LncRNAPVT1 is Associated with Cancer-Associated Fibroblasts Proliferation Through Regulating TGF-ßin Oral Squamous Cell Carcinoma.

INTRODUCTION: Human oral squamous cell carcinoma (OSCC) is the most common type of oral cancer and has a poor survival rate. Cell-cell communication between OSCC cells and cancer-associated fibroblasts (CAFs) plays important roles in OSCC progression. We previously demonstrated that CAFs promote OSCC cell migration and invasion. However, how OSCC cells influence CAFs proliferation is unknown. METHODS: Knockdown of PVT1 was confirmed using lentivirus infection technique. CAFs in tissues were identified by staining the cells with α-SMA using immunohistochemical technique. CCK-8 assay was used to evaluate cell proliferation. The mRNA level of a gene was measured by qRT-PCR. Secreted TGF-ß were detected using ELISA assay. RESULTS: We found that knockdown of the long non-coding RNA (lncRNA) plasmacytoma variant translocation 1 (PVT1) was associated with a low density of CAFs in xenograft tumors in mice; further analysis revealed that PVT1 in OSCC cells induced CAF proliferation through transforming growth factor (TGF)-ß. DISCUSSION: Our results demonstrate that lncRNA PVT1 in tumor cells participates in CAF development in OSCC by regulating TGF-ß. This study revealed a new mechanism by which PVT1 regulates OSCC progression and PVT1 is a potential therapeutic target in OSCC.

Autophagy inhibitors enhance biomolecular delivery efficiency of extracellular vesicles.

In recent years, extracellular vesicles (EVs) were loaded with therapeutic molecules to be delivered to recipient cells, so the possibility of utilizing EVs for drug delivery has been investigated in various models. Nonetheless, most EVs are degraded through the autophagy pathway after being up-taken by recipient cells, resulting in a low delivery efficiency. Here we introduced a strategy to overcome inefficient delivery of EVs. We demonstrated that autophagy inhibitors, used for reducing lysosomal degradation of EVs, enhanced the protein or plasmid DNA delivery efficiency of EVs in recipient cells without influencing the uptake of EVs by recipient cells. Moreover, autophagy inhibitors could also improve gene-editing efficiency of EV-loaded CRISPR/Cas9 system.

CAR19/22 T cell therapy in adult refractory Burkitt's lymphoma.

The treatment of refractory Burkitt's lymphoma (BL) is still a challenge. Although CAR-T cell therapy has achieved good responses in diffuse large B cell lymphoma, there is no case series report about the efficacy of CAR-T cell therapy in adult Burkitt's lymphoma. In this study, we evaluate the efficacy and safety of CAR19/22 T cell therapy in six refractory Burkitt's lymphoma cases with poor genetic prognostic factors. After CAR-T cell therapy, five cases had grade 1 and one had grade 3 cytokine release syndrome. Three patients achieved an objective response (3/6 50%), including two partial remission and one complete remission. One CR patient received allogeneic hematopoietic stem cell transplantation (HSCT) and one PR patient received CAR22/19-T cells following auto-HSCT, and they were still in remission at 37 and 22 months of follow-up, respectively. Interestingly, patients with bulky disease (case 2, 4 and 5) had higher levels of serum IL-2R, which was secreted by regulatory T cells, lower CAR lentiviral amplification and poorer prognosis with shorter survival time than cases with non-bulky disease. It is suggested that high tumor burden, more immune suppressive cells and limited CAR-T cell expansion might affect the efficacy of CAR-T cell therapy. CAR-T cell therapy in adult BL patients whose best response cannot achieve CR may need to bridge to other treatments (such as HSCT) early.

Clinical characteristics of hematological patients concomitant with COVID-19.

The rapid spread of coronavirus disease 2019 (COVID-19) represented the most serious issue to public health globally. Hematological patients as immunocompromised hosts are vulnerable to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. There is little information available regarding the clinical features of hematological patients concomitant with COVID-19. In this study, 9 concomitant patients were analyzed for their clinical manifestations, laboratory data, radiological findings, and immunologic features. The median age was 50 years (range, 17-68 years) and 6 patients were male. Seven patients were infected through hospital-associated transmission and other 2 through community-associated transmission. Onset of COVID-19 in all patients occurred during routine treatments for their hematological diseases. Eight patients were classified as moderate and 1 patient as critically ill COVID-19. Four patients died, 1 from leukemia progression, 2 from life-threatening secondary infection, and the other from respiratory failure caused by COVID-19. Abruptly elevated levels of cytokines were often correlated with progressive hematological disease or concurrent bacterial infections. Two patients had atypical computed tomography (CT) imaging findings of COVID-19. The median interval from the first CT scan imaging to improvement in survivors was 40 days (range, 14-51 days). Four of 5 survivors had negative serological tests 1 month after symptom onset. Positive viral load in 4 survivors lasted longer than 45 days. Our results indicated concomitant patients formed a distinct subgroup characterized by atypical clinical features, defective viral clearance, and lower level of SARS-CoV-2-specific Abs. Targeted therapies that impair host humoral immunity should be avoided. These findings will be helpful to tailor appropriate management for the concomitant patients.

Efficacy and toxicity for CD22/CD19 chimeric antigen receptor T-cell therapy in patients with relapsed/refractory aggressive B-cell lymphoma involving the gastrointestinal tract.

Gastrointestinal (GI) tract is the most common site of extranodal involvement in non-Hodgkin lymphoma. Life-threatening complications of GI may occur because of tumor or chemotherapy. Chimeric antigen receptor (CAR) T-cell therapy has been successfully used to treat refractory/relapse B-cell lymphoma, however, little is known about the efficacy and safety of CAR-T cell therapy for GI lymphoma. Here, we reported the efficacy and safety of CAR-T cell therapy in 14 patients with relapsed/refractory aggressive B-cell lymphoma involving the GI tract. After a sequential anti-CD22/anti-CD19 CAR-T therapy, 10 patients achieved an objective response, and seven patients achieved a complete response. CAR transgene and B-cell aplasia persisted in the majority of patients irrespective of response status. Six patients with partial response or stable disease developed progressive disease; two patients lost target antigens. Cytokine release syndrome (CRS) and GI adverse events were generally mild and manageable. The most common GI adverse events were diarrhea (4/14), vomiting (3/14) and hemorrhage (2/14). No perforation occurred during follow-up. Infection is a severe complication in GI lymphoma. Two patients were infected with bacteria that are able to colonize at GI; one died of sepsis early after CAR-T cells infusion. In conclusion, our study showed promising efficacy and safety of CAR-T cell therapy in refractory/relapsed B-cell lymphoma involving the GI tract. However, the characteristics of CAR-T-related infection in GI lymphoma should be further clarified to prevent and control infection.

SLC39A6/ZIP6 is essential for zinc homeostasis and T-cell development in zebrafish.

Zinc (Zn) is an essential trace element that modulate innate and acquired immune responses, and its deficiency triggers lymphopenia. However, the precise mechanisms underlying zinc-mediated lymphocyte maintenance have not been well clarified. Here, we have successfully generated a zip6-null mutant zebrafish line using TALENs. The Zip6-null mutant zebrafish developed normally during gastrulation. Loss of zip6 in zebrafish resulted in significant T lymphocyte reduction and a decrease in intracellular Zn levels. And the zip6 deficiency increases caspase-related cell apoptosis in both zebrafish cells and human T cells. Our results suggest that ZIP6 plays a critical part in T cell development, and enhance our understanding of Zn homeostasis and immune system maintenance.

PAX5 haploinsufficiency induce cancer cell dormancy in Raji cells.

PAX5 mutations have important role in leukemogenesis and leukemia relapse, cancer cell dormancy participates in cancer relapse, but there was no report about PAX5 mutation inducing cancer cell dormancy. we constructed the PAX5 deletion Raji cell lines using gene editing technology, evaluated dormancy biological characteristics of cell lines. Our results showed PAX5 haploinsufficiency restrained the proliferation of Raji cells, induced G0/G1 arrest of Raji cells, reduced chemotherapy sensitivity. The tumor formation rate reduced in PAX5 mutation Raji cells. Our results showed PAX5 insufficiency induced cancer cell dormancy in Raji cell.

Establishment of xenotransplantation model of human CN-AML with FLT3-ITD (mut) /NPM1 (-) in NOD/SCID mice.

Patients with FLT3-ITD (mut) /NPM1 (-) cytogenetically normal acute myeloid leukemia (CN-AML), as high-risk molecular group in CN-AML, are associated with a worse prognosis than other CN-AML patients. It is beneficial to generate xenotransplantation model of FLT3-ITD (mut) /NPM1 (-) CN-AML to better understand the pathogenesis and therapeutic strategies of such AML subtype. The purpose of present study was to establish the xenotransplantation model in NOD/SCID mice with FLT3-ITD (mut) /NPM1 (-) CN-AML primary cells. The FLT3-ITD (mut) /NPM1 (-) CN-AML primary cells from 3 of 7 cases were successfully transplanted into NOD/SCID mice, and human CD45 positive cells were detected in the peripheral blood, spleen and bone marrow of mice by using flow cytometry. Infiltration of human leukemia cells in various organs of mice was observed by using immunohistochemistry. Gene analysis confirmed sustained FLT3/ITD mutation without NPM1 mutation in mice. By performing serial transplantation, it was found that characteristics of the leukemia cells in secondary and tertiary generation models remained unchanged. Moreover, in vivo cytarabine administration could extend survival of NOD/SCID mice, which was consistent with clinical observation. In conclusion, we successfully established xenotransplantation model of human FLT3-ITD (mut) /NPM1 (-) CN-AML in NOD/SCID mice. The model was able to present primary disease and suitable to evaluate the curative effects of new drugs or therapy strategies.

Extracellular Vesicles Expressing CD19 Antigen Improve Expansion and Efficacy of CD19-Targeted CAR-T Cells.

Background: CAR-T cell therapy is effective in the treatment of certain hematological malignancies, and the expansion and functional persistence of CAR-T cells in vivo are crucial to clinical efficacy. The aim of this study was to investigate the potential of extracellular vesicles (EVs) modified with the CAR antigen to promote the efficacy of CAR-T cells in vivo. Methods: We generated HEK293T-derived EVs to present the CD19 antigen as the CAR target. In vitro, EVs expressing CD19 antigen (CD19 EVs) were co-incubated with anti-CD19 CAR-T cells. Then, proliferation, cytokine secretion, CD107a expression, tumor killing, subsets, and immune checkpoint expression were measured to assess CAR-T cell function. After infusion of CD19 EVs pretreated CAR-T cells into a lymphoma xenograft mouse model, flow cytometry and digital PCR were used to measure the expansion of CAR-T cells, and tumor volumes were continuously monitored to assess the anti-tumor efficacy of CAR-T cells in vivo. Another mouse model was created to investigate the effect of in vivo injection of CD19 EVs on the functional persistence of CAR-T cells, and safety was determined by histopathology of the main organs. Results: CD19 EVs activated CAR-T cells in an antigen-specific and dose-dependent manner and promoted the selective expansion and cytokine secretion of co-cultured CAR-T cells. Specifically, CD19 EVs preferably increased the expansion of the CAR-T subpopulation with a high surface CD19-CAR density and consequently enhanced the anti-tumor activity of CAR-T cells. Futhermore, CD19-EVs-primed CAR-T cells achieved superior proliferation and anti-tumor effects in a mouse model with lymphoma xenograft. In vivo administration of CD19 EVs promoted the functional persistence of CAR-T cells in the xenograft mouse model. Conclusion: Our findings indicate that antigen-expressing EVs can be utilized as a boost to improve CAR-T cell efficacy in vitro and in vivo.

Clinical characteristics and prognosis of 16 relapsed/refractory B-cell malignancy patients with CAR T-cell-related hyperferritinaemia.

With the success of chimeric antigen receptor-modified (CAR) T-cell therapy for relapsed/refractory (r/r) B-cell malignancies, severe complications after CAR T-cell infusion have emerged as nonnegligible prognosis-related factors. However, the prognosis of patients with CAR T-cell-related hyperferritinaemia (HFA) is unclear. We report the efficacy and safety of CAR T-cell therapy in 16 r/r B-cell malignancy patients with CAR T-cell-related HFA. The rates of serum ferritin levels above 10,000 ng/ml during CAR T-cell therapy were 6.2% and 14.3% in B-cell non-Hodgkin's lymphoma (B-NHL) and acute B lymphocyte leukemia (B-ALL), respectively. These patients were characterized by an extremely high tumor burden and a high rate of extranodal involvement. In lymphoma, the complete remission (CR) rate was 37.5% (3/8), which was lower than that in the control group with the lowest value of ferritin (CR was 87.5% (7/8), P=0.0406), and it could also be seen that the OS of the control group (1-year OS rate 100%) had a better trend than HFA group (1-year OS rate 50%). In the B-ALL patients, the OS of the control group (1-year OS rate 100%) was higher than HFA group (1-year OS rate 45%, P=0.0189), although there was no significant difference in CR rate. High-grade CRS (≥3) occurred in 56.25% of the patients, and the mortality rate was 56.25%, which was significantly higher than control group (12.5% and 12.5%, P=0.009). The peak serum ferritin level in the patients who died of CRS was significantly higher than others (P=0.0168). Regardless of whether the CAR T-related MAS diagnostic criteria were met, there was no significant difference in ORR and OS in HFA group, however patients with MAS showed a higher rate of high-grade CRS. Interestingly, in our study, glucocorticoid intervention in HFA group showed little impact on expansion of CAR-T cells, whether compared with control group or compared within HFA group by dividing patients into high and low dosage subgroups based on the median dose of glucocorticoid. High mortality was observed in patients with CAR T-cell-related HFA. Early glucocorticoid intervention might be worth trying to improve the safety of CAR T therapy in these patients.

Corrigendum to "Human MLL-AF9 Overexpression Induces Aberrant Hematopoietic Expansion in Zebrafish".

[This corrects the article DOI: 10.1155/2018/6705842.].

Clinical Characteristics of Hemophagocytic Lymphohistiocytosis Associated with Non-Hodgkin B-Cell Lymphoma: A Multicenter Retrospective Study.

BACKGROUND: Hemophagocytic lymphohistiocytosis (HLH) associated with B-cell lymphoma is a highly aggressive disease with unclear clinical features and has no standard treatment. PATIENTS AND METHODS: We analyzed the clinical characteristics of 31 patients from two individual centers. RESULTS: The median overall survival was only 1.5 months. Both univariate and multivariate analyses, based on lymphoma or HLH-related characteristics, revealed that patients with high Epstein-Barr virus (EBV) DNA load and ≥ 2 extranodal lesions, or hypofibrinogenemia, respectively, showed significantly poorer overall survival. Interestingly, some patients with high EBV DNA load had EBV-positive natural killer (NK) and/or T cells, which may be related to the coexistence of immunodeficiency and/or chronic active EBV infection. Molecular genetics examination confirmed that 47.4% (9/19) of patients had complex karyotypes, 37.5% (3/8) of patients had TP53 deletions, and 21.34% (3/14) of patients had TP53 mutation or alteration of malignancy-related pathways, including BCR/NF-κB, JAK-STAT, and epigenetic regulatory pathways, which may provide clues to choose targets for therapy. Treatment regimens containing etoposide, anti-CD20 monoclonal antibodies, or anthracyclines improved patient prognosis (P = .0183, .025, and .0436, respectively). Patients with infections had significantly shorter survival than those without infections (P = .00019). CONCLUSION: The patients' performance status, number of extranodal lesions, high EBV DNA load, and hypofibrinogenemia are poor prognostic factors for HLH associated with B-cell lymphoma. Molecular genetic high-risk factors are of particular importance because these factors can provide information for prognosis prediction, treatment decisions, and disease surveillance.

Case report: simultaneous occurrence of multiple myeloma and non-Hodgkin lymphoma treated by CAR T therapy.

RATIONALE: B cell lymphoma can co-occur with multiple myeloma (MM), and the prognosis in this case is usually poor. We propose the combination of CD19-chimeric antigen receptor (CAR) T cells and BCMA-CAR T cells for the treatment of such patients to obtain a superior prognosis. PATIENT CONCERNS: We present a 50-year-old patient with previous B cell lymphoma and subsequent multiple myeloma (MM). DIAGNOSIS: A diagnosis of B cell lymphoma and MM was made. INTERVENTIONS: The patient was treated with a combination of haploidentical CD19-chimeric antigen receptor (CAR) T cells and BCMA-CAR T cells. OUTCOMES: After CAR T cell therapy, the monoclonal plasma cells in the bone marrow and M protein disappeared. LESSONS: The combination therapy of CD19- and BCMA-CAR T cells is an effective measure to treat patients with concomitant or borderline cases of B cell lymphoma and MM.

CAR T-Cell Therapy Is Effective but Not Long-Lasting in B-Cell Lymphoma of the Brain.

Advanced central nervous system (CNS) lymphoma is an exclusion criterion for most chimeric antigen receptor (CAR) T-cell studies due to the associated levels of neurotoxicity. In this study, we described five patients with chemorefractory B-cell CNS lymphoma who received CAR19 and CAR22 T-cell "Cocktail" therapy and follow-up for 6-16 months. All patients experienced cytokine release syndrome (CRS). Two patients experienced CAR T-cell-related encephalopathy syndrome (CRES), which was controllable. The best response was observed in two patients, who successfully achieved complete remissions (CR), and the other three patients achieved partial remissions (PR). Four patients had progressive disease (PD) after remission. In addition, one CR patient and one PD patient accepted CAR T-cell infusion following hematopoietic stem cell transplantation therapy in the 3rd month and were in ongoing remission for 14 and 6 months of follow-up, respectively. The targeted antigens in two patients were still positive, and CAR T-cells were reboosted in the cerebrospinal fluid (CSF) after PD, but a small number of CD3-positive T-cells were observed to infiltrate into the tumor. Our study indicates the efficacy of CAR T-cell therapy for CNS lymphoma with an acceptable safety profile; however, the remission did not last long, perhaps due to the tumor immunosuppressive microenvironment (TME) of the CNS. CAR T-cell therapy should be combined with other treatments to help improve the TME of cerebral lymphoma.

Detection and Quantification of Chimeric Antigen Receptor Transgene Copy Number by Droplet Digital PCR versus Real-Time PCR.

Chimeric antigen receptor (CAR) T-cell immunotherapy is a new strategy for the treatment of refractory B-cell malignancies; therefore, the rapid and accurate quantification of CAR transgene copy number is essential. Real-time PCR was used for quantifying the copy number of chimeric antigen receptor transgene. Droplet digital PCR (ddPCR) is an absolute quantification method that does not require a standard curve. In this study, key performance parameters of the ddPCR and real-time PCR methods were assessed, including linearity, detection range, the lower limit of detection, repeatability, reproducibility, and accuracy, using a series of gradient diluted standards and clinical peripheral blood samples from CAR T-cell patients. The two platforms showed a good correlation for the standards (Pearson R2 = 0.9966; P < 0.0001) and clinical samples (Pearson R2 = 0.8952; P < 0.0001), and both showed good linearity (R2 = 0.9996 for ddPCR; R2 = 0.9984 for real-time PCR) over the detection range. Compared with real-time PCR, ddPCR showed lower intra-assay and interassay CVs for the series of diluted standards, which indicated ddPCR has better repeatability and reproducibility. The limit of detection of ddPCR was lower compared with that of real-time PCR. The combined results suggest that ddPCR is a more promising tool for the detection and quantification of the chimeric antigen receptor transgene copy number.

Combined inhibition of Notch and FLT3 produces synergistic cytotoxic effects in FLT3/ITD+ acute myeloid leukemia.

Internal tandem duplication (ITD) mutations of FMS-like tyrosine kinase-3 (FLT3) are the most frequent genetic alterations in acute myeloid leukemia (AML) and predict a poor prognosis. FLT3 tyrosine kinase inhibitors (TKIs) provide short-term clinical responses, but the long-term prognosis of FLT3/ITD+ AML patients remains poor. Notch signaling is important in numerous types of tumors. However, the role of Notch signaling in FLT3/ITD+ AML remains to be elucidated. In the current study, we found that Notch signaling was activated upon FLT3-TKI treatment in FLT3/ITD+ cell lines and primary cells. As Notch signaling can be blocked by γ-secretase inhibitors (GSIs), we examined the combinatorial antitumor efficacy of FLT3-TKIs and GSIs against FLT3/ITD+ AML and explored the underlying molecular mechanisms. As a result, we observed synergistic cytotoxic effects, and the treatment preferentially reduced cell proliferation and induced apoptosis in FLT3/ITD+ AML cell lines and in primary AML cells. Furthermore, the combination of FLT3-TKI and GSI eradicated leukemic cells and prolonged survival in an FLT3/ITD+ patient-derived xenograft AML model. Mechanistically, differential expression analysis suggested that CXCR3 may be partially responsible for the observed synergy, possibly through ERK signaling. Our findings suggest that combined therapies of FLT3-TKIs with GSI may be exploited as a potential therapeutic strategy to treat FLT3/ITD+ AML.

A good response of refractory mantel cell lymphoma to haploidentical CAR T cell therapy after failure of autologous CAR T cell therapy.

BACKGROUND: The aggressive form of Mantle cell non-hodgkin B cell lymphoma (MCL) has a dismal prognosis. Dual targeting BTK and BCL2 with ibrutinib and venetoclax has improved outcomes in MCL patients who were predicted not to respond to conventional therapy, but it is unlikely to be curative. Chimeric antigen receptor-modified T (CAR T) cells exhibit very effective function in elimination of relapsed/refractory B-cell lymphoid malignancies, we investigated their use in a patient with relapsed MCL. CASE PRESENTATION: Here, we report a case of a refractory MCL in a patient who had relapsed after conventional chemotherapy and autologous CAR T cell therapy. The patient received multiple molecularly targeted therapies, including targeting BTK and BCL2, and haplo-identical CAR T (haplo-CAR T) cells from her daughter without previous allo-hematopoietic stem cell transplantation. Haplo-CAR T cells could effectively proliferate in vivo and had a clinically significant antitumor activity without serious side effects. The patient achieved a partial remission, with minimal residual disease. CONCLUSIONS: This case suggests that haplo-CAR T cell therapy can be effective in controlling lymphoma that failed to respond to autologous CAR T cell therapy and overcome limitation of autologous CAR T cells, thus may be one possible regimen before the era of off-the-shelf "universal" CAR T cell therapy. TRIAL REGISTRATION: ChiCTR-OPN-16008526. http://www.chictr.org.cn/showproj.aspx?proj=13798 ; ChiCTR1800019385. http://www.chictr.org.cn/showproj.aspx?proj=32805 ; ChiCTR1800019449. http://www.chictr.org.cn/showproj.aspx?proj=32778 .

Human MLL-AF9 Overexpression Induces Aberrant Hematopoietic Expansion in Zebrafish.

The 11q23 of the mixed lineage leukemia 1 (MLL1) gene plays a crucial role in early embryonic development and hematopoiesis. The MLL-AF9 fusion gene, resulting from chromosomal translocation, often leads to acute myeloid leukemia with poor prognosis. Here, we generated a zebrafish model expressing the human MLL-AF9 fusion gene. Microinjection of human MLL-AF9 mRNA into zebrafish embryos resulted in enhanced hematopoiesis and the activation of downstream genes such as meis1 and hox cluster genes. Embryonic MLL-AF9 expression upregulated HSPC and myeloid lineage markers. Doxorubicin and MI-2 (a menin inhibitor) treatments significantly restored normal hematopoiesis in MLL-AF9-expressing animals. This study provides insight into the role of MLL-AF9 in zebrafish hematopoiesis and establishes a robust and efficient in vivo model for high-throughput drug screening.

Identification of Significant Pathways Induced by PAX5 Haploinsufficiency Based on Protein-Protein Interaction Networks and Cluster Analysis in Raji Cell Line.

PAX5 encodes a transcription factor essential for B-cell differentiation, and PAX5 haploinsufficiency is involved in tumorigenesis. There were few studies on how PAX5 haploinsufficiency regulated genes expression to promote tumorigenesis. In this study, we constructed the cell model of PAX5 haploinsufficiency using gene editing technology in Raji cells, detected differentially expressed genes in PAX5 haploinsufficiency Raji cells, and used protein-protein interaction networks and cluster analysis to comprehensively investigate the cellular pathways involved in PAX5 haploinsufficiency. The clusters of gene transcription, inflammatory and immune response, and cancer pathways were identified as three important pathways associated with PAX5 haploinsufficiency in Raji cells. These changes hinted that the mechanism of PAX5 haploinsufficiency promoting tumorigenesis may be related to genomic instability, immune tolerance, and tumor pathways.