Hematogenous dissemination is thought to be a late event in cancer progression. We showed recently that pancreas cells can be detected in the bloodstream before tumor formation, in a genetic model of pancreatic ductal adenocarcinoma (PDAC). To confirm these findings in humans, we used microfluidic geometrically enhanced immunocapture to detect circulating pancreas epithelial cells (CECs) in patient blood samples. We captured >3 CECs/ml in 7 of 21 (33%) of patients with cystic lesions and no clinical diagnosis of cancer (Sendai criteria negative), 8 of 11 (73%) with PDAC, and in 0 of 19 patients without cysts or cancer (controls). These findings indicate that cancer cells are present in the circulation of patients before tumors develop, which might be used in risk assessment.

We have developed and optimized a microfluidic device platform for the capture and analysis of circulating pancreatic cells (CPCs) and pancreatic circulating tumor cells (CTCs). Our platform uses parallel anti-EpCAM and cancer-specific mucin 1 (MUC1) immunocapture in a silicon microdevice. Using a combination of anti-EpCAM and anti-MUC1 capture in a single device, we are able to achieve efficient capture while extending immunocapture beyond single marker recognition. We also have detected a known oncogenic KRAS mutation in cells spiked in whole blood using immunocapture, RNA extraction, RT-PCR and Sanger sequencing. To allow for downstream single-cell genetic analysis, intact nuclei were released from captured cells by using targeted membrane lysis. We have developed a staining protocol for clinical samples, including standard CTC markers; DAPI, cytokeratin (CK) and CD45, and a novel marker of carcinogenesis in CPCs, mucin 4 (MUC4). We have also demonstrated a semi-automated approach to image analysis and CPC identification, suitable for clinical hypothesis generation. Initial results from immunocapture of a clinical pancreatic cancer patient sample show that parallel capture may capture more of the heterogeneity of the CPC population. With this platform, we aim to develop a diagnostic biomarker for early pancreatic carcinogenesis and patient risk stratification.

Current microfluidic techniques for isolating circulating tumor cells (CTCs) from cancer patient blood are limited by low capture purity, and dielectrophoresis (DEP) has the potential to complement existing immunocapture techniques to improve capture performance. We present a hybrid DEP and immunocapture Hele-Shaw flow cell to characterize DEP's effects on immunocapture of pancreatic cancer cells (Capan-1, PANC-1, and BxPC-3) and peripheral blood mononuclear cells (PBMCs) with an anti-EpCAM (epithelial cell adhesion molecule) antibody. By carefully specifying the applied electric field frequency, we demonstrate that pancreatic cancer cells are attracted to immunocapture surfaces by positive DEP whereas PBMCs are repelled by negative DEP. Using an exponential capture model to interpret our capture data, we show that immunocapture performance is dependent on the applied DEP force sign and magnitude, cell surface EpCAM expression level, and shear stress experienced by cells flowing in the capture device. Our work suggests that DEP can not only repel contaminating blood cells but also enhance capture of cancer cell populations that are less likely to be captured by traditional immunocapture methods. This combination of DEP and immunocapture techniques to potentially increase CTC capture purity can facilitate subsequent biological analyses of captured CTCs and research on cancer metastasis and drug therapies. V C 2014 AIP Publishing LLC. [http://dx

There has been an association between post solid organ transplant (SOT) hypogammaglobulinemia and infections, but the benefit of immunoglobulin (Ig) replacement has been less clear. We hypothesize that (1) only a subset of patients with post-SOT hypogammaglobulinemia have impaired antibody responses; (2) empiric Ig replacement before checking antibody responses may commit some patients to unnecessary therapy; and (3) patients with impaired antibody responses have fewer infections after replacement. METHODS: Epic Slicer Dicer was used to retrospectively identify patients with lung or kidney transplants evaluated by the Immunodeficiency Clinic at Johns Hopkins (2010-2019) for hypogammaglobulinemia (n5 59). Pneumococcal conjugate vaccine challenge titers to assess antibody responses were obtained. If titers revealed protection to fewer than 6/14 serotypes (cutoff 1.0 ug/ml) then patients were started on replacement. Data on severity, type, frequency of infections was collected. RESULTS: 27/36 SOT patients with hypogammaglobulinemia (median Ig level: 445 mg/dl, range: 199-634 mg/dl) had impaired antibody responses, and started replacement. They had a mean of 2.18 infections/year and median of 1 infection/year before replacement. After replacement they had a mean of 1.08 infections/year and median of 1 infection/year. 9/36 SOT patients with hypogammaglobulinemia (median Ig level: 458 mg/dl, range 354-511 mg/dl) had normal antibody responses and did not start replacement. They had a mean of 1.22 infections/year and median of 1 infection/year. CONCLUSIONS: Only a subset of patients with post-SOT hypogammaglobulinemia have evidence of impaired humoral response necessitating Ig replacement. This parameter can help identify patients who may benefit most from Ig replacement to reduce infectious complications.