Ultra-deep next-generation sequencing of plasma cell-free DNA in patients with advanced lung cancers: results from the Actionable Genome Consortium.

BACKGROUND: Noninvasive genotyping using plasma cell-free DNA (cfDNA) has the potential to obviate the need for some invasive biopsies in cancer patients while also elucidating disease heterogeneity. We sought to develop an ultra-deep plasma next-generation sequencing (NGS) assay for patients with non-small-cell lung cancers (NSCLC) that could detect targetable oncogenic drivers and resistance mutations in patients where tissue biopsy failed to identify an actionable alteration. PATIENTS AND METHODS: Plasma was prospectively collected from patients with advanced, progressive NSCLC. We carried out ultra-deep NGS using cfDNA extracted from plasma and matched white blood cells using a hybrid capture panel covering 37 lung cancer-related genes sequenced to 50 000× raw target coverage filtering somatic mutations attributable to clonal hematopoiesis. Clinical sensitivity and specificity for plasma detection of known oncogenic drivers were calculated and compared with tissue genotyping results. Orthogonal ddPCR validation was carried out in a subset of cases. RESULTS: In 127 assessable patients, plasma NGS detected driver mutations with variant allele fractions ranging from 0.14% to 52%. Plasma ddPCR for EGFR or KRAS mutations revealed findings nearly identical to those of plasma NGS in 21 of 22 patients, with high concordance of variant allele fraction (r = 0.98). Blinded to tissue genotype, plasma NGS sensitivity for de novo plasma detection of known oncogenic drivers was 75% (68/91). Specificity of plasma NGS in those who were driver-negative by tissue NGS was 100% (19/19). In 17 patients with tumor tissue deemed insufficient for genotyping, plasma NGS identified four KRAS mutations. In 23 EGFR mutant cases with acquired resistance to targeted therapy, plasma NGS detected potential resistance mechanisms, including EGFR T790M and C797S mutations and ERBB2 amplification. CONCLUSIONS: Ultra-deep plasma NGS with clonal hematopoiesis filtering resulted in de novo detection of targetable oncogenic drivers and resistance mechanisms in patients with NSCLC, including when tissue biopsy was inadequate for genotyping.

Plasma HPV cell-free DNA monitoring in advanced HPV-associated oropharyngeal cancer.

Background: Measuring cell-free (cf)DNA in blood and tissues holds significant potential as a minimally invasive method for disease monitoring in cancer. Cancers arising in the oropharynx and causally linked to human papillomavirus (HPV) represent an ideal model in which to interrogate these methods. Patients and methods: We designed an ultrasensitive and quantitative droplet digital (dd)PCR assay to detect the five dominant high-risk HPV subtypes linked to oropharyngeal cancer (OPC). We enrolled a pilot observational cohort of 22 patients with advanced HPV+ OPC to evaluate the clinical utility of our assay and explore its predictive and prognostic potential. Results: Total tumor burden (TTB) strongly correlated with HPV cfDNA levels (R = 0.91, P = 2.3×10-6) at this cohort size, and in most cases more distant anatomic disease locations predicted increasing HPV cfDNA levels. All participants demonstrated a corresponding change in their HPV cfDNA levels at a median of 16 days (range 12-38) before restaging scans confirming treatment response or progression. Patients with locoregional disease in the head and neck or pulmonary-only metastases had worse outcomes (P = 0.01). Both TTB and median plasma HPV cfDNA levels negatively correlated with survival (R=-0.65, P = 0.01; R=-0.48, P = 0.05, respectively). Conclusion(s): Plasma HPV cfDNA monitoring recapitulates fluctuations in disease status. While blood-based HPV DNA monitoring does not currently have a role in managing HPV+ OPC, these data speak to their broad clinical potential in an era of precision medicine.

Amplicon-based next-generation sequencing of plasma cell-free DNA for detection of driver and resistance mutations in advanced non-small cell lung cancer.

Background: Genomic analysis of plasma cell-free DNA is transforming lung cancer care; however, available assays are limited by cost, turnaround time, and imperfect accuracy. Here, we study amplicon-based plasma next-generation sequencing (NGS), rather than hybrid-capture-based plasma NGS, hypothesizing this would allow sensitive detection and monitoring of driver and resistance mutations in advanced non-small cell lung cancer (NSCLC). Patients and methods: Plasma samples from patients with NSCLC and a known targetable genotype (EGFR, ALK/ROS1, and other rare genotypes) were collected while on therapy and analyzed blinded to tumor genotype. Plasma NGS was carried out using enhanced tagged amplicon sequencing of hotspots and coding regions from 36 genes, as well as intronic coverage for detection of ALK/ROS1 fusions. Diagnostic accuracy was compared with plasma droplet digital PCR (ddPCR) and tumor genotype. Results: A total of 168 specimens from 46 patients were studied. Matched plasma NGS and ddPCR across 120 variants from 80 samples revealed high concordance of allelic fraction (R2 = 0.95). Pretreatment, sensitivity of plasma NGS for the detection of EGFR driver mutations was 100% (30/30), compared with 87% for ddPCR (26/30). A full spectrum of rare driver oncogenic mutations could be detected including sensitive detection of ALK/ROS1 fusions (8/9 detected, 89%). Studying 25 patients positive for EGFR T790M that developed resistance to osimertinib, 15 resistance mechanisms could be detected including tertiary EGFR mutations (C797S, Q791P) and mutations or amplifications of non-EGFR genes, some of which could be detected pretreatment or months before progression. Conclusions: This blinded analysis demonstrates the ability of amplicon-based plasma NGS to detect a full range of targetable genotypes in NSCLC, including fusion genes, with high accuracy. The ability of plasma NGS to detect a range of preexisting and acquired resistance mechanisms highlights its potential value as an alternative to single mutation digital PCR-based plasma assays for personalizing treatment of TKI resistance in lung cancer.

Loss of annexin 1 correlates with early onset of tumorigenesis in esophageal and prostate carcinoma.

Annexin I protein expression was evaluated in patient-matched longitudinal study sets of laser capture microdissected normal, premalignant, and invasive epithelium from human esophageal squamous cell cancer and prostatic adenocarcinoma. In 25 esophageal cases (20 by Western blot and 5 by immunohistochemistry) and 17 prostate cases (3 by Western blot and 14 by immunohistochemistry), both tumor types showed either complete loss or a dramatic reduction in the level of annexin I protein expression compared with patient-matched normal epithelium (P < or = 0.05). Moreover, by using Western blot analysis of laser capture microdissected, patient-matched longitudinal study sets of both tumor types, the loss of protein expression occurred in premalignant lesions. Concordance of this result with immunohistochemical analysis suggests that annexin I may be an essential component for maintenance of the normal epithelial phenotype. Additional studies investigating the mechanism(s) and functional consequences of annexin I protein loss in tumor cells are warranted.

Reverse phase protein microarrays which capture disease progression show activation of pro-survival pathways at the cancer invasion front.

Protein arrays are described for screening of molecular markers and pathway targets in patient matched human tissue during disease progression. In contrast to previous protein arrays that immobilize the probe, our reverse phase protein array immobilizes the whole repertoire of patient proteins that represent the state of individual tissue cell populations undergoing disease transitions. A high degree of sensitivity, precision and linearity was achieved, making it possible to quantify the phosphorylated status of signal proteins in human tissue cell subpopulations. Using this novel protein microarray we have longitudinally analysed the state of pro-survival checkpoint proteins at the microscopic transition stage from patient matched histologically normal prostate epithelium to prostate intraepithelial neoplasia (PIN) and then to invasive prostate cancer. Cancer progression was associated with increased phosphorylation of Akt (P<0.04), suppression of apoptosis pathways (P<0.03), as well as decreased phosphorylation of ERK (P<0.01). At the transition from histologically normal epithelium to PIN we observed a statistically significant surge in phosphorylated Akt (P<0.03) and a concomitant suppression of downstream apoptosis pathways which proceeds the transition into invasive carcinoma.

Characterization of intracellular prostate-specific antigen from laser capture microdissected benign and malignant prostatic epithelium.

The proportion of unbound serum prostate-specific antigen (PSA; percent-free PSA) is reported to be lower in men with prostate cancer compared to men with benign prostates (U. H. Stenman et al., Cancer Res., 51: 222-226, 1991; H. Lilja et al., Clin. Chem., 37: 1618-1625, 1991; D. L. Woodrum et al., J. Urol., 159: 5-12, 1998; W. J. Catalona et al., J. Am. Med. Assoc., 279: 1542-1547, 1998). The majority of immunoreactive PSA in serum is complexed to alpha-1-antichymotrypsin (ACT). Two major mechanistic questions have previously been unknown: (a) Does PSA in human prostate cancer cells in tissue exist in a free or bound form? and (b) Is PSA produced by malignant cells in the free form because it has lost the ability to form a complex with ACT? Laser capture microdissection (LCM) enables the acquisition of pure populations of defined cell types from tissue (M. R. Emmert-Buck et al., Science, 274: 998-1001, 1996; R. F. Bonner et al., Science, 278: 1481-1483, 1997). This technology provides a unique opportunity to study intracellular protein composition and structure from human cells. In this study, we used LCM to assess the bound versus free form of intracellular PSA in both benign and malignant epithelium procured from prostate tissue. One-dimensional and two-dimensional PAGE were performed on cellular lysates from LCM-procured benign and malignant prostate epithelium from frozen tissue specimens. Western blotting analysis of one-dimensional PAGE gels revealed a strong band at M(r) 30,000 (expected molecular weight of unbound PSA) in all cases demonstrating that the vast majority of intracellular tumor and normal PSA exists within cells in the "free" form. Binding studies showed that PSA recovered from LCM-procured cells retained the full ability to bind ACT, and two-dimensional PAGE Western analysis demonstrated that the PSA/ACT complex was stable under strong reducing conditions. We conclude that intracellular PSA exists in the "free" form and that binding to ACT occurs exclusively outside of the cell.

Proteomic patterns of nipple aspirate fluids obtained by SELDI-TOF: potential for new biomarkers to aid in the diagnosis of breast cancer.

Nipple aspirate fluid (NAF) has been used for many years as a potential non-invasive method to identify markers for breast cancer risk or early detection. Because individual markers have not been optimal, we are exploring the use of surface enhanced laser desorption and ionization time of flight (SELDI-TOF) mass spectrometry to identify patterns of proteins that might define a proteomic signature for breast cancer. SELDI-TOF was used to analyze a study set of NAF samples that included 12 women with breast cancer and 15 healthy controls (the latter included three women with an abnormal mammogram but subsequent normal biopsy). In this preliminary report, we present data showing that SELDI analysis of NAF is rapid, reproducible, and capable of identifying protein signatures that appear to differentiate NAF samples from breast cancer patients and healthy controls, including those with an abnormal mammogram who were later proven to be biopsy normal.

Laser capture microdissection: beyond functional genomics to proteomics.

Proteomics will drive biology and medicine beyond genomics, and can have a profound impact on molecular diagnostics. The posttranslational modifications of cellular proteins that govern physiology and become deranged in disease cannot be accurately portrayed by gene expression alone. Consequently, new technology is being developed to discover, and quantitatively monitor, proteomic changes that are associated with disease etiology and progression. In the past, proteomic technologies were restricted to tumor cell lines or homogenized bulk tissue specimens. This source material may not accurately reflect molecular events taking place in the specific cells of the tissue itself. This article describes a completely new class of proteomic-based approaches aimed at the identification and investigation of protein markers in the actual histologically defined cell populations that are immersed in heterogeneous diseased tissue. It is envisioned that these investigations will eventually lead to novel diagnostic, prognostic, or therapeutic markers that can be applied to monitor therapeutic toxicity or efficacy.

Micro RT-PCR.

This unit presents a collection of protocols for the microisolation, manipulation, and amplification of the RNA content of microdissected cells. Even though emphasis in these protocols is given for microdissected cells, these protocols have successfully been used for bulk tissue (i.e., less than 10 microg).

Laser capture microdissection.

This unit describes laser capture microdissection (LCM) using the Pixcell II as a technique to provide the scientific community with the opportunity to perform molecular analyses on pure cell populations procured directly from tissues. After identifying specific cells of interest, the cells are captured by firing a near infrared laser through a thermaplastic polymer film that rests on top of the cells. The cells are then ready for molecular analyses.

New technologies for biomarker analysis of prostate cancer progression: Laser capture microdissection and tissue proteomics.

The widespread use of serum markers during cancer screenings has led to the belief that there may be tumor markers yet to be discovered that offer better specificity and sensitivity than prostate-specific antigen (PSA). Proteomics, the analysis and characterization of global protein modifications, will add to our understanding of gene function and aid in biomarker and/or therapeutic target discovery. In the past, most proteomic studies were either performed using tumor cell lines or homogenized bulk tissue. Unfortunately, these approaches may not accurately reflect molecular events that take place in the actual ductal epithelium that change as a consequence of the malignant process. This report describes alternative proteomic-based approaches aimed at the identification of protein markers in the actual premalignant and frankly malignant epithelium.

New approaches to proteomic analysis of breast cancer.

Proteomic based approaches are beginning to be utilized to study the natural history and treatment of breast cancer. A variety of proteomics approaches are under study, and are summarized herein. Two-dimensional gel electrophoresis (2D-PAGE) is still the foundation of most proteomics studies. We present an analysis of 2D-PAGE studies reported to date in breast cancer, including those examining normal/tumor differences and selected populations of breast cells. Newer technologies such as laser capture microdissection and highly sensitive mass spectrometry methods are currently being used together to identify greater numbers of lower abundance proteins that are differentially expressed between defined cell populations. Novel technologies still in developmental phases will enable identification of validated targets in small biopsy specimens, including high density protein arrays, antibody arrays and lysate arrays. Surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) analysis enables the high throughput characterization of lysates from very few tumor cells and may be best suited for clinical biomarker studies. We present SELDI-TOF data herein to show the accuracy of the method in a small cohort of breast tumors, as well as its potential discriminatory capability. Such technologies are expected to supplement our armamentarium of mRNA-based assays, and provide critical information on protein levels and post-translational modifications.

Proteomic analysis of laser capture microdissected human prostate cancer and in vitro prostate cell lines.

Specific populations of normal and malignant epithelium from three radical prostatectomy tissue specimens were procured by laser capture microdissection (LCM) and analyzed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Six proteins that were only seen in malignant cells and two proteins that were only seen in benign epithelium were reproducibly observed in two of two cases examined. Furthermore, these proteins were not observed in the 2-D PAGE profiles from the patient-matched microdissected stromal cell populations, but were seen in the protein profiles from the undissected whole cryostat sections. One of these proteins was determined to be prostate-specific antigen (PSA) by Western blot analysis, and intriguingly the remaining protein candidates were found to be at least as abundant as the PSA protein. Comparison of 2-D PAGE profiles of microdissected cell with matched in vitro cell lines from the same patient, and metastatic prostate cancer cell lines (LnCaP and PC3) showed striking differences between prostate cells in vivo and in vitro with less than 20% shared proteins. The data demonstrate that 2-D PAGE analysis of LCM-derived cells can reliably detect alterations in protein expression associated with prostate cancer, and that these differentially expressed proteins are produced in high enough levels which could allow for their clinical utility as new targets for therapeutic intervention, serum markers, and/or imaging markers.

An approach to proteomic analysis of human tumors.

A strategy for proteomic analysis of microdissected cells derived from human tumor specimens is described and demonstrated by using esophageal cancer as an example. Normal squamous epithelium and corresponding tumor cells from two patients were procured by laser-capture microdissection and studied by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Fifty thousand cells resolved approximately 675 distinct proteins (or isoforms) with molecular weights ranging between 10 and 200 kDa and isoelectric points of pH 3-10. Comparison of the microdissected protein profiles showed a high degree of similarity between the matched normal-tumor samples (98% identical). However, 17 proteins showed tumor-specific alterations, including 10 that were uniquely present in the tumors and seven that were observed only in the normal epithelium. Two of the altered proteins were characterized by mass spectrometry and immunoblot analysis and were identified as cytokeratin 1 and annexin I. Acquisition of 2D-PAGE protein profiles, visualization of disregulated proteins, and subsequent determination of the identity of selected proteins through high-sensitivity MS-MS microsequencing are possible from microdissected cell populations. These separation and analytical techniques are uniquely capable of detecting tumor-specific alterations. Continued refinement of techniques and methodologies to determine the abundance and status of proteins in vivo holds great promise for future study of normal cells and associated neoplasms. Mol.