Whole Exome Sequencing of Cell-Free DNA for Early Lung Cancer: A Pilot Study to Differentiate Benign From Malignant CT-Detected Pulmonary Lesions

Introduction: Indeterminate pulmonary lesions (IPL) detected by CT pose a significant clinical challenge, frequently necessitating long-term surveillance or biopsy for diagnosis. In this pilot investigation, we performed whole exome sequencing (WES) of plasma cell free (cfDNA) and matched germline DNA in patients with CT-detected pulmonary lesions to determine the feasibility of somatic cfDNA mutations to differentiate benign from malignant pulmonary nodules.Methods: 33 patients with a CT-detected pulmonary lesions were retrospectively enrolled (n = 16 with a benign nodule, n = 17 with a malignant nodule). Following isolation and amplification of plasma cfDNA and matched peripheral blood mononuclear cells (PBMC) from patient blood samples, WES of cfDNA and PBMC DNA was performed. After genomic alignment and filtering, we looked for lung-cancer associated driver mutations and next identified high-confidence somatic variants in both groups.Results: Somatic cfDNA mutations were observed in both groups, with the cancer group demonstrating more variants than the benign group (1083 ± 476 versus 553 ± 519, p < 0.0046). By selecting variants present in >2 cancer patients and not the benign group, we accurately identified 82% (14/17) of cancer patients.Conclusions: This study suggests a potential role for cfDNA for the early identification of lung cancer in patients with CT-detected pulmonary lesions. Importantly, a substantial number of somatic variants in healthy patients with benign pulmonary nodules were also found. Such “benign” variants, while largely unexplored to date, have widespread relevance to all liquid biopsies if cfDNA is to be used accurately for cancer detection

Identification of Potential Prognostic Biomarkers in Patients with Untreated, Advanced Pancreatic Cancer from a Phase III Trial (CALGB 80303)

Patients with advanced stage adenocarcinoma of the pancreas have a poor prognosis. The identification of prognostic and/or predictive biomarkers may help stratify patients so that therapy can be individualized

Characterising phase variations in MALDI-TOF data and correcting

Abstract: The use of MALDI-TOF mass spectrometry as a means of analyzing the proteome has been evaluated extensively in recent years. One of the limitations of this technique that has impeded the development of robust data analysis algorithms is the variability in the location of protein ion signals along the x-axis. We studied technical variations of MALDI-TOF measurements in the context of proteomics profiling. By acquiring a benchmark data set with five replicates, we estimated 76% to 85% of the total variance is due to phase variation. We devised a lobster plot, so named because of the resemblance to a lobster claw, to help detect the phase variation in replicates. We also investigated a peak alignment algorithm to remove the phase variation. This operation is analogous to the normalization step in microarray data analysis. Only after this critical step can features of biological interest be clearly revealed. With the help of principal component analysis, we demonstrated that after peak alignment, the differences among replicates are reduced. We compared this approach to peak alignment with a model-based calibration approach in which there was known information about peaks in common among all spectra. Finally, we examined the potential value at each point in an analysis pipeline of having a set of methods available that includes parametric, semiparametric and nonparametric methods; among such methods are those that benefit from the use of prior information

Enhanced CDC of B cell chronic lymphocytic leukemia cells mediated by rituximab combined with a novel anti-complement factor H antibody

<div><p>Rituximab therapy for B cell chronic lymphocytic leukemia (B-CLL) has met with mixed success. Among several factors to which resistance can be attributed is failure to activate complement dependent cytotoxicity (CDC) due to protective complement regulatory proteins, including the soluble regulator complement factor H (CFH). We hypothesized that rituximab killing of non-responsive B-CLL cells could be augmented by a novel human monoclonal antibody against CFH. The B cells from 11 patients with B-CLL were tested <i>ex vivo</i> in CDC assays with combinations of CFH monoclonal antibody, rituximab, and a negative control antibody. CDC of rituximab non-responsive malignant B cells from CLL patients could in some cases be augmented by the CFH monoclonal antibody. Antibody-mediated cytotoxicity of cells was dependent upon functional complement. In one case where B-CLL cells were refractory to CDC by the combination of rituximab plus CFH monoclonal antibody, additionally neutralizing the membrane complement regulatory protein CD59 allowed CDC to occur. Inhibiting CDC regulatory proteins such as CFH holds promise for overcoming resistance to rituximab therapy in B-CLL.</p></div

A Therapeutic Antibody for Cancer, Derived from Single Human B Cells

Summary: Some patients with cancer never develop metastasis, and their host response might provide cues for innovative treatment strategies. We previously reported an association between autoantibodies against complement factor H (CFH) and early-stage lung cancer. CFH prevents complement-mediated cytotoxicity (CDC) by inhibiting formation of cell-lytic membrane attack complexes on self-surfaces. In an effort to translate these findings into a biologic therapy for cancer, we isolated and expressed DNA sequences encoding high-affinity human CFH antibodies directly from single, sorted B cells obtained from patients with the antibody. The co-crystal structure of a CFH antibody-target complex shows a conformational change in the target relative to the native structure. This recombinant CFH antibody causes complement activation and release of anaphylatoxins, promotes CDC of tumor cell lines, and inhibits tumor growth in vivo. The isolation of anti-tumor antibodies derived from single human B cells represents an alternative paradigm in antibody drug discovery. : Bushey et al. clone antibodies against complement factor H (CFH) from single human B cells. CFH protects tumor cells from complement-dependent cytotoxicity (CDC). The authors demonstrate that a recombinant CFH antibody induces CDC of tumor cells, inhibits tumor growth in vivo, and stimulates infiltration of the tumor by lymphocytes

Patient treatment history.

<p>Patient treatment history.</p

The effect of anti-CD59 antibody on CDC of RTX/CFH MAb nonresponder B-CLL cells with high mean CD59 expression levels (Patient #6).

<p>CDC reactions were carried out with NHS as a complement source. For each condition, the mean percent CDC was divided by the mean percent CDC of the αCon antibody to obtain "fold cytotoxicity". The CDC assay for the α-CFH + RTX condition was performed on a different day from the other conditions.</p

CDC of B-CLL cells.

<p>(A) CDC of B-CLL cells in the presence and absence of rituximab (RTX). B-CLL cells from each of 11 CLL patients were treated with RTX or left untreated, with NHS added as a source of complement. Mean percent CDC in the presence of RTX was divided by the mean percent CDC in the absence of RTX to obtain "fold cytotoxicity" for each patient. (B) CDC of B-CLL cells from patient 11 treated with a CFH mAb or control mAb in the presence or absence of RTX. B-CLL cells from this patient were treated with CFH mAb7968 or isotype-matched negative control antibody 7B2 with or without RTX, with NHS added as a source of complement. Mean percent CDC was divided by the mean percent CDC of the “no antibody” control to obtain "fold cytotoxicity" for each condition.</p

Patient demographics, B-CLL status, and B cell characterization.

<p>Patient demographics, B-CLL status, and B cell characterization.</p

Antibody-induced CDC of B-CLL cells from five CLL patients in the presence of PS or NHS.

<p>Panels show B-CLL cell cytotoxicity with the autologous patient serum (PS) from patients 1–5, respectively, in comparison with cytotoxicity using NHS. Cytotoxicity was induced in the presence of RTX plus the addition of either CFH mAb7968 (α-CFH) or isotype-matched negative control antibody 7B2 (α-Con). For each patient and serum type, the mean percent CDC in the presence of the αCFH antibody was divided by the mean percent CDC in the presence of the αCon antibody to obtain "fold cytotoxicity". The p values of the difference between response in PS vs. NHS are shown on the figure. The p values for fold cytotoxicity as a result of α-CFH addition compared to α-Con addition were 0.00017, 0.00060, n.s., n.s., n.s in PS, and 0.00016, 0.00021, 0.000012, 0.00001, and 0.00025 in NHS, for patients 1–5 respectively (n.s = not significant).</p