Establishing a diagnosis in patients suspected of having a myelodysplastic syndrome (MDS) can be challenging and could be informed by the identification of somatic mutations. We performed a ...prospective study to examine the frequency and types of mutations encountered in 144 patients with unexplained cytopenias. Based on bone marrow findings, 17% were diagnosed with MDS, 15% with idiopathic cytopenias of undetermined significance (ICUS) and some evidence of dysplasia, and 69% with ICUS and no dysplasia. Bone marrow DNA was sequenced for mutations in 22 frequently mutated myeloid malignancy genes. Somatic mutations were identified in 71% of MDS patients, 62% of patients with ICUS and some dysplasia, and 20% of ICUS patients and no dysplasia. In total, 35% of ICUS patients carried a somatic mutation or chromosomal abnormality indicative of clonal hematopoiesis. We validated these results in a cohort of 91 lower-risk MDS and 249 ICUS cases identified over a 6-month interval. Mutations were found in 79% of those with MDS, in 45% of those with ICUS with dysplasia, and in 17% of those with ICUS without dysplasia. The spectrum of mutated genes was similar with the exception of SF3B1 which was rarely mutated in patients without dysplasia. Variant allele fractions were comparable between clonal ICUS (CCUS) and MDS as were mean age and blood counts. We demonstrate that CCUS is a more frequent diagnosis than MDS in cytopenic patients. Clinical and mutational features are similar in these groups and may have diagnostic utility once outcomes in CCUS patients are better understood.
•Over 30% of patients with unexplained cytopenias who do not meet diagnostic criteria for MDS carry MDS-associated somatic mutations.•Clonal cytopenias of undetermined significance are more common than MDS and show comparable variant allele frequencies and blood counts.
We have shown previously that the hypertension-related, calcium-regulated gene (HCaRG) is involved in the control of renal cell proliferation and differentiation (Devlin AM, Solban N, Tremblay S, ...Gutkowska J, Schurch W, Orlov SN, Lewanczuk R, Hamet P, and Tremblay J. Am J Physiol Renal Physiol 284: F753-F762, 2003). To determine whether HCaRG plays a role in kidney repair after injury, we extended our studies on the cellular function of HCaRG by comparing cell migration of two kidney cell lines HEK293 and Madin-Darby canine kidney (MDCK)-C7 stably transfected with the plasmid alone or with a plasmid containing HCaRG cDNA. HCaRG-expressing HEK293 cells, which undergo lower proliferation, migrated faster than control cells and presented greater adhesiveness to the extracellular matrix. Faster migration was also observed for the MDCK-C7 cells, after they were stably transfected with HCaRG cDNA. HCaRG overexpression induced major morphological changes in HEK293 cells, including the formation of lamellipodia. Expression microarrays of HCaRG-expressing HEK293 cells revealed the elevated expression of several genes known to be involved in cell migration and lamellipodia formation, including transforming growth factor-alpha (TGF-alpha), galectins, autotaxins and fibronectin. These cells exhibited augmented synthesis and release of activated TGF-alpha. Conditioned medium from HCaRG-expressing cells stimulated the migration and induced significant morphological changes in control cells, in part, through activation of the TFG-alpha/EGF receptor. Together, these data support a role for HCaRG in kidney repair after injury through its effect on renal cell migration and TGF-alpha secretion.
Background:
Establishing the diagnosis of myelodysplastic syndromes requires excluding benign causes of cytopenias and is based largely on morphologic criteria subject to substantial interobserver ...variability. Clonal hematopoiesis defined by the presence of a typical karyotype abnormality can serve as presumptive evidence of MDS in the absence of other diagnostic criteria, however these lesions are absent in the majority of cases. Somatic mutations are much more common in patients who meet the diagnostic criteria for MDS. However, the frequency of clonal somatic mutations in patients with meaningful cytopenias who lack morphologic evidence of MDS is not known and could help identify those at increased risk of progressive disease.
Methods:
We conducted a prospective study enrolling patients with unexplained cytopenias, suspected of having MDS into one of three groups (positive for MDS, equivocal, or negative) based on traditional diagnostic methods of morphology, cytogenetics and flow cytometry. We conducted massively parallel amplicon-based sequencing using the Illumina MiSeq platform on bone marrow samples from consenting patients to examine the mutation status of 21 genes implicated in MDS (SF3B1, SRSF2, U2AF1, ZRSR2, TET2, IDH1, IDH2, DNMT3A, EZH2, ASXL1, SETBP1, TP53, PHF6, RUNX1, ETV6, CBL, NRAS, KIT, JAK2, MPL, NPM1). The minimum coverage was 500x, and only variants with minor allele fractions >5% were reported.
Results:
Eleven community oncology practices in the US enrolled 145 patients with cytopenias suspected of having MDS. Of these, 86 have been sequenced to date. The ages of the sequenced patients ranged from 31 to 97 years with an average age of 71. Bone marrow material from these 86 patients were examined by two hematopathologists and enrolled into three arms based on their findings; confirmed MDS (n=8), equivocal evidence of MDS (n=12), and non-MDS (n=66). We identified clinically significant MDS-associated somatic variants in all three categories. Variants were found in 5 of 8 confirmed MDS, in 9 of 12 with equivocal MDS, and in 16 of the 66 non-MDS patients. The finding of clinically significant variants in 16 of 66 (24%) in the non-MDS group was unexpected and included 6 patients with mutations in the TET2 gene,6 patients with mutations in the TP53 gene, as well as patients with mutations in RUNX1 (n=2), DNMT3A, SETBP1, ASXL1 and ZRSR2. There were no differences in age or blood counts between patients with mutations and those without them within the non-MDS group. Nor were there any significant differences in these measures between diagnostic groups.
Discussion:
Somatically acquired variants in the non-MDS group may be the result of early, low grade MDS without sufficient observable pathological characteristics, may indicate the presence of another disease affecting hematopoietic development, or may be incidental age-related somatic mutations. Our study may underestimate the fraction of patients with such mutations as genes not sequenced here may provide evidence of clonal hematopoiesis in more patients. Surprisingly, the number of patients without clear evidence of MDS greatly outnumbered those with a firm morphologic diagnosis in this prospective study. The large fraction of these patients with somatic mutations suggests that clonal cytopenias may be much more common than estimates based on the prevalence of MDS would indicate.
Additional follow up may allow us to determine how these patients evolve clinically over time. Sequencing of the remaining 59 patients and additional analyses are ongoing and will include the association of somatic variants with age, type and severity of cytopenia, and features of the bone marrow morphology. All 145 patients, including 103 in the arm without evidence of MDS will be included in the study when presented at the ASH meeting.
Conclusion:
Somatic mutations in genes typically associated with MDS can be found in a substantial fraction of patients with little or no morphologic evidence of the disease. Identification of clonal cytopenias may help exclude benign alternative diagnoses and impact how these patients are followed clinically over time.
Hall:Genoptix Medical Laboratory: Employment. Al Hafidh:Genoptix Medical Laboratory: Employment. Balmert:Genoptix Medical Laboratory: Employment. Dabbas:Genoptix, Inc., a Novartis company: Employment, Equity Ownership. Vaupel:Genoptix Medical Laboratory: Employment. El Hader:Genoptix Medical Laboratory: Employment. McGinniss:Genoptix Medical Laboratory: Employment. Beruti:Genoptix Medical Laboratory: Employment. Bejar:Genoptix Medical Laboratory: Consultancy, Honoraria, Licensed IP, no royalties Patents & Royalties, Membership on an entity’s Board of Directors or advisory committees; Celgene: Membership on an entity’s Board of Directors or advisory committees.
Abstract only
e11568
Background: Estrogen (ER), progesterone (PR) receptors and HER2 are important markers for breast cancer (BC) treatment planning. These markers are often measured by ...immunochemistry (IHC) but IHC is subject to limitations of operator subjectivity and equivocal results making selection of appropriate therapy difficult and ultimately affecting patient outcome. Methods: 135 females with BC were recruited in Puerto Rico between Sept and Dec 2012. Median patient age was 57 and 95 % had invasive ductal carcinoma. Our aim was to compare discordance rates of ER, PR and HER2 between IHC and AQUA, an automated fluorescent technology that quantifies antigen expression, in a population of Hispanic women and to investigate whether specimen type or tumor grade influence discordance rate between the two technologies. Results: 103 of 135 patient samples were found to be ER+ by both technologies and 28 samples were ER- by both IHC and AQUA. 4 samples (3%) were discordant; 2 each were AQUA+/IHC- and AQUA-/IHC+. 73 of 135 samples expressed PR by both technologies and 55 samples were PR- by both IHC and AQUA. 14 samples (10%) were discordant; 7 each were AQUA+/IHC- and AQUA-/IHC+. 87 of 135 samples were HER2- and 13 patients were found to be HER2+ by both technologies. However 35 samples (26%) were discordant for HER2 status; 31 were AQUA+/IHC- while 4 AQUA-/IHC+. 100 of the HER2 results were scored 0-1 (negative) by IHC and 25 % of these cases were overturned by AQUA to HER2 +. 19 of the HER2 results were scored 2 (borderline) by IHC. 32 % of these cases were positive by AQUA. Conclusions: Discordance rate were 3% for ER, 10 % for PR but 26% for HER2 between IHC and AQUA. The Cohen’s Kappa coefficient for ER and PR were 0.914 and 0.785 respectively but only 0.299 for HER2 (p values < 0.00001 for all 3 values). Needle/core biopsy yielded more HER2 discordance than lumpectomy/mastectomy (31% vs. 16%). HER2 discordance between IHC and AQUA was more common in higher grade tumors. The large number of HER2 negative IHC cases scored positive by AQUA, and considering the data demonstrating that HER2 by AQUA better predicts benefit from trastuzumab (Slamon D, SABCS 2012) suggests that a significant additional number of patients could benefit from such targeted therapy.
We have shown previously that the hypertension-related, calcium-regulated gene (HCaRG) is involved in the control of renal cell proliferation and differentiation (Devlin AM, Solban N, Tremblay S, ...Gutkowska J, Schurch W, Orlov SN, Lewanczuk R, Hamet P, and Tremblay J. Am J Physiol Renal Physiol 284: F753-F762, 2003). To determine whether HCaRG plays a role in kidney repair after injury, we extended our studies on the cellular function of HCaRG by comparing cell migration of two kidney cell lines HEK293 and Madin-Darby canine kidney (MDCK)-C7 stably transfected with the plasmid alone or with a plasmid containing HCaRG cDNA. HCaRG-expressing HEK293 cells, which undergo lower proliferation, migrated faster than control cells and presented greater adhesiveness to the extracellular matrix. Faster migration was also observed for the MDCK-C7 cells, after they were stably transfected with HCaRG cDNA. HCaRG overexpression induced major morphological changes in HEK293 cells, including the formation of lamellipodia. Expression microarrays of HCaRG-expressing HEK293 cells revealed the elevated expression of several genes known to be involved in cell migration and lamellipodia formation, including transforming growth factor-alpha (TGF-alpha), galectins, autotaxins and fibronectin. These cells exhibited augmented synthesis and release of activated TGF-alpha. Conditioned medium from HCaRG-expressing cells stimulated the migration and induced significant morphological changes in control cells, in part, through activation of the TFG-alpha/EGF receptor. Together, these data support a role for HCaRG in kidney repair after injury through its effect on renal cell migration and TGF-alpha secretion. PUBLICATION ABSTRACT
Introduction
An important process in renal regeneration after injury is the conversion of tubular cells to a migratory phenotype; surviving cells migrate to denuded areas, proliferate and ...differentiate in order to restore nephron structure and function. Beside its effect on renal cell proliferation and differentiation, our previous studies support a role for HCaRG (hypertension‐related calcium‐regulated gene) in the motile behavior increment of kidney cells.
Results
To better understand the evolvement of HCaRG in this phenomenon, we used the Yeast 2–hybrid technique to screen human kidney proteins that interact with HCaRG. Screening using HCaRG as bait revealed its interaction with β‐actin, Na+/K+ATPase and NKCC. This direct protein interaction was confirmed by several techniques, and their co‐localization at the leading edge of migrating cells was found by immunocytochemistry. This stimulatory effect of HCaRG on migration could be due to actin reorganisation coupled to ions transport activation. We produced two stable kidney cell lines (HEK293 and MDCK‐C7) overexpressing HCaRG to help us identify its function. We used oligonucleotide microarray analysis to determine changes in gene expression between HCaRG over expressing cells and their controls. Microarray results for selected genes were confirmed by quantitative RT‐PCR. HCaRG affects the expression of several genes implicated in cell motility.
Conclusion
A better comprehension of the mechanisms and molecular pathways involved in the functions of HCaRG will enable us to conceive better means of stimulating the regeneration of renal function after a lesion.
Supported by CIHR.
The process of kidney regeneration recapitulates many aspect of development; it involves cell migration, proliferation and differentiation. Our previous studies point to the involvement of HCaRG ..."Hypertension-related calcium-regulated gene" in two major processes contributing to kidney repair, i.e. control of cell proliferation and differentiation. We extended our studies on the cellular function of HCaRG by comparing cell migration of two kidney cell lines. HCaRG expressing HEK293 cells, which undergo lower proliferation, migrated faster than control cells and presented greater adhesiveness to the extracellular matrix. Faster migration was also observed for the MDKC-C7 cells, after stably transfecting them with HCaRG cDNA. Screening of a human kidney cDNA library with HCaRG as bait revealed its interaction with several ionic transporters, among them Na+,K+,2Cl- cotransporter (NKCC) and Na,K-ATPase (NK pump). HCaRG overexpression induced major morphological changes of HEK293 cells including the formation of lamellipodia. An interaction and a co-localization were further found between HCaRG and actin at the leading edge of migrating cells. Increased activities of the NK pump and NKCC were observed in MDCK-C7 cells expressing HCaRG. These cells displayed higher content of intracellular Na+, water, and total proteins. Ouabain and bumetanide dose-dependently suppressed cells migration with keeping a higher migratory potential for the HCaRG-expressing HEK293 cells. Expression microarrays of HCaRG clones cells resulted in a profile of differential regulation of molecules involved in cell proliferation, differentiation and migration as well as molecules involved in morphogenesis and cytoskeleton organization. Among the quantitatively most up-regulated genes was the transforming growth factor-alpha (TGF-a) which has been associated with normal renal development and recovery. HCaRG-expressing cells exhibited augmented synthesis and release of activated TGF-a and
Le processus de la régénération rénale récapitule beaucoup d'aspects du développement; Il comporte la migration, la prolifération et la différentiation cellulaire. Nos études précédentes démontrent l'implication de HCaRG "Hypertension-related calcium-regulated gene" dans 2 processus principaux contribuant à la réparation des reins; le contrôle de la prolifération et de la différentiation cellulaires. Nous avons prolongé nos études sur la fonction cellulaire de HCaRG en comparant la migration de deux lignées de cellules rénales. Les cellules HEK293 qui expriment HCaRG, et qui se caractérisent par une prolifération réduite, ont migré plus rapidement que les cellules contrôles et ont présenté une plus grande adhérence à la matrice extracellulaire. On a également observé une migration plus rapide pour les cellules MDKC-C7, après la transfection stable avec l’ADNc de HCaRG. Le criblage d'une banque d'ADNc humaine de rein avec HCaRG indiquait son interaction avec plusieurs transporteurs ioniques, parmi eux le co-transporteur Na+, K+, 2Cl- (NKCC) et la pompe Na+,K+-Atpase. L'expression de HCaRG a induit des changements morphologiques majeurs des cellules HEK293, ceci comprend la formation des lamellipodes. Une interaction et une co-localisation entre HCaRG et actine ont ensuite été retrouvées au bord des cellules en migration. On a observé une augmentation de l'activité de la pompe et du co-transporteur dans les cellules MDCK-C7 qui expriment HCaRG. Ces cellules ont montré une teneur plus élevée de Na+ intracellulaire, d'eau, et de protéines. La Ouabaïne et le bumétanide ont supprimé d'une façon dose dépendante la migration de cellules en gardant un potentiel migrateur plus élevé pour les cellules HEK293 qui expriment HCaRG. L'analyse des "microarrays" des clones qui expriment HCaRG a indiqué un profil de régulation différentiel des molécules impliquées dans la prolifération, la diff
The process of kidney regeneration recapitulates many aspect of development; it involves cell migration, proliferation and differentiation. Our previous studies point to the involvement of HCaRG ...“Hypertension-related calcium-regulated gene” in two major processes contributing to kidney repair, i.e. control of cell proliferation and differentiation. We extended our studies on the cellular function of HCaRG by comparing cell migration of two kidney cell lines. HCaRG expressing HEK293 cells, which undergo lower proliferation, migrated faster than control cells and presented greater adhesiveness to the extracellular matrix. Faster migration was also observed for the MDKC-C7 cells, after stably transfecting them with HCaRG cDNA. Screening of a human kidney cDNA library with HCaRG as bait revealed its interaction with several ionic transporters, among them Na+,K +,2Cl− cotransporter (NKCC) and Na,K-ATPase (NK pump). HCaRG overexpression induced major morphological changes of HEK293 cells including the formation of lamellipodia. An interaction and a co-localization were further found between HCaRG and actin at the leading edge of migrating cells. Increased activities of the NK pump and NKCC were observed in MDCK-C7 cells expressing HCaRG. These cells displayed higher content of intracellular Na+, water, and total proteins. Ouabain and bumetanide dose-dependently suppressed cells migration with keeping a higher migratory potential for the HCaRG-expressing HEK293 cells. Expression microarrays of HCaRG clones cells resulted in a profile of differential regulation of molecules involved in cell proliferation, differentiation and migration as well as molecules involved in morphogenesis and cytoskeleton organization. Among the quantitatively most up-regulated genes was the transforming growth factor-alpha (TGF-α) which has been associated with normal renal development and recovery. HCaRG-expressing cells exhibited augmented synthesis and release of activated TGF-α, and conditioned medium from these cells stimulated the migration and induced significant morphological changes of control cells in part through activation of the TFGα/EGF receptor. Taken together, these results reveal that HCaRG plays an important role in renal cell migration by induction of TGF-α secretion, its interaction with actin and its modulation of the intracellular ionic milieu required for optimal operation of the cellular migration machinery. It further provides a molecular basis for our previous and current findings that HCaRG-dependent intrarenal mechanisms enable tubule cell repair and regeneration. Keywords: Kidney, HCaRG, actin, ionic transporters, TGF alpha.
Thesis (Ph.D.).
Written for the Division of Experimental Medicine. Title from title page of PDF (viewed 2007/08/29). Includes bibliographical references.
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