Multiplexed, single-molecule, epigenetic analysis of plasma-isolated nucleosomes for cancer diagnostics.

The analysis of cell-free DNA (cfDNA) in plasma provides information on pathological processes in the body. Blood cfDNA is in the form of nucleosomes, which maintain their tissue- and cancer-specific epigenetic state. We developed a single-molecule multiparametric assay to comprehensively profile the epigenetics of plasma-isolated nucleosomes (EPINUC), DNA methylation and cancer-specific protein biomarkers. Our system allows for high-resolution detection of six active and repressive histone modifications and their ratios and combinatorial patterns on millions of individual nucleosomes by single-molecule imaging. In addition, our system provides sensitive and quantitative data on plasma proteins, including detection of non-secreted tumor-specific proteins, such as mutant p53. EPINUC analysis of a cohort of 63 colorectal cancer, 10 pancreatic cancer and 33 healthy plasma samples detected cancer with high accuracy and sensitivity, even at early stages. Finally, combining EPINUC with direct single-molecule DNA sequencing revealed the tissue of origin of colorectal, pancreatic, lung and breast tumors. EPINUC provides multilayered information of potential clinical relevance from limited (<1 ml) liquid biopsy material.

Liquid biopsy reveals collateral tissue damage in cancer.

Cancer inflicts damage to surrounding normal tissues, which can culminate in fatal organ failure. Here, we demonstrate that cell death in organs affected by cancer can be detected by tissue-specific methylation patterns of circulating cell-free DNA (cfDNA). We detected elevated levels of hepatocyte-derived cfDNA in the plasma of patients with liver metastases originating from different primary tumors, compared with cancer patients without liver metastases. In addition, patients with localized pancreatic or colon cancer showed elevated hepatocyte cfDNA, suggesting liver damage inflicted by micrometastatic disease, by primary pancreatic tumor pressing the bile duct, or by a systemic response to the primary tumor. We also identified elevated neuron-, oligodendrocyte-, and astrocyte-derived cfDNA in a subpopulation of patients with brain metastases compared with cancer patients without brain metastasis. Cell type-specific cfDNA methylation markers enabled the identification of collateral tissue damage in cancer, revealing the presence of metastases in specific locations and potentially assisting in early cancer detection.

ChIP-seq of plasma cell-free nucleosomes identifies gene expression programs of the cells of origin.

Cell-free DNA (cfDNA) in human plasma provides access to molecular information about the pathological processes in the organs or tumors from which it originates. These DNA fragments are derived from fragmented chromatin in dying cells and retain some of the cell-of-origin histone modifications. In this study, we applied chromatin immunoprecipitation of cell-free nucleosomes carrying active chromatin modifications followed by sequencing (cfChIP-seq) to 268 human samples. In healthy donors, we identified bone marrow megakaryocytes, but not erythroblasts, as major contributors to the cfDNA pool. In patients with a range of liver diseases, we showed that we can identify pathology-related changes in hepatocyte transcriptional programs. In patients with metastatic colorectal carcinoma, we detected clinically relevant and patient-specific information, including transcriptionally active human epidermal growth factor receptor 2 (HER2) amplifications. Altogether, cfChIP-seq, using low sequencing depth, provides systemic and genome-wide information and can inform diagnosis and facilitate interrogation of physiological and pathological processes using blood samples.

Recurrent TP53 missense mutation in cancer patients of Arab descent.

Hereditary cancer comprises more than 10% of all breast cancer cases. Identification of germinal mutations enables the initiation of a preventive program that can include early detection or preventive treatment and may also have a major impact on cancer therapy. Several recurrent mutations were identified in the BRCA1/2 genes in Jewish populations however, in other ethnic groups in Israel, no recurrent mutations were identified to date. Our group established panel sequencing in cancer patients to identify recurrent, founder, and new mutations in the heterogeneous and diverse populations in Israel, We evaluated five breast cancer patients of Arab descent diagnosed with cancer before the age of 50 years and identified the previously described TP53 mutation, c.541C>T, R181C (rs587782596), in two women from unrelated Arab families. The two probands were diagnosed with breast cancer at a young age (27 and 34 years) and had significant family history spanning a wide range of tumors (breast cancer (BC), papillary thyroid cancer, glioblastoma multiform (GBM), colon cancer and leukemia). The R181C variant is expected to disrupt p53 at the ASPP2 binding domain but not the DNA binding domain and is defined by Clinvar as likely pathogenic and in HGMD as disease mutation. We further tested 85 unrelated Arab cancer patients and father of a BC carrier patient for TP53 c.541C>T using a real time polymerase chain reaction (RT-PCR) approach and identified four additional carriers, two with BC one with lung cancer, and the father of a BC carrier patient, diagnosed with GBM. Another carrier suffering from BC was identified using a Myriad panel, suggesting a recurrent mutation in this population with a frequency of 5/42 (11.9%) of our selected BC patients. We suggest testing Arab women with a breast cancer at a young age, Arab patients with multiple malignancies, or with suggestive family history for TP53 c.541C>T.

An observational cohort study of bevacizumab and chemotherapy in metastatic colorectal cancer patients: safety and efficacy with analysis by age group.

Bevacizumab improves survival when added to chemotherapy in metastatic colorectal cancer (mCRC). We assessed the safety and efficacy of bevacizumab in mCRC patients ≥70 years old (YO) vs. those <70 YO. mCRC patients treated from 2005-2012 who received chemotherapy (physician's choice) plus bevacizumab were included. The primary end point was safety; secondary objectives were progression-free survival (PFS) and overall survival (OS). Data was collected retrospectively. Three-hundred eight patients (92 ≥70 YO, 216 <70 YO) with 20.5 month median follow-up were included. Of the patients, 1.9 % died due to bevacizumab-related adverse effects; all were <70 YO. Grades 3-5 adverse events of interest for bevacizumab in patients ≥70 YO included hypertension (37.0 %), venous thromboembolism (6.5 %), wound-healing complications (5.4 %), bleeding (7.6 %), fistula (4.3 %), arterial thromboembolism (3.3 %), congestive heart failure (2.2 %), and proteinuria (grades 1-2 only, 14.1 %). Treatment was stopped due to adverse effects in 6.0 % of older patients. Older patients had significantly more ischemic heart disease and hypertension at baseline, and were treated less with FOLFOX and more with 5FU/LV monotherapy; nevertheless, OS and PFS were similar in younger and older patients. Compared to younger patients, in older patients, rates of proteinuria (all grades 1-2) were significantly higher (14.1 vs. 5.6 %, p=0.012) and rates of treatment-related hypertension (grades 3-5) were marginally higher (37 vs. 25.9 %, p=0.053); rates of other adverse events were similar in the two groups. In our patient population, bevacizumab was safe and effective in older as well as younger patients.

Hypertension as a predictive biomarker in bevacizumab treatment for colorectal cancer patients.

Bevacizumab treatment is associated with an increased risk of hypertension (HTN), a potential marker for effectiveness. We aimed to assess whether grades 2-3 HTN during bevacizumab treatment was associated with increased overall survival (OS) or progression-free survival (PFS). One hundred and eighty-one patients with metastatic colorectal cancer (CRC), who were treated in our Department from January 2009-February 2011 were included. Bevacizumab was administered jointly with standard first- or second-line chemotherapy protocols. Blood pressure was measured before each treatment. HTN was graded using common toxicity criteria. There were 181 CRC patients. Grades 2-3 HTN developed in 81 patients (44.75 %) but not in 100 patients (55.25 %); no patient developed grades 4-5 HTN. Median follow-up was 15.2 months. HTN was associated with better OS in HTN-positive versus HTN-negative patients (median not reached vs. 36.8 months, p = 0.029) and better PFS (29.9 vs. 17.2 months, p = 0.024, respectively). Bevacizumab-related HTN may represent a biomarker for clinical benefit in metastatic colorectal cancer patients.