Makrigiorgos, Gerassimos

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Makrigiorgos, Gerassimos

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Makrigiorgos

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Gerassimos

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Makrigiorgos, Gerassimos

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Image-guided radiotherapy platform using single nodule conditional lung cancer mouse models
(2014) Herter-Sprie, Grit S.; Korideck, Houari; Christensen, Camilla L.; Herter, Jan M.; Rhee, Kevin; Berbeco, Ross; Bennett, David G.; Akbay, Esra A.; Kozono, David; Mak, Raymond; Makrigiorgos, Gerassimos; Kimmelman, Alec C.; Wong, Kwok-Kin
Close resemblance of murine and human trials is essential to achieve the best predictive value of animal-based translational cancer research. Kras-driven genetically engineered mouse models of non-small cell lung cancer faithfully predict the response of human lung cancers to systemic chemotherapy. Due to development of multifocal disease, however, these models have not been usable in studies of outcomes following focal radiotherapy (RT). We report the development of a preclinical platform to deliver state-of-the-art image-guided RT in these models. Presence of a single tumour as usually diagnosed in patients is modelled by confined injection of adenoviral Cre recombinase. Furthermore, three-dimensional conformal planning and state-of-the-art image-guided dose delivery are performed as in humans. We evaluate treatment efficacies of two different radiation regimens and find that Kras-driven tumours can temporarily be stabilized upon RT, whereas additional loss of either Lkb1 or p53 renders these lesions less responsive to RT.
DISSECT Method Using PNA-LNA Clamp Improves Detection of EGFR T790m Mutation
(Public Library of Science, 2013) Guha, Minakshi; Castellanos-Rizaldos, Elena; Makrigiorgos, Gerassimos
Non-small cell lung cancer (NSCLC) patients treated with small molecule EGFR inhibitors, such as gefitinib, frequently develop drug resistance due to the presence of secondary mutations like the T790M mutation on EGFR exon 20. These mutations may originate from small subclonal populations in the primary tumor that become dominant later on during treatment. In order to detect these low-level DNA variations in the primary tumor or to monitor their progress in plasma, it is important to apply reliable and sensitive mutation detection methods. Here, we combine two recently developed methodologies, Differential Strand Separation at Critical Temperature (DISSECT), with peptide nucleic acid-locked nucleic acid (PNA-LNA) polymerase chain reaction (PCR) for the detection of T790M EGFR mutation. DISSECT pre-enriches low-abundance T790M EGFR mutations from target DNA prior to implementing PNA-LNA PCR, a method that can detect 1 mutant allele in a background of 100–1000 wild type alleles. The combination of DISSECT and PNA-LNA PCR enables the detection of 1 mutant allele in a background of 10,000 wild type alleles. The combined DISSECT-PNA-LNA PCR methodology is amenable to adaptation for the sensitive detection of additional emerging resistance mutations in cancer.
COLD-PCR Amplification of Bisulfite-Converted DNA Allows the Enrichment and Sequencing of Rare Un-Methylated Genomic Regions
(Public Library of Science, 2014) Castellanos-Rizaldos, Elena; Milbury, Coren A.; Karatza, Elli; Chen, Clark C.; Makrigiorgos, Gerassimos; Merewood, Anne
Aberrant hypo-methylation of DNA is evident in a range of human diseases including cancer and diabetes. Development of sensitive assays capable of detecting traces of un-methylated DNA within methylated samples can be useful in several situations. Here we describe a new approach, fast-COLD-MS-PCR, which amplifies preferentially un-methylated DNA sequences. By employing an appropriate denaturation temperature during PCR of bi-sulfite converted DNA, fast-COLD-MS-PCR enriches un-methylated DNA and enables differential melting analysis or bisulfite sequencing. Using methylation on the MGMT gene promoter as a model, it is shown that serial dilutions of controlled methylation samples lead to the reliable sequencing of un-methylated sequences down to 0.05% un-methylated-to-methylated DNA. Screening of clinical glioma tumor and infant blood samples demonstrated that the degree of enrichment of un-methylated over methylated DNA can be modulated by the choice of denaturation temperature, providing a convenient method for analysis of partially methylated DNA or for revealing and sequencing traces of un-methylated DNA. Fast-COLD-MS-PCR can be useful for the detection of loss of methylation/imprinting in cancer, diabetes or diet-related methylation changes.
Methylation-sensitive enrichment of minor DNA alleles using a double-strand DNA-specific nuclease
(Oxford University Press, 2017) Liu, Yibin; Song, Chen; Ladas, Ioannis; Fitarelli-Kiehl, Mariana; Makrigiorgos, Gerassimos
Abstract Aberrant methylation changes, often present in a minor allelic fraction in clinical samples such as plasma-circulating DNA (cfDNA), are potentially powerful prognostic and predictive biomarkers in human disease including cancer. We report on a novel, highly-multiplexed approach to facilitate analysis of clinically useful methylation changes in minor DNA populations. Methylation Specific Nuclease-assisted Minor-allele Enrichment (MS-NaME) employs a double-strand-specific DNA nuclease (DSN) to remove excess DNA with normal methylation patterns. The technique utilizes oligonucleotide-probes that direct DSN activity to multiple targets in bisulfite-treated DNA, simultaneously. Oligonucleotide probes targeting unmethylated sequences generate local double stranded regions resulting to digestion of unmethylated targets, and leaving methylated targets intact; and vice versa. Subsequent amplification of the targeted regions results in enrichment of the targeted methylated or unmethylated minority-epigenetic-alleles. We validate MS-NaME by demonstrating enrichment of RARb2, ATM, MGMT and GSTP1 promoters in multiplexed MS-NaME reactions (177-plex) using dilutions of methylated/unmethylated DNA and in DNA from clinical lung cancer samples and matched normal tissue. MS-NaME is a highly scalable single-step approach performed at the genomic DNA level in solution that combines with most downstream detection technologies including Sanger sequencing, methylation-sensitive-high-resolution melting (MS-HRM) and methylation-specific-Taqman-based-digital-PCR (digital Methylight) to boost detection of low-level aberrant methylation-changes.
Elimination of unaltered DNA in mixed clinical samples via nuclease-assisted minor-allele enrichment
(Oxford University Press, 2016) Song, Chen; Liu, Yibin; Fontana, Rachel; Makrigiorgos, Alexander; Mamon, Harvey; Kulke, Matthew; Makrigiorgos, Gerassimos
Presence of excess unaltered, wild-type (WT) DNA providing no information of biological or clinical value often masks rare alterations containing diagnostic or therapeutic clues in cancer, prenatal diagnosis, infectious diseases or organ transplantation. With the surge of high-throughput technologies there is a growing demand for removing unaltered DNA over large pools-of-sequences. Here we present nuclease-assisted minor-allele enrichment with probe-overlap (NaME-PrO), a single-step approach with broad genome coverage that can remove WT-DNA from numerous sequences simultaneously, prior to genomic analysis. NaME-PrO employs a double-strand-DNA-specific nuclease and overlapping oligonucleotide-probes interrogating WT-DNA targets and guiding nuclease digestion to these sites. Mutation-containing DNA creates probe-DNA mismatches that inhibit digestion, thus subsequent DNA-amplification magnifies DNA-alterations at all selected targets. We demonstrate several-hundred-fold mutation enrichment in diverse human samples on multiple clinically relevant targets including tumor samples and circulating DNA in 50-plex reactions. Enrichment enables routine mutation detection at 0.01% abundance while by adjusting conditions it is possible to sequence mutations down to 0.00003% abundance, or to scan tumor-suppressor genes for rare mutations. NaME-PrO introduces a simple and highly parallel process to remove un-informative DNA sequences and unmask clinically and biologically useful alterations.
Metastasis-associated MCL1 and P16 copy number alterations dictate resistance to vemurafenib in a BRAFV600E patient-derived papillary thyroid carcinoma preclinical model
(Impact Journals LLC, 2015) Duquette, Mark; Sadow, Peter; Husain, Amjad; Sims, Jennifer N.; Antonello, Zeus A.; Fischer, Andrew H.; Song, Chen; Castellanos-Rizaldos, Elena; Makrigiorgos, Gerassimos; Kurebayashi, Junichi; Nose, Vania; Van Hummelen, Paul; Bronson, Roderick; Vinco, Michelle; Giordano, Thomas J.; Dias-Santagata, Dora; Pandolfi, Pier Paolo; Nucera, Carmelo
BRAFV600E mutation exerts an essential oncogenic function in many tumors, including papillary thyroid carcinoma (PTC). Although BRAFV600E inhibitors are available, lack of response has been frequently observed. To study the mechanism underlying intrinsic resistance to the mutant BRAFV600E selective inhibitor vemurafenib, we established short-term primary cell cultures of human metastatic/recurrent BRAFV600E-PTC, intrathyroidal BRAFV600E-PTC, and normal thyroid (NT). We also generated an early intervention model of human BRAFV600E-PTC orthotopic mouse. We find that metastatic BRAFV600E-PTC cells elicit paracrine-signaling which trigger migration of pericytes, blood endothelial cells and lymphatic endothelial cells as compared to BRAFWT-PTC cells, and show a higher rate of invasion. We further show that vemurafenib therapy significantly suppresses these aberrant functions in non-metastatic BRAFV600E-PTC cells but lesser in metastatic BRAFV600E-PTC cells as compared to vehicle treatment. These results concur with similar folds of down-regulation of tumor microenvironment–associated pro-metastatic molecules, with no effects in BRAFWT-PTC and NT cells. Our early intervention preclinical trial shows that vemurafenib delays tumor growth in the orthotopic BRAFWT/V600E-PTC mice. Importantly, we identify high copy number gain of MCL1 (chromosome 1q) and loss of CDKN2A (P16, chromosome 9p) in metastatic BRAFV600E-PTC cells which are associated with resistance to vemurafenib treatment. Critically, we demonstrate that combined vemurafenib therapy with BCL2/MCL1 inhibitor increases metastatic BRAFV600E-PTC cell death and ameliorates response to vemurafenib treatment as compared to single agent treatment. In conclusion, short-term PTC and NT cultures offer a predictive model for evaluating therapeutic response in patients with PTC. Our PTC pre-clinical model suggests that combined targeted therapy might be an important therapeutic strategy for metastatic and refractory BRAFV600E-positive PTC.
Nanoparticle Mediated Tumor Vascular Disruption: A Novel Strategy in Radiation Therapy
(American Chemical Society (ACS), 2015) Kunjachan, Sijumon; Detappe, Alexandre; Kumar, Rajiv; Ireland, Thomas; Cameron, Lisa; Biancur, Douglas; Motto-Ros, Vincent; Sancey, Lucie; Sridhar, Srinivas; Makrigiorgos, Gerassimos; Berbeco, Ross
More than 50% of all cancer patients receive radiation therapy. The clinical delivery of curative radiation dose is strictly restricted by the proximal healthy tissues. We propose a dual-targeting strategy using vessel-targeted-radiosensitizing gold nanoparticles and conformal-image guided radiation therapy to specifically amplify damage in the tumor neoendothelium. The resulting tumor vascular disruption substantially improved the therapeutic outcome and subsidized the radiation/nanoparticle toxicity, extending its utility to intransigent or nonresectable tumors that barely respond to standard therapies.
Enrichment of Mutations in Multiple DNA Sequences Using COLD-PCR in Emulsion
(Public Library of Science, 2012) Castellanos-Rizaldos, Elena; Milbury, Coren Audrey; Makrigiorgos, Gerassimos
Background: Multiplex detection of low-level mutant alleles in the presence of wild-type DNA would be useful for several fields of medicine including cancer, pre-natal diagnosis and infectious diseases. COLD-PCR is a recently developed method that enriches low-level mutations during PCR cycling, thus enhancing downstream detection without the need for special reagents or equipment. The approach relies on the differential denaturation of DNA strands which contain Tm-lowering mutations or mismatches, versus ‘homo-duplex’ wild-type DNA. Enabling multiplex-COLD-PCR that can enrich mutations in several amplicons simultaneously is desirable but technically difficult to accomplish. Here we describe the proof of principle of an emulsion-PCR based approach that demonstrates the feasibility of multiplexed-COLD-PCR within a single tube, using commercially available mutated cell lines. This method works best with short amplicons; therefore, it could potentially be used on highly fragmented samples obtained from biological material or FFPE specimens. Methods: Following a multiplex pre-amplification of TP53 exons from genomic DNA, emulsions which incorporate the multiplex product, PCR reagents and primers specific for a given TP53 exon are prepared. Emulsions with different TP53 targets are then combined in a single tube and a fast-COLD-PCR program that gradually ramps up the denaturation temperature over several PCR cycles is applied (temperature-tolerant, TT-fast-eCOLD-PCR). The range of denaturation temperatures applied encompasses the critical denaturation temperature \((T_c)\) corresponding to all the amplicons included in the reaction, resulting to a gradual enrichment of mutations within all amplicons encompassed by emulsion. Results: Validation for TT-fast-eCOLD-PCR is provided for TP53 exons 6–9. Using dilutions of mutated cell-line into wild-type DNA, we demonstrate simultaneous mutation enrichment between 7 to 15-fold in all amplicons examined. Conclusions: TT-fast-eCOLD-PCR expands the versatility of COLD-PCR and enables high-throughput enrichment of low-level mutant alleles over multiple sequences in a single tube.
Differential strand separation at critical temperature: A minimally disruptive enrichment method for low-abundance unknown DNA mutations
(Oxford University Press, 2012) Guha, Minakshi; Castellanos-Rizaldos, Elena; Liu, Pingfang; Mamon, Harvey; Makrigiorgos, Gerassimos
Detection of low-level DNA variations in the presence of wild-type DNA is important in several fields of medicine, including cancer, prenatal diagnosis and infectious diseases. PCR-based methods to enrich mutations during amplification have limited multiplexing capability, are mostly restricted to known mutations and are prone to polymerase or mis-priming errors. Here, we present Differential Strand Separation at Critical Temperature (DISSECT), a method that enriches unknown mutations of targeted DNA sequences purely based on thermal denaturation of DNA heteroduplexes without the need for enzymatic reactions. Target DNA is pre-amplified in a multiplex reaction and hybridized onto complementary probes immobilized on magnetic beads that correspond to wild-type DNA sequences. Presence of any mutation on the target DNA forms heteroduplexes that are subsequently denatured from the beads at a critical temperature and selectively separated from wild-type DNA. We demonstrate multiplexed enrichment by 100- to 400-fold for KRAS and TP53 mutations at multiple positions of the targeted sequence using two to four successive cycles of DISSECT. Cancer and plasma-circulating DNA samples containing traces of mutations undergo mutation enrichment allowing detection via Sanger sequencing or high-resolution melting. The simplicity, scalability and reliability of DISSECT make it a powerful method for mutation enrichment that integrates well with existing downstream detection methods.
FLAG Assay as a Novel Method for Real-Time Signal Generation During PCR: Application to Detection and Genotyping of KRAS Codon 12 Mutations
(Oxford University Press, 2007) Amicarelli, Giulia; Shehi, Erlet; Makrigiorgos, Gerassimos; Adlerstein, Daniel
Real-time signal generation methods for detection and characterization of low-abundance mutations in genomic DNA are powerful tools for cancer diagnosis and prognosis. Mutations in codon 12 of the oncogene KRAS, for example, are frequently found in several types of human cancers. We have developed a novel real-time PCR technology, FLAG (FLuorescent Amplicon Generation) and adapted it for simultaneously (i) amplifying mutated codon 12 KRAS sequences, (ii) monitoring in real-time the amplification and (iii) genotyping the exact nucleotide alteration. FLAG utilizes the exceptionally thermostable endonuclease PspGI for real-time signal generation by cleavage of quenched fluorophores from the 5′-end of the PCR products and, concurrently, for selecting KRAS mutations over wild type. By including peptide-nucleic-acid probes in the reaction, simultaneous genotyping is achieved that circumvents the requirement for sequencing. FLAG enables high-throughput, closed-tube KRAS mutation detection down to ∼0.1% mutant-to-wild type. The assay was validated on model systems and compared with allele-specific PCR sequencing for screening 27 cancer specimens. Diverse applications of FLAG for real-time PCR or genotyping applications in cancer, virology or infectious diseases are envisioned.