The next-generation pan-RAF inhibitor, KIN-2787, is active in class II and class III BRAF mutant models.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 3116-3116
Author(s):  
Aleksandra Franovic ◽  
Nichol Miller ◽  
Paul Severson ◽  
Toufike Kanouni ◽  
Noelito Timple ◽  
...  
Keyword(s):  
Class Ii ◽  
Class I ◽  
Braf Inhibitors ◽  
Class Iii ◽  
Braf Gene ◽  
Braf Mutations ◽  
Braf Mutant ◽  
Gene Alterations

3116 Background: Oncogenic BRAF gene alterations, leading to aberrantly activated BRAF monomers (Class I mutations) or dimers (Class II and Class III mutations), are observed in approximately 6% of all human cancers. First-generation BRAF inhibitors targeting Class I BRAF mutants, including dabrafenib, encorafenib, and vemurafenib, provide significant clinical benefit to patients with BRAF V600 mutation-driven melanoma and select solid tumors as monotherapies or in combination with other targeted therapies. The currently approved BRAF inhibitors have not, however, proven to be effective in patients with Class II or III BRAF alterations which account for a large proportion (34%) of BRAF mutations. KIN-2787 is an orally available, potent and selective small molecule pan-RAF inhibitor specifically designed to inhibit Class II and III BRAF dimers, in addition to Class I mutants. Methods: The efficacy and tolerability of the pan-RAF inhibitor, KIN-2787, was evaluated in vitro and in vivo in Class I, II, and III BRAF mutation-driven human cancer models. Results: In biochemical assays, KIN-2787 showed low nanomolar to picomolar potency against RAF1, BRAF, and ARAF (IC50 0.06-3.46 nM) with minimal activity towards non-RAF kinases. In cell-based assays, KIN-2787 inhibited RAF activity, as measured by inhibition of downstream ERK phosphorylation (pERK), across multiple BRAF mutant cancer cell lines. Class II and III BRAF mutant cell lines were the most responsive when treated with KIN-2787 (IC50 < 50 nM); 19- and 7-fold more sensitive compared to cells harboring wild-type BRAF, respectively. Dose-dependent inhibition of A-375 (Class I), BxPC-3 (Class II), and WM3629 (Class III) BRAF mutant human xenograft tumor growth was attained with daily KIN-2787 treatment and was well-tolerated. A trend towards greater tumor responses was observed with twice daily (BID) compared to once daily (QD) dosing of KIN-2787; however, the two dosing regimens led to similar tumor growth inhibition (TGI) and regressions (mean TGI up to 101-118%; p ≤0.0001) at equivalent total daily doses. Furthermore, KIN-2787 led to a significant in vivo pharmacodynamic response using either regimen, however, prolonged target coverage, as measured by pERK, was achieved with BID dosing. The impact of KIN-2787 treatment on additional biomarkers, including transcriptional changes and MAPK pathway modulation in cell-based models and patient-derived samples, will be presented at the meeting. Conclusions: KIN-2787 is a next-generation pan-RAF inhibitor with pronounced in vitro and in vivo activity against human cancers driven by Class II and III BRAF mutations. A phase 1 dose escalation and expansion clinical trial evaluating the safety and efficacy of KIN-2787 monotherapy in patients with advanced or metastatic solid tumors harboring BRAF gene alterations, including Class II and III mutations, is expected to initiate in 2021.

Assessment of BRAF class I/II/III mutations, demographics, and treatment outcomes in NSCLC.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. e21016-e21016
Author(s):  
Julian Andres Marin-Acevedo ◽  
James Kevin Hicks ◽  
Ram Thapa ◽  
Dung-Tsa Chen ◽  
ErinMarie Kimbrough ◽  
...  
Keyword(s):  
Demographic Data ◽  
Pairwise Comparison ◽  
Class Ii ◽  
Class I ◽  
Smoking History ◽  
Rank Test ◽  
Class Iii ◽  
Braf Mutations ◽  
Kaplan Meier ◽  
Braf Mutant

e21016 Background: BRAF mutations are grouped (class I, II or III) according to kinase activity, dependence on RAS, and dimerization. BRAF class I mutations are targeted with BRAF/MEK inhibitors. The management of class II/III mutations has not been fully elucidated. We aimed to further characterize BRAF-mutant NSCLC. Methods: Moffitt patients with BRAF-mutant NSCLC were identified through retrospective review of a clinical molecular database and electronic health record from 1/1/2012 - 03/31/2020. Demographics, PD-L1 expression, responses to chemotherapy/immunotherapy, and overall survival were collected. The correlation between BRAF mutation classes and these variables was determined using a pairwise comparison of p-values and corrected for testing using the Benjamini-Hochberg method. For survival analysis, we used the Kaplan-Meier method with log-rank test. Results: 153 patients were included. Table summarizes the demographic data. 14 evaluable patients with class I mutations received anti-BRAF therapy. The DCR was 79% (11/14) and 83% when PD-L1 ≥1% (5/6). 36 response-evaluable patients received immunotherapy. The DCR was 79% (11/14) for patients with class I, 69% (9/13) for class II, and 78% (7/9) for class III mutations (p=0.89). The DCR was 73% in those with PD-L1 ≥1% (22/30) and 44% in those with PD-L1 <1% (7/16; p= 0.02). 43 response-evaluable patients received chemotherapy. The DCR was 86% (18/21) for patients with class I, 69% (9/13) for class II, and 78% (7/9) for class III mutations (p=0.49). The mOS was 26.2 months for all patients. The mOS was 31.2 months for class I, 15.6 months for class II, 28.8 months for class III, 34.8 months for AMP, and 24 months for VUS (p=0.6). Conclusions: To our knowledge, this is the largest retrospective study of patients with BRAF-mutant NSCLC. BRAFmutations were more common in patients with adenocarcinoma and those with a smoking history. PD-L1 levels ≥1% were seen more frequently in all BRAF-mutant patients. Class I mutations were significantly more common than other mutations and responded well to BRAF-targeted therapy, particularly when PD-L1 ≥1%. All BRAF-mutant classes responded similarly to immunotherapy and better responses were seen among those with PD-L1 ≥1%. Responses to chemotherapy were similar across all classes. Improved OS in class I-mutant disease was seen. These findings were not statistically significant. Additional research is needed to better characterize these patients to individualize management and improve outcomes.[Table: see text]

scholarly journals GGA proteins associate with Golgi membranes through interaction between their GGAH domains and ADP-ribosylation factors

2002 ◽  
Vol 365 (2) ◽  
pp. 369-378 ◽  
Author(s):  
Hiroyuki TAKATSU ◽  
Kaori YOSHINO ◽  
Kyoko TODA ◽  
Kazuhisa NAKAYAMA
Keyword(s):  
Membrane Trafficking ◽  
Class Ii ◽  
Small Gtpases ◽  
Class I ◽  
Class Iii ◽  
Limited Region ◽  
Adp Ribosylation ◽  
Early Endosomes

ADP-ribosylation factors (ARFs) are a family of small GTPases that are involved in various aspects of membrane trafficking events. These include ARF1—ARF6, which are divided into three classes on the basis of similarity in the primary structure: Class I, ARF1—ARF3; Class II, ARF4 and ARF5; and Class III, ARF6. Previous studies identified a novel family of potential ARF effectors, termed GGA1—GGA3, which interact specifically with GTP-bound ARF1 and ARF3 and are localized to the trans-Golgi network (TGN) or its related compartment(s) (GGA is an abbreviation for Golgi-localizing, γ-adaptin ear homology domain, ARF-binding protein). In the present study we have shown that ARF proteins belonging to the three classes, ARF1, ARF5 and ARF6, can interact with all GGA proteins in a yeast two-hybrid assay, in vitro and in vivo. Segmentation of GGA proteins and isolation of GGA mutants defective in ARF binding have revealed that a limited region within the GGA homology domain, which is conserved in the GGA family, is essential for ARF binding. Expression in cells of GTPase-restricted mutants of ARF1 and ARF5 blocks dissociation of GGA proteins from membranes induced by brefeldin A. However, neither of the ARF mutants recruits GGA mutants defective in ARF binding. On the basis of these observations, we conclude that at least ARF1 (Class I) and ARF5 (Class II) in their GTP-bound state cause recruitment of GGA proteins on to TGN membranes. In contrast, on the basis of similar experiments, ARF6 (Class III) may be involved in recruitment of GGA proteins to other compartments, possibly early endosomes.

scholarly journals BRAF Mutant Melanoma Adjusts to BRAF/MEK Inhibitors via Dependence on Increased Antioxidant SOD2 and Increased Reactive Oxygen Species Levels

Cancers ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 1661 ◽  
Author(s):  
Long Yuan ◽  
Rosalin Mishra ◽  
Hima Patel ◽  
Samar Alanazi ◽  
Xin Wei ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Braf Inhibitor ◽  
Reactive Oxygen ◽  
Oxygen Species ◽  
Braf Mutations ◽  
Mek Inhibitors ◽  
Braf Mutant ◽  
Resistant Cells

B-Rapidly Accelerated Fibrosarcoma (BRAF) mutations are found in about 50% of melanoma patients. Treatment with Food and Drug Administration (FDA)-approved BRAF and MAP/ERK kinase (MEK) inhibitors has improved progression free and overall survival of patients with BRAF mutant melanoma. However, all responders develop resistance typically within 1 year of treatment with these inhibitors. Evidence indicates that reactive oxygen species (ROS) levels are elevated after BRAF pathway inhibition treatment. We aim to decipher the role of mitochondrial antioxidant proteins relative to ROS levels and BRAF pathway inhibitor resistance. We observed BRAF mutant melanoma cells treated with the combination of a MEK inhibitor (trametinib) and a BRAF inhibitor (dabrafenib), exhibited elevated ROS levels, both in in vitro and in vivo melanoma models. We next generated trametinib- and dabrafenib-resistant (TDR) cells and found increased ROS levels after acquisition of resistance. An immunofluorescence experiment showed an increase of DNA damage in TDR cell lines. Furthermore, we observed that TDR cells increased superoxide dismutase 2 (SOD2), an antioxidant, at both mRNA and protein levels, with the upregulation of the transcription factor Nuclear Factor (NF)-κB. Knockdown of SOD2 significantly reduced the growth of BRAF pathway inhibitor-resistant cells. In addition, the results indicate that TDR cells can be re-sensitized to BRAF pathway inhibitors by the ROS scavenger, N-Acetyl Cysteine (NAC). Overall, these data indicate that BRAF pathway inhibitor-resistant cells can compensate for elevated ROS via increased expression of the antioxidant SOD2.

scholarly journals Prevalence of class I–III BRAF mutations among 114,662 cancer patients in a large genomic database

2020 ◽  
pp. 153537022095965
Author(s):  
Jeff Owsley ◽  
Matthew K Stein ◽  
Jason Porter ◽  
Gino K In ◽  
Mohamed Salem ◽  
...  
Keyword(s):  
Braf Mutation ◽  
Kinase Activity ◽  
Human Cancer ◽  
Class Ii ◽  
Class I ◽  
Clinical Activity ◽  
Class Iii ◽  
Braf Mutations ◽  
Comprehensive Genomic Profiling ◽  
Relative Prevalence

BRAF mutations are relatively common in many cancers, particularly melanoma, colorectal cancer, and thyroid cancer and to a lesser extent in lung cancer. These mutations can be targeted by BRAF and MEK inhibitors, which exhibit good clinical activity. There are conflicting reports of the various relative rates of BRAF Class I mutations (V600 locus), defined as those that exhibit extremely strong kinase activity by stimulating monomeric activation of BRAF, Class II, define as non-V600 mutations that activate BRAF to signal as a RAS-independent dimer, and Class III mutations, defined as “kinase-dead” with low kinase activity as compared to wild type BRAF. Prospective studies have largely focused on patients with tumors harboring Class I BRAF mutations (limited to the V600 locus) where response rates up to 70% with BRAF plus MEK inhibition have been demonstrated. We report on the relative prevalence of various types of BRAF mutations across human cancers in a cohort of 114,662 patients that received comprehensive genomic profiling using next-generation sequencing. Of these patients, 4517 (3.9%) a pathogenic or presumed pathogenic BRAF mutation (3.9%). Of these, 1271 were seen in melanoma, representing 39.7% of all melanomas sequenced, representing the highest rate in all tumors. Class I (V600) mutations were seen overall in 2841 patients (62.1% of BRAF mutations, 2.4% of total cancers). Class II mutations were seen in 746 tumors (16.5% of BRAF mutant, 0.7% of total), and Class III mutations were seen in 801 tumors (17.7% of BRAF, 0.7% of total). Knowledge of the relative prevalence of these types of mutations can aid in the development of agents that might better address non-V600 mutations in cancer. Impact statement These data represent the largest aggregation of BRAF mutations within a single clinical database to our knowledge. The relative proportions of both BRAF V600 mutations and non-V600 mutations are informative in all cancers and by malignancy, and can serve as a definitive gold-standard for BRAF mutation cancer incidence by malignancy. The rate of BRAF mutation in human cancer in a real-world large database is lower than previously reported likely representing testing more broadly across tumor types. The relative percentages of Class II and Class III BRAF mutations are higher than previously reported, representing almost 35% of BRAF mutations in cancer. These findings provide support for the development of effective treatments for non-V600 BRAF mutations in cancer.

scholarly journals Uropathogenic Escherichia coli Flagella Aid in Efficient Urinary Tract Colonization

2005 ◽  
Vol 73 (11) ◽  
pp. 7657-7668 ◽  
Author(s):  
Kelly J. Wright ◽  
Patrick C. Seed ◽  
Scott J. Hultgren
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Class I ◽  
Class Iii ◽  
Epithelial Surface ◽  
Fitness Advantage ◽  
Regulatory Cascade ◽  
Tract Infections

ABSTRACT In the murine model of urinary tract infections (UTI), cystitis by uropathogenic Escherichia coli (UPEC) occurs through an intimate relationship with the bladder superficial umbrella cell entailing cycles of adherence, invasion, intracellular bacterial community (IBC) formation, and dispersal (fluxing) from the intracellular environment. IBC dispersal is a key step that results in the spread of bacteria over the epithelial surface to initiate additional rounds of IBC formation. We investigated the role of flagella in mediating adherence and motility during UTI, hypothesizing that the dispersion of the IBC would be incomplete in the absence of motility, thus interrupting the IBC pathway and attenuating the infection. Using gfp reporter fusions, the expression of the flagellar class I flhDC and class III fliC genes was monitored to track key points of regulation throughout the pathogenic cascade. In vitro, growth under conditions promoting motility resulted in the robust expression of both fusions. In contrast, only the class I fusion produced significant expression throughout early stages of IBC development including the dispersion stage. Thus, unlike in vitro modeling of motility, the regulatory cascade appeared incomplete in vivo. Throughout IBC formation, nonmotile ΔfliC mutants achieved the same number of IBCs as the wild-type (wt) strain, demonstrating that flagella are neither essential nor required for first- or second-generation IBC formation. However, in competition experiments between wt and ΔfliC strains, the wt was shown to have a fitness advantage in persisting throughout the urinary tract for 2 weeks, demonstrating a subtle but measurable role for flagella in virulence.

Large scale clinico-genomic analyses among patients with BRAF-mutated non-small cell lung cancers (NSCLC) identified by nationwide genomic screening project (LC-SCRUM-Japan).

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. 9590-9590
Author(s):  
Tetsuya Sakai ◽  
Shingo Matsumoto ◽  
Taku Nakagawa ◽  
Kadoaki Ohashi ◽  
Hidetoshi Itani ◽  
...  
Keyword(s):  
Clinical Outcomes ◽  
Large Scale ◽  
Gene Mutations ◽  
Class Ii ◽  
Small Cell ◽  
Class I ◽  
Class Iii ◽  
Small Cell Lung ◽  
Braf Mutations ◽  
Lung Cancers

9590 Background: BRAF mutations are functionally classified into three groups, comprisingV600-mutant kinase-activating monomers (class I), kinase-activating dimers (class II), kinase-inactivating heterodimers (class III). The difference of clinical outcomes and concomitant genetic alterations among the three classes in non-small cell lung cancers (NSCLC) are unclear. Methods: We have prospectively analyzed NSCLC patients (pts) for cancer-related genes by a next-generation sequencing system, Oncomine™ Comprehensive Assay, in a large-scale genome screening project in Japan (LC-SCRUM-Japan). The clinical characteristics and outcomes of pts with BRAF-mutated non-squamous (non-sq) NSCLC were comparatively evaluated among the three classes of BRAF mutations. Results: A total of 5166 non-sq NSCLC pts were enrolled into the LC-SCRUM-Japan from 2015 to 2019. BRAF mutations were detected in 176 pts (3%). Among the 176 pts, 153 (87%) were classified into the three classes according to the mutation variants, including 65 (42%) into class I, 52 (34%) into class II and 36 (24%) into class III. The remaining 23 were not classified into any of the three classes. Compared with class I, class II or class III was significantly associated with smoking (P = 0.02 and < 0.01, respectively). Concomitant RAS mutations were significantly more frequent in class II and class III than in class I (P < 0.01 and = 0.04, respectively). The frequency of concomitant STK11 mutations was significantly higher in class III than in others (P < 0.01, respectively). There was no significant difference in the frequency of other oncogene and tumor suppressor gene mutations among the three classes. In the 1st-line platinum-containing chemotherapies for advanced or recurrent cases, median progression-free survival (mPFS) of class III pts was shorter than class I or class II pts (4.2, 11.5 and 4.8 months, I vs III; P < 0.01, II vs III; P = 0.06). In the treatment with 2nd-4th line PD-1/PD-L1 inhibitors, mPFS was not significantly different among the three classes. Overall survival of class III pts was significantly shorter than class I pts (11.9 vs 35.2 months, P = 0.03). Conclusions: Concomitant gene mutations and clinical features are largely different among the BRAF mutation classes. Especially in class III, concomitant RAS and STK11 mutations are more frequent and clinical outcomes were significantly less favorable. These results suggest the need of novel therapeutic strategy based on the mutation class for BRAF-mutated lung cancers.

Endolyn (CD164) Interacts with CXCR4 and Modulates the CXCL12-Mediated Homing and Migration of Cord Blood CD133+ Hematopoietic Precusor Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1135-1135
Author(s):  
Suzanne M. Watt ◽  
Sinead Forde ◽  
Brit Jorgensen Tye ◽  
Sarah Newey ◽  
Maria Roubelakis
Keyword(s):  
Cord Blood ◽  
Leading Edge ◽  
Class Ii ◽  
Functional Class ◽  
Jurkat Cells ◽  
Temporal Association ◽  
Class Iii ◽  
Human Cd34

Abstract The sialomucin, endolyn or CD164, has been shown to act as an important regulator in the adhesion of human haemopoietic stem/precursor cells (HPC) to stromal niche cells, while also controlling the entry of primitive human CD34+CD38lo HPC into cycle. Here, we define a novel function for endolyn, by identifying its ability to modulate CD133+ cord blood HPC migration on fibronectin towards CXCL12 in vitro. Interestingly, CD133+ cell migration on fibronectin to CXCL12 was reduced 1) by engaging the functional class II glycosylation-dependent epitope on endolyn with the 103B2/9E10 class II but not N6B6 class III antibody; and 2) by RNAi knockdown of endolyn in both CD133+ HPC and Jurkat cells. The inhibition of migration was more pronounced in the more primitive CD34+CD38lo/− HPC subset than in the CD38+ subset. We show a direct and temporal association of endolyn with the CXCR4 receptor, at the leading edge of CD133+ HPC. When CXCL12 is presented on fibronectin, we first see an upregulation in the association of CXCR4 with the a-4 and a-5 integrins that is closely followed by recruitment of endolyn to this complex. This was confirmed by co-immunoprecipitation studies. Knock-down of endolyn using siRNAs revealed that signaling through CXCR4 via PKC-zeta and Akt pathways was significantly dampened, while leaving MAPK phosphorylation unaffected. Our current studies are aimed at examining the in vivo importance of endolyn in HPC homing to the bone marrow of NOD/SCID mice. Our findings support a novel association between three distinct families of cell surface receptors that regulate both cell migratory and proliferative responses and identify endolyn as a key regulator of CXCR4/CXCL12 function.

scholarly journals BRAF Mutations Classes I, II, and III in NSCLC Patients Included in the SLLIP Trial: The Need for a New Pre-Clinical Treatment Rationale

Cancers ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 1381 ◽  
Author(s):  
Jillian Wilhelmina Paulina Bracht ◽  
Niki Karachaliou ◽  
Trever Bivona ◽  
Richard B. Lanman ◽  
Iris Faull ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Kinase Activity ◽  
Synergistic Effects ◽  
Mutant Cell ◽  
Class I ◽  
Class Iii ◽  
Braf Mutations ◽  
Therapeutic Decisions ◽  
Nsclc Patients

BRAF V600 mutations have been found in 1–2% of non-small-cell lung cancer (NSCLC) patients, with Food and Drug Administration (FDA) approved treatment of dabrafenib plus trametinib and progression free survival (PFS) of 10.9 months. However, 50–80% of BRAF mutations in lung cancer are non-V600, and can be class II, with intermediate to high kinase activity and RAS independence, or class III, with impaired kinase activity, upstream signaling dependence, and consequently, sensitivity to receptor tyrosine kinase (RTK) inhibitors. Plasma cell-free DNA (cfDNA) of 185 newly diagnosed advanced lung adenocarcinoma patients (Spanish Lung Liquid versus Invasive Biopsy Program, SLLIP, NCT03248089) was examined for BRAF and other alterations with a targeted cfDNA next-generation sequencing (NGS) assay (Guardant360®, Guardant Health Inc., CA, USA), and results were correlated with patient outcome. Cell viability with single or combined RAF, MEK, and SHP2 inhibitors was assessed in cell lines with BRAF class I, II, and III mutations. Out of 185 patients, 22 had BRAF alterations (12%) of which seven patients harbored amplifications (32%) and 17 had BRAF mutations (77%). Of the BRAF mutations, four out of 22 (18%) were V600E and 18/22 (82%) were non-V600. In vitro results confirmed sensitivity of class III and resistance of class I and II BRAF mutations, and BRAF wild type cells to SHP2 inhibition. Concomitant MEK or RAF and SHP2 inhibition showed synergistic effects, especially in the class III BRAF-mutant cell line. Our study indicates that the class of the BRAF mutation may have clinical implications and therefore should be defined in the clinical practice and used to guide therapeutic decisions.

scholarly journals Growth factor gene activation and clonal heterogeneity in an autostimulatory myeloid leukemia.

1989 ◽  
Vol 9 (6) ◽  
pp. 2414-2423 ◽  
Author(s):  
K B Leslie ◽  
J W Schrader
Keyword(s):  
Growth Rate ◽  
Growth Factor ◽  
Leukemia Virus ◽  
Class I ◽  
Class Iii ◽  
Gm Csf ◽  
Rna Transcripts ◽  
Cell Densities

Cell lines were isolated from an in vivo-passaged myelomonocytic leukemia, WEHI-274, that arose in a mouse infected with the Abelson leukemia virus-Moloney leukemia virus complex. Clones were isolated in vitro in the presence or absence of a source of a hemopoietic growth factor, interleukin-3 (IL-3), and were divisible into three distinct classes. All three classes were leukemogenic in vivo. In vitro, the class I clone grew slowly at low cell density but responded with an increased growth rate to IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), and autoconditioned medium. Supernatants of these cultures contained a factor with the biological, biochemical, and antigenic properties of IL-3. Class II clones grew better in vitro at low cell densities than did the class I clone and also responded with an increased growth rate to IL-3, GM-CSF, and autoconditional medium but produced GM-CSF rather than IL-3. In contrast, class III clones died in vitro at all cell densities unless exogenous IL-3 or GM-CSF was added. Moreover, they produced no autostimulatory factors. In the class I and class II clones, one allele of the respective IL-3 or GM-CSF gene is rearranged, and in each case, grossly abnormal RNA transcripts of the rearranged gene are present. Neither rearrangements nor abnormal RNA transcripts of the IL-3 or GM-CSF gene were detected in the class III clones. All three classes exhibited a common rearrangement of the c-myb gene, which suggested that all were derived from the one ancestral cell. These experiments demonstrate that two distinct and independent autostimulatory events were involved in the progression of a single disease.

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