Matthew M. Rochefort

Quantitative ImmunoPET of Prostate Cancer Xenografts with 89Zr- and 124I-Labeled Anti-PSCA A11 Minibody

Prostate stem cell antigen (PSCA) is expressed on the cell surface in 83%–100% of local prostate cancers and 87%–100% of prostate cancer bone metastases. In this study, we sought to develop immunoPET agents using 124I- and 89Zr-labeled anti-PSCA A11 minibodies (scFv-CH3 dimer, 80 kDa) and evaluate their use for quantitative immunoPET imaging of prostate cancer. Methods: A11 anti-PSCA minibody was alternatively labeled with 124I- or 89Zr-desferrioxamine and injected into mice bearing either matched 22Rv1 and 22Rv1×PSCA or LAPC-9 xenografts. Small-animal PET data were obtained and quantitated with and without recovery coefficient–based partial-volume correction, and the results were compared with ex vivo biodistribution. Results: Rapid and specific localization to PSCA-positive tumors and high-contrast imaging were observed with both 124I- and 89Zr-labeled A11 anti-PSCA minibody. However, the differences in tumor uptake and background uptake of the radiotracers resulted in different levels of imaging contrast. The nonresidualizing 124I-labeled minibody had lower tumor uptake (3.62 ± 1.18 percentage injected dose per gram [%ID/g] 22Rv1×PSCA, 3.63 ± 0.59 %ID/g LAPC-9) than the residualizing 89Zr-labeled minibody (7.87 ± 0.52 %ID/g 22Rv1×PSCA, 9.33 ± 0.87 %ID/g LAPC-9, P < 0.0001 for each), but the 124I-labeled minibody achieved higher imaging contrast because of lower nonspecific uptake and better tumor–to–soft-tissue ratios (22Rv1×PSCA:22Rv1 positive-to-negative tumor, 13.31 ± 5.59 124I-A11 and 4.87 ± 0.52 89Zr-A11, P = 0.02). Partial-volume correction was found to greatly improve the correspondence between small-animal PET and ex vivo quantification of tumor uptake for immunoPET imaging with both radionuclides. Conclusion: Both 124I- and 89Zr-labeled A11 anti-PSCA minibody showed high-contrast imaging of PSCA expression in vivo. However, the 124I-labeled A11 minibody was found to be the superior imaging agent because of lower nonspecific uptake and higher tumor–to–soft-tissue contrast. Partial-volume correction was found to be essential for robust quantification of immunoPET imaging with both 124I- and 89Zr-labeled A11 minibody.

Circulating tumour cells as a biomarker for diagnosis and staging in pancreatic cancer

Background:Current diagnosis and staging of pancreatic ductal adenocarcinoma (PDAC) has important limitations and better biomarkers are needed to guide initial therapy. We investigated the performance of circulating tumour cells (CTCs) as an adjunctive biomarker at the time of disease presentation.Methods:Venous blood (VB) was collected prospectively from 100 consecutive, pre-treatment patients with PDAC. Utilising the microfluidic NanoVelcro CTC chip, samples were evaluated for the presence and number of CTCs. KRAS mutation analysis was used to compare the CTCs with primary tumour tissue. CTC enumeration data was then evaluated as a diagnostic and staging biomarker in the setting of PDAC.Results:We found 100% concordance for KRAS mutation subtype between primary tumour and CTCs in all five patients tested. Evaluation of CTCs as a diagnostic revealed the presence of CTCs in 54/72 patients with confirmed PDAC (sensitivity=75.0%, specificity=96.4%, area under the curve (AUROC)=0.867, 95% CI=0.798–0.935, and P<0.001). Furthermore, a cut-off of ⩾3 CTCs in 4 ml VB was able to discriminate between local/regional and metastatic disease (AUROC=0.885; 95% CI=0.800–0.969; and P<0.001).Conclusion:CTCs appear to function well as a biomarker for diagnosis and staging in PDAC.

Locally Advanced Pancreatic Cancer: Association Between Prolonged Preoperative Treatment and Lymph-Node Negativity and Overall Survival

IMPORTANCE Treatment of patients with locally advanced/borderline resectable (LA/BR) pancreatic ductal adenocarcinoma (PDAC) is not standardized. OBJECTIVE To (1) perform a detailed survival analysis of our institutions experience with patients with LA/BR PDAC who were downstaged and underwent surgical resection and (2) identify prognostic biomarkers that may help to guide a decision for the use of adjuvant therapy in this patient subgroup. DESIGN, SETTING, AND PARTICIPANTS Retrospective observational study of 49 consecutive patients from a single institution during 1992-2011 with American Joint Committee on Cancer stage III LA/BR PDAC who were initially unresectable, as determined by staging computed tomography and/or surgical exploration, and who were treated and then surgically resected. MAIN OUTCOMES AND MEASURES Clinicopathologic variables and prognostic biomarkers SMAD4, S100A2, and microRNA-21 were correlated with survival by univariate and multivariate Cox proportional hazard modeling. RESULTS All 49 patients were deemed initially unresectable owing to vascular involvement. After completing preoperative chemotherapy for a median of 7.1 months (range, 5.4-9.6 months), most (75.5%) underwent a pylorus-preserving Whipple operation; 3 patients (6.1%) had a vascular resection. Strikingly, 37 of 49 patients were lymph-node (LN) negative (75.5%) and 42 (85.7%) had negative margins; 45.8% of evaluable patients achieved a complete histopathologic (HP) response. The median overall survival (OS) was 40.1 months (range, 22.7-65.9 months). A univariate analysis of HP prognostic biomarkers revealed that perineural invasion (hazard ratio, 5.5; P=.007) and HP treatment response (hazard ratio, 9.0; P=.009) were most significant. Lymph-node involvement, as a marker of systemic disease, was also significant on univariate analysis (P=.05). Patients with no LN involvement had longer OS (44.4 vs 23.2 months, P=.04) than LN-positive patients. The candidate prognostic biomarkers, SMAD4 protein loss (P=.01) in tumor cells and microRNA-21 expression in the stroma (P=.05), also correlated with OS. On multivariate Cox proportional hazard modeling of HP and prognostic biomarkers, only SMAD4 protein loss was significant (hazard ratio, 9.3; P=.004). CONCLUSIONS AND RELEVANCE Our approach to patients with LA/BR PDAC, which includes prolonged preoperative chemotherapy, is associated with a high incidence of LN-negative disease and excellent OS. After surgical resection, HP treatment response, perineural invasion, and SMAD4 status should help determine who should receive adjuvant therapy in this select subset of patients.

Reality of Single Circulating Tumor Cell Sequencing for Molecular Diagnostics in Pancreatic Cancer

To understand the potential and limitations of circulating tumor cell (CTC) sequencing for molecular diagnostics, we investigated the feasibility of identifying the ubiquitous KRAS mutation in single CTCs from pancreatic cancer (PC) patients. We used the NanoVelcro/laser capture microdissection CTC platform, combined with whole genome amplification and KRAS Sanger sequencing. We assessed both KRAS codon-12 coverage and the degree that allele dropout during whole genome amplification affected the detection of KRAS mutations from single CTCs. We isolated 385 single cells, 163 from PC cell lines and 222 from the blood of 12 PC patients, and obtained KRAS sequence coverage in 218 of 385 single cells (56.6%). For PC cell lines with known KRAS mutations, single mutations were detected in 67% of homozygous cells but only 37.4% of heterozygous single cells, demonstrating that both coverage and allele dropout are important causes of mutation detection failure from single cells. We could detect KRAS mutations in CTCs from 11 of 12 patients (92%) and 33 of 119 single CTCs sequenced, resulting in a KRAS mutation detection rate of 27.7%. Importantly, KRAS mutations were never found in the 103 white blood cells sequenced. Sequencing of groups of cells containing between 1 and 100 cells determined that at least 10 CTCs are likely required to reliably assess KRAS mutation status from CTCs.

Unexpected Readmissions After Major Cancer Surgery: An Evaluation of Readmissions as a Quality-of-Care Indicator

Readmissions following major oncologic operation are common-affecting patient treatment, outcome, and hospital resources. The Center for Medicare and Medicaid Services mandates reporting of certain disease-specific readmissions and Congress is considering using individual hospital readmission rates as a performance measure. Studies using administrative data demonstrate that readmission rates following major cancer surgery are high. Administrative data cannot determine causes. Single-institution studies demonstrate length of hospital stay and comorbidities as risk factors. Discharge processes and outpatient healthcare utilization can be improved. Until studies on readmission rate are conducted, using readmission rates as a measure of quality should be pursued cautiously.

Applications of ImmunoPET: Using 124I-Anti-PSCA A11 Minibody for Imaging Disease Progression and Response to Therapy in Mouse Xenograft Models of Prostate Cancer

Purpose: Prostate stem cell antigen (PSCA) is highly expressed in local prostate cancers and prostate cancer bone metastases and its expression correlates with androgen receptor activation and a poor prognosis. In this study, we investigate the potential clinical applications of immunoPET with the anti-PSCA A11 minibody, an antibody fragment optimized for use as an imaging agent. We compare A11 minibody immunoPET to 18F-Fluoride PET bone scans for detecting prostate cancer bone tumors and evaluate the ability of the A11 minibody to image tumor response to androgen deprivation. Experimental Design: Osteoblastic, PSCA-expressing, LAPC-9 intratibial xenografts were imaged with serial 124I-anti-PSCA A11 minibody immunoPET and 18F-Fluoride bone scans. Mice bearing LAPC-9 subcutaneous xenografts were treated with either vehicle or MDV-3100 and imaged with A11 minibody immunoPET/CT scans pre- and posttreatment. Ex vivo flow cytometry measured the change in PSCA expression in response to androgen deprivation. Results: A11 minibody demonstrated improved sensitivity and specificity over 18F-Fluoride bone scans for detecting LAPC-9 intratibial xenografts at all time points. LAPC-9 subcutaneous xenografts showed downregulation of PSCA when treated with MDV-3100 which A11 minibody immunoPET was able to detect in vivo. Conclusions: A11 minibody immunoPET has the potential to improve the sensitivity and specificity of clinical prostate cancer metastasis detection over bone scans, which are the current clinical standard-of-care. A11 minibody immunoPET additionally has the potential to image the activity of the androgen signaling axis in vivo which may help evaluate the clinical response to androgen deprivation and the development of castration resistance. Clin Cancer Res; 20(24); 6367–78. ©2014 AACR.

An engineered anti-CA19-9 cys-diabody for positron emission tomography imaging of pancreatic cancer and targeting of polymerized liposomal nanoparticles

BACKGROUND Antibody-based therapeutics is a rapidly growing field. Small engineered antibody fragments demonstrate similar antigen affinity compared with the parental antibody but have a shorter serum half-life and possess the ability to be conjugated to nanoparticles. The goal of this study was to engineer an anti-carbohydrate antigen 19-9 (CA19-9) cys-diabody fragment in hopes of targeting nanoparticles to pancreatic cancer. METHODS The anti-CA19-9 cys-diabody was created by engineering a C-terminal cysteine residue into the DNA single-chain Fv construct of the anti-CA19-9 diabody and expressed in NS0 cells. Maleimide chemistry was used to conjugate the cys-diabody to polymerized liposomal nanoparticles (PLNs) through the cysteine residues. Flow cytometry was used to evaluate targeting of cys-diabody and cys-diabody-PLN conjugate to human pancreatic cancer cell lines. The cys-diabody was radiolabeled with a positron emitter ((124)I) and evaluated in a mouse model of CA19-9-positive and CA19-9-negative xenografts with micro-positron emission tomography/micro-computed tomography at successive time intervals after injection. Percentage of injected dose per gram of radioactivity was measured in blood and tumor to provide objective confirmation of the micro-positron emission tomographic images. RESULTS Tumor xenograft imaging of the anti-CA19-9 cys-diabody demonstrated an average tumor-to-blood ratio of 3.0 and positive-to-negative tumor ratio of 7.4. Successful conjugation of the cys-diabody to PLNs was indicated by flow cytometry showing specific binding of cys-diabody-PLN conjugate to human pancreatic cancer cells in vitro. CONCLUSIONS Our results show that the anti-CA19-9 cys-diabody targets pancreatic cancer providing specific molecular imaging in tumor xenograft models. Furthermore, the cys-diabody-PLN conjugate demonstrates target-specific binding of human pancreatic cancer cells with the potential to deliver targeted treatment.

Long-term survival in patients with pancreatic ductal adenocarcinoma.

BACKGROUND Long-term survival (LTS) is uncommon for patients with pancreatic ductal adenocarcinoma (PDAC). We sought to identify factors that predict 10-year, LTS after resection of PDAC. METHODS We identified all patients with PDAC who underwent resection at UCLA after 1990 and included all patients eligible for observed LTS (1/1/1990-12/31/2004). An independent pathologist reconfirmed the diagnosis of PDAC in patients with LTS. Logistic regression was used to predict LTS on the basis of patient and tumor characteristics. RESULTS Of 173 included patients, 53% were male, median age at diagnosis was 66 years, and median survival was 23 months. The rate of observed LTS was 12.1% (n = 21). Age, sex, number of lymph nodes evaluated, margin status, lymphovascular invasion, and adjuvant chemotherapy and radiation were not associated with LTS. The following were associated with LTS on bivariate analysis: low AJCC stage (Ia, Ib, IIa) (P = .034), negative lymph node status (P = .034), low grade (well-, moderately-differentiated) (P = .001), and absence of perineural invasion (P = .019). Only low grade (odds ratio 7.17, P = .012) and absent perineural invasion (odds ratio 3.28, P = .036) were independently associated with increased odds of LTS. Our multivariate model demonstrated good discriminatory power for LTS, as indicated by a c-statistic of 0.7856. CONCLUSION Absence of perineural invasion and low tumor grade were associated with greater likelihood of LTS. Understanding the tumor biology of LTS may provide critical insight into a disease that is typically marked by aggressive behavior and limited survival.

Metabolic exploitation of the sialic acid biosynthetic pathway to generate site-specifically labeled antibodies

Lack of a universal site-specific conjugation methodology for antibodies limits their potential to be developed as tumor-specific imaging agents or targeted therapeutics. A potential mechanism for site-specific conjugation involves utilization of the conserved N-glycosylation site in the CH2 domain. We sought to develop an antibody with an altered azido-sugar at this site whereby site-specific label could be added. The HB8059 hybridoma was cultured with peracetylated N-azidoacetlymannosamine (Ac4ManNAz). The resulting azido-sugar antibody was conjugated to phosphine-polyethylene glycol (PEG3)-biotin via a modified Staudinger reaction. Biochemical and functional characterization of the biotinylated antibody was performed. The azido-sugar antibody was also labeled with DyLight-650-Phosphine and injected into mice harboring pancreatic cancer xenografts. The tumors were dissected and imaged utilizing an IVIS fluorescent camera. The antibody was successfully produced in 100 μM Ac4ManNAz. The biotinylated antibody demonstrated a 50 kDa heavy and 25 kDa light chain on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but demonstrated a single band at 50 kDa on western blot. Treatment with a N-linked glycosidase extinguished the band. Flow cytometry demonstrated antigen-specific binding of CA19-9-positive cells and the antibody localized to the antigen-positive tumor in vivo. We successfully produced an antibody with an azido-sugar at the conserved CH2 glycosylation site. We were able to utilize this azide to label the antibody with biotin or fluorescent label and demonstrate that the label is added in a site-specific manner to the heavy chain, N-linked glycosylation site. Finally, we demonstrated functionality of our antibody for in vitro and in vivo targeting of pancreatic cancer cells.

Pancreatic cancer patients with lymph node involvement by direct tumor extension have similar survival to those with node-negative disease

Lymph node (LN) involvement is a well‐known poor prognostic factor in patients with pancreatic ductal adenocarcinoma (PDAC). However, there have been conflicting results on the significance of the mechanism of LN involvement, “direct” tumor invasion versus “metastatic,” disease on patient survival.