Epstein–Barr virus (EBV)-associated lymphoproliferative disorders have been garnering attention in recent years. EBV infects people primarily in their first years of life. The viral genome is maintained in a latent phase within the host cells, usually B-lymphocytes. The immunosuppression of different origins alters homeostasis between the virus and the host, enabling EBV-linked lymphoproliferative disorders to appear. Entities with different biological behaviors often share some morphological and phenotypic features, making the diagnosis complicated. Nodal and extra-nodal sites, including the gastrointestinal tract may be involved. This review, divided into three parts, aims to summarize the available clinical, pathological, molecular and therapeutic data on EBV-associated lymphoproliferative disorders involving the gastrointestinal tract. In this part of the review, we discuss plasmablastic lymphoma, extra-cavitary primary effusion lymphoma and Burkitt lymphoma.Epstein–Barr virus (EBV) is a common pathogen infecting people primarily early in life. The virus has the ability to persist throughout a person’s life, usually in B lymphocytes. Conditions of immunodeficiency as well as the introduction of immunosuppressive therapies and the advent of transplant technologies has brought immunodeficiency-associated lymphoproliferative disorders into view, which are often driven by EBV. The group of EBV-associated lymphoproliferative disorders includes different entities, with distinct biological features, ranging from indolent disorders, which may even spontaneously regress, to aggressive lymphomas requiring prompt and adequate treatment. These disorders are often diagnostically challenging due to their overlapping morphology and immunophenotype. Both nodal and extra-nodal sites, including the gastrointestinal tract, may be involved. This review, divided in three parts, summarizes the clinical, pathological, molecular features and treatment strategies of EBV-related lymphoproliferative disorders occurring in the gastrointestinal tract and critically analyzes the major issues in the differential diagnosis. In this part of the review, we discuss plasmablastic lymphoma, extra-cavitary primary effusion lymphoma and Burkitt lymphoma. EBV represents the most common persistent virus in humans, as after primary infection in the first years of life, the virus has the ability to latently persist in B lymphoid cells ,10,11,12While EBV is the common denominator of entities with different pathological features and clinical behaviors, other factors, including the state of the host immune system, iatrogenic immunosuppression (IS), age-related immune-senescence and genetic alterations, play important roles in the pathogenesis of EBV-associated LPDs ,10,11,12The incidence of different entities varies according to the geographical areas.Some EBV-positive LPDs, such as EBV-positive diffuse large B-cell lymphoma, not otherwise specified  and Burkitt lymphoma (BL), have a higher incidence in areas with high rates of EBV infection . DependiFor therapeutic reasons, the correct identification of different lymphoid entities linked to EBV is essential, and it may be diagnostically challenging as different entities share morphological and immunophenotypic features.EBV is associated with many tumors, including epithelial, mesenchymal and hematopoietic neoplastic proliferations.The EBV-associated lymphoid disorders are summarized in The gastrointestinal tract (GIT) is a common site of EBV-driven LPDs.In this review, we focus on LPDs that develop in the GIT, in which EBV detection is considered a diagnostic parameter. Some of these entities may even develop in inflammatory bowel disease (IBD). The mechanism underlying the association of IBD with EBV-associated hematopoietic disorders still remains not completely clarified ,24,25,26Our review consists of three parts. In this part, we describe the clinicopathological features of plasmablastic lymphoma (PBL), extra-cavitary primary effusion lymphoma (EC-PEL) and Burkitt lymphoma (BL).In part 1 of the review, we discussed EBVMCU, EBV-positive DLBCL, NOS and cHL .In part 3, we will analyze, post-transplant lymphoproliferative disorders (PTLDs); chronic active EBV (CAEBV) infection of T- and NK-cell types; and extra-nodal NK/T-cell lymphoma (ENKTL), nasal type .The exact role of EBV in the pathogenesis and development of many LPDs is often not completely elucidated.Among the entities discussed in the present paper, BL represents the first example of a virus-associated lymphoma. Due to the different associations of EBV in the three variants of BL and the restrictive latency pattern with only the expression of EBNA-1, some authors suggest that the virus may not be the initiating event for BL, with the oncogenic capacity of EBNA1 being controversial ,30. RecePEL is strictly linked to Kaposi Sarcoma Herpes Virus/Human Herpes Virus 8 (KSHV/HHV8), which is considered the main driving force in its pathogenesis; EBV is not present in all cases of PEL and it is supposed to play a cofactor role .MYC gene rearrangement [Similar to PEL, in PBL, EBV is identified in a proportion of cases and it is likely to have a pathogenetic role in conjunction with other factors such as angement . FurtherPBL, initially considered a morphological variant of diffuse large B-cell lymphoma (DLBCL), was identified as a distinct entity starting in the 2008 WHO classification .PBL is a high-grade lymphoma initially reported in the jaw and oral cavity of young, HIV-positive males ,34,35.It may develop even in the context of other causes of IS, including autoimmune disorders, post-transplant, iatrogenic IS and age-related immune-senescence ,37. ImmuThe disease shows a male prevalence of 4–5:1, occurring at any age, although HIV-positive individuals may develop PBL at a younger age (median 42 years) compared with PBL associated with other conditions of IS (median 60) ,38.PBL is predominantly an extra-nodal disease, with a predilection for the oral cavity in HIV-positive individuals, with the GIT being the second most common site. Other extra-nodal sites such as the skin, bone, genitourinary tract, sino-nasal tract, central nervous system (CNS), liver, lung and orbits are more rarely reported ,58,59,60Nodal involvement is rare, particularly in HIV-positive cases, whereas it is more frequently observed in post-transplant cases .EBV is likely to have a pathogenetic role in PBL occurrence. Particularly in the context of HIV infection, a large number of PBL cases harbor EBV as documented by EBV-encoded ribonucleic acid (EBER) in situ hybridization (ISH) . HoweverThe GIT represents the most common extraoral site, accounting for approximately 30% of extraoral PBL .In GI PBL, male patients are affected almost twice as often as female  and the The rectum and anal canal have been reported as the most commonly involved sites, particularly in HIV-positive patients ,43. In aThe association between IBD and non-Hodgkin lymphomas, including PBL, is well-known. The risk of lymphoma in IBD patients seems to be related to the treatment more than to the disease itself, in particular with thiopurines and to a lesser extent with anti- tumor necrosis factor α (anti-TNFα) ,44,45.In GIT, PBL presents, commonly, as a mass and/or a polypoid lesion  and, morPlasmablasts (PBs) are large cells with eccentrically located large nuclei, evident nucleoli, basophilic or amphophilic cytoplasm, often with a perinuclear clear halo.However, PBL shows a wide morphologic spectrum ranging from cohesive and monomorphic proliferation of large cells with immunoblastic, plasmablastic or anaplastic features to proliferation of cells with a clear-cut plasmacytic differentiation .Geographic necrosis can be present, and a starry sky pattern with apoptotic bodies and tingible body macrophages is common.The neoplastic cells express a plasma cell phenotype  .CD45 and B-cell markers (CD20 and PAX5) are generally negative  or weaklMYC-rearranged cases, suggesting alternative mechanisms of MYC activation and protein expression. EBER is positive in 60–75% of cases, more frequently in HIV-positive and post-transplant patients. In EBER-positive cases, LMP1 is rarely expressed, indicating type I latency pattern. The latency pattern type II is observed in PBL arising in HIV-positive individuals or in transplantation [MYC rearrangement and a better outcome than EBER-negative PBL [MYC protein is usually overexpressed and not limited to the antation . EBER-potive PBL ,59,60.c-MYC plays important roles in cellular proliferation, protein synthesis and apoptosis. About 50% of PBL cases show MYC rearrangement, generally with an Ig gene partner, and are associated with MYC protein overexpression by immunohistochemistry [MYC amplification and deletion are also detected in 11% and 4% of PBL cases, respectively [MYC rearrangement usually occurs in association with a positive EBER status, CD10 expression and PRDM1 gene mutation and in the absence of mutations in the MYC promoter region [PRDM1 gene, encoding for the BLIMP1 protein often expressed by PBL, has been found in some cases, and it acts as an MYC regulator [MYC in PBL plays an important pathogenetic role in association with EBV infection [Clonal immunoglobulin heavy chain (IGH) rearrangement is usually identified, even when immunoglobulin (Ig) expression is not demonstrable by immunohistochemistry. IGH variable region genes (IGHV) may have somatic hypermutation or may be unmutated with a germline configuration . c-MYC phemistry . MYC ampectively . MYC rear region ,49,50. Megulator . The oncIn the current WHO classification, there are distinct lymphomas with plasmablastic morphology and immunophenotype to be considered in the differential diagnosis with PBL, including EC-PEL, EBV-positive DLBCL, NOS, BL, anaplastic lymphoma kinase positive large B-cell lymphoma (ALK-positive LBCL) and plasmablastic myeloma (PM) ,61,62,63PEL, in its solid/extra-cavitary variant, usually involves lymph nodes, although it is reported even at extra-nodal sites, including the GIT. Similar to PBL, EC-PEL occurs often in immunocompromised patients and typically expresses plasma cell-related markers, CD30 and EMA, whereas B-cell antigens are often negative, although more frequently expressed in the extra-cavitary variant. Unlike in PBL usually expressing immunoglobulin (often IgG), cytoplasmic immunoglobulin are generally absent in PEL, although reported with higher expression in EC-PEL. In PEL, EBV infection is present mainly in HIV-positive individuals. KSHV/HHV8 expression in PEL allows for differential diagnosis with PBL, which is consistently HHV8-negative .Unlike PBL, EBV-positive DLBCL, NOS shows a pan B-cell phenotype, strongly expressing CD20 and PAX5, in the absence of plasma cell-associated markers ,53.MYC rearrangement. However, the phenotype is clearly distinct as BL expresses CD20 and CD10.BL shows some similarities with PBL such as frequent occurrence in HIV-positive hosts, the presence of plasmacytic differentiation, EBER positivity and ALK-positive LBCL is a high-grade lymphoma usually involving lymph nodes and mediastinum, although extra-nodal sites may be involved. Patients are usually diagnosed in the advanced stage. The neoplastic cells show an immunoblastic or plasmablastic morphology and a rather striking intrasinusoidal growth pattern. Similar to PBL, the neoplastic cells strongly express plasma cell-related markers and EMA and are negative or focally positive for B-cell associated markers and CD45. Unlike PBL, which is often CD30 and EBV positive, ALK-positive DLBCL is usually negative or weakly positive for CD30 and is always EBV-negative. The lymphoma cells are strongly positive for the ALK protein with a restricted granular cytoplasmic staining pattern, which is indicative of t or CTLC-ALK fusion protein. Few cases show cytoplasmic, nuclear and nucleolar ALK staining associated with the NPM-ALK fusion protein .PM and PBL share morphological and immunophenotypic features with the expression of plasma cell-related markers. The presence of IS and a positive EBV status favor PBL. On the other hand, the identification of multiple lytic bone lesions and renal dysfunction plus laboratory data such as monoclonal paraproteinemia, hypercalcemia and light chain proteins in urine support a diagnosis of PM. CD56 and cyclin-D1 expression by neoplastic cells is also in favor of PM. However, in some cases, especially with a solitary lesion, a definitive distinction cannot be made, and according to the current WHO, the use of a descriptive diagnosis such as “plasmablastic neoplasm consistent with PBL or PM” is acceptable .In some PBL cases, the aberrant expression of T-cell markers represents a potential pitfall which that lead to an erroneous diagnosis of T-cell lymphoma.The main distinguishing features of PBL, PEL and BL are summarized in MYC abnormalities; therefore, targeted therapies of oncogenic MYC are considered a potential treatment worthy of evaluation [PBL shows a dismal outcome with a median survival of 6–11 months. Currently, there is no standard therapy for PBL patients. Due to the frequent CD20 negativity, rituximab has no role in PBL, and the CHOP  scheme, used in DLBCL, is considered inadequate. More intensive regimens such as hyper-CVAD-MA , CODOX-M/IVAC , COMB  and EPOCH  are used . Furtheraluation . Both EBaluation . Anti-PDPEL is an aggressive large B-cell lymphoma classically presenting exclusively as serous effusions in the pleural, pericardial or peritoneal cavities in the absence of an identifiable tumor mass . AlthougIn a minority of cases (approximately 5%), lymphomas indistinguishable for PEL present as solid tumor masses outside body cavities and have been recognized as EC-PEL in the current WHO classification ,73,74,75KSHV/HHV8 is the pathogen involved in all cases of PEL in both the serous-based classic PEL and EC-PEL; HHV8 detection is a required diagnostic criterion for the disease.Similar to classic PEL, the extra-cavitary variant most often occurs in HIV-infected individuals or in severe immunodeficiency of some kind. Besides the context of HIV infection, IS may be a consequence of either drugs, for instance following transplantation, or aging ,76. The In the context of HIV infection, the neoplastic cells are often co-infected by HHV8 and EBV, whereas co-infection is documented more infrequently in HIV-negative cases ,73,74,75Similar to EBV, KSHV/HHV8 is a gamma human herpes virus that is lymphotropic and oncogenic.Unlike EBV, which is ubiquitous, KSHV/HHV8 is endemic in certain geographic areas such as sub-Saharan Africa, Latin America, the Caribbean, the Mediterranean and Middle Eastern countries.The virus is transmitted through saliva, but it may also be transmitted sexually and even vertically through breast milk. Other possible routes of transmission are blood transfusion, intravenous drug use and transplant ,76.Epidemiologic evidence supports a fecal–oral route of spread and viral DNA has been identified in rectal mucosa from HIV-positive individuals .Similar to EBV, HHV8 may infect cells such as endothelial cells and lymphocytes, persisting throughout a person’s life in a latent form. When the host immune control is altered, HHV8 may reactivate the lytic replicative cycle producing viremia .The virus was initially identified as having a pathogenetic role in Kaposi sarcoma (KS) , and subEC-PEL may occur in any part of the GIT, including the anorectal site, although the stomach and large bowel are more often involved ,73,74,75The tumor may present as a mass; a polypoid lesion; or a thickening of the gastrointestinal (GI) wall, with or without nodal involvement ,73,74,75In GIT, EC-PEL have also been reported before the development of effusions  and follThe histology of EC-PEL is indistinguishable from classic PEL.The neoplastic cells are large-sized resembling immunoblasts (IBs) or PBs or displaying an anaplastic morphology .PEL in its classic form usually fails to express pan-B-cell markers such as CD19, CD20, CD79α and PAX5 as well as surface and cytoplasmic immunoglobulin. The classic form usually expresses CD45/LCA.The extra-cavitary variant may express B-cell markers (25% vs. <5%), although not in all cases , as wellThe neoplastic cells generally express plasma cell-related markers, including CD138, MUM18  and MUM1The lymphoma cells are positive for HHV8 in all cases. HHV8 is usually demonstrated by immunohistochemistry, detecting the expression of HHV8-encoded latency-associated nuclear antigen 1 (LANA-1) protein .HHV8 can also be documented by in situ hybridization or polymerase chain reaction (PCR) analyses.EBV co-infection is identified by EBER in 50–80% of PEL. In non-HIV infected individuals, PEL may be EBV-negative. Despite positivity for EBV by EBER, EBV-latent membrane protein 1 (LMP1) is usually absent, showing a type I latency pattern, similar to PBL.MYC, BCL2 and BCL6 loci are usually absent. Structural alterations in the MYC gene are absent; however, MYC protein overexpression may be seen, probably due to the activity of HHV8-encoded latent proteins [Clonal immunoglobulin (IG) gene rearrangement is present, indicating a B-cell origin. In some cases, T-cell receptor (TCR) gene rearrangement may be found in addition to IG genes. HHV8 viral genomes are present in all cases. Rearrangements at the proteins   and activation of the NF-kB, JAK/STAT and PI3K/AKT/mTOR pathways involved in the survival of PEL [HHV8-encoded latent proteins, including latency-associated nuclear antigen-1 (LANA-1), LANA-2/vIRF-3, viral cyclin, viral FLICE inhibitory protein (v-FLIP) and kaposin, are considered to play significant roles in PEL. These proteins are related to inhibition of apoptosis ; EBV-positive DLBCL, NOS; PBL; HHV8-positive DLBCL, NOS; ALK-positive LBCL; and PM.The anaplastic morphology and the intrasinusoidal growth observed in some cases of PEL  in combiPerforming a panel of immunostainings including plasma cell-related markers and, in particular, HHV8 helps to avoid missing the diagnosis of EC-PEL; molecular analysis may further support the diagnosis.Unlike the classic form of PEL, which is usually negative for B-cell markers, EC-PEL often expresses some B-cell markers and should be differentiated from EBV-positive DLBCL, NOS. HHV8 positivity represents the diagnostic criterion for PEL.The distinction of EC-PEL from PBL has been discussed in the section on PBL.PEL, in both the classic and extra-cavitary variants, belongs to the spectrum of HHV8-related lymphoid disorders including HHV8-positive MCD ; GLPD 8,82,83; aAmong these HHV8-linked entities, HHV8-positive DLBCL, NOS is differentially diagnosed with EC-PEL.HHV8-positive DLBCL, NOS is an aggressive lymphoma usually developing in HIV-positive hosts, mainly in the setting of HHV8-positive MCD and involves lymph nodes, the spleen and the liver, although extra-nodal sites and BM may be affected [The neoplastic cells of HHV8-positive DLBCL, NOS have the same MCD phenotype with characteristic expressions of IgM lambda, MUM1/IRF4 and CD38, and is absent or weak positivity for B-cell markers, CD30 and CD138.Unlike HHV8-positive DLBCL, NOS, PEL usually expresses CD138, EMA and CD30 and lacks cytoplasmic immunoglobulin.In contrast with PEL, ALK-positive LBCL is always negative for HHV8 and EBER, and CD30 is negative or only weakly and focally positive; ALK staining confirms the diagnosis of ALK-positive LBCL.The distinction between PEL and PM frequently relies on clinical correlation. The presence of lytic bone lesions and a serum monoclonal protein favors the diagnosis of PM.The outcome of PEL is dismal with a median survival of less than 6 months despite intensive chemotherapy.EC-PEL in HIV-positive hosts seems to have a slightly better outcome than patients with classic PEL, with a median survival of 11 months vs. 3 months . The stuBL is a highly aggressive B-cell lymphoma, named after the surgeon who first identified it in the 1950s in Uganda as a rapidly growing neoplasm in children with jaw and abdominal masses ,89,90.In the current WHO classification, there are three epidemiological subtypes of BL: endemic, sporadic and immunodeficiency-related .MYC oncogene [BL is the first historical example of a virus-associated human neoplasm and shows activation of the oncogene . The strEndemic BL occurs in certain geographical area  with a strict epidemiological correlation with malaria and the EBV genome is identified in >95% of cases of endemic BL .Plasmodium falciparum and even with other viruses, such as herpesviruses HHV5 and HHV8 [Local environmental factors play a contributory role in BL development ,92. In eand HHV8 ,92.Endemic BL is the most common pediatric malignancy in equatorial Africa and Papua New Guinea, with a male predilection (M:F = 2:1) and a median age of 7 years at presentation . The earSporadic BL is observed worldwide, generally in children and young adults, although three peaks of age at 10, 40 and 70 years are observed; there is a male prevalence (M:F = 2–3:1) ,95. In EImmunodeficiency-related BL occurs in middle-aged HIV-positive patients, often early in the disease course when the count of CD4+ T cells is still high and the use of HAART has not decreased BL incidence . ImmunodBL involves mainly extra-nodal sites with some differences among the three forms.In endemic BL, the most frequently involved site at presentation is the head and neck area , although the ileocecal region is also frequently involved.In sporadic BL, the disease more frequently presents as an abdominal mass  and more uncommonly in the facial region. Other sites involved in both sporadic and endemic forms are the gonads, kidneys and breasts. Lymph node presentation is rare but observed more frequently in adults.The immunodeficiency-associated BL shows nodal and BM involvement more often. The presentation of the disease as acute leukemia is rare in the endemic form. All of the variants of BL may involve the CNS.BL often affects the GIT, although the frequency of GIT involvement varies among the three forms.Primary GI involvement is observed more frequently in the sporadic form.The most frequently involved GI site is the small bowel at the ileo-cecal region. The stomach and appendix are more rarely involved.Due to the significantly elevated proliferation rate of BL, patients generally present with a bulky disease.BL of the bowel appears as a mass or segmental wall thickening. The intestinal tract involvement usually presents with intussusception, leading to signs of obstruction; bleeding and sometimes malnourishment may be present ,98. In tBL typically consists of a diffuse cohesive proliferation of monomorphic medium-sized cells with round nuclei, finely clumped chromatin and multiple small nucleoli. The cytoplasm is deeply basophilic and contains lipid vacuoles more easily seen in imprint preparations .Frequent mitotic figures and apoptosis are present; the starry sky pattern is indicative of the high proliferative index and is due to the presence of numerous tingible-body macrophages.Some cases have a more variable morphology with greater nuclear irregularity, whereas a subset of cases show plasmacytic differentiation, especially in HIV-positive patients.BL cells have a full B-cell phenotype with strong positivity for CD20, CD79alpha, PAX5, CD19 and IgM expression with light chain restriction. The cells express germinal center markers (CD10 and BCL6) and are usually negative for BCL2. In some cases, there is an atypical phenotype with the absence of CD10, CD5 positivity and weak BCL2 expression. The proliferation rate is very high (ki67 > 95%) and there is strong MYC protein overexpression. EBER is positive  in the mMYC proto-oncogene and one of the IG genes is identified in BL, irrespective of the epidemiological form and of whether or not BL carries EBV.The reciprocal chromosomal translocation between the MYC translocations may occur even in other types of lymphoma and are not specific of BL.However, MYC gene, is involved in the three most common translocations. In the majority of BL (about 80%), there is the translocation ) that transposes the telomeric region of chromosome 8 to the IGH on chromosome 14.A breakpoint in the long arm of chromosome 8 at the 8q24 locus, next to or within the Other translocations such as t  and t  are present in a subset of cases.MYC, whereas in the endemic form, the breakpoints are dispersed several hundred kilobases further upstream of the gene [MYC rearrangement [MYC-negative cases, stringent clinical, morphological and immunophenotypic criteria need to be used to rule out lymphomas that may simulate BL. Some of these cases lacking MYC rearrangement represent the provisional entity Burkitt-like lymphoma with 11 q aberration [In sporadic and immunodeficiency-related BL, the breakpoints are usually within or close to the gene ,100. Aboangement . Accordierration .MYC, some direct consequences are cell growth and proliferation, which may cause lymphoma genesis [From the activation of oncogenes such as  genesis .MYC may also induce cell death by apoptosis and an irreversible block to proliferation named senescence [However, activated nescence . LymphomMYC translocation, leading to the inhibition of the proapoptotic signaling [EBV promotes epigenetic changes of the proapoptotic protein named Bim, inhibiting Bim function and, hence, favoring cell survival ,100. In ignaling ,100.MYC and BCL2 and/or BCL6 rearrangements, so called double-hit and triple-hit lymphomas.BL need to be differentiated from high-grade B-cell lymphoma with A subset of these lymphomas (about 50%) shows morphological features closely mimicking BL or has features in between DLBCL and BL.The diffuse proliferation of monomorphic, medium-sized cells simulates BL although the cytoplasm is probably less basophilic than those in BL and the cytoplasmic vacuoles are absent. BL can be ruled on the basis of the immunophenotype (often strong BCL2 expression in contrast to the absent or weak BCL2 expression in BL) and molecular genetic findings.A subset of BL shows plasmacytic features and needs to be distinguished from other lymphomas with plasma cell differentiation such as PBL and PEL. Unlike these neoplasms often lacking B-cell markers, BL shows a pan-B-cell phenotype.MYC rearrangements. Compared with BL, this subset of lymphomas often involves lymph nodes and shows a certain degree of nuclear pleomorphism.Of note, the provisional entity of Burkitt-like lymphoma with 11 q aberration has to be excluded in cases resembling BL but lacking Current treatments employ intensive, multi-agent regimens that have significantly improved the overall survival of BL patients to almost 90% of cases .The chemotherapeutic schemes involve CODOX-M/IVAC  and DA-EPOCH-R . Modified Berlin–Frankfurt–Münster (BMF) chemotherapeutic regimens plus rituximab have been adopted in adult patients affected by BL, with high rates of complete response (CR) and durable responses . Due to Clinicians and pathologists should be aware of the complex and expanding spectrum of EBV-linked LPDs that may arise even in the GIT. These entities are often difficult to correctly diagnose not only due to their rarity but also due to their overlapping morphologic and immunophenotypic features shared by different entities.Their diagnoses should be based on the evaluation of multiple parameters such as clinical data, morphology, immunophenotyping and genetic analysis.


R = 0.953–0.999) and in women (R = 0.813–0.982), except for VT1–V1 compared with FT1–F1, which demonstrated a moderate correlation in women . Only VT1–V1 and FT1–F1 in women displayed a significant difference (p = 0.001). Therefore, this is the first study to simultaneous record VFSS and noninvasive signals by FSR sensor. The correlation of temporal parameters between these two tests was strong. This finding is valuable for future applications of this noninvasive swallowing study tool.Small flexible force-sensing resistor (FSR) sensors can detect laryngeal excursion during swallowing, but the detected laryngeal excursion has not been correlated with videofluoroscopic swallowing study (VFSS) results. Here, we tested the correlation of temporal parameters between the laryngeal excursion recording by FSR sensor and the hyoid motion recording by VFSS under simultaneously swallowing test recordings. Swallowing measurements were recorded in a radiological suite by simultaneously using VFSS and FSR sensors to detect hyoid motion and laryngeal excursion, respectively. Volunteers sat with their head vertical to the Frankfort plane. Two FSR sensors, each for detecting thyroid cartilage excursion and thumb pressing, were placed. VFSS images and FSR sensor signals during single 5-mL barium liquid (30% wt/volume %) bolus swallowing were collected and analyzed for four swallows per participant. In total, 15 men (28.0 ± 4.1 years old); 14 women (28.4 ± 4.2 years old) were recruited. Temporal parameters between VFSS and noninvasive system demonstrated a strong correlation by Pearson’s correlation analysis: in men ( Noninvasive swallowing studies, a new trend, have several advantages. Because it does not involve radiation exposure, it is suitable for studying early, subtle, or mild dysphagia , 2, and Recently, noninvasive oropharyngeal swallowing study tools are being used, and various noninvasive sensors are used to detect and record thyroid cartilage excursion from the skin surface. These sensors include piezoelectric sensors, accelerators, bend sensors, force-sensing resistor (FSR) sensors , 4–9, anOur team used piezoelectric , 9 and FDetecting thyroid cartilage excursion is a key feature of noninvasive swallowing study. A study demonstrated that excursion of thyroid cartilage is synchronized with hyoid motion during VFSS . When swThe results from noninvasive swallowing studies using FSR sensors to detect the thyroid cartilage excursion, however, were not correlated with those from VFSS. Therefore, our team synchronized these two systems to verify if the temporal parameters between the systems were correlated and postulated that laryngeal excursion detected using FSR sensors is synchronized with the hyoid motion during VFSS. This study aimed to identify the relationship between the temporal swallowing parameters of laryngeal excursion signals detected using FSR sensors and hyoid motion images detected during VFSS, which were recorded simultaneously in young volunteers without dysphagia.The Ethics Committee of Chang-Gung Memorial Hospital approved the study protocol (No. 201602016B0C101). Each participant was introduced to and thoroughly explained the study’s aims and procedures and asked to sign the informed consent form.All participants were young and healthy. Exclusion criteria included a history of dysphagia, cardiopulmonary disease, neurological disease, chronic indigestion disorder, diseases of the head and neck, and smoking in the previous 5 years. All volunteers were administered the Functional Oral Intake Scale (FOIS)  and EatiThe ambulatory noninvasive swallowing respiration assessment system based on the MP150 system  was sent to radiological suites and set up for simultaneous recording by using a VFSS equipment .Two FSR sensors were used—one to detect thumb pressing and the other to detect thyroid cartilage excursion. Noninvasive swallowing and respiration coordination signal monitoring based on the Biopac system was set up in our laboratory when FSR sensors were used to detect thyroid cartilage excursion in previous studies , 3, 7–9.For the VFSS, the recording speed was 30 frames/s in the lateral view and the time duration was 1/30-s interval per image.To overcome the problem of synchronizing the two systems Biopac system and VFSS, another FSR sensor, on the upper end of a handheld stick designed to detect a thumb pressing, was used. These thumb pressing reference points enabled the analysis of timepoints and durations between the two systems. The noninvasive oropharyngeal swallowing signals, including those detected using FSR sensors, were simultaneously recorded during VFSS and analyzed offline later using LabView .Acqknowledge  for the MP150 system was used for recording swallowing signals, and LabView was used for the subsequent offline analysis.To validate FSR sensor detection of temporal swallowing parameters, an ambulatory noninvasive swallowing study system (based on the MP150 system) was used and a VFSS was performed simultaneously in radiological suites. In this study, a specially designed FSR sensor on one side of a small stick was held in the palm to detect thumb pressings as the reference time events for analysis. Volunteers were equipped with two ECG adhesive electrodes for submental sEMG, a nasal cannula for respiration, an FSR sensor for the laryngeal excursion, and an FSR sensor for thumb pressing—all of which were connected onto the MP150 system. These validation study procedures included an FSR sensor held in the volunteer’s hand, which was pressed with the thumb twice—before swallowing and once after swallowing—and another FSR sensor was fixed onto the anterior neck at thyroid cartilage level to detect thyroid cartilage excursion during 5-mL barium swallowing in VFSS. During VFSS, the volunteers sat on a chair with their head vertical to the Frankfort plane. Few small water boluses at room temperature were dispensed to volunteers for the setting up a noninvasive measurement system and to familiarize the volunteers with the test procedure. Liquid barium  bolus was administered using a 20-mL disposable syringe into the mouth of the volunteers. All participants were instructed to hold the FSR sensor fixed on one side of small stick in their hands and hold the fed 5-mL barium bolus in their mouths; they were then asked to press the FSR sensor and swallow the barium bolus on verbal instruction by the radiologist in the following order: “press” (thumb press the FSR sensor on the stick), “press,” “swallow,” and “press”. In total, four separate single boluses of 5-mL barium were recorded each individually by using the two systems simultaneously. In the lateral view in the VFSS, the dynamic images of oropharyngeal swallowing were recorded with 30 images/s. All VFSS videos were recorded and saved in an picture archiving and communication system (PACS). After the study was completed, the copies of VFSS videos from PACS were exported for later offline analysis. The recorded swallowing and respiration signals were exported for subsequent offline analysis  during VFSS  along wi, and V3 FSR sensors were used to detect thyroid cartilage excursion during swallowing. A W-shaped waveform with two “V”s was recorded. F1 is the onset time of W, F2 is the turning point of W, and F3 is the offset time. A semiautomatic analysis software based on LabView was used, which detected and analyzed all three swallowing-related signals  locked in the same frame of three different traces  and combThe point VT1 in VFSS and FT1 by FSR sensor were defined as timepoint zero Fig. . Consecup-value of < 0.01 for a two-tailed T test.Statistical analyses were performed using SPSS statistical package . To determine the correlation between the hyoid motion during VFSS and thyroid cartilage excursion by using FSR in a noninvasive system, Pearson’s correlation coefficients (ϒs) were calculated. Statistical significance between the parameters of the two study systems was taken as a R = 0.838–0.990, all p < 0.001; data not shown). No significant differences were noted in the temporal parameters between the two tests for each corresponding time duration , except for the onset of hyoid motion (VT1–V1) and of thyroid cartilage excursion (FT1–F1), which demonstrated a significant difference .The temporal parameters hyoid motion detected during VFSS and thyroid cartilage excursion detected using FSR sensors demonstrated highly significant correlation . In women, the correlation was strong for all , except VT1–V1 and FT1–F1, which displayed moderate correlation . There were no significant differences in temporal parameters between the two systems, except for that in one temporal parameter (VT1–V1 to FT1–F1) in women that displayed a statistically significant difference but a high correlation. Unlike in the study on the correlation of bend sensor and VFSS , we simuFindings on the effects of gender on swallowing thus far have been inconclusive: some have indicated differences in swallowing parameters between genders, whereas have not –33. ThesWhen simultaneously recording the swallowing signals of a noninvasive FSR sensor swallowing study system and VFSS, some technical issues need to be solved using hardware and software. To synchronize the two systems, a handheld stick with an FSR sensor, connected to one of the multiple MP150 system channels, was used in this study. Our handheld stick design could be used in future swallowing studies to create timepoint references to aid in synchronizing FSR with VFSS or other recording methods.This noninvasive swallowing study method using FSR sensors to detect thyroid cartilage excursion of swallowing is reliable and efficient at indirectly detecting the temporal parameters of oropharyngeal swallowing, which has uses in clinical application and future studies. This system has multiple channels to simultaneously record, and time-lock, swallowing-related physiological signals including submental sEMG, nasal airflow, and uncalibrated measurement of global laryngeal excursion, which is adequate to detect a swallowing event. Swallowing events were analyzed but the detailed parameters were not. The reliability of the detection of swallowing events has been demonstrated, and thus, this noninvasive system can be used to analyze piecemeal deglutition and dysphagia limits . These fSwallowing study objective instruments all have various advantages and limitations. The numerous facets of swallowing function studies include temporal parameters , 29, dynThe noninvasive swallowing study without radiation exposure is suitable for posttherapy monitoring of the immediate effects. Furthermore, the advantages of noninvasive swallowing study systems include the possibility of a larger quantity of swallowing events data that can be collected for a cross-section study, a larger number of repeated measurements over a short period in follow-up studies, and studies for swallowing data collection over a long follow-up duration. It is also useful in long-term objective follow-up studies. Another valuable potential application of a noninvasive swallowing system is in feedback training , 38. OurNoninvasive systems are, however, not without limitations. The noninvasive system cannot record through the oropharyngeal anatomic structures and hyoid displacement, UES relaxation, bolus passage, penetration, aspiration, and residuals after swallowing were recorded during VFSS . These fDeveloping automatic analysis software systems to analyze the dynamic images and temporal parameters of swallowing would be valuable in both VFSS and noninvasive swallowing study systems. Using artificial intelligence to solve this semiautomatic analysis problem may lead noninvasive swallowing studies to another frontier.This is the first study to concurrently record noninvasive swallowing signals and perform VFSS. We provided a new method for researchers to conduct correlation studies between noninvasive swallowing studies and VFSS.Our results demonstrated a strong correlation in the temporal parameters of swallowing between noninvasive surface sensors and VFSS. This is crucial for further usage of this noninvasive swallowing study tool in studies and clinically.


Aminophenoxazinones are degradation products resulting from the metabolism of different plant species, which comprise a family of natural products well known for their pharmacological activities. This review provides an overview of the pharmacological properties and applications proved by these compounds and their structural derivatives during 2000–2021. The bibliography was selected according to our purpose from the references obtained in a SciFinder database search for the Phx-3 structure (the base molecule of the aminophenoxazinones). Compounds Phx-1 and Phx-3 are among the most studied, especially as anticancer drugs for the treatment of gastric and colon cancer, glioblastoma and melanoma, among others types of relevant cancers. The main information available in the literature about their mechanisms is also described. Similarly, antibacterial, antifungal, antiviral and antiparasitic activities are presented, including species related directly or indirectly to significant diseases. Therefore, we present diverse compounds based on aminophenoxazinones with high potential as drugs, considering their levels of activity and few adverse effects. Secondary metabolites are natural products produced by organisms to participate in their biological processes. As they are the result of natural selection through evolution, they are highly optimized structures. Thus, their affinity with the proteins to be synthesized and modified, or their influence on pathological processes , have made of these natural products an increasingly choice for the development of new drugs, either by themselves, or more frequently, through structural synthetic derivatives. These substances are excellent lead compounds in terms of selectivity, specificity, effectiveness and pharmacokinetics. Besides, their structural diversity allow one to secure suitable chemotheques .Aminophenoxazinones are degradation products resulting from the metabolism of different plant species. They comprise a family of natural products well known for their pharmacological activities. In fact, this particularly high interest on these compounds started more than a century ago, as their different industrial and medical applications as pigments, pesticides or drugs were being discovered. Aminophenoxazinones are tricyclic structures with double bonds in aromatic systems containing oxygen and nitrogen atom, which facilitates the development of synthetic derivatives to enhance the properties of these molecules.This review provides an overview of the research works that have focused on the pharmacological properties and applications of aminophenoxazinones and its structural derivatives. The most relevant previous review related to aminophenoxazinones was published in 2013 and was focused on Phx-3 [50) provided in the bibliography for the different assays have been included. The compounds named Phx-1, Phx-2 and Phx-3 have been the most evaluated in the literature, especially Phx-3, having shown a number of promising properties.First, we provide information about the biosynthesis of aminophenoxazinones in plants. Then, their anticancer activities will be presented, including the most relevant results that have allowed us to expand knowledge about their mechanism(s) of action. Antibacterial, antiviral and antiparasitic properties are subsequently detailed. To quantify the levels of activity and facilitate a comparison of the compounds, the values of the half maximal inhibitory concentrations  and 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) . Those, Because of the diversity of products that can be obtained from the degradation of hydroxylated benzoxazinones depending on their cultivation soil  or the eTriticum aestivum L., whereas DIBOA-Glc is mainly concentrated in S. cereale L. [H-phenoxazin-3-one (AMPO) through the action of edaphic microorganisms when submerged into aqueous solutions [H)-one (BOA) that turns into to Phx-3 . However, DIBOA has a high persistence and could have a more important role in chemical defence mechanisms than DIMBOA in plants that produce large amounts of hydroxamic acids [DIBOA and DIMBOA can be detected inside plants as their β-D-glucosides DIBOA-Glc and DIMBOA-Glc. β-D-Glucosides are transformed into DIBOA and DIMBOA when plants are damaged, by enzymatic mechanisms catalyzed by a β-glucosidase . The latreale L. . DIMBOA olutions . This faic acids .Arabidopsis thaliana) are related to the inhibition of histone deacetylases, which provokes locus-specific alterations in histone acetylation processes [In comparison to their precursors with benzoxazinone structures, aminophenoxazinones like Phx-3 are much stable and easier to extract. These advantages, which have enabled the development of numerous studies on their biological activities  and moderocesses . In certrocesses . Anotherrocesses .The degradation process of benzoxazinones functionalized with hydroxamic acid groups leads to benzoxazolinones . BOA andAminophenoxazines are an interesting family of tricyclic compounds related to the previously mentioned benzoxazinones and benzoxazolinones. They are originated as a result of the decomposition of benzoxazolinones into their corresponding 2-aminophenol and its subsequent dimerization.A wide variety of aminophenoxazines has been developed because of their pharmacological interest . Their antiviral , antitumo-aminophenol with catechol [N-heterocyclic (4-phenyl-piperazin-1-yl)methanones derivatives showed excellent inhibition growth activities against different tumor cell lines, being remarked the activity levels of compound 1  have been developed for in vivo tumor detection by formation of fluorescent polymer nanoparticles . SimilarAminophenoxazines  were firThe increasing number of the studies that demonstrated the pharmacological properties of aminophenoxazines made the main focus of attention to move from its use as a dye to the biological field. In fact, these tricyclic compounds are the structural base of several pharmacotherapeutic agents with a wide range of biological properties.Streptomyces antibioticus [A recent study on the biological activities of certain synthetic aminophenoxazinones rendered highly promising results regarding the development of new drugs with antibacterial, antifungal, anticancer, reversal resistance as well as anti-inflammatory, antiviral or antifilarials . Moreovebioticus . This anbioticus . Actinombioticus .50 = 8.87 ± 3.49 µM) [The study of structure-activity relationships (SAR) makes possible to define optimal structural characteristics to improve the properties of the molecules. That is the case of the natural product chandrananimycin A , whose h3.49 µM) . A secon3.49 µM) . More inIn general terms, cancer is an uncontrolled cellular division process that provokes the aggregation of cells and, subsequently, the formation of tumors. A number of additional factors such as genetic mutations, improper diet, smoking or the ingestion of heavy metals and other contaminants may also have some influence on this process by altering the signaling pathways ,40. The Gastric cancer is the third cause of cancer-related death worldwide and a common pathology with a high incidence rate , being n+/H+ exchanger called NHE-1 [The intracellular pH (pHi) varies in accordance with the acidification of the extracellular medium as homeostatic response. However, cancer cells do not decrease this pHi as expected, despite of tumors lead to the acidification of the extracellular pH. The regulatory system commonly concluded to be related is the membrane protein Naed NHE-1 . Severaled NHE-1 . Some exed NHE-1 ,48.2-Aminophenoxazin-3-one (Phx-3) in particular, stands out according to the numerous studies that have proven its effectiveness to change the pHi of different cancer cell lines . In the Kasuga et al.  determin50 value between 6–12 μM) and pro-apoptotic effects in these lines. Phx-3 also generates cytotoxic effects on HT-29 (IC50 = 16.7 μM) [50 = 20.03 ± 4.98 μM) [Colorectal cancer  is the third most frequent cancer and the second leading overall cause of cancer deaths . The in 16.7 μM)  and LoVo4.98 μM) . In the The review of Tomoda et al.  gatheredThus, studies like the ones detailed in this section support that Phx-3 would be an accurate agent itself to treat gastric and colon cancers. Regarding in vivo evaluations of adverse effects, experiments in which mice were treated with  Phx-3 showed that, in addition, this compound causes low adverse effects, since they did not show gastrointestinal lesions or diarrhea during 4 weeks of administration .N-(2-hydroxyphenyl)-2-phenazinamine, known as NHP (50 = 27.82 µg/mL). Moreover, NHP was active against other human lines: COC1 , HepG2  and A549 . At comparing the activity of NHP with that found for Phx-3 against two of these lines in other studies, it is worth highlighting that the corresponding IC50 value of NHP is significantly higher than that shown by Phx-3 against A549 (5.48 ± 0.38 µg/mL), whereas a value of 6.58 ± 0.61 µM was obtained for the HepG2 line. On the other hand, the value of NHP against A549 improves that of the aminophenoxazinone Phx-1 (78.29 ± 5.11 µg/mL) [Even though the Phx family (mainly Phx-3) has been the most studied, it is possible to find evaluations of other derivatives. This is the case of n as NHP , a secon1 µg/mL) ,61. The Glioblastoma is the most common type of aggressive brain tumor in adults, whose treatment is considered a high challenge for oncology since surgery, chemotherapy or radiotherapy are not enough to prevent the tumor progression. One of the main difficulties is achieving the cross of sufficient doses of the chemotherapeutic agents through the blood-brain barrier . The abiGlioblastoma is a complex disorder that appears as a response to a particular form of cellular damage by which cells’ signaling pathways get altered. Cell differentiation is then affected, and as different cell growth factors are over-expressed, the glioblastoma cells get activated and the oncogenesis process, the process by which a normal cell becomes a carcinogen, is promoted . Two of 50 values were 2.602 ± 0.087 µM (24 h) and 1.655 ± 0.093 µM (48 h), which confirms the potent inhibitory effect of Phx-3 at low concentrations against LN229 cell line, being one of the lowest values ever measured until 2013. As an example of the previous study on the effect of Phx-3 on glioblastoma cell lines, let us point out the remarkable cell growth inhibition confirmed for U251MG cell [Focusing on the aminophenoxazinone Phx-3, Che et al.  studied 1MG cell .Che et al. also clarified the molecular mechanisms of the apoptosis induced by Phx-3, by attending to the ERK and JNK signaling pathways. The levels of these phosphorylated pathways increased significantly (6 h) after treatment with Phx-3, being maintained for 20 h. The activation of JNK was suggested to be mediated by the generation of reactive oxygen species (ROS). On the contrary, the phosphorylation of AKT and mTOR  were suppressed. All these effects would contribute with the apoptosis induced by Phx-3, and the relevance of their roles was studied by the evaluation of the activity of Phx-3 in the presence or absence of specific inhibitors. The results showed as the phosphorylation of ERK would not represent a major pathway for the induction of apoptosis, whereas AKT would act under ERK signaling. In the case of JNK, its activation would play a key role in the induction of apoptosis. Long exposure to Phx-3 would activate alternative apoptotic mechanisms by mitochondrial demoralization, like the inhibition of mTOR activity.Previous studies confirmed that treatment of the lung adenocarcinoma cell line A549 with Phx-3 resulted in the depolarization of the mitochondria, causing a decrease of pHi and the generation of ROS . The supIn summary, all the information gathered indicates that Phx-3 depolarizes mitochondria and decreases pHi, which causes the generation of ROS in mitochondria. Then, ROS activates the JNK pathway, which is a signaling pathways highly related to glioblastoma, and also the main generator of apoptosis in cancer cells by caspase cascade. The long exposure to Phx-3 would activate alternative mechanisms.The incidence of melanoma has increased at a higher rate than other solid tumors, being currently the third most common skin cancer. When at advanced stages it represents a serious threat when it spreads to internal organs, which is the major cause of death in skin-cancer patients. This disease may be due to different causes, including both genetic and environmental factors. Several factors have already been identified as significant influencers on both the incidence and the clinical and oncogenic characteristics of this disease. These factors are UV exposure, use of tanning beds, a family history of melanoma, and phenotypic characteristics of fair skin and hair color ,74.Phx-3 has been considered as effective for inhibiting the proliferation of malignant B16 melanoma cells in mice in vitro and in vivo, causing few adverse effects . In the In a subsequent study , it was From the results mentioned in this section, Phx-3 could be proposed as suitable candidate drug for the treatment or prevention of melanoma, including its cosmetic use to treat hyperpigmentation and to prevent melanogenesis.50 values, the sensitivity of the mentioned cancer cell lines after 72 h of treatment with Phx-3 showed how almost all of them were vulnerable to Phx-3 at 10 µM (IC50 lower than 8 µM). KLM-1, Lovo-1 and Y79 lines were the exceptions (IC50 close to 20 µM). The normal cell lines HEL (embryonic pulmonary fibroblast) and HUVEC  were also tested, being obtained high IC50 values , which indicates that these cells are less sensitive to Phx-3 than cancer cells.First of all, we should highlight the study that Che et al. carried out to evaluate the cytotoxicity of Phx-3 on different types of cancer cells, such as MCF-7, A431, KCP-4, A549, KLM-1, MIA PaCa-2, ACHN, LoVo-1, U251MG and Y-79 lines . In the Regarding the pHi decrease, significant results were obtained for all cancer cell lines , being comparable to those of the normal lines. The decrease was proved dose-dependent for MCF-7 (breast cancer) and A431 (skin cancer) cell lines for 30 min and ranging between 0 and 100 µM . TherefoMCF-7 and A431 lines were further evaluated to provide conclusions of the mechanism of action of Phx-3, attending at the reduction of the mitochondrial membrane potential (first and irreversible step towards apoptosis). In other study, the cytotoxic and pro-apoptotic effects of Phx-3 on hepatocellular carcinoma dRLh-84 (rat) and HepG2 (human) cell lines, and the normal hepatocellular RLN-10 (rat) cell line have been studied . Phx-3 r50 = 12.09 ± 3.29 µM) [The list of cancer lines with cell whose growth can be inhibited by Phx-3 may be completed with HeLa  , U266 (m3.29 µM)  lines. TPhx-1  is an amRegarding the pHi reduction in MCF-7 and A431 cancer cell lines, similar or rather close results as those from Phx-3 were observed at the lowest concentrations (5–10 µM) were applied. However, Phx-1 proved to be significantly less active than Phx-3 at 50 µM, and concentration had to be increased up to 100 µM for any major differences to appear in the A431 line.The study of Phx-1 on diverse cell lines . These results are in agreement with the pHi changes undergone by the cells treated either Phx-1 or Phx-3 that have been summarized in the previous paragraph. This could represent a certain advantage regarding cancer treatment, since healthy cells would suffer a lesser damage.50 value of Phx-1 for Y-79 cells  is the highest, previous studies [Although the IC studies  showed iIt is worth highlighting that Phx-1 inhibited the proliferation and induced the apoptosis of diverse human leukemia cell lines  in a dose-dependent manner. This study also proved that Phx-1 reduced in vivo the tumor growth rate in mice, whereas just few adverse effects were found on weight loss and white blood cell .Once confirmed the ability of Phx-1 and Phx-3 to induce the apoptosis of diverse cancer cell lines, Tabuchi et al. studied the effects of these compounds to induce apoptotic cell death in human neutrophils, as this kind of cells are related with the mitochondrial depolarization and reduction of pHi. Both aminophenoxazines caused apoptosis or the loss of the morphology of neutrophils, while lymphocytes and monocytes did not undergo this process. These results would suggest that Phx-1 and Phx-3 are specific drugs to induce apoptotic cell death of neutrophils, being potential preventive anti-inflammatory drugs .50 value of Phx-3 much lower (0.5 µM vs. 20 µM) [The antitumor activity of Phx-1 and Phx-3 on NB-1 (neuroblastoma cell line) was demonstrated by another study. It was thereby confirmed that both aminophenoxazinones induced apoptosis and necrosis, being the IC. 20 µM) .From the structural point of view, the differences in the bioactivity exhibited by Phx-1 and Phx-3 would be associated to the methyl group in Phx-1, even this premise is still to be confirmed. Molecular dynamics simulations could cast some light on the solubility of these compounds in cell membranes, which would allow to determine how much their structural differences affect cross-membrane processes and, thereby, explain the differences in their activity levels. In this line, certain recent studies have proven that the replacement of hydroxyl groups by fluorinated esters (in the eudesmanolide structure), improves the cytotoxicity of these compounds to cancer cells  .Recent studies have tested the activity of different derivatives with an indole group on different cancer cell lines. Indole groups are heterocyclic compounds formed by a benzene atom linked to a pyrrole compound, that have a pair of free electrons in the nitrogen atom of their aromatic ring. They are fairly common components in perfumes, drug candidates and hormones (like melatonin) . Some naTaking into account the potential pharmacological effects of indoles, as well as those already recognized of phenoxazines and the synergistic effects exhibited by some pharmacophoric hybrids , Nunewar50 values. The most inhibited cell line was A549, by the derivative 2 (IC50 = 3.71 ± 0.57 µM), characterized by two isopropyl substituents (3 (IC50 = 4.43 ± 0.64 µM). In addition, 2 was the only derivative with the previously explained DNA intercalation capacity. These results confirm that hybrid compounds are a promising alternative in the search for new anticancer drugs.Therefore, the cytotoxicity of certain indol derivatives was tested by Nunewar et al. on A549 (lung), MG-63 (bone), BT-474 (breast), Hep G2 (liver) and HCT116 (colon) human cancer cell lines, along with a normal line of lung epithelial tissue (L-132). All the derivatives tested showed remarkable ICtituents , and folN-terminal group of L-lysine was linked to the chromophore through an amide bond  conjugated with the amino acid L-lysine, which were designed and synthesized with the aim of developing novel drug compounds with anticancer potential . Similaride bond .4–7 and 4′–7′ were tested on cancer cell lines of leukemia , colon (HT-29), breast cancer (MCF-7), cervical cancer (HeLa), papillary renal cell carcinoma (ACHN), and melanoma (SKMEL-28 and G-361). All the products inhibited the proliferation of a panel of human liquid and solid neoplastic cell lines, the latter being more sensitive to antiproliferative effects (only compound 7 showed similar activity in both states). The IC50 values of the derivatives with the L-lysine side chain attached to C-10 were higher than their corresponding derivative functionalized at C-9. The IC50 values of 4–7 were comparable to that of AMD and even lower than those of Doxo and VP-16 . It is worth highlighting the lowest values of IC50, all in the range of 0.001–0.007 µM, achieved by 4 and 7 for HT-29, SKMEL-28, MCF-7 and G-361 lines. The authors proved that both compounds are strong DNA intercalators, and possess the capacity to selectively target the topoisomerase Topo IIα.Products The difference in activity between the two series of derivatives was related to structure-activity correlations. Thus, the position of the nitrogen atom in D-ring would play an important role in the antiproliferative activity, whereas the position of the L-lysine side chain and the type of amine groups affect the cytotoxic activity both between the two series and within each series.4 and 7 in particular, provide really interesting opportunities for the development of new DNA-targeting anticancer drugs.All the mentioned properties indicate that this new series of pyridophenoxazinones conjugated to L-lysine have a great antitumor therapeutic potential. Products The emergence of resistant bacteria and its impact on health, together with the inappropriate use of antibiotics, has become a pressing problem for patient safety. Infectious diseases are one of the main causes of global morbidity and mortality, especially in developing countries.Mycobacterium scrofulaceum attending to minimum inhibitory concentration (MIC) values obtained (1.4–2.8 µg/mL). Phx-3 also inhibited Mycobacterium marinum and Mycobacterium intracellulare, with slightly higher MIC values, whereas Mycobacterium kansasii showed the best inhibition levels for Phx-1 and Phx-2 . Phx-3 was suggested for the treatment of T cell-mediated autoimmune diseases, and for those chronic-inflammatory diseases induced by bacteria.In the case of Phx-3, high antituberculosis activity levels have been also reported (MIC = 3.91 µM) , as wellHelicobacter pylori, which affects approximately 50% of the world population, is a causative agent of chronic gastroduodenal ulcer and, possibly, gastric cancer. The triple therapy that comprises amoxicillin, clarithromycin (CAM) and a proton pump inhibitor, has proven to be effective to eradicate this bacterium. However, resistance is a common issue that appears when applied to the control of bacteria like H. pylori. As an example, the recovery rate when this triple therapy treatment is applied in Japan is as low as 20% [w as 20% . This low as 20% .H. pylori  and against three clinical strains . According to the results regarding cell-mediated immunity (CMI), none of the strains, including TK 1029, which has proven to be resistant to the triple therapy, grow in the presence of Phx-3 at concentrations of 1 µg/mL or higher. After 48 h, the bacterial populations were below their detection limit. Phx-3 has also been tested against H. musterae (ATCC 43772), and 0.5 µg/mL CMI value was reported.This resistance issue has brought about the development of alternative drugs over the last decade, being Phx-3 one of the drugs of interest for this purpose . Phx-3 wH. pylori have the capacity to implement certain morphological changes [H. pylori and H. mustelae strains.Inhibitors of  changes . Such chH. pylori infections to gastric lymphomas and adenocarcinomas (currently known as type 1 carcinogens) [Information provided herein would be quite relevant for the development of new drugs for the control of H. pylori based on Phx-3. Some reports associate inogens) . TherefoHelicobacter, the appearance of certain antimicrobial resistance represents a menace to the prevention and treatment of infections caused by bacteria, parasites, viruses and fungi. The diseases associated to these organisms are the third leading cause of death in developed countries [Fusarium spp. (fungi) in plants is conditioned by the resistance generated by the detoxification of certain drugs, like benzoxazolinones BOA and MBOA (plant defense compounds). This process was found to be catalyzed by the fungal γ-lactamase enzyme encoded by FDB1 locus [Similarly to the genera ountries . For insB1 locus . TherefoFusarium verticilloides [Bacillus subtilis and Staphylococcus aureus, as well as on the fungus Candida albicans, whereas no activity against Escherichia coli was observed. The derivative hydroxylated at C-6 was active for the two first species, with slightly better results [S. aureus the article by Tse et al. [S. aureus, that was attributed to the release of silver ions into the cells after getting dissociated from the complex. Like Phx-3, this complex was inactive against E. coli, which was probably explained by the obstacle posed by the double layer cell wall that characterizes gram-negative species [Phx-3 has shown high levels of toxicity to the species illoides . Tested  results . We woule et al. . Later o species .Mucor miehei and, in the case of the first, also on B. subtilis [S. aureus, C. albicans, Chlorella vulgaris, Chlorella sorokiniana and Scenedesmus suspicatus. The better activity of chandrananimycin C in the concentration tested (20 µM) would be related to its structure, which presents the scaffold of chandrananimycin B with an additional fourth ring (cyclic amine). None of the three compounds were active on E. coli.Chandrananimycin A  and B arsubtilis . In the C. albicans (MIC = 64 µg/mL) [The natural product NHP , in addi4 µg/mL) .8–15) of phenoxazines against the bacteria Listeria ivanovii, E. coli, S. aureus and Klebsiellapneumoniae spp., and against the fungi C. albicans and Aspergillusniger spp. For the synthesis of the compounds, in a first stage, 5 was synthesized, then, the rest of compounds were obtained by arylation (compounds 9–12) or amination (compounds 13–15) reactions of 8  exhibited activity against E. coli, which was more selective. In the case of S. aureus, K. pneumoniae, C. albicans and A. niger, it should be highlighted that only compound 9 presented some activity against all these species. It should also be noted that compound 8 (scaffold of 9–15) showed sensitivity to all the organisms except S. aureus and L. ivanovii, both of which are gram-positive bacteria. Therefore, it could be concluded, with regard to molecular structures, that the compounds obtained either through the arylation or amination of compound 8 causes the inactivation of the molecule on fungi, but exhibits an increased activity when applied to gram-positive bacteria.The agar well diffusion method was employed for the sensitivity study of 11 and 15 on L. ivanonii (14.42 µg/mL), being a significantly lower value than that of the reference drug (21.30 µg/mL). This higher activity than currently commercialized drugs, let us think that new derivatives could be developed to a more efficient control of microorganisms.The data obtained from the experiments show how most of the compounds are active at low concentrations. The best minimum active concentration observed was for derivatives Klebsialla sp., Bacillus sp., Proteus vulgaris, Salmonella sp., Shigella flexneri, S. aureus, Streptococcus pneumoniae and Vibrio cholerae, with compound 16 being remarkable  by detecting β-alanyl aminopeptidase activity in clinical samples [The final study presented in this section will be the finding of new chromogen aminophenoxazinone derivatives with applicability for the detection of  samples . This faIn vivo antiviral studies of aminophenoxazinones Phx-1, Phx-2 and Phx-3 in mice allowed to propose the three compounds as potential drugs for preventing the replication of HSV and the aggravation of lesions that these viruses cause. Their activity on herpes simplex virus type-1 (HSV-1) was highlighted, whereas Phx-2 was the only that showed inhibitory effects against HSV-2 (moderately). Another point of interest regarding HSV-2 was the improvement of the survival rates after the treatment with Phx-1, Phx-2 or Phx-3  . Phx-2 aThe antiviral potential of the three compounds is also promising for the human T-lymphotropic virus type-1 (HTLV-1), which causes adult T-cell leukemia (ATL). Phx-1, Phx-2 and Phx-3 prevent the cell-to-cell transmission of this virus in model cell lines, as well as the induction of apoptosis in MT-1 cells in doses of 10 μg/mL . The samRecently, Phx-3 was computationally studied by docking to evaluate its pharmacokinetic related with the inhibition against MMP9 enzymes, whose overexpression is caused by the infection of hepatitis B virus . As resuAnopheles mosquitoes [Malaria is a parasitic disease that causes high fevers, chills, flu-like symptoms and anemia. It is caused by a parasite and is transmitted to humans through the bite of infected squitoes .17, 18 should also be highlighted. However, this compound presented the disadvantage of its shorter life; probably due to the shorter chains of the tertiary amine.Ge et al.  evaluate17 was considered a suitable candidate for the development of antimalarial drugs. In the first place, it exhibited an IC50 of 7.6 nM it against Plasmodium falciparum as well as a noticeable higher selectivity when compared to the other synthesized derivatives. Moreover, the in vivo experiments that were carried out on mice infected with Plasmodium berghei  achieved the cure after three daily doses for three consecutive days.Compound Leishmania major is a parasitic species that causes cutaneous leishmaniasis, a tropical disease that generates a large number of adverse effects on human health, mainly affecting the skin and mucous membranes. Human infections occur after the bite of sand fly (Phlebotomus). A number of haloacetamines derivated from N-substituted phenoxazines, as well as other related structures such as phenothiazines, have been studied through in vitro essays [L. major. The lowest IC50 values were obtained by the products shown in 19), 9.5 µM (20) and 9.6 µM (21).o essays . The halH. pylori or F. verticilloides are examples of high sensitive species to these aminophenoxazinones. Moreover, it should be remarked their activity against herpes simplex virus type-1 (HSV-1) and human T-lymphotropic virus type-1 (HTLV-1). Remarkable antiproliferative capacity have been proven for some structural derivatives, such as those conjugated to indole groups 2 and 3 , the pyridophenoxazinones 4 and 7 , compounds 8, 9, 11, 15 and 16 , 17  and 19–21 (against Leishmania major).Our compendium of relevant studies from the literature leads us to conclude that aminophenoxazinones possess a number of promising properties like anticarcinogenic, antifungal, antiparasitic, antibacterial or antimicrobial activities. Aminophenoxazinones are degradation products that present really interesting pharmacological properties, selectivity and minimal toxicity that make them suitable for the treatment of certain current and relevant diseases as explained in this review. Phx-1, Phx-2 and Phx-3 are the most studied aminophenoxazinones in the literature, especially Phx-3, whose activities and modes of action have been detailed herein. In the case of their cytotoxicity against cancer cell lines, Phx-3 stands out as promising anticancer drug, due to the capacity to decrease the pHi in cancer cells and inhibit the proliferation of cell lines like the related to gastric and colon cancer, glioblastoma or melanoma, among others. The activity of Phx-3 has been compared to that of anticancer drugs like camptothecin, known for its side effects. In this context, in vivo studies have shown that Phx-1 and Phx-3 cause low or null adverse effects, which indicates that they would be less aggressive drugs. Regarding their antibacterial and antimicrobial activities, Phx-1, Phx-2 and Phx-3 have proven to have the desirable characteristics that would allow certain drug resistance issues to be overcome. Some non-tuberculous mycobacteria, Therefore, the active compounds mentioned herein could be considered for the development or improvement of therapeutic treatments, from the perspective of the activity levels. This conclusion supports the research studies that are currently being conducted on this family of compounds. In the authors’ opinion, it is necessary the seek and evaluate new derivatives that could allow one to define the structural features that lead to the effects observed in the different types of assays. Even though different in vivo studies have been presented in this review, we consider that the proportion of this type of assays should increase over the next years, which would bring firm conclusions for the future use of aminophenoxazinones and related compounds as drugs.


The need to produce immortal, food-relevant cell lines is one of the most pressing challenges of cellular agriculture, the field which seeks to produce meat and other animal products via tissue engineering and synthetic biology. Immortal cell lines have a long and complicated story, from the first recognized immortal human cell lines taken from Henrietta Lacks, to today, where they are used to assay toxicity and produce therapeutics, to the future, where they could be used to create meat without harming an animal. Although work in immortal cell lines began more than 50 years ago, there are few existing cell lines made of species and cell types appropriate for cultured meat. Cells in cultured meat will be eaten by consumers; therefore, cultured meat cell lines will also require unique attributes not selected for in other cell line applications. Specifically, cultured meat cell lines will need to be approved as safe for consumption as food, proliferate and differentiate efficiently at industrial scales, and have desirable taste, texture, and nutrition characteristics for consumers. This paper defines what cell lines are needed, the existing methods to produce new cell lines and their limitations, and the unique considerations of cell lines used in cultured meat. Cellular agriculture is an emerging field that aims to produce animal products from cell cultures rather than whole animals. One of the anticipated products of cellular agriculture is cultured meat made in vitro from animal cell cultures, also known as cell-based meat, cultivated meat, in vitro meat, clean meat, artificial meat, or lab-grown meat. Multiple names exist due to a lack of consensus on a term that is both informative to consumers and accurate by technical and regulatory standards ,2,3,4. GAlthough primary cell cultures may be used to study the mechanisms of cultured meat production over short time scales, primary cells can only undergo a finite number of cell divisions before they lapse into senescence or arrest of the cell cycle. This makes longer-term studies and commercial-scale production difficult. Using primary cells for cultured meat production requires that donor herds of animals be kept to provide biopsies, and that these biopsies are regularly acquired and approved for use in food production. Unlike primary cell cultures, immortal cell lines do not undergo senescence, and can exhibit infinite divisions. They could, therefore, be easier to study, and allow for safer and more consistent cultured meat without ongoing need for animal biopsies . Currently, however, there are no cultured-meat-appropriate cell lines available to researchers and developers. The culinarily appropriate components of animal meat are primarily skeletal muscle and adipose tissue ; relevanEfforts have only recently begun in developing repositories of cell lines specifically appropriate for cultured meat in order to facilitate the research and development of novel food. One example is the partnership between the Good Food Institute (GFI) and Kerafast that aims to curate a repository of standardized terrestrial and aquatic cell lines appropriate for cultured meat research ,13. ThusAs no commercially available, agriculturally relevant, immortal cell lines have been confirmed food-safe and initial repositories are seeking deposits, this paper defines the existing methods to produce new cell lines and their limitations, and the unique considerations of cell lines for use in cellular agriculture.Cell lines become immortal when they lose their cell cycle checkpoint pathways and circumvent the process of senescence. There are currently three methods to establish immortal cell lines: the discovery of spontaneously immortalized cell lines, expression of the catalytic subunit of telomerase (TERT), or induction by viral genes that inactivate p53/p14/Rb. Each method utilizes either telomerase expression or inactivation/bypassing of the cell cycle, or both, as summarized in In rare events, most often in cancer, cells will spontaneously immortalize. These cells can then be isolated. Tissue must be sampled by biopsy and then dissociated, and the cell types must be isolated and tested for proliferative capacity and identity. This was the original method to obtain cell lines beginning with the first immortal cell line derived in the 1940s from mouse fibroblasts, as well as the HeLa cell line isolated from the cervical cancer of Henrietta Lacks. Immortality of the HeLa cells could be attributed to the cells’ infection with human papillomavirus 18, which may have either degraded the tumor-suppressor protein p53 or caused chromothripsis, a chromosome-shattering and rearrangement associated with 2–3% of all cancers and which changes the expressions of thousands of protein-coding genes ,15. In addition to being discovered in samples, spontaneous immortalization can be coordinated by scientists. Cancer can be induced by radiation or chemical carcinogens. A cell line can also be put under serial passaging to select for clones with immortalization markers, high TERT expression or low p15/p16/Rb expression. One example of spontaneously immortalized cell lines in cultured meat is the chicken fibroblast line used by the company Future Meat. The Future Meat cell line was created by culturing fibroblasts isolated from a chick embryo, and isolating, concentrating, and expanding the foci of more rapidly growing cells until there was a culture of uniform morphology that was able to survive past the 20–30 divisions undergone by an unmodified somatic cell [Spontaneous immortalization has its limitations, and may be more suitable for some situations and less suitable for others. For example, spontaneously immortalized cells would likely not be considered genetically modified (GM), which could allow them access to European markets that currently have strict regulations on GM foods . Under oIn a normal cell, chromosome telomeres shorten with every replication of DNA. Repeated shortening eventually exposes the chromosome ends to damage, which leads to cell senescence. The upper limit of divisions that a cell can reach before it enters senescence is known as the Hayflick Limit, and is typically between 20 and 30 divisions . Germ liThe introduction of telomerase has been successful in immortalizing cell lines, because telomere elongation helps cells escape senescence triggered by telomere shortening. This has been performed by ectopic expression of the catalytically active subunit of telomerase (TERT), or by the overexpression of TERT. Despite the checkpoints controlling human cell immortalization , human fTelomerase expression has not been as commonly successful in immortalization as methods that inactivate or bypass the p53/p16/Rb stress response  large T-antigen (TAg), which binds to and inactivates p53/Rb as well as other tumor suppressor factors in a number of species and organ types ,29,30. IIn many cases, TERT expression or inactivation of p15/p16/Rb alone are not sufficient to immortalize a cell line, suggesting that both telomere shortening and the p53/p16/Rb stress response must be bypassed. Myoblasts have previously been shown to require the bypass of both senescence triggers to become immortal . In 2016Cellular agriculture requires cell lines made from organisms that are agriculturally relevant rather than well-characterized; therefore, some cell lines have been difficult to establish. In particular, no permanent cell line is available for marine invertebrates, such as mollusks or snails or crustaceans, perhaps due to a lack of information regarding their physiology and biochemistry and understanding of the variables required for their immortalization. To create these cell lines, scientists must first characterize their stem cell expression markers and ideal culture conditions, and must also develop methods for inducing myogenesis in the created cell lines . CurrentOne of the most common concerns about cultured meat from consumers is the safety of ingesting cell lines. Currently, cultured animal cell lines are not commonly eaten by consumers. An immortal cultured meat cell line may contain expressed oncogenes, and if so, food products produced with these cells will need to be confirmed not to have tumorigenicity ,44. AlthIn the case that a cell line is confirmed safe for consumption, it will still need to be regularly monitored for contamination and genetic drift. Contamination of cell lines with other cell lines and adventitious agents can be common in cell culture ,46,47. ARegulatory agencies are beginning to adapt pathways for cultured meat products to be examined regarding safety for consumption. To some degree, this will involve updating the existing regulations on cell-derived products for relevance to consumption as food ,52,53. ARegulatory bodies in a few other regions have begun to adapt their regulations to new cultured meat products .The immortalization method itself may be an important factor in the decision of consumers to accept cultured meat. To the best of the authors’ knowledge, no study has yet compared the consumer acceptance of cultured meat made of primary cells and cultured meat made of immortalized cells. There have, however, been initial studies analyzing the acceptance of genetically modified and non-genetically modified cultured meat. These studies found that consumers are more willing to purchase non-genetically modified cultured meat compared to genetically modified cultured meat ,60. ProdThere are, however, some differences across applications and among consumers in how genetic engineering is evaluated that may inform decisions on the immortalization of cell lines for cultured meat. Consumers appear to be more concerned when genes are exchanged between different species, and see gene insertion as more unnatural than gene deletion ,65,66. UAs mentioned in Furthermore, some cell lines may be prohibited from consumption under religious food restrictions. Jewish kosher laws and Islamic halal laws do not allow the consumption of conventional pork and other meats , and it Cell lines for cultured meat production will not only be from animals and cell types not previously established, but will also require different attributes from those previously typical for immortal cell lines. Specifically, they should be food-safe, able to proliferate stably and efficiently in a large-scale production environment with minimum costs, and have desirable taste, texture, and nutrition. Multiple companies have filed patents on ways to overcome the unique challenges of growing food-relevant cells at scale. The cultured meat company Wild Type has filed one patent on a footprint-free method of genetic modifications to control cell differentiation and proliferation with a small genetic footprint . AnotherThe sensory experience of cultured meat may be affected by the cell types used. A scientific paper on the sensorial qualities of cultured meat compared to animal-sourced meat has not been published thus far . AlthougThe sensory experience of cultured meat may additionally be affected by certain attributes to be selected for in cell lines. Consumer surveys have found that many consumers expect cultured meat to be less tasty than conventional meat, and this perception contributes to their unwillingness to consume cultured meat ,12. The Cultured meat should be designed to provide similar, if not enhanced, available levels of nutrients compared to conventional meat. Meat provides 26% of the global protein supply, 24% of the global fat supply, and 9% of the global calorie supply per capita per day, as well as essential micronutrients such as iron, zinc, and vitamins A and B12 . For culOther cultured meats could be engineered for desired differences from the nutritional profile of conventional meat. As an initial exploration of nutritional engineering for cultured meat, Stout et al. have incorporated a biosynthetic pathway for carotenoids, a class of antioxidants native to some plants but not to animals, into primary bovine satellite cells (BSCs) . They deAlthough there are several techniques used to establish cell lines appropriate for bio-logical or medical research, there are still several unanswered questions about how to establish immortalized cell lines suitable for human consumption as cultured meat and which of these existing techniques are appropriate or would need to be modified for use in cultured meat. The lack of readily available cell lines is a current barrier to conducting research in cultured meat. Most academic cultured meat research, from media composition to bioscaffolding to bioreactor projects, must currently begin by creating cell lines for study. Cultured meat will require unique cell lines that are safe, appropriate for industrial-scale production, and desirable to consumers. Although development of these cell lines can be based on established immortalization methods, they will need to be applied to cell types appropriate to meat. In addition, the properties of food safety, industrial production, and consumer desirability have only begun to be explored in cell lines for cultured meat. There is still much work to be done before a collection of cell lines appropriate to cultured meat can be established and readily available for the use of researchers and developers.


CDKN2A, CDKN2B), expression of fusion genes  or of genes targeted by point mutations . MEF2D-BCL9 and PAX R38H mutations in cell lines have hitherto been undescribed, suggesting that YCUB-4 (MEF2D-BCL9), PC-53 (PAX R38H) and their sister cell lines will be useful models to elucidate the function of these genes. All aberrations mentioned above occur in both sister cell lines, demonstrating that the sisters derive from a common ancestor. However, we also found mutations that are specific for one sister cell line only, pointing to individual subclones of the primary tumor as originating cells. Our data show that sequential sister cell lines can be used to study the clonal development of tumors and to elucidate the function of common and clone-specific mutations.For many years, immortalized tumor cell lines have been used as reliable tools to understand the function of oncogenes and tumor suppressor genes. Today, we know that tumors can comprise subclones with common and with subclone-specific genetic alterations. We sequenced DNA and RNA of sequential sister cell lines obtained from patients with pre-B acute lymphoblastic leukemia at different phases of the disease. All five pairs of cell lines carry alterations that are typical for this disease: loss of tumor suppressors ( For many decades, cell lines have enabled the modeling of human disease in cell culture for a better understanding of a plethora of pathophysiological processes and also, most importantly, for drug screening. Clearly, cell lines represent a useful in vitro tool owing to their easy manipulability and reduced culture costs; furthermore, they constitute a renewable resource, and, provided they are grown under recommended conditions, they retain their characteristic features and the results are reproducible. Nevertheless, a detailed and fully annotated characterization is fundamental before their use. In fact, in many cancer research fields, cell lines offer the most comprehensively characterized platform ,2.Additionally, in the domain of hematopoietic tumors, cell lines represent vital and powerful models in experimental systems to unravel the pro-leukemogenic roles of specific genetic mutations. As all leukemia cell lines inevitably carry molecular alterations, it is also essential here for the utility of these models to appropriately detail these genomic changes .Despite the achievement of a complete remission in the vast majority of patients with acute lymphoblastic leukemia (ALL), ALL relapse remains a leading cause of childhood cancer-related death . RelapseTo obtain further insight into the relative impact of founding genomic alterations and acquired genetic alterations, we set out to examine the gene mutation status and gene expression of paired cell lines procured from ALL patients at diagnosis and at subsequent relapse or at other consecutive stages of their disease, aiming at a better understanding of the ALL relapse processes.Cell lines NALM-20 and NALM-21 were taken from the stock of the cell lines bank . All other cell lines were supplied for research purposes. Cell lines were authenticated by DNA profiling. Detailed references and cultivation protocols for NALM-20, NALM-27 and PC-53 have been described previously . www.bioinformatics.babraham.ac.uk/projects/fastqc, accessed on 28 May 2019 and aligned by STAR (2.5.3a) . NA. NABCR apositive  24]. NA. NABCR aMEF2D translocations were described in pre B-ALL, with BCL9 being the most common fusion partner of MEF2D [MEF2D exon 5/BCL9 exon 9 fusion transcript, resulting from t  . These c of PAX5 . PAX5 deLL cases .PAX5 is also a partner of translocations, and it is the target of point mutations in this disease [PAX5 R38H mutation  along with their sister cell lines (IKZF1 deletions and deletions of CDKN2A and CDKN2B are markers of disease recurrence in adolescent and adult Philadelphia chromosome-negative pre-B ALL [BCR-ABL1-positive cases and a lower proportion (<5–25% depending on the subtype) of BCL-ABL1-negative cases carry the IKZF1 deletion [IKZF1 deletion are from adults and carry the BCR-ABL1 fusion —NUCKS1 (ex 2) fusion is expressed in the cell line PC-53 but not in the sister cell line PC-53A  analysis showed that in PC-53 both genes, NSMAF and NUCKS1, were located in the middle of the transition between two copy numbers ) fusion transcript in PC-53 and not in PC-53A.The e PC-53A . This fu numbers . These d numbers . Thus, tBecause the CGH analysis was suitable for detecting molecular differences between the sister cell lines PC-53 and PC-53A, we performed copy number analyses to elucidate whether other sister cell lines carried clone-specific copy number differences as well. Most aberrations detected by the CGH analysis were found in both of the sister cell lines (KRAS A146T (Gca/Aca) or with NRAS G12C (Ggt/Tgt) mutations   were mutant. In AT-2, 3% were mutant  . HoweverGca/Aca) . Furthere mutant . KRAS homozygously wild-type (0/0)/NRAS heterozygously mutant (0/1) and “clone B” being KRAS (0/1)/NRAS (0/0). The easiest explanation for these results is that both sister cell lines comprise two subclones, “clone A” being BCL2) and clone-specific (MYC) alterations [BCL6 [We have previously shown that B-lymphoma cell lines can comprise subclones . Twelve erations . The difns [BCL6 . The immns [BCL6 . CDKN2A, CDKN2B, ETV6, IKZF1 and PAX5, all of them recurrent deletions in pre-B ALL —BCL9 (ex 9) in YCUB-4 and YCUB-4R was especially noteworthy. BCL9 is the most frequent fusion partner of MEF2D in pre-B ALL [MEF2D-BCL9 fusion transcript. What is also novel is the PAX5 R38H mutation (COSM5986423) in cell lines PC-53 and PC-53A  and NRAS G12C , were detected in each of the sister cell lines, albeit at different percentages and not with the 50% wt vs. 50% mu read proportion that would be expected if a cell clone carried a wild-type and mutant version of a gene (KRAS A146T and the other with the NRAS G12C mutation, had already existed in the patient. AT-1 and AT-2, established at first and second relapse, comprise both subclones, albeit at different percentages (Without DNA from the primary tumor, we could not formally exclude that the sister cell specific aberrations had developed in vitro. However, the WES analysis of f a gene  . It is centages .MEF2D-BCL9 fusion in YCUB-4 and YCUB-4R and the PAX5 R38H mutation in PC-53 and PC-53A are noteworthy. Mutations specifically occurring in one of the sisters suggest a derivation from individual clones of the primary tumor, whereas common mutations point to the common ancestor. The study shows that sequential sister cell lines allow one to study common and clone-specific mutations.In sum, the results of the RNA-seq, WES and CGH analyses of five pairs of sequential pre-B ALL cell lines show aberrations characteristic for the disease. Because they are unreported in cell lines so far, the