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Hughes T.P.,SA Pathology | Hochhaus A.,Universitatsklinikum Jena | Branford S.,Molecular Pathology | Branford S.,University of Adelaide | And 13 more authors.
Blood | Year: 2010

This study examines the prognostic significance of early molecular response using an expanded dataset in chronic myeloid leukemia patients enrolled in the International Randomized Study of Interferon and STI571 (IRIS). Serial molecular studies demonstrate decreases inBCR-ABL transcripts over time. Analyses of event-free survival (EFS) and time to progression to accelerated phase/blast crisis (AP/BC) at 7 years were based on molecular responses using the international scale (IS) at 6-, 12-, and 18-month landmarks. Patients with BCR-ABL transcripts > 10% at 6 months and > 1% at 12 months had inferior EFS and higher rate of progression to AP/BC compared with all other molecular response groups. Conversely, patients who achieved major molecular response [MMR: BCR-ABL (IS) ≤ 0.1%] by 18 months enjoyed remarkably durable responses, with no progression to AP/BC and 95% EFS at 7 years. The probability of loss of complete cytogenetic response by 7 years was only 3% for patients in MMR at 18 months versus 26% for patients with complete cytogenetic response but not MMR (P < .001). This study shows a strong association between the degree to which BCR-ABL transcript numbers are reduced by therapy and long-term clinical outcome, supporting the use of time-dependent molecular measures to determine optimal response to therapy. This study is registered at www.clinicaltrials.gov as NCT00006343. © 2010 by The American Society of Hematology.


Adult cells, such as skin or blood cells, have a cellular "memory," or record of how the cell changes as it develops from an uncommitted embryonic cell into a specialized adult cell. Now, Harvard Stem Cell Institute researchers at Massachusetts General Hospital (MGH) in collaboration with scientists from the Research Institutes of Molecular Biotechnology (IMBA) and Molecular Pathology (IMP) in Vienna have identified genes that when suppressed effectively erase a cell's memory, making the cell more susceptible to reprogramming and, consequently, making the process of reprogramming quicker and more efficient. The study was recently published in Nature. "We began this work because we wanted to know why a skin cell is a skin cell, and why does it not change its identity the next day, or the next month, or a year later?" said co-senior author Konrad Hochedlinger, PhD, an HSCI Principal Faculty member at MGH and Harvard's Department of Stem Cell and Regenerative Biology, and a world expert in cellular reprogramming. Every cell in the human body has the same genome, or DNA blueprint, explained Hochedlinger, and it is how those genes are turned on and off during development that determines what kind of adult cell each will become. By manipulating those genes and introducing new factors, scientists can unlock dormant parts of an adult cell's genome and reprogram it into another cell type. However, "a skin cell knows it is a skin cell," said IMBA's Josef Penninger, even after scientists reprogram those skin cells into induced pluripotent stem cells (iPS cells) - a process that would ideally require a cell to "forget" its identity before assuming a new one. Cellular memory is often conserved, acting as a roadblock to reprogramming. "We wanted to find out which factors stabilize this memory and what mechanism prevents iPS cells from forming," Penninger said. To identify potential factors, the team established a genetic library targeting known chromatin regulators—genes that control the packaging and bookmarking of DNA, and are involved in creating cellular memory. Hochedlinger and Sihem Cheloufi, co-first author and a postdoc in Hochedlinger's lab, designed a screening approach that tested each of these factors. Of the 615 factors screened, the researchers identified four chromatin regulators, three of which had not yet been described, as potential roadblocks to reprogramming. In comparison to the three to four fold increase seen by suppressing previously known roadblock factors, inhibiting the newly described CAF1 (chromatin assembly factor 1) made the process 50 to 200 fold more efficient. Moreover, in the absence of CAF1 reprogramming turned out to be much faster: While the process normally takes nine days, the researchers could detect the first iPS cell after four days. "The CAF1 complex ensures that during DNA replication and cell division daughter cells keep their memory, which is encoded on the histones that the DNA is wrapped around," said Ulrich Elling, a co-first author from IMBA. "When we block CAF-1, daughter cells fail to wrap their DNA the same way, lose this information and covert into blank sheets of paper. In this state, they respond more sensitively to signals from the outside, meaning we can manipulate them much more easily." By suppressing CAF-1 the researchers were also able to facilitate the conversion of one type of adult cell directly into another, skipping the intermediary step of forming iPS cells, via a process called direct reprogramming, or transdifferentiation. Thus, CAF-1 appears to act as a general guardian of cell identity whose depletion facilitates both the interconversion of one adult cell type to another as well as the conversion of specialized cells into iPS cells. In finding CAF-1, the researchers identified a complex that allows cell memory to be erased and rewritten. "The cells forget who they are, making it easier to trick them into becoming another type of cell," said Sihem Cheloufi. CAF-1 may provide a general key to facilitate the "reprogramming" of cells to model disease and test therapeutic agents, IMP's Johannes Zuber explained. "The best-case scenario," Zuber said, "is that with this insight, we hold a universal key in our hands that will allow us to model cells at will." More information: Sihem Cheloufi et al. The histone chaperone CAF-1 safeguards somatic cell identity, Nature (2015). DOI: 10.1038/nature15749


News Article
Site: www.biosciencetechnology.com

They say we can’t escape our past — no matter how much we change, we still have the memory of what came before. The same can be said of our cells. Mature cells, such as skin or blood cells, have a cellular “memory,” or record of how the cell changed as it developed from an uncommitted embryonic cell into a specialized adult cell. Now, Harvard Stem Cell Institute researchers at Massachusetts General Hospital (MGH), in collaboration with scientists from the Institutes of Molecular Biotechnology (IMBA) and Molecular Pathology (IMP) in Vienna, have identified genes that, when suppressed effectively, erase a cell’s memory, making it more susceptible to reprogramming and, consequently, making the process of reprogramming quicker and more efficient. The study was recently published in Nature. “We began this work because we wanted to know why a skin cell is a skin cell, and why does it not change its identity the next day, or the next month, or a year later?” said co-senior author Konrad Hochedlinger, an HSCI principal faculty member at MGH and Harvard’s Department of Stem Cell and Regenerative Biology, and a world expert in cellular reprogramming. Every cell in the human body has the same genome, or DNA blueprint, explained Hochedlinger, and it is how those genes are turned on and off during development that determines what kind of adult cell each becomes. By manipulating those genes and introducing new factors, scientists can unlock dormant parts of an adult cell’s genome and reprogram it into another cell type. However, “a skin cell knows it is a skin cell,” said IMBA’s Josef Penninger, even after scientists reprogram those skin cells into induced pluripotent stem cells (iPS cells) — a process that would ideally require a cell to “forget” its identity before assuming a new one. Cellular memory is often conserved, acting as a roadblock to reprogramming. “We wanted to find out which factors stabilize this memory and what mechanism prevents iPS cells from forming,” Penninger said. To identify potential factors, the team established a genetic library targeting known chromatin regulators — genes that control the packaging and bookmarking of DNA, and are involved in creating cellular memory. Hochedlinger and Sihem Cheloufi, co-first author and a postdoc in Hochedlinger’s lab, designed a screening approach that tested each of these factors. Of the 615 factors screened, the researchers identified four chromatin regulators, three of which had not yet been described, as potential roadblocks to reprogramming. In comparison to the three- to fourfold increase seen by suppressing previously known roadblock factors, inhibiting the newly described chromatin assembly factor 1 (CAF1) made the process 50- to 200-fold more efficient. Moreover, in the absence of CAF1, reprogramming turned out to be much faster: While the process normally takes nine days, the researchers could detect the first iPS cell after four days. “The CAF1 complex ensures that during DNA replication and cell division, daughter cells keep their memory, which is encoded on the histones that the DNA is wrapped around,” said Ulrich Elling, a co-first author from IMBA. “When we block CAF1, daughter cells fail to wrap their DNA the same way, lose this information, and covert into blank sheets of paper. In this state, they respond more sensitively to signals from the outside, meaning we can manipulate them much more easily.” By suppressing CAF1 the researchers were also able to facilitate the conversion of one type of adult cell directly into another, skipping the intermediary step of forming iPS cells, via a process called direct reprogramming, or transdifferentiation. Thus, CAF1 appears to act as a general guardian of cell identity whose depletion facilitates both the interconversion of one adult cell type to another as well as the conversion of specialized cells into iPS cells. In finding CAF1, the researchers identified a complex that allows cell memory to be erased and rewritten. “The cells forget who they are, making it easier to trick them into becoming another type of cell,” said Cheloufi. CAF1 may provide a general key to facilitate the “reprogramming” of cells to model disease and test therapeutic agents, IMP’s Johannes Zuber explained. “The best-case scenario,” he said, “is that with this insight, we hold a universal key in our hands that will allow us to model cells at will.”


Meldrum C.,Molecular Pathology | Meldrum C.,Peter MacCallum Cancer Center | Doyle M.A.,Peter MacCallum Cancer Center | Tothill R.W.,Peter MacCallum Cancer Center
Clinical Biochemist Reviews | Year: 2011

Next-generation sequencing (NGS) is arguably one of the most significant technological advances in the biological sciences of the last 30 years. The second generation sequencing platforms have advanced rapidly to the point that several genomes can now be sequenced simultaneously in a single instrument run in under two weeks. Targeted DNA enrichment methods allow even higher genome throughput at a reduced cost per sample. Medical research has embraced the technology and the cancer field is at the forefront of these efforts given the genetic aspects of the disease. World-wide efforts to catalogue mutations in multiple cancer types are underway and this is likely to lead to new discoveries that will be translated to new diagnostic, prognostic and therapeutic targets. NGS is now maturing to the point where it is being considered by many laboratories for routine diagnostic use. The sensitivity, speed and reduced cost per sample make it a highly attractive platform compared to other sequencing modalities. Moreover, as we identify more genetic determinants of cancer there is a greater need to adopt multi-gene assays that can quickly and reliably sequence complete genes from individual patient samples. Whilst widespread and routine use of whole genome sequencing is likely to be a few years away, there are immediate opportunities to implement NGS for clinical use. Here we review the technology, methods and applications that can be immediately considered and some of the challenges that lie ahead.


Chatterjee S.,Tata Memorial Center | Malhotra R.,Tata Memorial Center | Varghese F.,Tata Memorial Center | Bukhari A.B.,Tata Memorial Center | And 5 more authors.
PLoS ONE | Year: 2013

Background: Human sodium iodide symporter (hNIS) gene over-expression is under active consideration worldwide as an alternative target molecule for breast cancer (BC) diagnosis and targeted radio-iodine treatment. However, the field demands better stratified analysis of endogenous hNIS expression across major BC subtypes. Therefore, we have analyzed subtype-specific variation of hNIS overexpression in breast tumor tissue samples by immunohistochemistry (IHC) and also report the development of a homogeneous, quantitative analysis method of digital IHC images. Methods: hNIS expression was analyzed from 108 BC tissue samples by IHC. Sub-cellular localization of hNIS protein was analyzed by dual immunofluorescence (IF) staining method using hNIS and HER2 antibodies. An ImageJ based two-step digital analysis method was developed and applied for the bias-free analysis of the images. Results: Staining of the tumor samples show 70% cases are hNIS positive indicating high incidence of hNIS positive cases in BC. More importantly, a subtype specific analysis done for the first time shows that hNIS expression is overly dominated in estrogen receptor (ER) positive cases than the receptor negative cases. Further, 56% of the ER+ve, PgR+ve, HER2-ve and 36% of ER+ve, PgR+ve, HER2+ve cases show highest intensity staining equivalent to the thyroid tissue. A significant positive correlation is also observed between hNIS and estrogen receptor expression (p = 0.0033, CI = 95%) suggesting hNIS mediated targeted radio-iodine therapy procedures may benefit both ER+ve, PgR+ve, HER2-ve as well as HER2+ve cases. Further, in a few cases, hNIS and HER2 protein localization is demonstrated by overlapping membrane co-expression. ImageJ based image analysis method shows over 70% match with manual pathological scoring method. Conclusion: The study indicates a positive link between hNIS and ER expression in BC. The quantitative IHC image analysis method reported here will further help in patient stratification and potentially benefit global clinical assessment where hNIS mediated targeted 131I radio-ablative therapy is aimed. © 2013 Chatterjee et al.

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