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.


PRCC) is the most common nonclear cell RCCs and is known to comprise two histological subtypes. PRCC2 is more aggressive and is molecularly distinct from the other subtypes. Despite this, PRCCs are treated together as one entity, and they show poor response to the current therapies that do not target pathways implicated in their pathogenesis. We have previously detected ABCC2 (an ABC transporter), VEGF, and mTOR pathways to be enriched in PRCC2. In this study, we assess the therapeutic potential of targeting these pathways in PRCC2. Twenty RCC cell lines from the Cancer Cell Encyclopedia were compared to the Cancer Genome Atlas PRCC cohort (290), to identify representative PRCC2 cell lines. Cell lines were further validated in xenograft models. Selected cell lines were treated in vitro and in vivo (mice models) under five different conditions, untreated, anti‐VEGF (sunitinib), ABCC2 blocker (MK571), mTOR inhibitor (everolimus) and sunitinib + MK571. Sunitinib +ABCC2 blocker group showed a significant response to therapy compared to the other treatment groups both in vitro (P ≤ 0.0001) and in vivo (P = 0.0132). ABCC2 blockage resulted in higher sunitinib uptake, both in vitro (P = 0.0016) and in vivo (P = 0.0031). Everolimus group demonstrated the second best response in vivo. The double‐treatment group showed the highest apoptotic rate and lowest proliferation rate. There is an urgent need for individualized therapies of RCC subtypes that take into account their specific biology. Our results demonstrate that combined targeted therapy with sunitinib and ABCC2 blocker in PRCC2 has therapeutic potential. The results are likewise potentially significant for other ABCC2 high tumors. However, the results are preliminary and clinical trials are needed to confirm these effects in PRCC2 patients.Papillary renal cell carcinoma ( ABC transportersATP binding cassetteABCC2ABC binding cassette subfamily C member 2CCRCCclear cell renal cell carcinomaChrchromosomePRCCpapillary renal cell carcinomaet al., et al., Renal cell carcinomas (RCCs) are tumors that arise from renal tubules. They represent ~90% of adult kidney tumors and one of the most prevalent malignancies worldwide  (~75%), and the second most common are papillary RCC (PRCC) (10–15%) which is comprised of multiple subtypes  tyrosine kinase inhibitors (TKIs), for example, sunitinib and pazopanib, and second‐line therapies the mammalian target of rapamycin (mTOR) inhibitors (everolimus and temsirolimus), and the recently approved PD‐1 immune checkpoint inhibitor nivolumab , with high statistical significance , and CCLE (Cancer Cell Line Encyclopedia‐Broad Institute). Gene expressions  were downloaded for the KIRP 290 PRCC cohort from the TCGA Web site (our published study)  mice (4 mice). Tumors were harvested and sections were histologically examined for cell lines morphology and IHC profiles and compared to those of PRCC1. Similar to PRCC1, the histology of the mice tumors showed a tubulo‐papillary architecture of small low‐grade tumor cells, linear nuclear arrangement, and negative staining for ABCC2 IHC  as a PRCC1 cell line based on literature evidence of its papillary nature  and the Deutsche Sammlung von Mikroorganismen und Zellkulturen , respectively. The former was grown in McCoy cell media with 10% fetal bovine serum (FBS), while the latter was grown in DMEM cell media with 15% FBS, 0.4 μg·mL3 cells per well in a 96‐well plate and after 24 h grouped into the five treatment conditions, untreated, treated sunitinib 1.0 μm, treated with MK‐571 25 μm (ABCC2 blocker) . The plate was scanned with image express analyzer to detect the number of cells with dye uptake. Subsequently, the cells were fixed with paraformaldehyde (PFA), subjected to the Hoechst dye again, and rescanned with the image express.nu) mice and NOD SCID (severe combined immunodeficiency) gamma mice.Mice bought from ‘The Jackson laboratory’ were the immunocompromised strains outbred athymic nude 2 x π /6, where length is the longest dimension and diameter is the shortest dimension , sunitinib only (50 mg·kg−1), everolimus only (2.5 mg·kg−1), sunitinib + MK571, and everolimus + MK571.CAKI‐2 cells were grown to 80% confluency and trypsinized, into single‐cell suspension. 10et al., Immunohistochemistry was performed using Ki67 and ABCC2 antibodies. The Ki67 was performed using a Ventana automated system. Ki67 is a marker of cell proliferation (detected by nuclear staining), as it is present in all active phases of the cell cycle .Mice tumor slides stained with Ki67 were scanned and analyzed with the et al., et al., graphpad prism 7 and spss statistical software .From the PRCC TCGA cohort, a distinct PRCC2 mRNA signature of 500 genes (compared to PRCC1) was elicited , as well as best correlation with the ARE pathway genes  Figs Of the 19 RCC cell lines, CAKI‐2 showed excellent correlation with PRCC2 top matched genes  mice (6 mice). Tumors were harvested and sections were histologically examined for cell lines morphology and IHC profiles and compared to those of PRCC2. All of the six injected nude mice developed tumors with papillary morphology and had large eosinophilic cells, with nuclear pseudostratification and prominent nucleoli, consistent with what is known as a PRCC2 morphology  ± MK571. The group of cells that were exposed to the dual treatment with both sunitinib and blocker was the most sensitive to treatment  and everolimus did not produce significant differences in cell viability versus the blocker alone  treated groups were stained with the cell permeable Hoescht 33342 DNA dye and then subjected to image screening and visualization using the Xpress image analyzer. MK571‐treated cells showed considerable dye uptake (indicating dye retention), whereas untreated cells failed to stain indicating an active export of the Hoescht dye by ABCC2. Then, we proceeded to fix the cells with paraformaldehyde (PFA) to terminate the ABCC2 drug transporter pump effect. Fixed untreated cells showed a significant increase in staining after blocking the ABCC2 effect with fixation, et al., To further confirm that ABCC2 contributes to drug resistance through pumping out medications, we measured sunitinib uptake in CAKI‐2 cells through the detection of its known spectral range in flow cytometry  was observed in the group that was exposed to the sunitinib and MK571 treatment  and 3686 mm3 in the control untreated group (P = 0.0132). The growth curves of everolimus‐treated group showed the second highest response to therapy, volume of 1225 mm3. Both everolimus and sunitinib + MK571 arms showed significant differences from the sunitinib treated arm. Sunitinib only and MK571 only treated groups showed similar responses to their corresponding therapy  with no significant differences between the two treatment arms.Tumor mouse model was optimized in immunocompromised athymic nude and NOD SCID gamma mice. 10P = 0.0045) compared to other treatment groups  , which was significantly higher , followed by sunitinib (14%), everolimus (7.5%) and then MK571 and untreated groups (2–5%). Upon comparing the distant organ metastasis among the different groups, the sunitinib + MK571 and everolimus groups showed the lowest level of metastasis among the treated mice (25–30%) (number of mice showing distant organ metastasis in their group). Even though the everolimus group showed a low level of lung metastasis, it was less tolerated by mice as 50% (2/4) were deceased halfway through the treatment period. The sunitinib and MK571 groups showed 50% and 75% metastasis, respectively Table .in vivo analysis yielded similar results to what was detected with in vitro treatment, with a significant increase in intracellular sunitinib among the group that was additionally treated with the ABCC2 blocker. sunitinib median fluorescent intensity (MFI) was 11 250 in the sunitinib + MK571 treated mice versus 6870 in the sunitinib only treated mice (P = 0.028) . After tumor dissociation into a single‐cell suspension, the sunitinib content of each mouse tumor was measured by flow cytometry as described in previous sections. Our et al., et al., et al., et al., et al., In the era of personalized medicine, we are getting to understand more about the individualized biology of different tumors and our approach to this management should reflect this knowledge. While there are many drugs under development toward that goal, we are not yet there for the different RCC subtypes  in PRCC2  and enrichment in the mTOR pathway, consistent with TCGA findings  did not show an increase in sunitinib uptake upon ABCC2 blockage. The results ascertain what was shown in other studies regarding an increase in drug uptake upon ABC transporter blockage, especially in cancer with a high level of transporters  have attempted to compare between sunitinib and everolimus treatment in PRCCs . These data highlight potential benefit in subtyping PRCCs in clinical trials. Large subtype‐specific clinical trials are required to formulate better guidelines  RCC cell line. in vitro setting and in mouse models. However, our findings would pave the way for clinical trials that can confirm these results and lead to a more accurate tumor‐specific treatment for the patients of PRCC2 and similar tumors.We believe our study uncovers extremely pertinent information regarding PRCC2 treatment. Discoveries on the individualized nature of tumor biology should direct the clinical field toward similarly individualized treatments. We assessed the efficacy of targeting tumor‐specific pathways in a preclinical setting and showed that these treatment modalities show great promise. We highlight the importance of targeting ABC transporters, as ABCC2 in PRCC2 and in other high drug transporter tumors. We acknowledge that the current study has a number of limitations, particularly its experimental design in cell lines and the nature of the assessment in in vivo and in vitro experiments and analyzed data. ZL guided and performed experiments, and interpreted results. FB reviewed and helped interpret the tumor pathology data. JB helped with the in vivo experiments. GB reviewed data and guided the tumor therapeutic experiments, and helped interpret data. AF reviewed and helped interpret the clinical data. FB performed experiments and immunohistochemical stains. MD: reviewed and helped interpret the tumor pathology data. GS, senior scientist, supervised the project, guided the experimental framework, helped interpret the results, and reviewed the final work and the manuscript.RS designed the project, performed the experiments, analyzed the data, and wrote the manuscript. MF performed Fig. S1. The CAL‐54 RCC cell line represents PRCC1.Fig. S2. The CAKI‐2 cell line represents PRCC2.Table S1. Histological examination of mice tumor models.Click here for additional data file.


Additionally, analogues of perillyl alcohol, a monoterpene with known anticancer activity, were evaluated to identify the molecular characteristics which contribute to their cytotoxicity, which was tested against OVCAR-8, HCT-116, and SF-295 human tumor cell lines, using the MTT assay. The results of this study showed that (−)-perillaldehyde 8,9-epoxide exhibited the highest percentage inhibition of cell proliferation (GI = 96.32%–99.89%). Perillyl alcohol exhibited high cytotoxic activity (90.92%–95.82%), while (+)-limonene 1,2-epoxide (GI = 58.48%–93.10%), (−)-perillaldehyde (GI = 59.28%–83.03%), and (−)-8-hydroxycarvotanacetone (GI = 61.59%–94.01%) showed intermediate activity. All of the compounds tested were less cytotoxic than perillyl alcohol, except (−)-perillaldehyde 8,9-epoxide (IC50 = 1.75–1.03 µL/mg). In general, replacement of C-C double bonds by epoxide groups in addition to the aldehyde group increases cytotoxicity. Furthermore, stereochemistry seems to play an important role in cytotoxicity. We have demonstrated the cytotoxic influence of chemical substituents on the p-menthane structure, and analogues of perillyl alcohol.Compounds isolated from essential oils play an important role in the prevention and treatment of cancer. Monoterpenes are natural products, and the principal constituents of many essential oils. The aim of this study was to investigate the cytotoxic potential of  Cancer is a disease in which certain cells in the body grow in an uncontrolled way. It is one of the world’s most serious illnesses, and together with heart attacks, it kills more people than any other disease. Its hallmarks constitute an organizing principle for rationalizing the complexities of neoplastic diseases. They include sustained proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and metastasis .Nature is an important source of new candidates for therapeutic compounds; a large chemical diversity is found in several species of plants, animals, and microorganisms . PrakashVepris macrophylla and Myristica fragrans in vitro  [14 [15  exhibit rocesses ,35,36.50 of compound 16, besides presenting the highest percentage of inhibition of cell proliferation, 16 was more potent than 3, showing high cytotoxicity in all three cell lines, with IC50 values ranging from 1.03–1.75 µg/mL, as shown in Evaluating the IC16 showed high cytotoxicity in tumor cells, it was tested for its ability to induce lysis in mouse erythrocytes (data not shown). The mechanical stability of the erythrocyte membrane is a good indicator of in vitro damage in cytotoxicity assays, since many drugs can alter this delicate structure [16 was not hemolytic even at the highest tested concentration (500 μg/mL). This suggests that the cytotoxicity mechanism is probably related to a more specific pathway.Since compound tructure . Red blotructure ,39,40,4116, ½ IC50 (0.32 µg/mL), IC50 (0.64 µg/mL) and 2 × IC50 (1.28 µg/mL) were chosen against HL-60. The anti-proliferative effects of 16 were demonstrated through the trypan blue dye exclusion test (Three concentrations of compound ion test . It show16), in cancer cells, two tests were performed, fluorescence microscopy using acridine orange/ethidium, and hematoxylin-eosin coloration analyzed by optical microscopy. After 24-h incubation, the effects of 16 were evaluated based on cell morphology using hematoxylin-eosin. All concentrations and showed severe drug-mediated changes. The hematoxylin-eosin stained HL-60 cells treated with 16 presented a morphology consistent with apoptosis, including a reduction in cell volume, chromatin condensation, and nuclei fragmentation, as well as, necrosis with membrane disruption, cell swelling and rupture leading to inflammation. The acridine orange/ethidium bromide stained and treated cells also displayed a morphology consistent with apoptosis and necrosis, in a concentration-dependent manner (p < 0.05) (Attempting to ascertain the cellular death process (as induced by  < 0.05) .16 caused cell death by apoptosis in the three concentrations tested, as shown in Compound 16 presented necrotic cell death simultaneously. This type of cell death is characterized by swelling of cellular organelles, cell membrane rupture, cell lysis (unlike apoptosis), and the core becomes distended yet substantially intact. Necrotic death is typically followed by inflammatory reactions [16 is increased.Besides the apoptotic process in doses of 0.64 and 1.28, compound eactions . This ap16 as a possible candidate for in vivo tests, even with the two processes of cell death responsible for its in vitro cytotoxicity.Various chemical agents such as β-lapachone described by Li and collaborators , induce This evaluation is important due to the fact that perillyl alcohol and its analogues are structurally similar to many of the chemical constituents of essential oils and other natural products. Therefore the results obtained in this study may well provide a reference for the development of novel cytotoxic agents having an appropriate biological profile.15) displayed good cytotoxic activity, with a GI value of 59.28%–83.03%. Compounds 14 , and 15  were less cytotoxic  than 16 . This indicates that the presence of an epoxide and/or aldehyde function in the molecule is important for cytotoxicity. Further, the anti-proliferative activity was even more pronounced when these functional groups  are both in the chemical structure, since (+)-limonene 1,2-epoxide (12) (GI = 58.48%–93.10%), which contains only the epoxide group, was less bioactive than 16 (GI = 96.32%–99.89%). On the other hand, comparing (+)-limonene 1,2-epoxide (12) with (−)-carvone epoxide (5), it appears that the endocyclic ketone function does not contribute to cytotoxicity, as compound 12 (GI = 58.48%–93.10%) was approximately 2.5- to 11-fold more cytotoxic than 5 (GI = 8.21%–29.24%).The presence of an exocyclic aldehyde carbonyl and the conjugated C-C double bond seem to contribute to cytotoxicity, as (−)-perillaldehyde (14) and (−)-perillaldehyde (15) differ from each other only by a functional group. Compound 14 has a hydroxyl group whereas 15 presents an aldehyde function, both located at the same position in the p-menthane skeleton (C-7). Perillyl alcohol (14) (GI = 90.92%–95.82%) exhibited higher cytotoxicity compared to (−)-perillaldehyde (15) (GI = 59.28%–83.03%), suggesting that a hydroxyl group at C-7 position produces in a more potent inhibition of cell proliferation than an aldehyde group at the same position.The monoterpenes perillyl alcohol (1) (GI = 2.28%–12.28%), (+)-carvone (2) (GI = 34.39%–48.07%), (+)-pulegone (9) (GI = 10.25%–27.44%), and (−)-trans-isopulegone (11) (GI = 5.98%–18.96%). In the general, the evaluated ketones showed moderate to weak cytotoxicity, but considerable differences in their potencies were observed. We also noted that compounds 9  and 11 (saturated ketone) displayed different cytotoxic effects against the three tumor cell lines.To investigate the effect of the disposition of the ketone carbonyl group in cytotoxic activity, we compared the oxygenated monoterpenes (−)-carvone (1) (GI = 2.28%–12.28%) with (−)-carvone epoxide (5) (GI = 8.21%–29.24%) and (+)-pulegone (9) (GI = 10.25%–27.44%) with (R)-pulegone oxide (10) (GI = 16.02%–43.21%). Similar results were found comparing the compounds (+)-carveol epoxide (6) and (−)-cis-carveol (7), and (−)-perillaldehyde (15) and (−)-perillaldehyde 8,9-epoxide (16). Replacement of a non-conjugated C-C double bond by an epoxide function in 7 (GI = 3.61%–21.16%) and 15 (GI = 59.28%–83.03%), resulting in the respective compounds 6 (GI = 4.58%–35.35%) and 16 (GI = 96.32%–99.89%), produces a subtle increase in cytotoxicity.Replacement of a carbon-carbon double bond, conjugating the carbonyl with an epoxide group resulted in a modest enhancement of anti-proliferative effect. Improvement of the biological potency was observed thru comparison of (−)-carvone (5) with (R)-pulegone oxide (10). Compounds 5 and 10 present a ketone and epoxide group in different positions in the p-menthane skeleton, and different biological effects for these monoterpenes was observed. (R)-Pulegone oxide (10) was more bioactive (GI = 16.02%–43.21%) than (−)-carvone epoxide (5) (GI = 8.21%–29.24%), suggesting that the position of these groups in the p-menthanic structure influence the cytotoxic activity.To ascertain the influence of the disposition of the epoxide and ketone groups in cytotoxicity, we compared (−)-carvone epoxide (1) was compared with (−)-8-hydroxycarvotanacetone (3), and (+)-carvone (2) with (+)-8-hydroxycarvotanacetone (4). Compound 1 (GI = 2.28%–12.28%) was significantly less cytotoxic than 3 (GI = 61.59% to 94.01%), indicating that the presence of a hydroxyl group causes an enhancement of cytotoxicity. However, (+)-carvone (2) (GI = 34.39%–48.07%) was more bioactive than its corresponding monoterpene alcohol (+)-8-hydroxycarvotanacetone (4) (GI = 3.12%–16.36%). In addition, we also evaluated the effect of adding a second hydroxyl group to the p-menthane structure. It was observed that (−)-sobrerol (13) (two hydroxyl groups) showed a significant decrease in the inhibition rate of cell proliferation as compared to (−)-8-hydroxycarvotanacetone (3) (one hydroxyl group), with GI values of 9.78%–41.10% and 61.59%–94.01%, respectively. Thus, these results revealed that the more polar monoterpenes, such as (−)-sobrerol (13), are less effective inhibitors of cellular proliferation.To examine whether the introduction of a hydroxyl group (hydroxylation of a double bond) in the molecular structure affects the cytotoxic activity, (−)-carvone (3) with (−)-acetoxycarvotanacetone (8), and (S)-(−)-perillyl alcohol (14) with both (−)-perillyl acetate (17), and (S)-perillyl benzoate (18). (−)-8-hydroxycarvotanacetone (3) (GI = 61.59%–94.01%) presented more potent cytotoxic activity than its acetate derivative, (−)-8-acetoxycarvotanacetone (8) (GI = 1.62%–30.47%), suggesting that the replacement of a hydroxyl group by an acetate causes a marked decrease in the cytotoxicity. Additionally, compound 14 was more cytotoxic (GI = 90.92%–95.82%) than 17 (GI = 3.06%–16.41%) and 18 (GI = 2.86%–5.02%), probably due to its lower lipophilicity. (S)-Perillyl benzoate (18), the most lipophilic compound, was found to be the least potent inhibitor of cellular proliferation for all of the cell lines evaluated.To investigate whether the presence of an ester function in the chemical structure alters the cytotoxicity, we compared (−)-8-hydroxycarvotanacetone (1) and (+)-carvone (2), and (−)-8-hydroxycarvotanacetone (3) and (+)-8-hydroxy- carvotanacetone (4). (+)-Carvone (2) (GI = 34.39%–48.07%) was more cytotoxic than its enantiomer (−)-carvone (1) (GI = 2.28%–12.28%) in all the cancer lines tested. Similarly, (−)-8-hydroxy- carvotanacetone (3) exhibited better cytotoxic effect than its enantiomeric form (+)-8-hydroxy- carvotanacetone (4), with respective GI values of 61.59%–94.01% and 3.12%–13.36%. The findings suggest an association between the cytotoxicity of the monoterpenes tested and their stereochemistry.To evaluate the influence of chirality on cytotoxicity, we compared the pairs of enantiomers (−)-carvone (R)-pulegone oxide [cis-carveol [trans-isopulegone [S)-perillyl benzoate [1H- and 13C-NMR. (+)-Pulegone, (−)- and (+)-carvone, and (S)-(−)-perillyl alcohol were purchased from Sigma-Aldrich Co. . The chemical structures of the evaluated compounds are shown in The compounds -carne oxide , (−)-cis-carveol , (+)-car-carveol , (+)-lim-carveol , (−)-per-carveol , (−)-per-carveol , (−)-per-carveol , (−)-trapulegone , (+)- anpulegone , (−)-8-apulegone , (S)-perbenzoate , and (−)benzoate  were pre2 atmosphere.The cytotoxicity of the 18 compounds was tested against OVCAR-8 (ovarian adenocarcinoma), HCT-116 (colon carcinoma), SF-295 (glioblastoma), and HL-60 (leukemia) human cancer cell lines, all obtained from the National Cancer Institute, Bethesda, MD, USA. Cells were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 µg/mL streptomycin, and 100 U/mL penicillin, and incubated at 37 °C in a 5% COH-tetrazolium bromide  to a purple formazan product, as described by Mossman [6 cells/mL in 100 μL medium). After 24 h, the 18 compounds (25 μg/mL), were dissolved in 0.7% dimethyl sulfoxide (DMSO), and were added to each well . Then, the cells were incubated for 72 h at 37 °C in a 5% CO2 atmosphere. The experiment was performed as three independent experiments, using DMSO at 1%, and doxorubicin at 100 μg/mL as negative and positive controls, respectively. Doxorubicin, purity >98%; Sigma Chemical Co.).Tumor cell growth was determined by the ability of living cells to reduce the yellow dye 3--2,5-diphenyl-2 Mossman . For allAt the end of incubation, the plates were centrifuged, and the medium was replaced by fresh medium (150 μL) containing 0.5 mg/mL MTT. Three hours later, the formazan product was dissolved in 150 μL DMSO, and absorbance was measured using a multiplate reader . The treatment effects were expressed as the percentage of control absorbance of reduced dye at 595 nm. All absorbance values were converted into a cell growth inhibition percentage (GI%) by the following formula:50). The same protocol for the same cells was used to determine the IC50, varying only the concentration of the compound tested from 0 to 25 μg/mL, to verify the most potent compound. In addition, the IC50 was determined against HL-60 only for the most cytotoxic compound 16. Compounds with an IC50 value lower than 4 μg/mL were considered promising for the search for new anticancer agents [The compounds that caused greater degree of cell growth inhibition and which have not been described in the literature for cytotoxic activity against tumor cell lines were evaluated for their median inhibitory concentration able to induce 50% of maximal effect  were added to the suspension of red blood cells obtained from mice according to methodology adapted from Kang and collaborators [16 erythrocyte mixtures were incubated on a mixer for 60 min and then centrifuged at 3000 rpm for 5 min. Mixtures were incubated on a mixer for 60 min and then centrifuged at 3000 rpm for 5 min. After incubation at room temperature for 30 min and centrifugation, the supernatant was removed and the liberated hemoglobin was measured spectrophotometrically as the absorbance at 540 nm.The test was performed in 96-well plates using a 2% mouse erythrocyte suspension in 0.85% NaCl containing 10 mm CaClborators . Variousborators , Pita anborators , and Bezborators . The tub6 cells/mL) in absence or presence of different concentrations of compound 16 . After the incubation period of 24 h, 90 µL were removed from the cell suspension and added to 10 µL of trypan blue. Viable and non-viable cells were differentiated and counted in a Neubauer chamber [The cell viability was determined by the trypan blue dye exclusion test. HL-60 cells were seeded  was added, and the cells were observed under a fluorescence microscope. Three hundred cells were counted per sample and classified as viable, apoptotic or necrotic .6 cells/mL) in the absence or presence of different concentrations of compound 16 . After an incubation period of 24 h, 50 µL of cell suspension were transferred to cytospin slides, fixed with metanol for 60 s, and stained with hematoxylin-eosin [For morphological cell analysis, we used hematoxylin-eosin and examined using light microscopy. HL-60 cells were seeded  were obtained by nonlinear regression. Differences among the experimental groups were compared by one-way variance analysis (ANOVA), followed by Newman-Keuls test (p < 0.05). All analyses were carried out using the Graphpad program .Data are presented as mean ± SEM (or SD) or IC16 was the p-menthane derivative with the highest cytotoxic activity against the cancer cells lines tested. The process induced apoptotic and necrotic cell death. In addition the analysis of structure-activity relationships demonstrated that the greater anti-proliferative activity of 16 was determined by chemical aspects, such as presence of functional groups and their positions on p-menthane skeleton, suggesting that using appropriate structural modifications in these compounds, it may be possible to develop new cytotoxic agents. Therefore, compound 16 is a potential anticancer drug, yet requiring further tests to determine its in vivo antitumor activity.Based on the data of this study, we can conclude that among the 18 derivative compounds of perillyl alcohol tested, compound


Clonal VDJ rearrangement of B/T cell receptors (B/TCRs) occurring during B/T lymphocyte development has been used as a marker to track the clonality of B/T cell populations.We systematically profiled the B/T cell receptor repertoire of 936 cancer cell lines across a variety of cancer types as well as 462 Epstein-Barr Virus (EBV) transformed normal B lymphocyte lines using RNA sequencing data.Rearranged B/TCRs were readily detected in cell lines derived from lymphocytes, and subclonality or potential biclonality were found in a number of blood cancer cell lines. Clonal BCR/TCR rearrangements were detected in several blast phase CML lines and unexpectedly, one gastric cancer cell line (KE-97), reflecting a lymphoid origin of these cells. Notably, clonality was highly prevalent in EBV transformed B lymphocytes, suggesting either transformation only occurred in a few B cells or those with a growth advantage dominated the transformed population through clonal evolution.Our analysis reveals the complexity and heterogeneity of the BCR/TCR rearrangement repertoire and provides a unique insight into the clonality of lymphocyte derived cell lines.The online version of this article (10.1186/s12885-018-4840-5) contains supplementary material, which is available to authorized users. Clonal V(D)J  rearrangement which occurs during development of B/T lymphocytes has been used as a marker to track the clonality of B/T cell populations , 2. ThisIn this study, we systematically profiled the B/T cell receptor repertoire of 936 cancer cell lines across a variety of cancer types, as well as 462 Epstein-Barr Virus (EBV) transformed normal B lymphocyte lines, using RNA sequencing data from the Cancer Cell Line Encyclopedia (CCLE) . This stTranscriptome sequencing data were downloaded from the CCLE and Geuvadis RNA sequencing databases; and the B/T cell receptor repertoire of each cell line was analyzed using MiXCR . The 936n = 25), mantle cell lymphoma (n = 4), Burkitt lymphoma (n = 10) and chronic lymphocytic leukemia-small lymphocytic lymphoma  etc. High expression of both IGH (heavy chain) and IGK/L (light chain) rearrangement are detected in all of the mantle cell lymphoma, Burkitt lymphoma and CLL-small lymphocytic lymphoma cell lines , except two , express high levels of clonal rearrangement of both IGH and IGL/K  and CCLE. The analysis of the expression of major lineage genes also suggests that these two cell lines should not be grouped together with other DLBCL cell lines, as none of them express any typical B cell genes such as CD19, CD20 or CD79. Notably, cell line A4/Fuk also displays an abnormal expression pattern of major lineage specific genes. Conclusions of experiments reached from this cell line for DLBCL research may also need to be interpreted with caution.Potential subclonality was also found in diffuse large B cell lymphoma (DLBCL) cell lines Pfeiffer and Toledo  also express high level of heavy and light chain BCR rearrangements. Cell line BCP-1 shows an inconsistent gene expression pattern as compared with other B cell lymphoma lines , suggesting that it might need to be reclassified into other blood cancer types. In contrast to the finding of subclonality in Burkitt lymphoma or DLBCL, cell lines NU-DUL-1 and JM1 contain more than 3 different IGLV rearrangements  were derived from either mature B cells  or post GC B cells), but are also known to have a global down-regulation of B cell gene expression and a general loss of B cell phenotype [Hodgkin lymphoma cell lines (henotype . Consistn = 15) express low levels of IGH rearrangement as their dominant clonotypes. Consistent with the notion that most of the ALL cells had arrested in early stages of B/T cell development [In contrast to the aforementioned cell lines arising from mature B cells, most of the B-ALL cell lines  with an identical CDR3 sequence . This disease is a neoplasia of T lymphocytes often possessing helper/inducer cell surface phenotype . ExpressWith the exception of the cell line ALL-SIL, which has a TCR gamma rearrangement (TRGV7-TRGJ1), the other 14 T-ALL cell lines express TCR beta rearrangements as their dominant clonotypes (TRBV-TRBD-TRBJ or TRBV-TRBJ). Most of the T-ALL cell lines express biallelic-rearrangements –30 or pon = 31; blast phase CML, n = 12; CML, n = 2; essential thrombocythaemia, n = 1), less than 50–100 BCR/TCR reads were detected in the dominant rearrangement (CPM < 0.5). A few rearrangements appear to be commonly detected in a number of solid cancer and AML cell lines with very low read counts. For example, the rearrangements TRAV8–7 -TRAJ19  were commonly found in many cell lines with extremely low read count (< 30). These barely expressed rearrangements may be caused by non-specific sequencing noise or possibly due to the trace amount of cross contamination during sample preparation and sequencing [Cell lines derived from solid tumors and myeloid cells do not undergo B/TCR rearrangements. In almost all of the solid tumor cell lines (except for the few which would be discussed here) and myeloid derived cancer cell lines  carry clonal rearrangements . This cell line was derived from a mucinous gastric adenocarcinoma from a 52-year-old Japanese male . ConsideThe same bioinformatics pipeline was used to analyze 462 EBV transformed normal B lymphocyte cell lines which were immortalized from healthy donor’s B cells. Rearrangements of both IGH and IGK/L were readily detected in almost all of the lymphocyte cell lines Fig. . IGH reaWe hypothesize that EBV mediated transformation occurred in a number of B cells, and during culture, some clones outcompeted the others and gradually became dominant clones. Therefore, diversity was lost and the population became oligoclonal or even monoclonal , 40. In https://www.genecards.org). In addition, the feasibility of profiling BCR repertoires from RNA sequencing data have recently been explored in CLL [https://www.ncbi.nlm.nih.gov/sra). This suggests that the BCR/TCR repertoire pattern may be used as an alternative/complement authorization method for lymphocyte derived cell lines. The advantage of this approach is that when RNA sequencing data is available , the cell lines (need to be lymphocyte derived cells) used in these studies can be simultaneously validated based on the BCR/TCR profile. As such, the RNA sequencing data deposited in the online public database  can be further checked and authenticated independently by any researchers. Crucially, we noticed that poly A selection before sequencing is important for examining the BCR/TCR repertoire, as poly A enriched samples generated 100-fold more BCR/TCR sequencing reads as compared to samples which were not subjected to  poly A selection but were sequenced at similar sequencing depth. Nonetheless, RNA sequencing of samples without poly A selection can still discover the majority of BCR/TCR rearrangements. However, accuracy of clonotype fraction of subclonal rearrangements may be significantly affected.One of the potential pitfalls of profiling the BCR/TCR repertoire using RNA sequencing is that non-transcribed rearrangements may not be detected, and the clonality analysis may be biased by the expression level of BCR/TCR loci. Nevertheless, in comparison to traditional PCR based method which is labor intensive and requires well trained staffs, NGS/RNA sequencing based analysis is more standardized and can be simply outsourced to big sequencing centers. In contrast to PCR based analysis which is limited by the number of loci that can be feasibly examined, NGS based profiling can comprehensively detect all expressed TCR/BCR rearrangements, including some rearrangements which are not usually included in PCR based analysis . The BCR/TCR repertoire identified using RNA sequencing were consistent with previous studies using BIOMED-2 PCR Sanger sequencing , 30 . However, in the B cell lymphoma cell lines NU-DUL-1, JM1 and the Multiple myeloma cell line AMO-1, more than three different IGL  or IGH (cell lines AMO-1) rearrangements were detected, suggesting independent biclonality in these cell lines. As cancer cell lines are generally regarded as of monoclonal origin, the potential biclonality at these cell lines is interesting and may require further detailed study. On the other hand, our observation of high clonality and potential clonal selection/evolution in EBV transformed normal B lymphocytes suggests that careful experimental design and interpretation of the result may be required when using EBV transformed lines as a model to study normal B cell population, B cell gene expression or quantitative trait loci (QTL) , 46.In summary, we comprehensively profiled the B/T cell receptor repertoire in 936 cancer cell lines and 462 samples of EBV transformed normal B lymphocytes. The relative “pure” feature of cancer cell lines circumvents the problem of tumor infiltrating T/B and stroma cells in primary tumor samples. Our analysis provides unique insights into the BCR/TCR rearrangement repertoire and clonality of cell lines derived from lymphocyte cells.Additional file 1:Figure S1. Expression of CD19/CD20, CD79A/B and RAG1/RAG2 in blood cancer cell lines. X axis: 164 blood cancer cell lines grouped base on the disease types (rectangle color bar below the X axis), each dot in X axis represent one cell line, and the Y axis, expression level  of indicated genes. Figure S2. Clonal fraction (filtered by >30 reads) of B-ALL, multiple myeloma, diffuse large B cell lymphoma, Burkitt lymphoma, B cell lymphoma (unspecified) and mantle cell lymphoma base on IGH or IGHK/L. Blue color indicates the clonotype fraction of the most dominant clone, Red color indicate the clonotype fraction of second dominant clone, Yellow color indicate the third dominant clone, while any smaller subclones were aggregated and labelled in grey. Figure S3. Clonal fraction (filtered by >30 reads) of T-ALL and anaplastic large cell lymphoma base on TRCA or TRCB. Blue color indicates the clonotype fraction of dominant clone, red color inidicate the clonotype fraction of second dominant clone, while gray color indicate the third dominant clone. Figure S4. Heatmap showing the usage of IGK/L V genes (A), IGK/L J genes (B), and constant region (C) in 462 samples of EBV transformed normal B lymphocytes. Figure S5. The phylogenetic tree inferred based on the rearrangement of the CDR3 region of IGH, IGK and IGL of EBV transformed B lymphocyte samples ERR188025, ERR188358 and ERR188212. These three cell lines have much higher number of rearrangement types than the other B lymphocyte lines. Clonal Fraction (upper panel) and the read counts (lower panel) of the dominant clone of 462 samples of EBV transformed normal B lymphocytes. (ZIP 1290 kb)


Ferula L. includes perennial flowering plants belonging to the Apiaceae family. This genus is a rich source of biologically active phytochemicals such as sulfur-containing derivatives, coumarins, sesquiterpenes, sesquiterpene lactones, sesquiterpene coumarins, glucuronic acid, galactose, arabinose, rhamnose, and daucane esters. Over the last decade, considerable attention has been paid to biological activities of these compounds; it is assumed that the most prominent biological features of the genus Ferula are their cytotoxic effects. This article discusses cytotoxic activity of the genus Ferula and their important compounds.The genus Ferula and their important compounds, the type of cell line used in vitro, concentrations of the extracts/active compound that were used, and the underlying mechanisms of action through which Ferula-related chemicals induced cytotoxicity. In addition, we explained different mechanisms of action through which the active constituents isolated from Ferula, could decrease cellular growth.In this mini-review article, papers published from 1990 to April 2016 were included and the following information was discussed; cytotoxic activity of the genus Ferula plants and found to be appropriate as adjuvant therapy for certain diseases, should be identified. Also, the versatile biological activities of sesquiterpene coumarins suggest them as promising agents with a broad range of biological applications to be used in the future.It is highly recommended that potent and effective compounds that were isolated from  Ferula includes perennial flowering plants belonging to the family Apiaceae (Umbelliferae). This genus consists of about170 species which are distributed worldwide. Out of 30 species of Ferula that could be found in Iran, 16 plants are endemic. Different species of the genus Ferula are broadly distributed in arid areas from the eastern Mediterranean regions to central Asia  Ferula species, showed cytotoxic activity by inhibition of the growth of human M4Beu metastatic pigmented malignant melanoma cells through induction of cell cycle arrest in G1 and caspase-dependent apoptosis   showed cytotoxic activities against human melanoma cell line SK-MEL-28  cells . Similar results were found for feselol, a sesquiterpene coumarin isolated from the fruits of F. badrakema , IGROV-1 and OVCAR-3 (human ovarian cancer cell line), respectively  against MCF-7 and K562 cancer cells F. oopoda showed significant cytotoxicity. For example, dehydrooopodin revealed significant cytotoxicity with IC50 values of 5 and 15 µM against K562 and MCF7cancer cell lines, respectively , K562 (leukemia), MCF7, and COLO 205 (coloncarcinoma) cell lines, as well as the multidrug-resistant human cancer cell lines KB-C2 (colchicine-resistant KB) and K562/ADR (Adriamycin-resistant K562). These compounds showed moderate cytotoxicity with IC50 values ranging from 24.7 to 56.9 μg/mL F. gummosa extract has specific cytotoxic effects mainly against MCF7 and oral cancer cell lines  and one normal (MDBK) cell lines. In another study, the cytotoxicity of some of the Iranian medicinal Ferula species was examined and all the extracts and oleo-gum resins of F. assa-foetida showed dose-dependent cytotoxicity , cancer cell lines. They revealed that hexane and chloroform fractions of these plants have cytotoxic effects at concentration up to 100 μg/ml. They also reported that the cytotoxicity of F. persica extracts was higher than that of F. hezarlalezarica extracts  F. assa-foetida extract displayed neuroprotective effects in a glutamate-induced neurotoxicity model  was reported  showed that this activity is dependent on the type of solvent (methanolic>ethanolic) and its concentration (higher methanolic content>lower methanolic content)  and 800 mg/mL  were significantly decreased. Also, the F. gummosa root extract reduced the viability of GP-293 cells at concentration of 750 mg/mL  Some other cell-based assaysF. szowitsiana revealed cytotoxic properties. Umbelliprenin showed the highest inhibitory activity against M4Beu melanoma cell line (IC50=12.4±0.5 µM) compared to cisplatin (23.1±0.8 µM)   exhibited tubulin-polymerizing activity. Under Ca2+-free conditions, ferulenol appeared to be equipotent as Taxol in promoting tubulin assembly  Ferula, showed apoptosis-inducing activities in cancerous cells by induction of sub-G1 peak as revealed by PI staining . Farnesiferol C could be considered a potential chemotherapeutic agent; its anticancer effects are partly mediated via inducing tumor cells apoptosis by increasing the Bax/Bcl-2 ratio . They revealed that auraptene induced a sub-G1 peak in the flow cytometry histogram of treated cells compared to control cells. In this study, DNA fragmentation was suggested as one of the underlying mechanisms of auraptene-induced apoptosis. Also, western blot analysis showed that auraptene significantly up-regulated Bax expression in MCF-7 cells compared to untreated controls F. ferulaeoides (Steud.) Korov. Induced C6 glioma cell apoptosis and endoplasmic reticulum (ER) stress, via mitochondrial and death-receptor-mediated pathways  . They also reported that F. gummosa extracts inhibited AGS cell line proliferation in a dose-dependent manner with IC50 values of 37.47 µg/mL for flower and 32.99 µg/mL for leaf extracts. F. gummosa extracts also induced apoptosis, as reflected by DNA fragmentation and plasma membrane translocation of phosphatidyl serine , reflecting the possible presence of anti-angiogenic compounds .The ethyl acetate fraction of Mechanisms of actionFerula genus, showed both growth inhibitory and cytotoxic activities in different cancerous cell lines  through cell cycle arrest in G1 phase -induced proliferation and inhibited VEGF-induced migration and tube formation in human umbilical vein endothelial cells (HUVECs). These effects were accompanied by decreased phosphorylation of p38-mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), and AKT, and decreased expression of VEGFR targets endothelial nitric oxide synthase (eNOS) and cyclin D1 in VEGF-treated HUVECs  plants and their main constituents. This review aimed to highlight cytotoxic activities of Ferula species and their phytochemicals . We also It is assumed that the most prominent biological features of the genus Ferula are their cytotoxic effects. Previous reports proposed that Ferula phytochemicals have different activities. This probably suggests that much effort still remains to be made to identify potent and effective Ferula compounds that could be appropriate to be used as adjuvant therapy along with the conventional antibiotics. It is ultimately suggested that considering the versatile biological activities of sesquiterpene coumarins, these compounds may have an even broader range of biological applications in the future.