Aviad Zick

Identification of tissue-specific cell death using methylation patterns of circulating DNA

Significance We describe a blood test for detection of cell death in specific tissues based on two principles: (i) dying cells release fragmented DNA to the circulation, and (ii) each cell type has a unique DNA methylation pattern. We have identified tissue-specific DNA methylation markers and developed a method for sensitive detection of these markers in plasma or serum. We demonstrate the utility of the method for identification of pancreatic β-cell death in type 1 diabetes, oligodendrocyte death in relapsing multiple sclerosis, brain cell death in patients after traumatic or ischemic brain damage, and exocrine pancreas cell death in pancreatic cancer or pancreatitis. The approach allows minimally invasive monitoring of tissue dynamics in humans in multiple physiological and pathological conditions. Minimally invasive detection of cell death could prove an invaluable resource in many physiologic and pathologic situations. Cell-free circulating DNA (cfDNA) released from dying cells is emerging as a diagnostic tool for monitoring cancer dynamics and graft failure. However, existing methods rely on differences in DNA sequences in source tissues, so that cell death cannot be identified in tissues with a normal genome. We developed a method of detecting tissue-specific cell death in humans based on tissue-specific methylation patterns in cfDNA. We interrogated tissue-specific methylome databases to identify cell type-specific DNA methylation signatures and developed a method to detect these signatures in mixed DNA samples. We isolated cfDNA from plasma or serum of donors, treated the cfDNA with bisulfite, PCR-amplified the cfDNA, and sequenced it to quantify cfDNA carrying the methylation markers of the cell type of interest. Pancreatic β-cell DNA was identified in the circulation of patients with recently diagnosed type-1 diabetes and islet-graft recipients; oligodendrocyte DNA was identified in patients with relapsing multiple sclerosis; neuronal/glial DNA was identified in patients after traumatic brain injury or cardiac arrest; and exocrine pancreas DNA was identified in patients with pancreatic cancer or pancreatitis. This proof-of-concept study demonstrates that the tissue origins of cfDNA and thus the rate of death of specific cell types can be determined in humans. The approach can be adapted to identify cfDNA derived from any cell type in the body, offering a minimally invasive window for diagnosing and monitoring a broad spectrum of human pathologies as well as providing a better understanding of normal tissue dynamics.

Trypanosomes Have Six Mitochondrial DNA Helicases, with One Controlling Kinetoplast Maxicircle Replication

Kinetoplast DNA (kDNA), the trypanosome mitochondrial DNA, contains thousands of minicircles and dozens of maxicircles interlocked in a giant network. Remarkably, Trypanosoma bruceis genome encodes 8 PIF1-like helicases, 6 of which are mitochondrial. We now show that TbPIF2 is essential for maxicircle replication. Maxicircle abundance is controlled by TbPIF2 level, as RNAi of this helicase caused maxicircle loss, and its overexpression caused a 3- to 6-fold increase in maxicircle abundance. This regulation of maxicircle level is mediated by the TbHslVU protease. Previous experiments demonstrated that RNAi knockdown of TbHslVU dramatically increased abundance of minicircles and maxicircles, presumably because a positive regulator of their synthesis escaped proteolysis and allowed synthesis to continue. Here, we found that TbPIF2 level increases following RNAi of the protease. Therefore, this helicase is a TbHslVU substrate and an example of a positive regulator, thus providing a molecular mechanism for controlling maxicircle replication.

Assigning functions to genes: identification of S-phase expressed genes in Leishmania major based on post-transcriptional control elements

Assigning functions to genes is one of the major challenges of the post-genomic era. Usually, functions are assigned based on similarity of the coding sequences to sequences of known genes, or by identification of transcriptional cis-regulatory elements that are known to be associated with specific pathways or conditions. In trypanosomatids, where regulation of gene expression takes place mainly at the post-transcriptional level, new approaches for function assignment are needed. Here we demonstrate the identification of novel S-phase expressed genes in Leishmania major, based on a post-transcriptional control element that was recognized in Crithidia fasciculata as involved in the cell cycle-dependent expression of several nuclear and mitochondrial S-phase expressed genes. Hypothesizing that a similar regulatory mechanism is manifested in L.major, we have applied a computational search for similar control elements in the genome of L.major. Our computational scan yielded 132 genes, of which 33% are homologues of known DNA metabolism genes and 63% lack any annotation. Experimental testing of seven of these genes revealed that their mRNAs cycle throughout the cell cycle, reaching a maximum level during S-phase or just prior to it. It is suggested that screening for post-transcriptional control elements associated with a specific function provides an efficient method for assigning functions to trypanosomatid genes.

Unique Characteristics of the Kinetoplast DNA Replication Machinery Provide Potential Drug Targets in Trypanosomatids

Kinetoplast DNA (kDNA) is a remarkable DNA structure found in the single mitohondrion of flagellated protozoa of the order Kinetoplastida. In various parasitic species of the family Trypanosomatidae, it consists of 5,000-10,000 duplex DNA minicircles (0.5-10 kb) and 25-50 maxicircles (20-40 kb), which are linked topologically into a two dimensional DNA network. Maxicircles encode for typical mitochondrial proteins and ribosomal RNA, whereas minicircles encode for guide RNA (gRNA) molecules that function in the editing of maxicircles’ mRNA transcripts. The replication of kDNA includes the duplication of free detached minicircles and catenated maxicircles, and the generation of two progeny kDNA networks. It is catalyzed by an enzymatic machinery, consisting of kDNA replication proteins that are located at defined sites flanking the kDNA disk in the mitochondrial matrix (for recent reviews on kDNA see refs. 1-8).

A case of metastatic adamantinoma responding to treatment with pazopanib.

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Treatment inferred from mutations identified using massive parallel sequencing leads to clinical benefit in some heavily pretreated cancer patients

Abstract Molecular portraits of numerous tumors have flooded oncologists with vast amounts of data. In parallel, effective inhibitors of central pathways have shown great clinical benefit. Together, this promises potential clinical benefits to otherwise end-stage cancer patients. Here, we report a clinical service offering mutation detection of archived samples using the ion Ampliseq cancer panel coupled with clinical consultation. A multidisciplinary think tank consisting of oncologists, molecular-biologists, genetic counselors, and pathologists discussed 67 heavily pretreated, advanced cancer patient cases, taking into account mutations identified using ion Ampliseq cancer panel, medical history, and relevant literature. The team generated a treatment plan, targeting specific mutations, for 41 out of 64 cases. Three patients died before results were available. For 32 patients, the treating oncologists chose not to include the panel recommendation in the treatment plan for various reasons. Nine patients were treated as recommended by the panel, 5 with clinical benefit, and 4 with disease progression. This study suggests that routine use of massive parallel tumor sequencing is feasible and can judiciously affect treatment decisions when coupled with multidisciplinary team-based decision making. Administration of personalized based therapies at an earlier stage of disease, expansion of genetic alterations examined, and increased availability of targeted therapies may lead to further improvement in the clinical outcome of metastatic cancer patients.

Comprehensive human cell-type methylation atlas reveals origins of circulating cell-free DNA in health and disease

Methylation patterns of circulating cell-free DNA (cfDNA) contain rich information about recent cell death events in the body. Here, we present an approach for unbiased determination of the tissue origins of cfDNA, using a reference methylation atlas of 25 human tissues and cell types. The method is validated using in silico simulations as well as in vitro mixes of DNA from different tissue sources at known proportions. We show that plasma cfDNA of healthy donors originates from white blood cells (55%), erythrocyte progenitors (30%), vascular endothelial cells (10%) and hepatocytes (1%). Deconvolution of cfDNA from patients reveals tissue contributions that agree with clinical findings in sepsis, islet transplantation, cancer of the colon, lung, breast and prostate, and cancer of unknown primary. We propose a procedure which can be easily adapted to study the cellular contributors to cfDNA in many settings, opening a broad window into healthy and pathologic human tissue dynamics.

Specific detection of cell-free DNA derived from intestinal epithelial cells using methylation patterns

Epithelial cells of the intestine undergo rapid turnover and are thought to be cleared via stool. Disruption of tissue architecture, as occurs in colorectal cancer (CRC), results in the release of material from dying intestinal epithelial cells to blood. This phenomenon could be utilized for diagnosis and monitoring of intestinal diseases, if circulating cell-free DNA (cfDNA) derived from intestinal cells could be specifically identified. Here we describe two genomic loci that are unmethylated specifically in intestinal epithelial cells, allowing for sensitive and specific detection of DNA derived from such cells. As expected, intestinal DNA is found in stool, but not in plasma, of healthy individuals. Patients with inflammatory bowel disease (IBD) have minimal amounts of intestinal cfDNA in the plasma, whereas patients with advanced CRC show a strong signal. The intestinal markers are not elevated in plasma samples from patients with pancreatic ductal adenocarcinoma (PDAC), and a combination of intestine- and pancreas-specific markers allowed for robust differentiation between plasma cfDNA derived from CRC and PDAC patients. Intestinal DNA markers provide a mutation-independent tool for monitoring intestinal dynamics in health and disease.

Intratumoral immune-biomarkers and mismatch repair status in leiyomyosarcoma -potential predictive markers for adjuvant treatment: a pilot study

Leiomyosarcoma is the second most frequent soft-tissue sarcoma. Tumor lymphocytic infiltration (TIL) and programed cell death ligand-1 (PD-L1) have been associated with prognosis in different malignancies while DNA mismatch-repair deficiency (MMR-D) has been associated with response to check-point inhibitors. In this pilot study, we sought to examine TIL, PD-L1 and mismatch-repair (MMR) protein expression in 11 leiomyosarcoma and its association with outcome as potential biomarkers for adjuvant treatment. Eleven primary leiomyosarcoma archived-tissues were analyzed for expression of MMR proteins (MSH2, MLH1, MSH6 and PSM2), PD-L1 expression and PD-1, CD3 or CD8. MMR-D was detected in tumor tissue from 2/11 leiomyosarcoma patients. CD3 T-cells were present in all samples, whereas CD8 staining was positive in all but one. PDL-1 was positive in 4/11 and PD-L1 in 6/11. Interestingly, the three patients with the poorest outcome had strongly positive staining for PD-L1 and CD8 while in the two patients who are alive and recurrence-free, both PD-L1 and CD8 infiltration were lacking. We found an association between tumor infiltrating CD8 cytotoxic lymphocytes, strong PD-L1 staining and survival; suggesting a role as biomarkers for treatment decisions regarding peri-operative chemotherapy. We also identified MMR-D in two patients with leiomyosarcoma comprising 18% of our sample.

Defects in homologous recombination repair genes are associated with good prognosis and clinical sensitivity to DNA-damaging agents in pancreatic cancer: A case report

Tumor genome sequencing is important for increasing our understanding of the development of cancer, which may be affected by different therapies. In the present study, genomic evolution was investigated in a patient with stage IV pancreatic cancer bearing a germline breast cancer 2 (BRCA2) mutation. The patient received cisplatin, a DNA cross-linking agent, which led to a long-lasting complete response. Eventually the patient developed brain metastasis, suggesting the acquisition of resistance to cisplatin. He subsequently underwent brain lesion resection, radiofrequency ablation and chemotherapy, again resulting in long-lasting response. Samples of blood, pancreatic tumor tissue and brain metastases were collected and the extracted DNA was sequenced. The pancreatic and brain lesions, when compared with the blood samples, exhibited mutations in the BRCA1 and checkpoint kinase 2 genes, in addition to the germline BRCA2 mutation. The brain lesion, when compared with the primary tumor, harbored no additional mutations or copy-number variations. These findings suggest that the isolated relapse in the brain was due to pharmacological sanctuary rather than genomic alterations. It may be suggested that the presence of defects in the homologous recombination repair pathways are associated with a good prognosis and clinical sensitivity to agents that damage the DNA in pancreatic cancer.