GP, Whiting SH, Meric-Bernstam F. Phase 1 study of CB-839, a first-in-class oral inhibitor of glutaminase, in combination with paclitaxel in patients with advanced triple negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD3-13.