O6-methylguanine DNA methyltransferase (MGMT) strongly influences the effectiveness of cancer treatment with chemotherapeutic alkylating agents, and MGMT status in cancer cells could potentially contribute to tailored therapies for individual patients. However, the promoter methylation and immunohistochemical assays presently used for measuring MGMT in clinical samples are indirect, cumbersome and sometimes do not accurately report MGMT activity. Here we directly compare the accuracy of 6 analytical methods, including two fluorescent reporter assays, against the in vitro MGMT activity assay that is considered the gold standard for measuring MGMT DNA repair capacity. We discuss the relative advantages of each method. Our data indicate that two recently developed fluorescence-based assays measure MGMT activity accurately and efficiently, and could provide a functional dimension to clinical efforts to identify patients who are likely to benefit from alkylating chemotherapy.The DNA repair protein  O6-methylguanine DNA Methyltransferase  repairs DNA damage induced by endogenous, environmental, and therapeutic alkylating agents, and is the most important pathway for repairing O6-alkylguanine adducts in human cells [O6-methylguanine (O6-MeG) cytotoxic DNA lesions induced by the cancer chemotherapy agents Temozolomide and Dacarbazine, and cytotoxic O6-chloroethylguanine lesions induced by cross-linking agents such as BCNU [an cells , 2. MGMT as BCNU . These c as BCNU , and are as BCNU , highligAlthough MGMT diminishes the effectiveness of cancer therapies, it also plays a critical role protecting normal tissues from DNA damage. Over 10-fold inter-individual variation in MGMT activity has been observed in normal tissues , 7, and Presently, MGMT status is assessed in clinical samples primarily using MGMT promoter methylation as a proxy for MGMT expression, and in some cases using immunohistochemistry; however, both methods can fail to reflect MGMT levels accurately. For example high levels of MGMT expression are possible in tumors with MGMT promoter hypermethylation due to expression of a previously unrecognized enhancer element . Such obSeven B-lymphoblastoid cell lines including TK6 (RRID CVCL_0561) , TK6+MGM7 cells were collected, washed twice with PBS, and suspended in 400 μL of lysis buffer comprising 50 mM Tris, pH 7.5, 1 mM EDTA, 1 mM DTT, 5% glycerol, 50 mM NaCl, and 1 mM AEBSF (protease inhibitor). Cells were disrupted by sonication, cell debris was pelleted by centrifugation at 14,000g for 30 minutes at 4°C, supernatants were collected, and total protein concentration was determined using a BCA assay. Cell lysates were incubated at 37°C for 30 minutes with 4 pmoles of a 32P 5’-end labeled duplex comprising an oligonucleotide, GAACTXCAGCTCCGTGCTGGCCC, in which X represents O6MeG, and the corresponding complementary oligonucleotide. The reaction products were then purified by phenol/chloroform extraction and ethanol precipitation, dissolved, and finally digested using PstI restriction enzyme. O6MeG blocks PstI cleaveage, providing the basis for a gel-shift assay for the extent of MGMT-dependent lesion removal. Digests were analyzed by SDS PAGE followed by autoradiographic imaging, and densitometry was used to calculate the percentage of cleaved oligonucleotide. The linear range of the MGMT assay was established for each cell line by varying the amount cell lysate (between 10 and 400 μg) incubated with the duplex. MGMT activity in each cell lysate was calculated from the slope of a linear best fit of the percentage of oligonucleotide cleaved versus total protein concentration.Oligonucleotide cleavage assays were performed as described previously , and illPreparation of cell lysates and immunoblotting were performed as described previously . BrieflyqPCR data were described previously . Total RO6MeG-containing oligonucleotides that were annealed to single-stranded plasmid DNA, followed by primer extension and ligation. The DNA lesion induces transcriptional errors that result in expression of functional mPlum fluorescent protein, unless repair removes O6MeG, the source of transcriptional errors. As a result, cells that efficiently repair O6MeG express relatively low levels of mPlum fluorescent protein, whereas MGMT deficient cells express relatively high levels of mPlum fluorescent protein. Transient transfection, flow cytometric analysis, and calculation of DNA repair capacity were described previously [FM-HCR assays have been published previously . Brieflyeviously .6 cells from cell lines using a QIAamp DNA Kit, and bisulfite conversion of 1 microgram of the resulting gDNA was carried out using an EpiTect Bisulfite Kit (Qiagen). For PCR detection of unmethylated MGMT promoter sequences, the following primers were used: 5’TTTGTGTTTTGATGTTTGTAGGTTTTTGT-3’, 5’AACTCCACACTCTTCCAAAAACAAAACA-3’. For detection of methylated DNA, the following primers were used: 5’TTTCGACGTTCGTAGGTTTTCGC-3’, 5’GCACTCTTCCGAAAACGAAACG-3’. Approximately 100 ng of gDNA was combined with primers at a final concentration of 400 nM, and amplified with 1 unit of AmpliTaq Gold DNA polymerase for 35 cycles . PCR products were analyzed on a 3% agarose gel visualized with ethidium bromide  comprising a short DNA oligomer containing a fluorophore and an O6-benzylguanine nucleoside that is modified with a quencher dye [Cell lysates were prepared from approximately 10cher dye . Repair For each method, error bars represent standard deviation from three biological replicates . One-way ANOVA with Tukey’s multiple comparisons test was used to determine the ability of assays to distinguish MGMT levels or activity among the cell lines. All statistical analyses were carried out in Graphpad Version 7.0c.O6MeG generating alkylating agents observed previously for the larger set of cell lines [A panel of 24 lymphoblastoid cell lines derived from apparently healthy individuals from diverse genetic backgrounds , Coriellll lines , which cll lines .The gold standard radiolabeled oligonucleotide-based biochemical MGMT assay revealed an approximately 100-fold range of MGMT activity in the samples, with the following rank order established using the biochemical MGMT assay: TK6 < Coriell #5 < Coriell #4 < Coriell #12 < Coriell #14 < Coriell #16 < TK6+MGMT. The available quantitative methods for assessing MGMT status in human lymphoblastoid cell lines have been compared with this biochemical assay . All sixIn vitro biochemical MGMT activity assays can be regarded to be the “gold standard” for measuring MGMT activity because they directly measure the level of repair activity of MGMT protein using a chemically defined substrate with a radiolabel that permits detection of very low levels of repair activity. However, the assay has several technical drawbacks. Assay conditions must be optimized to determine the range of cell lysate protein concentrations that produce a linear response, which is cell-line dependent (Compare x-axes in panel C of FM-HCR assays distinguished cells with low MGMT activity (Cell lines #4 and #5) from cells that lack MGMT activity (TK6), and thus exhibited the same dynamic range as the gold standard biochemical assay . The sensitivity of FM-HCR is achieved in part because of the ability to detect individual cells harboring unrepaired DNA lesions that lead to transcriptional errors and fluorescent protein expression; whereas a minor subpopulation of repair deficient cells may be lost in ensemble measurements, they can be readily detected by FM-HCR. Together with qPCR and the fluorescent NR-1 probe assay (below), FM-HCR required the least amount of active laboratory time (1.5 hours) for analysis of up to 4 samples in parallel.32P-oligonucleotide-based biochemical assay, FM-HCR and qPCR, were below the limit of detection by western blotting. Western blots also required approximately twice the active laboratory time  as the least labor-intensive assays.As has been observed by others , MGMT prMGMT transcript levels measured by qPCR analysis, reported previously , correlaMGMT activity as measured with the NR-1 probe correlated well with activity measured using the biochemical MGMT assay, however two cell lines (#4 and #5), which had the lowest activity as judged by the biochemical assay, were below the limit of detection. Analysis using the NR-1 probe required approximately 1.5 hours for analysis of up to 4 samples in parallel.O6-benzylguanine [Promoter methylation was detected, as expected, in TK6, previously shown to exhibit MGMT promoter methylation, and in TK6+MGMT, which is derived from TK6 and expresses MGMT constitutively under a CMV promoter . Strikinlguanine . AnalysiThe time-sensitive nature of cancer treatment as well as the serious side effects and risk of therapy-related cancers in patients treated with radiotherapy and chemotherapy have motivated a search for biomarkers that can predict whether specific therapies will work for individual patients . The suc8) of the six assays used. These considerations render oligonucleotide-based assays too labor intensive for clinical use.Although the biochemical MGMT assay is considered a gold standard because it provides a sensitive quantitative measure of functional MGMT levels in cell lysates, it was by far the most time consuming  is larger than that of the NR-1 probe (4.3). Although the NR-1 probe failed to detect the very low levels of MGMT activity in extracts from cell lines #4 and #5, the probe did distinguish significant differences in activity among cell lines with higher MGMT activity , whereas FM-HCR did not . HoweverClinical translation will require studies in primary patient samples to determine how well these assays predict therapeutic outcomes assessed using standard measures such as the Response Evaluation Criteria in Solid Tumors (RECIST). For cell-based assays, optimization of tumor processing to maximize live cell content may be needed. Strategies for excluding signal from tumor stroma should be considered, for example using cell surface markers or immunomagnetic separation.Limitations of existing assays have usually resulted in binary classification of tumor MGMT status, making it difficult to assess whether the distinction between tumors with low MGMT and those that are completely lacking MGMT is clinically important. However, available data are consistent with a continuous relationship between MGMT activity and temozolomide sensitivity . QuantitMany cancers exhibit alterations in MGMT activity that may be exploited when treating patients with alkylating chemotherapeutic agents, and MGMT activity in normal tissues may predict inter-individual differences in alkylating agent toxicity. However, there remains a critical need for accurate, functional assays that could be used to identify individuals with MGMT-deficient cancers. The recently developed FM-HCR assays and fluorescent NR-1 probe both overcome the problems associated with currently used indirect methods of measuring MGMT activity, and merit consideration as alternatives for use in pre-clinical studies and in clinical trials involving cancers where MGMT status may be associated with therapeutic outcomes.S1 FigO6MeG DNA lesion results in an 8 nt 32P labeled fragment detectable by polyacrylamide gel electrophoresis. B) Polyacrylamide gel analysis of restriction digest products following treatment of 4 pmol of oligonucleotide with 10–400 μg of protein extracts from 7 lymphoblastoid cell lines for 30 minutes at 37 °C. C) Determination of linear range and calculation of MGMT activity. Slopes calculated from the data in these plots are reported in Panel A.A) Oligonucleotide digest assay for MGMT activity. Repair of a PstI cleavage blocking (PDF)Click here for additional data file.S2 FigProtein levels were below the limit of detection for Coriell #5, Coriell #4, and TK6.(TIFF)Click here for additional data file.S3 FigEach lane shows PCR products obtained from amplification of bisulfite converted genomic DNA from the indicated cell lines with primers specific for unmethylated DNA (U), or methylated DNA (M).(PDF)Click here for additional data file.S1 Table32P Oligo, fmoles cleaved oligonucleotide per microgram protein lysate; NR-1, Fluorescence signal, arbitrary units; FM-HCR, % Reporter Expression; Western Blot, MGMT protein levels as a percentage of actin protein levels; qPCR, MGMT transcript levels normalized to MGMT transcript levels in TK6+MGMT.Units are as follows: (DOCX)Click here for additional data file.S2 TableCell lines that were significantly different from one another for each MGMT assay are indicated with an asterisk “*”; those that were not significantly different are marked “ns”.(DOCX)Click here for additional data file.


Embryology of normal web space creation and the genetics of syndactyly in humans and experimental animals are well described in the literature. In this review, the author offers a 3-step pathway of pathogenesis for syndactyly. The first step is initiated either by the overactivation of the WNT canonical pathway or the suppression of the Bone Morphogenetic Protein (BMP) canonical pathway. This leads to an overexpression of Fibroblast Growth Factor 8 (FGF8). The final step is the suppression of retinoic acid in the interdigital mesenchyme leading to suppression of both apoptosis and extracellular matrix (ECM) degradation, resulting in syndactyly. Studies on developmental biology and embryology of the upper limb have greatly improved our understanding of the genetics and phenotypes of various congenital hand abnormalities .The embryology of normal web space creation  and the FIBULIN1 (OMIM 608180) [The genetics literature classifies syndactyly into nine types . However 608180) , the tri 608180) .MMP11 gene also contains retinoic acid responsive elements [A 3-step pathway could explain the pathogenesis of almost all types of syndactyly in humans and experimental animals . The firelements .  Table LRP4 will lead to signal activation and syndactyly  [APC gene lead to beta-catenin accumulation. In most cases, a familial adenomatous polyposis phenotype (OMIM 175100) is seen. However, APC mutations may occasionally result in a Cenani–Lenz syndactyly phenotype [FMN1 deletions or duplications encompassing the GREM1-FMN1 genes [LRP4 is a strong suppressor of WNT signaling, and hence, loss-of-function mutations of  212780) . Loss-ofe OMIM 17100 is sehenotype . SimilarN1 genes .th and the 22nd embryonic stages (47–54 intrauterine days) [RAB23, TWIST1, and GLI3 will be associated with a high SHH activity and syndactyly  . The resne days) . In humane days) , 24, 25.ne days) –28. Hencndactyly .GJA1 lead to loss of the functional activity of CX43, which in turn leads to reduced BMPs and syndactyly  [The last protein acting on Step I is the CX43 protein. Mutations of  186100) .FGFR1/FGFR2 are associated with an increased activity of FGF8 and syndactyly. Overexpression of other FGFs such as FGF2 in the cranium also occurs, leading to craniosynostosis. The severity of craniofacial abnormalities versus hand syndactyly will also depend on the type of mutation. A classic example is Apert syndrome (OMIM 101200). Hand surgeons classify patients with Apert syndrome with FGFR2 mutations into two groups according to the severity of syndactyly/craniofacial defects [FGFR2. The resulting gain-of-function of the receptor in this mutation leads to an increased functional effect of FGF2 which is mainly expressed in the craniofacial skeleton. The second group have “mildly affected” heads but “severely affected” hands and carry the mutation Pro253Arg in FGFR2. The resulting gain-of-function of the receptor in this mutation results in loss of the ligand-binding specificity in which the abnormal receptor is able to bind to FGF10, which results in upregulation of ectodermal FGF8 through the FGF10–FGF8 loop [In Step II, acrocephalo-syndactyly syndromes caused by gain-of-function mutations of  defects , 31. TheFBLN1 mutations, OMIM 608180) also occurs at Step II. Normally, the FIBULIN1 protein binds to FGF8 with high affinity, modulating its activity and expression [The pathophysiology of the synpolydactyly type 2b  is probably related to HOXD13-GLI3R interactions. Chen et al. [Hoxd13 directly binds to Gli3r (the repressor form of Gli3). The mutated Hoxd13 directs Gli3r for a premature degradation. This will result in polydactyly, similar to the polydactyly phenotype caused by depletion of Gli3r. Brison et al. [HOXD13 gene in humans with synpolydactyly confirms a novel functional domain in HOXD13 which regulates digit number through its interaction with GLI3R.ndactyly  includin autopod . Hence,  autopod  reviewedn et al.  have shon et al.  have alsAnimal models of syndactyly may also be classified according to the 3-step pathway of pathogenesis .Lrp4 mutations develop polysyndactyly in their forelimbs and hindlimbs [Lrp4 will lead to signal activation and syndactyly [Mice with indlimbs . The Lrpndactyly .Fmn1 deletions and show oligosyndactyly, renal defects, and radio-ulnar synostosis [GREM1 gene [The murine limb deformity (Ld) model is caused by nostosis . The phenostosis  is also EM1 gene .Bmpr1a in the limb bud was associated with upregulation of both Fgf8 and Fgf4, resulting in syndactyly [Other animal models of syndactyly were created by suppression of Bmp signaling. Overexpression of the Bmp antagonist Noggin in mice resulted in extensive soft-tissue syndactyly and postaxial polydactyly . Inactivndactyly . Bmp2-dendactyly . Mice landactyly . SMADs 1ndactyly .Shh promotor. This results in overexpression of interdigital Shh, secondary suppression of the Bmp signaling, and finally syndactyly. The pathophysiology of this secondary suppression of Bmp was recently shown by Mouri et al. [Finally, the hammertoe (Hm) mutant mouse model shows syndactyly of digits 2–5. In this spontaneous mouse mutation, a 150 kb noncoding DNA fragment from chromosome 14 is inserted upstream of the i et al. . The ovei et al. .Jagged2. This resulted in an increased expression of Fgf8 and fusion of the middle three digits. Since Notch is also expressed in the thymus and the craniofacial area, mutant mice also exhibited cleft palate, tongue fusion, and thymic defects [Suppression of notch signaling in mice results in increased expression of Fgf8 in the AER and syndactyly . Notch 1 defects .Msx1/Msx2 double-mutant mice show variable phenotypes including oligodactyly, polydactyly, and syndactyly. This is associated with extended Fgf activity in the AER [ the AER .FRAS1 gene encodes an extracellular matrix protein involved in the establishment of the epidermal basement membrane. Mutations in FRAS1 in humans cause Fraser syndrome (OMIM 219000) with eye, kidney, and craniofacial defects [Fras1. The primary defect in these animals was the decreased Msx2 expression [The  defects . Hines e defects  identifipression . As mentpression .Nid1 or Nid2 in mice does not affect basement membrane formation and animals show no abnormalities. Mice lacking both Nidogens have defective ectodermal basement membrane of multiple organs including aberrant AER formation, altered distribution of Fgf8, and soft-tissue syndactyly [Basement membranes are extracellular matrices underlying the epithelium and endothelium of various organs including the AER. All basement membranes contain at least one member of Laminin, type IV collagen, and Nidogen families . Two Nidndactyly . Since bndactyly . This moRaldh2 gene. The enzyme oxidizes retinaldehyde to retinoic acid. Retinoic acid functions as a ligand for nuclear retinoic acid receptors (known as RAR) to induce the transcription of target genes. Hence, suppression of retinoic acid activity in experimental mice may be done either by targeting the retinaldehyde dehydrogenase-2 enzyme or the RAR receptors. Raldh2–/– autopods show syndactyly secondary to suppression of Mmp11 . In this model, Fgf8 expression is normal, indicating that retinoic acid acts downstream of Fgf8 in the pathogenesis of syndactyly [RARb/RARg double-mutant mice also show syndactyly secondary to the suppression of Mmp11 [Animal models with suppressed retinoic acid activity show syndactyly , 18. Thendactyly . RARb/RAHoxd13). The authors showed that the mutated Hoxd13 has a direct suppressive effect on retinoic acid in the autopod [Kuss et al.  used the autopod . Intraut autopod .Bid/Bim/Puma triple knockout mice show reduced apoptosis and syndactyly. Besides Mmp11, the ADAMTS group of peptides is essential in interdigital ECM degradation. Versican (a proteoglycan) is an important component of the interdigital ECM. ADAMTS mediates the cleavage of versican. Hence, mice deficient in ADAMTS exhibit syndactyly [The proteins Bid, Bim, and Puma act to activate Bax and Bak which are essential to initiate the apoptotic pathway at the mitochondrial level. Hence, ndactyly .Akirin2 null embryos are not able to survive beyond embryonic day 9.5. Transgenic mouse models with knockout of Akirin2 in the limb epithelium leads to a loss of interdigital cell death and an increase in cell proliferation, resulting in the retention of the interdigital web and soft-tissue syndactyly [Akirins (Akirin 1 and 2) are small nuclear proteins that localize to promoter and enhancer regions of genes. They function as “bridge” proteins to coordinate gene expression patterns . Akirin2ndactyly .Limb development is a complex process involving the action of signaling centers that coordinate spatially and temporally to sculpt a limb . Digit fThe current review offers a unified pathway that could explain the pathogenesis of syndactyly in humans and experimental animals. The pathway is a 3-step pathway, and hence, human and animal syndactyly may be classified accordingly Tables  and 2. TNoggin-null mice, syndactyly of Sp6 mutant mice, and isolated 4/5 metacarpal fusion in humans). These types of syndactyly are summarized in The pathway proposed in the current review explains the pathogenesis of most types/models of syndactyly. Certain types of syndactyly have a different pathway of pathogenesis (such as Poland syndrome syndactyly) or the pathogenesis is yet to be determined  is characterized by the unilateral absence or hypoplasia of the sternocostal head of the pectoralis major muscle and ipsilateral symbrachydactyly. However, several other pectoral muscle and hand defects have also been described . One chaFibrillin2 (Fbn2) loss-of-function mutations [The mouse mutant Shaker-with-syndactylism (Sy) is caused by utations . Fibrillutations . Since FBHLHA9 result in split hand-foot malformation with long-bone deficiency type 3  [BHLHA9 lead to mesoaxial synostotic syndactyly with phalangeal reduction  [Fingerin causes syndactyly, both in vivo and ex vivo. However, the exact pathogenesis of syndactyly is still unknown, and two different theories have been proposed [The Basic Helix-Loop-Helix Member A9  is a transcription factor which is expressed in the distal hand/foot plates . Duplicaproposed . As mentproposed . The secNOGGIN in humans leads to various phenotypes including brachydactyly type B2 (OMIM 611377) and various forms of synostosis/ankylosis/symphalangism . In contrast, cutaneous syndactyly is a prominent feature in Noggin-null mice [Mutations in ull mice . The exaull mice .Sp8 mutant mice exhibit severe loss of Fgf8 expression in the AER leading to limb truncations. In contrast, Sp6 mutant mice show a mild loss of Fgf8 expression and a mild syndactyly phenotype [Sp6  and Sp8  are transcription factors implicated in AER induction and maintenance . They arhenotype . The pathenotype . As a rehenotype . This pahenotype , 68.FGF16 [Fgf16 knockdown in experimental animals is known to affect Fgf8 expression in the AER as well as the expression of Shh expression in the zone of polarizing activity [Isolated fusion of the 4/5 metacarpal has been classified as syndactyly type 8 (OMIM 309630). This is the only type of syndactyly that is inherited as X-linked and is caused by nonsense mutations in FGF16 . The patactivity .Fbn2 mutations in mice show syndactyly, while FBN2 mutations in humans are associated with congenital contractual arachnodactyly (OMIM 121050). Another example is the mouse model of Apert syndrome which showed most of the classic craniofacial features, but none of the animals exhibited syndactyly [Our review also demonstrates that mutations in humans and animals do not always result in similar phenotypes. ndactyly . A thirdndactyly . In humaLRP4 mutations. The classic syndrome is characterized by bilateral hand and feet syndactyly along with metacarpal/metatarsal synostosis and phalangeal disorganization. Hand function in these patients is poor even after surgical correction [LRP4 gene [LRP4 gene cause a very mild phenotype with simple syndactyly and well-developed digits, without metacarpal synostosis/phalangeal disorganization [LRP4 lead to the most severe oligosyndactyly phenotype and may even be lethal with intrauterine demise [HOXD13 mutations, and a severe syndactyly phenotype is seen with homozygous HOXD13 mutations [Another observation from the current review is the correlation between the degree of pathway abnormality and the severity of syndactyly phenotype. One example is seen in Apert patients. The “severely affected” hand group has a more severe syndactyly phenotype and higher FGF8 overactivity when compared to the “mildly affected” hand group. Another example is Cenani–Lenz syndrome (OMIM 212780) caused by rrection . This reRP4 gene . In contnization . Furthere demise –76. A thutations , 77.LRP4 and APC mutations  and those directly leading to persistence of GREM1 expression  will have a similar Cenani–Lenz phenotype. Hence, syndactyly related to mutations of LRP4, APC, FMN1, and GREM1 should be grouped together. In contrast, human syndactyly related to high SHH signaling has a different syndactyly phenotype and is associated with preaxial polydactyly, although the pathogenesis is still at Step I  loss-of-function mutations [The 3-step pathway of pathogenesis provides a novel look at the correlation between the genetics and pathogenesis of syndactyly. The syndactyly is best classified according to the pathogenesis since the phenotype is affected by the site along the pathway. The pathway may also help to guide research on syndactyly types with undetermined pathogenesis. For example, the mouse mutant Shaker-with-syndactylism (Sy) is caused by utations . Since Futations , the pat


In this review, we discuss the immunomodulatory properties supporting the therapeutic use of MSCs in GVHD and contextualize the main clinical findings of recent trials using these cells. Critical parameters for the clinical translation of MSCs, including consistent production of MSCs according to Good Manufacturing Practices (GMPs) and informative potency assays for product quality control (QC), are addressed.Graft versus host disease (GVHD) is a common condition in patients subjected to allogeneic hematopoietic stem cell transplantation (HSCT). The immune cells derived from the grafted stem cells attack recipient’s tissues, including those from the skin, liver, eyes, mouth, lungs, gastrointestinal tract, neuromuscular system, and genitourinary tract, may lead to severe morbidity and mortality. Acute GVHD can occur within few weeks after the allogeneic cells have engrafted in the recipient while chronic GVHD may occur any time after transplant, typically within months. Although treatable by systemic corticosteroid administration, effective responses are not achieved for a significant proportion of patients, a condition associated with poor prognosis. The use of multipotent mesenchymal stromal cells (MSCs) as an alternative to treat steroid-refractory GVHD had improved last decade, but the results are still controversial. Some studies have shown improvement in the life quality of patients after MSCs treatment, while others have found no significant benefits. In addition to variations in trial design, discrepancies in protocols for MSCs isolation, characterization, and  Allogeneic hematopoietic stem cell transplant (HSCT) is a treatment for high risk hematological and malignant diseases. Conditioning regimen, immunosuppressive strategies, supportive care and prophylaxis for infectious disease are improving, reducing mortality related to transplant . HoweverGraft versus host disease occurs when immunologically competent donor T lymphocytes recognize recipient’s tissue as foreign resulting in damage in many organs systems including skin, liver, gastrointestinal tract, and lung. Clinical manifestations are classified as acute or chronic GVHD. In the past, all clinical manifestations of GVHD occurring before 100 days of transplant where classified as acute GVHD. When clinical manifestations occurred later on, after 100 days of transplant, it was considered chronic GVHD. In 2005, the National Institutes of Health (NIH) Consensus Conference determined new criteria of diagnosis and scoring and abolished the 100 days criterion. NIH Consensus considered acute and chronic GVHD as two distinguished symptoms without restriction of time .Among all patients undergoing allogeneic HSCT 30–50% will have acute GVHD (grade 1–4) and 14% of patients exhibit chronic GVHD. The most important acute GVHD risk factor is human leukocyte antigen (HLA) mismatch . Transplin vitro and in vivo and natural killer cells (NK cells) have been shown to modulate GVHD in a clinical trial reducing incidence of GVHD . Four studies using umbilical cord-derived MSC to prevent GVHD and one study using adipose-derived MSCs to treat GVHD have also been reported. These studies have compared MSCs treatment only with conventional treatment or historical control. Thus far, no studies have compared the impact of different biological sources of MSC in clinical outcome. Variabilities in trial design is one main limitation for carrying out a meta-analysis to determine which biological source is better or if they are all equivalent in terms of adverse effects and therapeutic benefits .de novo malignancies have been reported, although the clinical follow-up in the examined trials were rather short, ranging from 3 to 60 months . Infusion was safe and well tolerated in all studies (doses ranging from 0.1 to 10 million cells per kg) and there was no report of oncogenesis in the follow-up period. Trials using MSCs prophylactically had a median follow-up of 10–60 months. Follow-up of trials using MSCs as treatment varied from 2 to 23 months .As immunosuppressive treatment can reduce graft-versus-leukemia (GVL) effect, two studies reported relapse of malignant disease in short follow up . HoweverSafety regarding the use of MSCs also involves evaluation of possible complications related to embolism in capillary-rich organs. Among the most common routes of MSCs delivery, including topical application, intramuscular or direct injection into organs, intravenous and intra-arterial infusion, the preferred route is the intravenous . DespiteA study by In another experimental model, Altogether these studies indicate that patients in cell therapy protocols should be monitored for possible embolism or thrombus events related to MSCs infusion and, when applicable, antithrombotic therapies could be applied. Preclinical studies like these are of utmost importance to prevent translational failure. To this end, critical parameters such as the therapeutic window, delivery route, type of cells, immunogenicity, comorbidity, and concomitant treatment, should be considered in these studies.Evidence of efficacy is difficult to determine since the clinical studies published so far are highly heterogeneous. Some studies use MSCs as prophylactic scheme, with infusion in pre-determined days. Six studies analyzed incidence of acute GVHD and other six studies evaluated chronic GVHD , 2. PossOnly two controlled trials evaluated efficacy in acute GVHD treatment without difference in clinical manifestation in both groups (MSCs versus no MSCs). In one study with pediatric patients, complete and partial responses were reported in 58 and 17% patients, respectively . One trial evaluated MSC for treatment of chronic GVHD in 40 patients. In this study, complete and partial responses were observed in 15 and one patients, respectively  .As for safety, the cell administration route is a variable potentially impacting therapeutic effects. MSC systemic delivery is one of the most common administration route in cell therapy since it does not require an invasive procedure but, on the other hand, it relies on a transendothelial cell migration process toward the lesion site, which could have a direct influence in the treatment efficacy Mesenchymal stromal cells should be able to exit circulation and migrate to tissues/organs in order to repair local lesions due to GvHD. This process initially involves a contact between the cells and the endothelium and, subsequently, transmigration toward the target tissue/organ. MSCs rolling depends on presence of platelets  and diffEnhancing migration and homing of MSCs to target tissue/organ could be achieved by genetic engineering these cells to increase the expression of chemokines, integrins or selectins . This ap6 cells. Since this pioneer study, many others have been performed over the last decade establishing an excellent safety profile for both, autologous and allogeneic MSC infusions, over a range of cell doses and different schemes  or low dose (2 × 106 MSC/kg). Patients were stratified for dose levels between grades II and grades III/IV of acute GVHD. Standard steroid therapy using glucocorticosteroids and cyclosporine or tacrolimus, was continued at therapeutic dose levels. Seventy seven percent of patients had complete response and 16% partial response  without difference in both arms . In these studies, patients were treated with MSCs doses ranging from 10MSC dose .The MSCs anti-inflammatory properties as well as homing to sites of inflammation, immunomodulatory properties, and trophic influence on tissue repair, have made these cells very popular for clinical studies . Up to Fex vivo for clinical purposes, as an alternative method for regenerative therapy in many diseases. In any case, regulatory issues related to the safety, efficacy and quality of MSC therapies must be considered while preparing a cell- or tissue-based product for clinical and commercial use. Quality assurance (QA) and quality control (QC) must be provided in any cell manipulation under good manufacturing practices (GMP) grade.Currently, distinct strategies are used to produce human MSCs Thus far, there is no consensus on the production of MSCs regarding GMP system. The QC standards may be discussed individually on each project following the local regulatory agency authority. In Brazil, the regulatory agency , establish specific criteria for advanced therapy products to assure the quality, safety and efficacy of cell therapy in the country (Resolutions: RDC 214/2018 and RDC 260/2018). Cell viability, cytogenetics, potency tests and sterility tests to verify contamination by mycoplasma, aerobic and anaerobic bacteria, fungus, and special microorganisms such as filamentous fungus, are routinely used to ensure the quality of the cell product. Main GMP issues addressed by regulatory agencies in different countries are comparable and can be assessed in ex vivo expansion. A study by Nikitina and colleagues estimated that around 10% of MSC samples contain chromosomal aberrations after expansion, although the clinical consequences of such aberrations are unknown (According to the Foundation for the Accreditation of Cell Therapy (FACT) and Brazil’s ANVISA, cytogenetic testing is mandatory to release a cell product. Most manufacturing units perform karyotyping on the final cell product and the results are an important component of the release criteria. In case of any chromosomal abnormalities in the manipulated cells, the incoming samples may be karyotyped to ensure that the donor does not have any constitutive chromosomal abnormalities and that the alteration has probably originated in a cell clone during  unknown .However, since the potential clinical impact of minor changes in karyotyping is difficult to evaluate, not all services consider the cytogenetic testing as a release criterion .There are many different variations in existing manufacturing protocols for MSC production that may influence the final characteristics of the cells. The type of media supplementation is a typical example. Fetal bovine serum (FBS) is the most common supplementation used. FBS concentration in media ranges from 2 to 20%, with 10% FBS being the most common concentration. This variability on FBS concentration may result in different amounts of growth factors to stimulate cell survival and proliferation . Xeno-frEx vivo MSC expansion may be performed by conventional cell culture techniques or by using bioreactors. Considering autologous use, it is possible to produce lower cell quantity and the scale-out approach can be performed, using planar culture systems with multiple flasks in cell factories. For allogeneic use, MSCs can be expanded to a large number of cells in bioreactor systems  and cryopreserved in cell banks for future use . There are several bioreactors available for cell expansion such as stirred tank bioreactor, rocking bioreactor, hollow fiber bioreactor, and fixed bed bioreactor. The choice of bioreactor will depend on the aim to be achieved, i.e., final amount of cells required for infusion, type of cell (adherent or suspension growing cells), mid/long term use, etc .After MSC expansion, cells are harvested by using an enzyme, usually trypsin. Since this enzyme is normally of porcine origin, alternative GMP recombinant enzymes are available and their use should be prioritized. Mechanical detachment of cells cultured under GMP facilities is also possible by using a cell scraper tool, although detachment must be done gently in order to avoid cell damage and death. While enzymatic MSC detachment can be adapted in expansion protocols using bioreactors, the same adaptation is harder to achieve for mechanical detachment techniques.When high MSC doses and multiple cell infusions are required, which is typically the case of GVHD treatment, cryopreservation of previously expanded cells is an optimal solution to have high amounts of GMP-grade cells readily available for infusion. Nonetheless, cryopreservation is a critical step in MSC manipulation. Although either cryopreservation bags or regular cryotubes could be used for this purpose, the use of the latter is limited by the low volume of cell preparation that can be stored per vial . Cryopreservation media usually contain DMSO as cryoprotectant but it can cause some adverse effects when infused in patients. Typically, cells are stored using a ratio of 10% DMSO and 90% of serum. Thus, after thawing, some laboratories centrifuge and wash cells to remove DMSO. An alternative is to use cryoprotectants containing methylcellulose, sucrose, trehalose, glycerol, hydroxyethyl starch, and polyvinylpyrrolidone. Some companies already developed serum-free and xeno-free cryopreservation media  to circumvent toxicity. Importantly, validation of progressive freezing and thawing cycles is required to avoid significant loss of cell viability.ex vivo expanded MSC need to meet the quality standards required by regulatory agencies.Despite the lack of consensus in critical steps of MSCs production under GMP conditions, different protocols are available to attain high yields of expanded MSCs. However, these Cellular products intended for clinical use must also pass functional evaluation, a key part of a GMP quality control program. Currently, there is no specific release criterion required by the authorities for testing MSC potency. The most used and accepted potency tests for MSCs evaluate their ability to differentiate into three different cell types , to inhibit T-lymphocyte proliferation or to promote endothelial tube formation.in vitro assays that can be easily adapted for routine screening of cell preparations. Nonetheless, adoption of different assays for extensive functional characterization of cells is expensive and time consuming, which may delay the availability of freshly produced MSCs for infusion in patients at critical clinical conditions. Alternatively, a key test could be performed to evaluate a specific MSC property that is correlated with the aimed therapeutic effect. To this end, knowledge of the mechanism of cell action in vivo is of fundamental importance, which reinforces the relevance of basic stem cell biology studies.All these tests involve For instance, exploration of MSC immunosuppressive potential is well established in immunological-based diseases such as diabetes, multiple sclerosis, and GVHD. For this type of application, assaying MSC immunomodulatory activity would better suit a potency test then assaying MSC multipotency. This could be achieved by co-culture assays with T cells to evaluate MSC effects on T cell proliferation and/or by cytokine release by MSC .+ and CD8+ cells. This study suggested that cell-cell contact between MSC and effector cells was not necessary to inhibit T-lymphocyte proliferation (in vitro (feration . Howeverferation . MSC do in vitro .The immuno-suppressive activity of MSCs may also be determined after exposure to IFN-gamma. In this assay, presence of primed MSC could be indicated by expression of MHC class I, MHC II, PDL-1, or other modulatory molecules. Release of certain chemokine receptors such as CXCR3, CXCR4, CXCR5, and CCR7 could also be assayed to indicate immunomodulatory active MSC . As prevlow/D7fib+ve/LNGF+ve cells in samples derived from adults compared with samples derived from children. MSCs obtained from elderly people also showed increased levels of ROS, p21, and p53 proteins. The authors suggested that active MSCs derived from bone marrow decrease in number with age and that these cells are not as potent as the ones isolated from younger patients. Such aging effect on MSC properties should be considered when defining inclusion criteria for MSC donors in cell therapy protocols.Specific MSC properties involved in their expected therapeutic effect may be affected by different factors, including cryopreservation . Thus, pex vivo expansion of cells should help minimize product variability. Also, the choice of potency test is critical in evaluating suitability of the final product, since the desirable therapeutic effect may differ among clinical trials. In the case of GVHD, potency tests addressing MSCs immunomodulatory activity are key factors for obtaining high quality MSCs for therapeutic purposes. Allogeneic MSCs transplantation seem highly appropriate for GVHD treatment, due to the poor clinical condition of patients for tissue donation and lack of necessity of HLA match. In this scenario, the clinical use of cryopreserved third party MSC products offers the additional advantage of faster cell product availability, compared with autologous transplantation. Additional tests to address the cytotoxicity of recipient’s cells toward allogeneic MSCs should help refining the selection of eligible patients. Distribution of MSC products stored in cryobanks to different hospitals is also feasible, allowing potential therapeutic benefits for a greater number of patients in need.Evidences in the literature support safety of MSCs treatment for GVHD, whereas efficacy of this type of cell therapy still needs further clarification. Efficacy of MSC-based treatment is more evident for chronic GVHD patients. Double-blind randomized controlled trials with steroid-refractory GVHD patients should be designed to better address MSCs efficacy. Well-defined inclusion/exclusion criteria for patient accrual and a standardized protocol for GMP production of MSCs should facilitate multicenter studies and acquisition of faster results for clinical outcome assessment. Regarding MSCs production, uniformity in donor age and automated All authors wrote the manuscript and revised the final submitted version.OO was a visiting scholar at Departamento de Hemoterapia e Terapia Celular, Hospital Israelita Albert Einstein.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


As a tumor immunotherapy, allogeneic hematopoietic cell transplantation with subsequent donor lymphocyte injection (DLI) aims to induce the graft-versus-tumor (GVT) effect but often also leads to acute graft-versus-host disease (GVHD). Plasma tests that can predict the likelihood of GVT without GVHD are still needed.We first used an intact-protein analysis system to profile the plasma proteome post-DLI of patients who experienced GVT and acute GVHD for comparison with the proteome of patients who experienced GVT without GVHD in a training set. Our novel six-step systems biology analysis involved removing common proteins and GVHD-specific proteins, creating a protein-protein interaction network, calculating relevance and penalty scores, and visualizing candidate biomarkers in gene networks. We then performed a second proteomics experiment in a validation set of patients who experienced GVT without acute GVHD after DLI for comparison with the proteome of patients before DLI. We next combined the two experiments to define a biologically relevant signature of GVT without GVHD. An independent experiment with single-cell profiling in tumor antigen–activated T cells from a patient with post–hematopoietic cell transplantation relapse was performed.The approach provided a list of 46 proteins in the training set, and 30 proteins in the validation set were associated with GVT without GVHD. The combination of the two experiments defined a unique 61-protein signature of GVT without GVHD. Finally, the single-cell profiling in activated T cells found 43 of the 61 genes. Novel markers, such as RPL23, ILF2, CD58, and CRTAM, were identified and could be extended to other antitumoral responses.Our multiomic analysis provides, to our knowledge, the first human plasma signature for GVT without GVHD. Risk stratification on the basis of this signature would allow for customized treatment plans. The lymphocytes in the donor graft recognize and eliminate residual tumoral cells through the graft-versus-tumor (GVT) effect, and thus, donor lymphocyte injection (DLI) often is used at the time of relapse post-HCT to induce GVT. However, GVT can occur in parallel with lymphocyte reactivity to normal host tissues, which gives rise to graft-versus-host disease (GVHD). Despite the correlation of GVT and GVHD, indirect evidence for a GVT reaction separate from GVHD has been reported in large cohorts of HCT patients or when DLI is administered to induce remission in HCT patients who have experienced a relapse.+ and CD8+ T cells.4 miHAgs can mediate GVT without inducing GVHD if they are only expressed by the recipient hematopoietic cells .6 Nonhistocompatibility antigen proteins expressed by tumor cells, called tumor-associated antigens  also can mediate GVT activity.7 Cytokines 3; checkpoint proteins, such as PD-1/PD-L18; and T-cell trafficking modulators9 are other possible mediators of GVT.The GVT effect is mediated by minor histocompatibility antigens (miHAgs) on recipient leukemic cells that are recognized by donor CD410 first in a training set and then in a validation set and added a novel systems biology pipeline to identify a signature of 61 proteins that are significantly expressed in the plasma of HCT patients who received DLI for tumor relapse. Furthermore, 43 (70%) of the 61 genes were found in tumor antigen (PRAME)–activated T cells from a patient with post-HCT relapse.Our study aim was to develop a proteomic signature to identify GVT without GVHD after allogeneic DLI. We used an intact-protein analysis system (IPAS) coupled with protein tagging as previously reported by usKey ObjectiveSeparating graft-versus-host disease (GVHD) from graft-versus-tumor (GVT) effect after hematopoietic cell transplantation (HCT) has proven difficult. We attempted to do so through plasma proteomics and systems biology analyses of patients in relapse after HCT who received donor lymphocytes as immunotherapy.Knowledge GeneratedOur novel six-step systems biology analysis involved the removal of common proteins and GVHD-specific proteins, creation of a protein-protein interaction network, calculation of relevance and penalty scores, and visualization of candidate biomarkers in gene networks to define a unique, biologically relevant 61-protein signature of GVHD-free GVT. Forty-three of the 61 genes also were found in an independent experiment using massive single-cell profiling of tumor antigen–activated T cells from a patient who experienced post-HCT relapse.RelevanceThis multiomic analysis provides the first human plasma signature for GVHD-free GVT to our knowledge. Novel, biologically relevant markers were identified and could be extended to other antitumoral responses. Risk stratification on the basis of the GVT without GVHD protein signature would allow for customized treatment plans after HCT.All patients or their legal guardians provided written informed consent to participate in this study, and the collection of samples for studying post-HCT complications was approved by the institutional review board of the University of Michigan. The methodology for this study is described in the Data Supplement.Our initial approach to identifying GVT-specific biomarkers was to undertake a proteomics analysis. We hypothesized that proteomes of GVT are distinct from those of GVHD. We reasoned that the best setting to observe a GVT effect without any effect of chemotherapy or preparative conditioning regimen would be after DLI is given for relapse of malignant disease. Our first proteomics experiment (IPAS-01) was designed to distinguish proteomes that predict GVHD-free GVT from those that predict GVT with GVHD after DLI in a training set. We compared a pool of plasma samples taken approximately 30 days after DLI from five patients without relapse and without GVHD  labeled with the heavy isotope and compared them with a pool of plasma samples at matched time points from 11 patients without relapse but with concomitant GVHD  labeled with the light isotope . In this11A total of 825 proteins were confidently identified and quantified using the SwissProt database (Data Supplement). Of these proteins, 218 were upregulated with a heavy/light ratio greater than 1.2 in GVT-positive, GVHD-negative samples . Similar results were obtained using the UniProt human proteome database (Data Supplement). A total of 755 proteins were identified, including 327 that were upregulated by more than 1.2-fold in GVT-positive, GVHD-negative samples. The threshold of 1.2-fold also was used in our most recent GVHD clinical proteomics, which has allowed the discovery of biologically relevant biomarkers.13 to fit the GVT experiment and includes generating abundance ratios from comparisons of plasma samples taken from GVT without GVHD, filtering out GVHD-specific proteins identified in previous experiments, generating protein-protein interaction (PPI) pairs with and without outer gene networks, selecting GVT-relevant proteins by relevance scores and penalty scores for unspecific proteins found in other diseases, and using a visual analytic software tool (Data Supplement). After this data processing, the IPAS-01 experiment provided a list of 46 proteins associated with a GVT signature without GVHD post-DLI in the training set (www.terrainatlas.com) visualization as shown in z-axis adds data obtained from the proteomics experiments to generate a 2-dimensional terrain heat map. The first node is centered around CD8-α (CD8A) and includes CD58, IL1A, Ly75, FAS, and GPNMB and a secondary node centered around RAF1 and containing GUK1, EPHB4, and CXCL12. For example, CD8A is expressed on the surface of most cytotoxic CD8+ T lymphocytes and is a coreceptor of the T-cell receptor. The relative weight of CD4+ and CD8+ T cells in GVHD and GVT responses remains undetermined. Our data with the CD8A-centered node strongly suggest that GVT is majorly mediated by CD8 T cells and GVHD less so, which provides an opportunity to separate GVT and GVHD by manipulating these two T-cell subsets differently. A second cluster comprises MAP3K6, DBNL, and FERMT3, which are all implicated in immune response pathway activity. Penalty scores are designed to enrich for the most relevant proteins without altering the primary data. As a proof of concept, we performed the experiment without applying these proteins to show that their final scores have not been significantly changed by the use of penalty scores  used a validation set that was designed to compare a pool of samples from five GVT-positive, GVHD-negative (labeled with the heavy isotope) patients with a pool of samples from the same patients taken before DLI when the patients were in relapse and thus GVT negative , have not yet integrated PPI networks for this protein.The next step was to combine the proteins found in the training and validation sets. Combined analysis of the two IPAS experiments yielded 61 proteins: 49 with a final combined score of greater than 1 and 12 with a final combined score of less than 1, all specific to GVT without GVHD . The Gen15 T-cell responses to RPL23 also are increased in autoimmune diseases,16 which suggests a role for the RAS/RAF/MAPK pathway in T cells that is currently underexplored. CD58 is the ligand of CD2 on T cells, and engagement of both lead to T-cell activation and adhesion. The CD58/CD2 axis is the primary costimulatory pathway for CD8+ T cells that lack CD28, which suggests an alternate activation mechanism during GVT for exhausted T cells.17 CRTAM is upregulated in CD4+ and CD8+ T cells and encodes a type 1 transmembrane protein with V and C1-like immunoglobulin domains.18 It has been shown to negatively regulate ZEB1 (zinc finger E-box–binding homeobox 1) in T cells19 and to determine the CD4+ cytotoxic T-lymphocyte lineage,14 and it might determine the CD8+ cytotoxic T-lymphocyte lineage as well. The expression profiles of all 61 GVHD-free GVT markers are shown in the Data Supplement. These results suggest that tumor-specific T cells serve as a source of cells that express the GVT signature but require additional validation in future studies.Because the signature contained mostly intracellular proteins and additional samples from the discovery sets were not available to us, we next used single-cell profiling on T cells sorted and stimulated by the tumor antigen PRAME from a patient who relapsed after HCT compared with T cells sorted and stimulated by the viral antigen cytomegalovirus (CMV) pp65a as a surrogate measurement for an antitumoral response post-HCT. A mean reads number of 381,079 per cell and median gene number of 1,091 per cell were analyzed for an average of 1,436 cells per condition. Among the 61 genes identified in the proteomic GVHD-free GVT signature, 11 were more highly expressed in PRAME-specific T cells compared with CMV-specific T cells or nonreactive T cells. Thirty-two genes were enriched in both CMV- and PRAME-specific T cells, which represent a general activation marker of T cells. In contrast, 18 genes were not expressed in T-cell populations. The function and main GeneGO  process for each protein are described in 20 This pipeline allowed us to identify a final plasma signature of 61 proteins from initially thousands of proteins. One systems biology novelty of our approach was the performance of a one-layer extension on PPI data using interactions one node away from the gene in the original list that are called outer genes. However, we found that this strategy did not significantly change the final signature of 61 proteins (data not shown). A strength of our approach is that we filtered out nonrelevant proteins using a penalty score, which led to a more-specific list of candidate proteins and avoided contaminants. This study favored a large-scale proteomics approach as opposed to a hypothesis-driven candidate approach.21 We have shown that for GVHD markers, this method is efficient in discovering new candidate markers.11 Compared with our previous studies, we experimentally removed the GVHD proteins by assigning them the light isotope. Of note, this approach showed that the proteins identified and their ratio have not been influenced much by the implementation of penalty scores. In an independent experiment with single-cell profiling of T cells from a patient with relapse after HCT that were activated in vitro, 43 of the 61 proteomic genes also were found in activated T cells, which suggests that the proteins identified are biologically relevant in different antitumoral responses.The current study has identified the first plasma proteomic signature for GVHD-free GVT after DLI using an in-depth tandem mass spectrometry–based analysis of plasma combined with a novel six-step systems biology approach. Previous studies have focused on cellular T-cell markers.22 and the activation of c-Jun NH(2) terminal kinase and transcription factor AP-1.23 Guanylate kinase 1 (GUK1) is an enzyme that catalyzes the transfer of a phosphate group from ATP to guanosine monophosphate (GMP) to form guanosine diphosphate (GDP) and is believed to be a good target for cancer chemotherapy.24 Its expression on tumor-specific T cells was not previously reported. PIN1  catalyzes the cis/trans isomerization of peptidyl-prolyl peptide bonds and thus catalytically regulates the postphosphorylation conformation of its substrates and is involved in the regulation of T-cell biology. In particular, its implication has been shown in systemic lupus erythematosus and T-cell acute lymphoblastic leukemia progression.27 WAPL cohesin release factor has been shown to restrict chromatin loop extension.28 Of note, it was part of a microRNA-mRNA network in allogeneic T-cell responses.29 Fermitin family member 3 (FERMT3) is a member of the kindlins that mediates PPI involved in integrin activation. Mutations in this gene cause the autosomal recessive leukocyte adhesion deficiency syndrome-3.30 Its role in T cells has not been studied. CD160 is another surface protein tightly expressed on peripheral cytotoxic CD8 T lymphocytes and natural killer cells,31 and soluble CD160 enhances CD8+ T cells, which results in increased interferon-γ, IL-2, and tumor necrosis factor-α secretion as well as cytolysis against tumor cells in vitro and in vivo.32The biology of the GVT markers not yet described is as follows. The function of TRPC4AP  has been shown to be involved in the ubiquitination of E3 ligase skp233 was also upregulated in this study. The Fas cell surface death receptor (FAS) that plays a critical role in the activation of the death-inducing signaling complex with Fas-associated death domain protein and triggers a downstream caspase cascade that leads to apoptosis34 also was upregulated.Not surprisingly, serglycin (SRGN), which serves as a mediator of granule-mediated apoptosis through the macromolecular complex of granzymes and perforin and determines the secretory granule repertoire of cytotoxic T lymphocytes,35 It is one of the rare proteins for which the final score was modified more than twice by the penalty score but is still included overall as a GVT protein. Several of the tripartite motif (TRIM) members encode for miHAgs.21 Although TRIM42, 22, and 37 are located on chromosomes 3, 11, and 17, respectively, TRIM39 is on chromosome 6 in the major histocompatability class I region and, in our study, showed global expression on activated T cells. RAF1 is the cellular homolog of viral raf proto-oncogene (v-raf) and is also a MAP3K that functions downstream of the Ras family of membrane-associated GTPases to which it binds directly. CSE1L is a RAS-related nuclear protein that binds strongly to nuclear localization signal-free importin-α, and this complex is then released in the cytoplasm by the combined action of RANBP1 and RANGAP1. The role of RAS/RAF/MAPK and phosphatidylinositol 3-kinase signaling in T cells remains understudied. One particular protein of non–T-cell origin, IL-1α, was found to be elevated in the plasma of patients with GVT response and may play a significant role in antitumor effects. Of note, IL-1α–engineered tumor cells rarely develop into tumors, and if they do, the tumors are quickly destroyed through a mechanism that involves CD8+ T cells and natural killer cells, and IL-1α enhances immunoediting in the tumor microenvironment.36The chemokine CXCL12 has been proposed to be able to distinguish immune cells that induce GVT going to the bone marrow from immune cells that induce GVHD.Although our approach identified several proteins for GVHD-free GVT, there are limitations in this study. First, plasma samples were pooled together. Second, only a small percentage of the whole circulating proteins are identified and quantified with tandem mass spectrometry. Third, the systems biology pipeline relies on knowledge from the published domain, which makes the application of this method to diseases that have not been well studied, such as GVT, difficult. Finally, because most of the proteins identified were intracellular, we could not apply immunoassays as we have done previously. As a result of the lack of samples available from additional DLI patients, we sought to look at the 61 genes from the proteomic signature in T cells from a patient who experienced relapse after HCT and that were stimulated by the tumor antigen PRAME as a surrogate measurement for an antitumoral response post-HCT. Forty-three of these 61 genes also were found with this technique. Although challenged by the paucity of samples available after DLI, validation of this proteomic signature in larger cohorts is warranted.


H3F3A mutation G34W. This point mutation is regarded as the driver mutation of GCTB. We have established three new stable H3F3A mutated GCTB cell lines: U-GCT1, U-GCT2, and U-GCT3M. MK-1775 is a Wee1-kinase inhibitor which has been used for blocking of sarcoma growth. In the cell lines we detected Wee1, Cdk1, Cyclin B1, H3K36me3, and Rrm2 as members of the Wee1 pathway. We analyzed the effect of MK-1775 and gemcitabine, alone and in combination, on the growth of the cell lines. The cell lines showed a significant reduction in cell proliferation when treated with MK-1775 or gemcitabine. The combination of both agents led to a further significant reduction in cell proliferation compared to the single agents. Immunohistochemical analysis of 13 GCTB samples revealed that Wee1 and downstream-relevant members are present in GCTB tissue samples. Overall, our work offers valuable new tools for GCTB studies and presents a description of novel biomarkers and molecular targeting strategies.The giant cell tumor of bone (GCTB) is a locally aggressive primary bone tumor that is composed of mononuclear stroma cells, scattered macrophages, and multinucleated osteoclast-like giant cells which cause pathologic osteolysis. The stroma cells represent the neoplastic population of the tumor and are characterized by the  Malignant transformation is rare in GCTB occurring in less than 1%, but may rise to 6,6% after radiation therapy3. The neoplastic and mitotically active mononuclear stroma cells are characterized by a mutation in H3F3A4. In the presence of macrophage colony–stimulating factor (M-CSF) produced by macrophages the neoplastic cells recruit and activate osteolytic osteoclast giant cells by constitutive expression of receptor activator of nuclear factor kappa-Β ligand (RANKL)6. Surgery is used as a standard treatment7. However, the tumor recurs in 5 to 25% of patients8. Denosumab is a humanized monoclonal antibody specific for the RANKL and is used in patients with advanced disease9. Recently, several cases of patients with GCTB have been described in which high-grade osteosarcomas arose after treatment with denosumab11.The GCTB is a benign but locally aggressive neoplasm. The tumor is predominantly localized in the epiphyseal region of long bones16. The Wee1 kinase controls the function of Cdk1 by phosphorylation at tyrosine 15, causing an inactivation of Cdk1 . H3F3A mutations in GCTB are known to be associated with an increase in H3K36me322. Severe Rrm2 depletion is thought to lead to dNTP starvation and to induce replication stress. For example, H3K36me3-deficient cell lines, like the kidney carcinoma cell lines A498, have been shown to be selectively killed by MK-1775 via dNTP starvation12. MK-1775, a specific Wee1 inhibitor, has been tested as a possible therapeutic option in sarcomas; e.g., Wee1 inhibition has been shown to sensitize osteosarcoma cells in vitro to chemotherapy or radiation at clinically feasible concentrations23. Compared to normal tissues, Wee1 is overexpressed in osteosarcomas23. In the breast cancer cell line CAL51, Wee1 is overexpressed and inhibition by MK-1775 is associated with a functional loss of Wee1 leading to cell death underlining the essential role of Wee1 in tumor cell viability24. In a Phase I pharmacological and pharmacodynamics study in patients with melanoma, lung cancer, ovarian cancer, breast cancer or colorectal cancer MK-1775 had a low toxicity profile both as monotherapy and in combination with DNA-damaging agents like gemcitabine 25. Gemcitabine is a prodrug that is di- or triphosphorylated inside the cell. The triphosphate form (dFdCTP) is a nucleoside analog of cytidine, inhibiting DNA synthesis26. The diphosphate form (dFdCDP) affects the enzyme ribonucleotide reductase and leads to a depletion of deoxycytidine triphosphate (dCTP) pool that potentiates the effects of the drug27.The Wee1 pathway has been explored in several types of cancer, such as kidney cancer, colorectal cancer, melanoma, and osteosarcomadk1 Fig. 17. The ain vitro. We demonstrate that the Wee1 pathway is active in GCTB cell lines and is inhibited by a specific Wee1 inhibitor, MK-1775.Based on these data we have investigated the effect of the inhibitor MK-1775 and gemcitabine on the H3F3A-mutated GCTB cell lines. Here, we show that Wee1, Cdk1, H3K36me3, and Rrm2, as crucial players in cell proliferation, are detectable in both GCTB tissue samples and H3F3A-mutated GCTBs with typical cytologic morphology of osteoclast-like giant cells and spindle-shaped mononuclear cells  analysis of the cell lines and the parental tumor confirmed the origin of the cell lines  comparable to published data for the A498 cell line12. Increasing concentrations of MK-1775 led to a reduction in viable cells in all the GCTB cell lines analyzed .2 cell culture flask  with Iscove’s Modified Dulbecco’s Medium with 25 mM HEPES  with 10% fetal bovine serum , 2 mM L-glutamine , and penicillin-streptomycin . Cells were detached with trypsin  before reaching a state of complete confluence. As control cells we used the commercially available cell lines A498, Jurkat, and HEK-293 . The cell line experiments were performed within 4 months after STR analysis and negative testing for mycoplasma by PCR. All cell lines were cultured at 37 °C in a 5% CO2 incubator applying the same protocol as mentioned above.After extraction, the tumor tissue was prepared for cell culture. The tissue was minced into small pieces and partially digested with collagenase : C9891) 1 mg/ml diluted in 50 nM Trizma Base  and 0.32 nM calcium chloride dihydrate , pH 7.4. Isolated cells were cultured in Nunc EasYFlask 25 cm41.DNA from the cell lines was extracted using the QIAamp DNA Mini Kit . STR analysis was performed comparing the parental tumors with the corresponding cell lines and genotyping them and the control cell lines by using a standard protocolCell lines were tested for the presence of mycoplasma using PCR. PCR amplification was done using specific primers for mycoplasma DNA .Gene sequencing (Sanger) was performed according to our diagnostic standard protocolTP53 were amplified using a Multiplex PCR Kit . Subsequently, PCR products were sequenced using the MiSeq-Sequencer  with a coverage of 5000x. The resulting sequences were compared to an hg19 as the reference genome using the MiSeq Reporter Software. The non-synonymous aberration was checked using the Integrated Genome Viewer. Genomic areas were reviewed for clinically relevant TP53-point mutations using http://cancer.sanger.ac.uk/cosmic.Exons of WEE1) or 58 °C (β-Actin and GAPDH) for 30 seconds and at 72 °C for 30 seconds. All experiments were performed in triplicate.RNeasy Mini-Kit  was used for extracting total RNA. A NanoDrop 2000 spectrophotometer  was used to control the RNA quality and quantity. Omniscript Reverse Transcription Kit  was used to synthesize single-strand cDNA from total RNA. QuantiTect SYBR Green PCR Kit  and the Light-cycler Rotor Gene Q  were used to perform the qPCR. The expression of the WEE1 gene was analyzed using the appropriate QuantiTect Primer Assays . β-actin and GAPDH served as housekeeping genes , Wee1 , Anti-Histone H3.3 G34W , Histone H3K36me3 antibody , Cdc2 p34 , Phospho-cdc2 Tyrosine 15 , and Cyclin B1 . As secondary antibody we used: Anti-Rabbit IgG , rabbit anti-goat IgG-HRP  and goat anti-mouse IgG-HRP . Anti-β-Actin antibody  or Anti-β-Tubulin  were used to detect β-actin or β-tubulin as a housekeeping protein.Protein isolation and Western blots were performed using standard protocols as described elsewhereFor inhibition assays, MK-1775  was diluted in DMSO  and gemcitabine hydrochloride  was diluted in PBS .2 of the cell lines were seeded in Nunclon Delta 96-Well MicroWell Plates . An initial incubation at 37 °C and 5% CO2 for 24 hours ensured complete adherence and spreading of the cells. The cells were then incubated for 72 hours under the above-mentioned cell culture conditions at eight different concentrations of the agents increasing in decimal power from 0.01 nM to 100 µM. Untreated cells served as controls. Five wells were prepared for each treatment. As the next step, 10 µl CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS)  were added to all wells and the absorption was measured after 3 hours using the Biokinetics Microplate Reader  with a 490 nm filter. The results were calibrated in Excel 2016 . The average of the medium data was subtracted from the other results and the inhibition data were then divided by the average of the dilution solution only-treated cells. The combination effect of MK-1775 and gemcitabine were established by comparing the detected IC25 values of the agents in single-dose and combination as described above.For MTS cell proliferation assays, 15000 cells/cm2 Cell Culture Flask  for 24 hours. The cells were then incubated with 3 mM of the MK-1775, gemcitabine, or both agents in 1 ml medium for 72 h at a concentration of 1 mM. HEK293 cells were treated with 5 ng/ml bortezomib , diluted in DMSO and 50 ng/ml TNF-α  diluted in medium for 24 hours and were used as positive controls42. Protein isolation and Western blot were conducted using the above-mentioned protocol and subsequently using the primary and secondary antibodies of the apoptosis Western blot cocktail  as previously described. Formalin-fixed and paraffin-embedded cell pellets were prepared using standard protocols. Ki-67  stainings were performed as described below and evaluated on 3 paraffin sections of cell pellet preparations. At least 100 cells were counted on each cytoblock section. Untreated controls were included in every experiment. All experiments were repeated at least three times.For apoptosis, Western blot and Ki-67 analysis, the cell lines were cultivated in 2 ml medium in a Nunc EasYFlask 25 cmH3F3A mutation from 13 patients were available from the Ulm tissue bank . The parental tumors of the cell lines were included  and was in compliance with the ethical principles of the WMA Declaration of Helsinki44.GCTB tissue samples with The alkaline phosphatase/RED detection system  was used for immunohistochemistry and immunochemistry on formalin-fixed and paraffin-embedded tissue or cell pellets via the avidin-biotin-complex-method. The following antibodies were used: Anti-Histone H3.3 G34W , Wee1 , Cdc2 p34 , Cyclin B1 , Histone H3K36me3 Antibody , R2 , and Ki-67 Antigen . As retrieval methodes all sides were pretreated by a steamer in EDTA pH 8. Consensus evaluation was done on multihead microscope .For statistical analysis, two-sided t-tests were performed. A p-value ≤ 0.05% was considered as significant. The graphs were generated with Prism Version 6.01 .All methods were performed in accordance with the relevant guidelines and regulations of good scientific practice of the University of Ulm and the Deutsche Forschungsgemeinschaft (DFG): 10.1002/9783527679188.oth1.Supplementary Data


We believe that adherence to a common set of GvHD assessment criteria is vitally important to improve the quality of data, compare results of retrospective studies and prospective clinical trials, and make therapeutic recommendations based on quality evidence.Several international recommendations address the assessment of graft-versus-host disease (GvHD) after hematopoietic cell transplantation (HCT). This position statement by GvHD experts from the European Society for Blood and Marrow Transplantation (EBMT), the National Institutes of Health (NIH) and the Center for International Blood and Marrow Transplant Research (CIBMTR) reviews the existing guidelines for both acute and chronic GvHD, addresses potential confusions that arise in daily practice and proposes consensus definitions for many key terms. We provide a historical perspective on the currently available guidelines and recommend the Mount Sinai Acute GvHD International Consortium (MAGIC) criteria for acute GvHD and the NIH 2014 criteria for chronic GvHD as the most comprehensive and detailed criteria available. This statement also offers practical guidance for the implementation of these recommendations and a set of consensus definitions for commonly used GvHD terms in order to facilitate future clinical and translational research. To assist the dissemination of these recommendations, a web-application based on this position statement is available ( Graft-versus-host disease (GvHD) refers to a clinical syndrome caused by the response of transplanted donor allogeneic cells to histocompatibility antigens expressed on tissues of the transplantation recipient. It is the most serious complication of allogeneic hematopoietic cell transplantation (HCT). Its recognition and control are key elements of a successful outcome. In fact, the World Health Organization stipulates that data collection and data analysis are integral parts of therapy .In practice, however, the application of basic concepts pertaining to the diagnosis and staging of this condition differs widely among HCT clinicians. The use of the templated data collection forms , the European Society for Blood and Marrow Transplantation (EBMT), and the National Institutes of Health/National Cancer Institute (NIH/NCI)) improves standardization by collecting data elements as proposed by published consensus documents but demands significant time from healthcare professionals and researchers.Several studies have shown a lack of adherence to recommendations and inconsistencies in GvHD evaluation –9. Weisdhttps://www.uzleuven.be/egvhd).Such discrepancies are concerning because they can significantly affect the interpretation of GvHD data in clinical trials. Misclassifications have been observed even among experienced HCT and GvHD professionals, and inaccuracies are therefore likely to be even more prominent among less experienced centers. In fact, a recent survey of practice patterns completed by transplant professionals during the annual 2017 EBMT conference showed wide variations in the types of reference guidelines used for GvHD assessments, and up to one third of the survey participants reported a lack of confidence in their ability to apply these guidelines . Of inteThe use of electronic tools to streamline and increase the reliability of the GvHD evaluation process has been advocated by several groups , 9–12, bAcute GvHD refers to the appearance of an allogeneic inflammatory response in exclusively three organs: the skin (inflammatory maculopapular erythematous skin rash), the liver (hyperbilirubinemia due to cholestatic jaundice), and the gastro-intestinal (GI) tract  –16. The The Glucksberg aGvHD classification was first proposed in the 1970s based on a cohort of 60 patients evaluated for aGvHD after myeloablative conditioning. This classification staged skin, lower gastrointestinal tract and liver, each on a scale of 0 (absent) to 4 (severe) points , to creaMost recently, the Mount Sinai Acute GvHD International Consortium (MAGIC) has revisited these criteria based on a review of their extensive database containing detailed clinical information on aGvHD, and recommended more precise definitions for grade IV aGvHD . SpecifiOf note, the MAGIC group also introduced the concept of diagnostic confidence levels for acute GvHD: “confirmed”, “probable”, “possible” and “negative” correlating with histological confirmation, initiation of treatment, resolution without therapeutic intervention, and definitive alternative histologic diagnosis, respectively. Further prospective validation of the confidence categories is underway to formally assess their predictive value and reliability.Chronic GvHD was originally defined in the early 1980s in a cohort of 20 Seattle patients, as any GvHD present beyond day 100. cGvHD severity was categorized as “limited”  or “extensive”  . 20 yearconfirmation (e.g. biopsy or other objective diagnostic test) in at least one target organ . The “overlap cGvHD sub-type” was defined by the diagnosis of cGvHD together with acute GvHD manifestations of the skin, liver or gut  evaluates exclusively three organs for aGvHD  and eight organs for cGvHD , based on the highest score of organ involvement as described above Tables  and 3. NFor both acute and chronic GvHD, a given organ is not considered in the overall GvHD grade if the manifestation is solely due to a non-GvHD cause . In the case of both GvHD and concomitant non-GvHD etiologies, it is useful to document the non-GvHD causes but there is currently no justification to downgrade an organ score due to concurrent additional causes (e.g. simultaneous liver GvHD and veno-occlusive disease) , 25.Acute GvHD typically only involves three organs: the skin, the liver, and the GI tract . AlloimmSome patients have atypical signs and symptoms that might be considered cGvHD but fall outside of the current diagnostic, staging and response criteria , 27. SucSimilarly, isolated increase of transaminases is relatively common during the taper of immunosuppression or after donor lymphocyte infusions. This increase should also be assigned to the “undefined other cGvHD” group, provided it is treated as GvHD in the absence of meeting NIH diagnostic criteria and no histopathological confirmation of liver GvHD has been obtained. Because of their invasive character, liver biopsies are rarely performed and the nature of hepatic enzyme disturbances remains therefore uncertain. This further emphasizes the need for prospective recording of such abnormalities .Overlap cGvHD is a subtype of cGvHD which has been associated with a poor prognosis , 30. It In cGvHD, MRI can sometimes be a useful tool to detect fascia involvement , yet disClinical practice rarely allows time and resources for an exhaustive patient evaluation of cGvHD at every visit. For example, pulmonary function tests (PFTs) and genital examinations typically require third-party input, which can be challenging to obtain on the same day.Although both the dyspnea and lung function scores should ideally be recorded, PFTs are the best way to describe lung involvement and should be obtained at diagnosis of GvHD and then minimally every 3–6 months thereafter in patients on systemic therapy for active cGvHD , 35. HowA formal genital exam or inspection should ideally be performed at diagnosis and at every GvHD evaluation thereafter in patients with active cGvHD. In clinical practice, this is not always feasible; therefore, we recommend this exam be performed within 3 months of cGvHD diagnosis followed by a regular follow-up every 9−12 months , 36. At Of note, both pulmonary and urogenital complications can go undetected if not specifically queried, with potentially dramatic clinical consequences , 37–39. In spite of the extensive harmonization effort of the cGvHD NIH consortium, some criteria would benefit from further clarification. For instance, weight loss is categorized based on the percentage decrease of bodyweight occurring over a 3-month period . It is uThree primary areas differ in the pediatric population with regards to GvHD assessment: (1) some criteria used in adults are difficult to apply in young children (e.g. PFTs and Schirmer’s test for children under the age of 6 ); (2) thCurrently, the only organs with specific pediatric modifications recommendations for GvHD assessment are: (1) adapted body surface area maps for skin involvement; (2) appropriate reference values for lung function; and (3) weight-adapted measures for diarrhea , 18. MorIn clinical practice, GvHD presentations can range from a rapidly progressive extensive inflammatory syndrome requiring immediate and aggressive systemic immune suppression, to purely fibrotic, cicatricial manifestations with fixed deficits that are unlikely to respond quickly or completely resolve with therapy . BetweenIn the setting of clinical trials, response to treatment compares disease burden at specific points in time, usually with regards to a particular treatment. It is based on a number of clinical findings, sometimes including fixed deficits. Classical categories of response are complete response (CR), partial response (PR), and lack of response , as established by the NIH consortium for chronic GvHD . For acuHowever, GvHD activity may be distinct from response if the disease burden includes fixed deficits that are no longer responsive to treatment. Identification of such deficits can be difficult but is essential to the accurate description of complex clinical phenotypes, particularly in cGvHD. Determination of GvHD activity is often the principal driver in therapeutic decisions  or discontinuation of immunosuppression) and is likely to be critical for biomarker validation. We therefore propose a classification of GvHD activity that incorporates both the presence of disease manifestations and the use of immunosuppression, consistent with the NIH Consensus task force model of GvHD physio-pathology .GvHD is considered “clinically active” if the patient has inflammatory or worsening manifestations (either acute or chronic) regardless of the use of immunosuppressive therapy. After the inflammation resolves, GvHD manifestations can either disappear without residua or fixed deficits may remain. Such fixed or irreversible deficits represent scars in the affected organ due to either permanent damage or aberrant tissue repair  that persist regardless of immunosuppressive treatment .Once all signs of clinical activity have disappeared, GvHD activity can be described in three different ways. If immunosuppression is still ongoing or has been discontinued for less than 12 weeks  or 24 weGvHD onset refers to the presentation of the first episode of clinically evident alloreactivity of the donor against the recipient host .“Classic acute GvHD” refers to the initial diagnosis of acute GvHD within the first 100 days following transplantation or DLI infusion (whichever happened last) . “Late aChronic GvHD is referred to as having “de novo onset” if cGvHD is diagnosed  for the There is currently no formal nomenclature to refer to the pattern of GvHD recurrence after an initial diagnosis. The term “flare” is sometimes used to define the reappearance or worsening of any signs of GvHD. Although this might reflect the natural course of the disease, this term currently lacks a validated definition. For written scientific communications, we recommend instead the precise terminology that refers to disease onset  or the cAcute GvHD steroid refractoriness or resistance is most often referred to as either (1) progression in any organ within 3 –71, 4 7–76, or 5Steroid dependence has been defined for aGvHD as the inability to taper prednisone under 2 mg/kg/day after an initially successful treatment of at least 7 days , 80, 81 Finally, the term “steroid intolerance” has not been formally validated but refers to the emergence of unacceptable toxicity  attributed to corticosteroids, as evaluated by a healthcare professional , 92.https://www.uzleuven.be/egvhd), are already developing more standardized and accurate methods to capture “real-world” GvHD data. This progress underlines the responsibility of transplantation societies to help clarify definitions, to facilitate comparisons of clinical research results and to set standards for clinical practice.This report stresses the critical importance of a common, international approach to describe the variety of GvHD clinical manifestations observed after HCT. In the era of electronic patient records and e-health applications, it is possible to apply complex algorithms at the bedside and follow internationally vetted guidelines in daily clinical practice. Several efforts in this direction , 9–12, sThis task force panel advocates the use of the MAGIC criteria for aGvHD and the NIH 2014 criteria for cGvHD as the most comprehensive and detailed criteria currently available. In addition, this statement provides consensus definitions for a lexicon of commonly used GvHD terms and concepts in order to facilitate GvHD clinical research.The standardization of GvHD assessments should be a dynamic process that can incorporate progress in new diagnostic and therapeutic approaches. Even as refined classifications improve communication among clinicians, they should also be prospectively evaluated for their predictive potential. Furthermore, in the absence of any pathognomonic signs or test for GvHD, subjective elements remain an integral part of the final clinical assessment. As prospective biomarkers that detect underlying GvHD pathophysiology are validated, they may assist clinicians by offering objective laboratory metrics in addition to clinical GvHD manifestations. But the formal validation of these markers requires accurate and reliable clinical assessment of GvHD severity in all organs.We hope that this position statement will serve as the cornerstone of a larger scale consensus project. Consistent adherence to common sets of criteria, such as those endorsed here, will help the transplantation community to improve the quality of data capture across all types of GvHD manifestations and therapeutic strategies. Harmonization of standards for the accurate assessment of GvHD is an essential prerequisite for the formulation of recommendations , 93 rega


Kinesin family member C1 (KIFC1) is a minus end-directed motor protein that plays an essential role in centrosome clustering. Previously, we reported that KIFC1 is involved in cancer progression in prostate cancer (PCa). We designed this study to assess the involvement of KIFC1 in docetaxel (DTX) resistance in PCa and examined the effect of KIFC1 on DTX resistance. We also analyzed the possible role of a KIFC1 inhibitor (CW069) in PCa. We used DTX-resistant PCa cell lines in DU145 and C4-2 cells to analyze the effect of KIFC1 on DTX resistance in PCa. Western blotting showed that KIFC1 expression was higher in the DTX-resistant cell lines than in the parental cell lines. Downregulation of KIFC1 re-sensitized the DTX-resistant cell lines to DTX treatment. CW069 treatment suppressed cell viability in both parental and DTX-resistant cell lines. DTX alone had little effect on cell viability in the DTX-resistant cells. However, the combination of DTX and CW069 significantly reduced cell viability in the DTX-resistant cells, indicating that CW069 re-sensitized the DTX-resistant cell lines to DTX treatment. These results suggest that a combination of CW069 and DTX could be a potential strategy to overcome DTX resistance. Prostate cancer (PCa) is the most prevalent cancer among men and the second leading cause of cancer-related death in developed countries . AndrogeIncreased centrosome number, called centrosome amplification (CA), is a hallmark of human cancer . Recent 2 at 37 °C. DTX cell lines were cultured under DTX at a dose of 2 ng/mL for DU145-DR and 5 ng/mL for C4-2-DR.Two PCa cell lines (DU145 and C4-2) and DTX-resistant DU145 cells (DU145-DR) and DTX-resistant C4-2 cells (C4-2-DR) were kindly provided by Dr. Masaki Shiota . The DU145 cell lines were maintained in MEM , and the C4-2 cells were maintained in RPMI 1640  containing 10% fetal bovine serum , 2 mM L-glutamine, 50 U/mL penicillin, and 50 g/mL streptomycin in a humidified atmosphere of 5% CODTX was obtained from Sanofi-Aventis and handled according to the manufacturer’s recommendations . CW069 wFor Western blotting analysis, cells were lysed as described previously . PrimaryTotal RNA was isolated from frozen cancer cell lines using Isogen , and 1 μg of total RNA was converted to cDNA with a first-strand cDNA synthesis kit . The qPCR was performed with a SYBR Select Master Mix  as described previously . ACTB-sp® Select  against KIFC1 was used for RNA interference. Two independent oligonucleotides and negative control small interfering RNA (siRNA)  were used. Transfection was performed using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions. Cells were used 48 h after transfection in each of the experiments and assays ,2322,23]To verify whether KIFC1 is involved in DTX resistance, we investigated the expression of KIFC1 in DU145-DR and C4-2-DR cells. Western blotting and qRT-PCR showed that KIFC1 was overexpressed in DU145-DR and C4-2-DR cells compared with the parental DU145 and C4-2 cells at both mRNA and protein levels A,B.Several studies have shown that KIFC1 is associated with an apoptosis pathway ,25. We uA recent study reported that CW069 is a novel and allosteric inhibitor of KIFC1 . To clarAs shown in DTX has been the first-line therapy for metastatic CRPC patients since 2004. Recent clinical studies have reported that early DTX treatment combined with androgen deprivation therapy results in improved overall survival in comparison to androgen deprivation therapy alone in patients with metastatic hormone-sensitive PCa ,29. ThisA recent study reported that CW069 was identified as a highly selective small-molecule KIFC1 inhibitor using a chemogenomics-based approach . CW069 iThere are some limitations in this study. First, so far, three KIFC1 inhibitors  have been reported ,38,39. AIn conclusion, we used DTX-resistant PCa cell lines to analyze the role of KIFC1 in DTX resistance. We found that the expression of KIFC1 was significantly upregulated in cells with DTX resistance. Inhibition of KIFC1 induced an apoptosis pathway and re-sensitized the cellular response to DTX. Additionally, CW069 re-sensitized DTX-resistant cells to DTX treatment. The data presented here emphasize the great potential of combination therapy with DTX and CW069 in the treatment of PCa.