Pralsetinib-associated pneumonia in RET fusion-positive non-small cell lung cancer.

OBJECTIVE: Oncogenic alternation in RET is one of the important targets of non-small cell lung cancer (NSCLC). Pralsetinib has shown great efficacy in RET fusion-positive NSCLC, but a series of adverse reactions will inevitably occur in the meantime. We aimed to explore the clinical characteristics of patients with pneumonia and recognition it in early stage, so patients could longer benefit from pralsetinib. METHODS: This is a multicenter, retrospective study. RET fusion-positive advanced NSCLC patients who developed pneumonia during pralsetinib treatment from January 2020 to December 2022 were included. Clinical data, time to onset of pneumonia, methods of pneumonia diagnosis, treatment with pneumonia, prognosis of pneumonia, and the effect of pneumonia on the efficacy of pralsetinib. RESULTS: A total of 8 patients with pneumonia were included in the study, most of which were non-smoking female patients and the main fusion gene was KIF5B (87.5%), which was consistent with the general characteristics of RET fusion population. The median occurrence time of pralsetinib-associated pneumonia was 2.15 (range 1.1-6.63) months. All patients were infected by opportunistic pathogens, and the most common pathogen was human herpesviruses and pneumospora yerbii. Fever was always the first symptom, and timely anti-infective treatment including antibiotics, antiviral drugs, and antifungal drugs was effective. Until February 28, 2023, the median follow-up time was 18.7 months, the mean PFS of patients was 17.4 months, and the median PFS was not reached. Fortunately, patients who restarted pralsetinib after infection control continued to benefit. CONCLUSIONS: Opportunistic infection may be a unique adverse effect of pralsetinib. During the treatment of pralsetinib, we should be vigilant about the occurrence of pneumonia and achieve early recognition and timely treatment.

GdX/UBL4A-knockout mice resist collagen-induced arthritis by balancing the population of Th1/Th17 and regulatory T cells.

Rheumatoid arthritis (RA) is an autoimmune disease associated with synovial hyperplasia and bone and cartilage destruction. T cells, notably T helper (Th)-1 and Th17 cells, play a critical role in the pathologic process of RA. However, it remains unclear how Th1 and Th17 cells are regulated during RA. In this study, we report that the small ubiquitin-like protein X-linked gene in the G6PD cluster at Xq28 (GdX) regulates the balance of Th17 and regulatory T (Treg) cells during collagen-induced arthritis (CIA). We discovered that the splenocytes of GdX-knockout (KO) mice were insensitive to T-cell stimulants. Correspondingly, GdX-KO mice showed alleviative Th1-mediated delayed-type hypersensitivity and were resistant to CIA compared with wild-type mice. GdX-KO mice showed fewer swollen paws, lower serum proinflammatory cytokine and anti-collagen IgG levels, and decreased synovial hyperplasia. Mechanistically, we observed that deletion of GdX decreased the transcription of proinflammatory cytokines and impaired the Th1 and Th17 differentiation but increased the Treg cell proliferation. Consistently, deletion of GdX decreased the transcription level of T-cell-specific T-box transcription factor and RAR-related orphan receptor-γ transcription factor but increased that of forkhead box P3 after being challenged with type-II collagen. These findings suggested that GdX functions as an important regulator of Th1 or Th17 and Treg cell balance during the inflammatory responses. Therefore, GdX may be a potential target for the therapy of RA.-Fu, Y., Liu, S., Wang, Y., Ren, F., Fan, X., Liang, J., Liu, C., Li, J., Ju, Y., Chang, Z. GdX/UBL4A-knockout mice resist collagen-induced arthritis by balancing the population of Th1/Th17 and regulatory T cells.

Insulin receptor substrate 1/2 (IRS1/2) regulates Wnt/ß-catenin signaling through blocking autophagic degradation of dishevelled2.

Wnt signaling plays a pivotal role in cell proliferation, tissue homeostasis, and tumorigenesis. Dishevelled (Dvl) is a central node of Wnt signaling. Insulin receptor substrates (IRSs), as a critical component of insulin signaling, are involved in cell proliferation, metabolism, and cancer development. In this study, we report that IRS1/2 promotes Wnt/ß-catenin signaling by stabilizing Dvl2. We found that IRS1/2 interacts with Dvl2. Overexpression of IRS1/2 increased the protein level of Dvl2 and promoted canonical Wnt signaling, as evidenced by the increased T cell-specific factor 4 transcriptional activity and the up-regulation of expression of CYCLIN D1 and c-MYC, two Wnt target genes critical for cell growth, whereas depletion of IRS1/2 reduced the level of Dvl2 and attenuated Wnt/ß-catenin signaling. Biochemical analyses revealed that IRS1/2 decreased Lys-63-linked ubiquitination of Dvl2 and stabilized Dvl2 protein via suppressing its autophagy-mediated degradation. We further revealed that IRS1/2 blocks autophagy-induced formation of the Dvl2-p62/SQSTM1 complex, resulting in disabled association of Dvl2 to autophagosomes. We demonstrated that IRS1/2 promoted the induction of epithelial-mesenchymal transition (EMT) and cell proliferation in response to Wnt stimulation, whereas depletion of Dvl2 impaired the IRS1/2-mediated EMT and cell growth. Our findings revealed that IRS1/2 promotes EMT and cell proliferation through stabilizing Dvl2.

Programmed Cell Death Protein 1 Inhibitor-Mediated Peripheral Neuropathy.

The discovery of immune checkpoint inhibitors (ICIs) has revolutionized the model of antitumor therapy. With the continuous deepening of the research on the mechanism of immunotherapy, ICIs, such as programmed cell death protein 1 (PD-1), programmed death-ligand 1 inhibitors and cytotoxic T lymphocyte-associated protein 4 inhibitors, have been widely used in a variety of tumors. Nevertheless, the use of ICI can also lead to a series of immune-related adverse events. Common immune-related adverse events include gastrointestinal toxicity, pulmonary toxicity, endocrine system toxicity, and skin toxicity. Neurologic adverse events are relatively rare, but they seriously affect the quality of life and shorten the survival time of patients. This article reports cases of peripheral neuropathy mediated by PD-1 inhibitors and retrieves the relevant literatures at home and abroad to summarize the neurotoxicity caused by PD-1 inhibitors, so as to strengthen the awareness of clinicians and patients on neurologic adverse reactions and mitigate potential adverse effects of implemented therapies.

Protein tyrosine phosphatase Meg2 dephosphorylates signal transducer and activator of transcription 3 and suppresses tumor growth in breast cancer.

INTRODUCTION: Signal transducer and activator of transcription 3 (STAT3) is over-activated or phosphorylated in breast cancers. The hyper-phosphorylation of STAT3 was attributed to either up-regulated phosphorylation by several tyrosine-kinases or down-regulated activity of phosphatases. Although several factors have been identified to phosphorylate STAT3, it remains unclear how STAT3 is dephosphorylated by PTPMeg2. The aim of this study was to determine the role of PTPMeg2 as a phosphatase in regulation of the activity of STAT3 in breast cancers. METHODS: Immunoprecipitation assays were used to study the interaction of STAT3 with PTPMeg2. A series of biochemistry experiments were performed to evaluate the role of PTPMeg2 in the dephosphorylation of STAT3. Two breast cancer cell lines MCF7 (PTPMeg2 was depleted as it was endogenously high) and MDA-MB-231 (PTPMeg2 was overexpressed as it was endogenously low) were used to compare the level of phosphorylated STAT3 and the tumor growth ability in vitro and in vivo. Samples from breast carcinoma (n = 73) were subjected to a pair-wise Pearson correlation analysis for the correlation of levels of PTPMeg2 and phosphorylated STAT3. RESULTS: PTPMeg2 directly interacts with STAT3 and mediates its dephosphorylation in the cytoplasm. Over-expression of PTPMeg2 decreased tyrosine phosphorylation of STAT3 while depletion of PTPMeg2 increased its phosphorylation. The decreased tyrosine phosphorylation of STAT3 is coupled with suppression of STAT3 transcriptional activity and reduced tumor growth in vitro and in vivo. Levels of PTPMeg2 and phosphorylated STAT3 were inversely correlated in breast cancer tissues (P = 0.004). CONCLUSIONS: PTPMeg2 is an important phosphatase for the dephosphorylation of STAT3 and plays a critical role in breast cancer development.

Mutation of the Nrf2 gene in non-small cell lung cancer.

Nrf2 (NFE2L2) is a transcription factor belonging to the Cap'N'Collar subfamily of basic-leucine zipper (bZIP) family of transcription factors, which plays a significant role in adaptive responses to oxidative stress. To investigate the relationship of between the mutation of Nrf2 gene and non-small cell lung cancer (NSCLC), in this study, we sequenced the Nrf2 gene from a total of 103 patients with NSCLC and corresponding blood samples. It is found that there is a discordance of Nrf2 mutations between NSCLC and corresponding blood samples. These differences may indicate that the variants in the Nrf2 gene are associated with an increased risk for lung cancer. In addition, the factor of smoking status is observed to play important roles in triggering the occurrence of the disorder.

Prolonged overall survival in metastatic gastric cancer treated with ipilimumab and lapatinib.

OBJECTIVE: Gastric cancer is one of the most important common tumors in the world. It remains the second leading cause of cancer-related death worldwide. The prognosis of patients with unresectable or metastatic gastric cancer remains poor. New targeted drugs were evaluated in clinic, such as lapatinib. The potential for making beneficial progress is to investigate innovative therapeutic strategies, such as immunotherapy. METHODS: We used combination therapy including immunotherapy for treating gastric cancer. We treated a patient of metastatic gastric cancer for which lapatinib plus ipilimumab was effective after progression on trastuzumab. RESULTS: The patient has prolonged overall survival after treatment. Positron emission tomography-computed tomography examination showed that some of the cervical lymph nodes and retroperitoneal lymph nodes reduced and glucose metabolism decreased. In blood examination, the tumor marker (carcinoembryonic antigen) decreased. The patient has been lived for 4 years after multiple lymph node metastasis. CONCLUSION: Targeted therapy combined with immunotherapy might be a useful option for metastatic gastric.

CREPT/RPRD1B associates with Aurora B to regulate Cyclin B1 expression for accelerating the G2/M transition in gastric cancer.

Gastric cancer, like most of other cancers, has an uncontrolled cell cycle regulated by cyclins and cyclin-dependent kinases (CDKs). In this study, we reported that gastric cancer cells showed an accelerated G2/M transition promoted by CREPT/RPRD1B and Aurora kinase B (Aurora B). We found that CREPT/RPRD1B and Aurora B were coordinately expressed during the cell cycle in gastric cancer cells. Deletion of CREPT/RPRD1B disturbed the cell progression and extended the length of cell cycle, leading to a significant accumulation of mitotic cells. Mechanistically, we revealed that CREPT/RPRD1B interacted with Aurora B to regulate the expression of Cyclin B1 in gastric cancer cells. Interestingly, Aurora B phosphorylates S145 in a well-conserved motif of CREPT/RPRD1B. We proposed that phosphorylation of CREPT/RPRD1B by Aurora B is required for promoting the transcription of Cyclin B1, which is critical for the regulation of gastric tumorigenesis. Our study provides a mechanism by which gastric tumor cells maintain their high proliferation rate via coordination of Aurora B and CREPT/RPRD1B on the expression of Cyclin B1. Targeting the interaction of Aurora B and CREPT/RPRD1B might be a strategy for anti-gastric cancer therapy in the future.

Nimotuzumab treatment and outcome analysis in patients with leptomeningeal metastasis from nonsmall cell lung cancer.

OBJECTIVE: Leptomeningeal metastasis (LM) leades a devastating consequence in patients with nonsmall cell lung cancer(NSCLC). Treatment is very limited for patients with LM. We introduced to use nimotuzumab (also known as h-R3) combined with methotrexate for treating LM in NSCLC patients. PATIENTS AND METHODS: In the present report, twenty patients with LM of NSCLC were included, and the clinicopathology information and outcomes after treatment were analyzed. RESULTS: All the twenty patients with LM were lung adenocarcinoma. Thirteen patients had poor Eastern Cooperative Oncology Group performance status (≥ 3) before treatment, fifteen patients received combined administration of h-R3 and methotrexate, and another five patients received h-R3 treatment alone. The median survival time after the diagnosis of LM was 5 months (range, 2.4-7.6 months) for these twenty patients. The mean cerebrospinal fluid opening pressure was 270mmH2O before treatment and decreased significantly after treatment (140 mmH2O) (P < 0.001). Associated symptoms were relieved quickly after one or two cycles of intrathecal therapy. CONCLUSION: These findings indicated that nimotuzumab might be a potential drug for treatment of LM in NSCLC patients.

Prolonged overall survival of patients with leptomeningeal carcinomatosis from nonsmall cell lung cancer.

Leptomeningeal metastasis (LM) carries a devastating prognosis. Treatment selection is limited for patients with LM. We introduced to use nimotuzumab (also known as h-R3) for treating LM of nonsmall cell lung cancer. Here, we report two patients in our treatment who had prolonged overall survival over 1 year each. The pressure of cerebrospinal fluid of the patients decreased remarkably after intrathecal therapy. Symptoms of the patients had been improved quickly after one or two times of intrathecal therapy. Nimotuzumab was well tolerated used in intrathecal therapy.

Clinicopathological features and prognosis of pseudomyxoma peritonei.

The aim of this study was to evaluate the effects of treatment and the factors influencing the postoperative recurrence and survival time for pseudomyxoma peritonei (PMP). A total of 39 patients with PMP who received treatment were analyzed in The General Hospital of PLA (Beijing, China) between 2002 and 2011. The patients received cytoreductive surgery (CRS) and 25 cases of PMP recurred. Seven patients received postoperative hyperthermic intraperitoneal chemoperfusion (HIPEC). The median follow-up was 40 months. There were eight mortalities in this period. The 5- and 10-year survival rates were 89.0 and 35.0%, respectively. The medians of overall survival (OS) and recurrence time were 37 and 4 months, respectively. Multivariate analyses revealed that pathological subtype was able to influence the recurrence (P=0.042) and OS (P=0.033) times, as an independent prognostic factor. HIPEC was significantly associated with postoperative recurrence time (P=0.017). Patients with disseminated peritoneal adenomucinosis had a more favorable prognosis. CRS combined with HIPEC was able to extend the postoperative recurrence time for patients with PMP.

[Expression and clinical significance of CD3, CD4 and COX-2 in non-small cell lung cancer].

AIM: To study expression and clinical significance of CD3, CD4 and COX-2 in non-small cell lung cancer (NSCLC). METHODS: Expression of COX-2, CD3 and CD4 was detected by immunohistochemical staining in 37 cases of NSCLC. The correlation of CD3, CD4, COX-2 expressions and overall survival(OS) was evaluated with spearman rank correlation analysis. RESULTS: The average surface density of CD3 and the average optical density of CD4 were significantly associated with OS of NSCLC patients after surgery (P<0.05). However, both two densities of COX-2 were correlated with survival time of patients (P>0.05). CONCLUSION: The expressions of CD3, CD4 were significantly associated with OS of NSCLC patients.

Antigenically dominant proteins within the human liver mitochondrial proteome identified by monoclonal antibodies.

Analysis of the mitochondrial proteome would provide valuable insight into the function of this important organelle, which plays key roles in energy metabolism, apoptosis, free radical production, thermogenesis, and calcium signaling. It could also increase our understanding about the mechanisms that promote mitochondrial disease. To identify proteins that are antigenically dominant in human liver mitochondria, we generated >240 hybridoma cell lines from native mitochondrial proteins after cell fusion, screening, and cloning. Antibodies that recognized mitochondrial proteins were identified by screening human liver cDNA expression libraries. In this study, we identified 6 major antigens that were recognized by at least 2 different monoclonal antibodies (mAbs). The proteins that were antigenically dominant were: acetyl-Coenzyme A acyltransferase 2 (mitochondrial 3-oxoacyl-Coenzyme A thiolase), aldehyde dehydrogenase 1 family member A1, carbamoyl phosphate synthetase 1, dihydrolipoamide S-acetyltransferase (E2 component of pyruvate dehydrogenase complex), enoyl coenzyme A hydratase 1, and hydroxysteroid (11-beta) dehydrogenase 1. We also determined the subcellular localizations of these enzymes within the mitochondria using immunohistocytochemistry. We believe that these well-characterized antibodies will provide a valuable resource for the Human Liver Proteome Project (HLPP), and will make studies aimed at investigating liver mitochondrial function far easier to perform in future. Our results provide strong evidence that, (i) depletion of dominant proteins from liver mitochondrial samples is possible and, (ii) the approaches adopted in this study can be used to explore or validate protein-protein interactions in this important organelle.

[Preparation and identification of monoclonal antibody against enoyl-CoA hydratase 1].

OBJECTIVE: To prepare monoclonal antibodies (mAbs) against enoyl-CoA hydratase 1 (ECH1). METHODS: Normal human liver tissues were homogenized, and the mitochondria were isolated by differential centrifugation. The total mitochondrial proteins were used to immunize BALB/c mice to prepare mAbs by routine hybridoma technique. The mAbs were characterized by ELISA, Western blotting and immunohistochemistry. The specificity of the antibody was identified by mass spectrometry (MS) following immunoprecipitation (IP) and confirmed by Uni-ZAP expression library screening. RESULTS: One clone of the hybridoma BGB095 secreting specific mAb against ECH1 was obtained. The mAb was identified to belong to Ig subclass IgG1 and could be used in ELISA, Western blotting, immunohistochemistry, and immunoprecipitation. CONCLUSION: A hybridoma cell line stably secreting specific mAb against ECH1 has been established. The specific mAb against ECH1 can be of great value for functional and distribution studies of ECH1.

[Preparation, characterization and application of monoclonal antibody against CPS1].

AIM: To prepare and characterize the monoclonal antibody (mAb) against human carbamyl phosphate synthetase I (CPSI) and make a study of its application. METHODS: Normal human liver tissues were homogenized, and their mitochondria were isolated by differential centrifugation. The total mitochondrial proteins were used to immunize BALB/c mice to prepare mAb using the routine hybridoma technique. The mAb was detected by ELISA, Western blot immunohistochemistry and immunofluorecent staining. The specificity of mAb was identified by mass spectrometry (MS) and immunoprecipitation (IP) and then confirmed by Uni-ZAP expression library screening. The antibody was used to isolate potential enzymatic complexes by immunocapturing. RESULTS: Three hybridoma cell lines BEH045, ACB271 and BFG021 secreting specific mAb against CPS1 were obtained. The Ig subclass of the mAb was IgG(1), which was used in ELISA, Western blot immunohistochemistry, immunoprecipitation, immunofluorecent staining and the isolation of potential enzymatic complexes. CONCLUSION: A hybridoma cell line which can secre specific mAb against CPSI stably has been established. The specific mAb against CPSI is of value to the research into the functions and distribution of CPSI.

[Preparation and identification of monoclonal antibody against UGP2].

The study was aimed to generate monoclonal antibodies (mAbs) against homo sapiens UDP-glucose pyrophosphorylase 2 (UGP2). Normal human liver tissues homogenized, and cytosolic proteins isolated by centrifugation were used to immunize BALB/c mice to generate mAbs by hybridoma technique. The mAbs were identified by ELISA, Western blot, and immunohistochemistry assay. The antibody specificity was confirmed by Uni-ZAP expression library screening. The results indicated that one hybridoma BAD062 secreting specific mAb against UGP2 was established. The Ig subclass of this mAb was IgG(2b) (kappa), and it could be used in ELISA, Western blot, immunohistochemistry assay. The antigen recognized by BAD062 mAb was localized in the hepatocyte cytoplasm, with molecular weight of 56 kD in the cytosolic proteins of human liver tissue. The BAD062 mAb was further confirmed by immunoscreening of Uni-ZAP XR liver cDNA expression library. It is concluded that a hybridoma cell line stably secretes specific mAb against UGP2. This mAb reacted with UGP2 in ELISA, Western blot, immunohistochemistry assay, and would be very useful for the UGP2 studies.

[Preparation and characterization of monoclonal antibody against DCXR].

AIM: To prepare monoclonal antibodies (mAbs) against Dicarbonyl/L-xylulose reductase (DCXR). METHODS: Normal human liver tissues were homogenized, and mitochondria were isolated by differential centrifugation. The total mitochondrial proteins were used to immunize BALB/c mice to prepare mAbs by routine hybridoma technique. The mAbs were detected by ELISA, Western blot and immunohistochemistry. The antibody specificities were identified by mass spectrometry (MS) following immunoprecipitation (IP) and confirmed by Uni-ZAP expression library screening. RESULTS: One clone of hybridoma AFF091 secreting specific mAb against DCXR was obtained. The Ig subclass of the mAb was IgG1 and it can be used in ELISA, Western blot, immunohistochemistry, and immunoprecipitation. CONCLUSION: A hybridoma cell line stably secreting specific mAb against DCXR was established. The specific mAb against DCXR would be very useful for investigation of DCXR functions and distribution.

Proteomics-based generation and characterization of monoclonal antibodies against human liver mitochondrial proteins.

Monoclonal antibodies (mAbs) have the potential to be a very powerful tool in proteomics research to determine protein expression, quantification, localization and modification, as well as protein-protein interactions, especially when combined with microarray technology. Thus, a large amount of well-characterized and highly qualified antibodies are needed in proteomics. Purified antigen, which is not always available, has proven to be one of the rate-limiting steps in mAb large-scale generation. Here we describe our strategies to establish a murine hybridoma cell bank for human liver mitochondria using unknown native proteins as the immunogens. The antibody-recognized mitochondrial proteins were identified by MS following immunoprecipitation (IP), and by screening of human liver cDNA expression library. We found that the established antibodies reacted specifically with a number of important enzymes in mitochondria. The subcellular localization of these antigens in mitochondria was further confirmed by immunohistocytochemistry. A panel of antibodies was also tested for their ability to capture and deplete the targeting proteins and complexes from the total mitochondrial proteins. We believe these well-characterized antibodies would be useful in various applications for Human Liver Proteome Project (HLPP) when the scale of this hybridoma cell bank is enlarged significantly in the near future.