Proteomic profiling of FFPE specimens: Discovery of HNRNPA2/B1 and STT3B as biomarkers for determining formalin fixation durations.

Recent advancements in proteomics technologies using formalin-fixed paraffin-embedded (FFPE) samples have significantly advanced biomarker discovery. Yet, the effects of varying sample preparation protocols on proteomic analyses remain poorly understood. We analyzed mouse liver FFPE samples that varied in fixatives, fixation duration, and storage temperature using LC/MS. We found that variations in fixation duration significantly affected the abundance of specific proteins, showing that HNRNPA2/B1 demonstrated a significant decrease in abundance in samples fixed for long periods, whereas STT3B exhibited a significant increase in abundance in samples fixed for long durations. These findings were supported by immunohistochemical analysis across liver, spleen, and lung tissues, demonstrating a significant decrease in the nuclear staining of HNRNPA2/B1 in long-duration acid formalin(AF)-fixed FFPE samples, and an increase in cytoplasmic staining of STT3B in long-duration neutral buffered formalin-fixed liver and lung tissues and granular staining in all long-duration AF-fixed FFPE tissue types. Similar trends were observed in the long-duration fixed HeLa cells. These results demonstrate that fixation duration critically affects the proteomic integrity of FFPE samples, emphasizing the urgent need for standardized fixation protocols to ensure consistent and reliable proteomic data. SIGNIFICANCE: The quality of FFPE samples is primarily influenced by the fixation and storage conditions. However, previous studies have mainly focused on their impact on nucleic acids and the extent to which different fixation conditions affect changes in proteins has not been evaluated. In addition, to our knowledge, proteomic research focusing on differences in formalin fixation conditions has not yet been conducted. Here, we analyzed FFPE samples with different formalin fixation and storage conditions using LC/MS and evaluated the impact of different fixation conditions on protein variations. Our study unequivocally established formalin fixation duration as a critical determinant of protein variation in FFPE specimens and successfully identified HNRNPA2/B1 and STT3B as potential biomarkers for predicting formalin fixation duration for the first time. The study findings open new avenues for quality assessment in biomedical research and diagnostics.

Predictive Model for Occurrence of Febrile Neutropenia after Chemotherapy in Patients with Diffuse Large B-Cell Lymphoma: A Multicenter, Retrospective, Observational Study.

Febrile neutropenia (FN) is a major concern in patients undergoing chemotherapy for diffuse large B-cell lymphoma (DLBCL); however, the overall risk of FN is difficult to assess. This study aimed to develop a model for predicting the occurrence of FN in patients with DLBCL. In this multicenter, retrospective, observational analysis, a multivariate logistic regression model was used to analyze the association between FN incidence and pretreatment clinical factors. We included adult inpatients and outpatients (aged ≥ 18 years) diagnosed with DLBCL who were treated with chemotherapy. The study examined 246 patients. Considering FN occurring during the first cycle of chemotherapy as the primary outcome, a predictive model with a total score of 5 points was constructed as follows: 1 point each for a positive hepatitis panel, extranodal involvement, and a high level of soluble interleukin-2 receptor and 2 points for lymphopenia. The area under the receiver operating characteristic curve of this model was 0.844 (95% confidence interval: 0.777-0.911). Our predictive model can assess the risk of FN before patients with DLBCL start chemotherapy, leading to better outcomes.

Etiology of Severe Acute Hepatitis in a Pediatric Transplant Center in Japan.

The proportion of pediatric cases with severe acute hepatitis of unknown etiology in the coronavirus disease 2019 era was higher than that in the pre-coronavirus disease 2019 era in Japan's largest pediatric transplant center. Further research and monitoring are essential.

Comprehensive genetic analysis of histological components of combined small cell carcinoma.

BACKGROUND: Combined small-cell lung cancer (cSCLC) is a rare type of small-cell lung cancer (SCLC) that includes both SCLC and non-small-cell lung cancer (NSCLC). The molecular biological mechanisms underlying the heterogeneity of histological types in combined or metachronously transformed SCLC (mtSCLC) remain unclear. This study aimed to investigate the relationship between genetic alterations and each histological component heterogeneously detected in cSCLC and mtSCLC. METHODS: This study included four cSCLC cases and one mtSCLC case. Formalin-fixed and paraffin-embedded sections of each histological component of these tumors were subjected to next-generation sequencing (NGS) and quantitative reverse transcription-polymerase chain reaction to investigate the genetic mutations and expression levels of neuroendocrine cell-specific transcription factors (achaete-scute homolog-1 [ASCL1], brain-2 [BRN2] also known as POU domain class 3 transcription factor 2, nuclear factor 1 B [NF1B], insulinoma-associated protein 1 [INSM1], and thyroid transcription factor-1 [TTF-1]). RESULTS: NGS analysis revealed that SCLC and NSCLC components share the same somatic mutations detected most frequently in TP53, and also in RB1 and EGFR. Gene expression analysis showed ASCL1 expression was significantly lower in the NSCLC component than in the SCLC component. CONCLUSION: We conclude that the morphological evolution of heterogeneous histological components in cSCLC may be associated with differences in ASCL1 expression levels, but not in acquired somatic gene mutations.

Outcomes of liver transplantation for mitochondrial respiratory chain disorder in children.

AIM: Mitochondrial respiratory chain disorder (MRCD) can cause acute liver failure (ALF), which may necessitate liver transplantation (LT). However, MRCD is often difficult to diagnose before LT and the indications of LT are controversial due to the likelihood of progressive neurological disease. The present study further characterized the patient population and described the outcomes. METHODS: Thirteen patients who underwent LT for MRCD from November 2005 to May 2020 were enrolled in this study. RESULTS: Six of 13 MRCD patients were diagnosed with a mitochondrial inner membrane protein 17-related mitochondrial DNA depletion syndrome (MTDPS). Overall, nine survived with a median follow-up of 1.8 years (IQR, 1.3-5.1 years); four died within 2 years. In the long-term, seven survivors showed no progression of hypotonia after LT and attended a normal kindergarten or primary school. Neurological abnormalities were observed in two survivors, including vison loss related to Leber's hereditary optic neuropathy in one patient and psychomotor retardation related to Leigh syndrome in the other. Three non-survivors after LT were diagnosed with MTDPS and died of severe pulmonary hypertension, which had developed at 8, 9, and 18 months after LT (n=1 each). The remaining patient died of postoperative respiratory infection with respiratory syncytial virus. CONCLUSION: The long-term results support the performance of LT in patients with MRCD, although a genetic diagnosis is preferable for determining the accurate indications for LT in these patients. Furthermore, care should be taken to avoid complications due to mitochondrial dysfunction during the long-term follow-up.

Versatile Mitogenic and Differentiation-Inducible Layer Formation by Underwater Adhesive Polypeptides.

Artificial materials have no biological functions, but they are important for medical devices such as artificial organs and matrices for regenerative medicine. In this study, mitogenic and differentiation-inducible materials are devised via the simple coating of polypeptides, which contain the sequence of epidermal growth factor or insulin-like growth factor with a key amino acid (3,4-dihydroxyphenylalanine) of underwater adhesive proteins. The adhesive polypeptides prepared via solid-phase synthesis form layers on various substrates involving organic and inorganic materials to provide biological surfaces. Through the direct activation of cognate receptors on interactive surfaces, the materials enable increased cell growth and differentiation compared to that achieved by soluble growth factors. This superior growth and differentiation are attributed to the long-lasting signal transduction (triggered by the bound growth factors), which do not cause receptor internalization and subsequent downregulation.

High serum free IL-18 is associated with decreased omalizumab efficacy: findings from a 2-year omalizumab treatment study.

OBJECTIVE: Omalizumab is more effective in severe allergic patients with eosinophilic asthma than those with non-eosinophilic asthma. IL-18, a unique cytokine involved in allergic but non-eosinophilic inflammation, might be associated with the latter condition. We aimed to clarify the roles of IL-18 related pathways in insufficient response to omalizumab treatment. METHODS: Patients with severe allergic asthma who completed 2-year omalizumab treatments at Kyoto University Hospital were included in this study (UMIN000002389). Associations between pretreatment levels of serum free IL-18 in addition to other mediators and asthma phenotypes including responses to omalizumab treatment were analyzed. Changes in serum free IL-18, periostin and total IgE levels during the treatment were also examined. RESULTS: Twenty-seven patients (19 females, average age of 55.7 years) were examined. Fifteen incomplete responders who experienced exacerbations in the second year, were significantly and more frequently obese and showed significantly earlier asthma onset, lower blood eosinophils and more exacerbations before omalizumab treatment than complete responders. Significantly more patients showed high baseline serum free IL-18 levels (≥141 pg/mL, a threshold for the highest tertile) among the incomplete responders than complete responders. Patients with high serum free IL-18 levels shared similar characteristics with incomplete responders, showing significant reductions in serum total IgE levels during omalizumab treatment. Finally, serum free IL-18 levels negatively correlated with serum periostin levels at baseline and in change ratios. CONCLUSIONS: High baseline serum free IL-18 levels may predict reduced omalizumab efficacy in severe allergic patients with type-2 low asthma, regarding reduction of exacerbations.

Prospective study of live attenuated vaccines for patients receiving immunosuppressive agents.

Patients receiving immunosuppressive agents are at risk of life-threatening infections. However, live vaccines are generally contraindicated in them. We conducted a prospective study regarding live attenuated vaccines for them. Patients elder than one year of age with immunosuppressive agents who showed negative or borderline antibody titers (virus-specific IgG levels < 4.0) against one or more of measles, rubella, varicella, and mumps and fulfilled the criteria (CD4 cell counts ≥ 500/mm3, stimulation index of lymphocyte blast transformation by PHA ≥ 101.6, serum IgG level ≥ 300 mg/dl, no steroid use or prednisolone < 1 mg/kg/day or < 2 mg/kg/2 days, trough levels of tacrolimus or cyclosporine were < 10 ng/ml or < 100 ng/ml and under good control of primary disease) were enrolled. Sixty-four vaccinations were administered to 32 patients. The seroconversion rates for measles, rubella, varicella, and mumps were 80.0%, 100.0%, 59.1%, and 69.2%, respectively. No life-threatening adverse events were observed, although one patient suffered from vaccine-strain varicella who showed cellular and humoral immunodeficiency (CD4 cell counts = 511/mm3, stimulation index of lymphocyte blast transformation by PHA = 91.1, serum IgG level = 208 mg/dl). This girl was immunized before we established the criteria for vaccination. Immunization with live attenuated vaccines for patients receiving immunosuppressive agents might be effective and safe if their cellular and humoral immunological parameters are within normal levels. However, determining the criteria for vaccination by immunological parameters should be established to guarantee the safety of live vaccines in the future. Clinical Trial Registration: UMIN Clinical Trials Registry (UMIN-CTR) UMIN000007710. The date of registration: 2012/4/13.

Clinical and molecular basis of hepatocerebral mitochondrial DNA depletion syndrome in Japan: evaluation of outcomes after liver transplantation.

BACKGROUND: Hepatocerebral mitochondrial DNA depletion syndrome (MTDPS) is a disease caused by defects in mitochondrial DNA maintenance and leads to liver failure and neurological complications during infancy. Liver transplantation (LT) remains controversial due to poor outcomes associated with extrahepatic symptoms. The purposes of this study were to clarify the current clinical and molecular features of hepatocerebral MTDPS and to evaluate the outcomes of LT in MTDPS patients in Japan. RESULTS: We retrospectively assessed the clinical and genetic findings, as well as the clinical courses, of 23 hepatocerebral MTDPS patients from a pool of 999 patients who were diagnosed with mitochondrial diseases between 2007 and 2019. Causative genes were identified in 18 of 23 patients: MPV17 (n = 13), DGUOK (n = 3), POLG (n = 1), and MICOS13 (n = 1). Eight MPV17-deficient patients harbored c.451dupC and all three DGUOK-deficient patients harbored c.143-307_170del335. The most common initial manifestation was failure to thrive (n = 13, 56.5%). The most frequent liver symptom was cholestasis (n = 21, 91.3%). LT was performed on 12 patients, including nine MPV17-deficient and two DGUOK-deficient patients. Among the 12 transplanted patients, five, including one with mild intellectual disability, survived; while seven who had remarkable neurological symptoms before LT died. Five of the MPV17-deficient survivors had either c.149G > A or c.293C > T. CONCLUSIONS: MPV17 was the most common genetic cause of hepatocerebral MTDPS. The outcome of LT for MTDPS was not favorable, as previously reported, however, patients harboring MPV17 mutations associated with mild phenotypes such as c.149G > A or c.293C > T, and exhibiting no marked neurologic manifestations before LT, had a better prognosis after LT.

Comparison of characteristics and outcomes of late acute and NIH chronic GVHD between Japanese and white patients.

Although differences in the incidence of chronic graft-versus-host disease (GVHD) across the races have been suggested, these have not been systematically investigated. This study compared the incidence, sites, severity, and outcomes of late acute GVHD and chronic GVHD according to National Institutes of Health (NIH) consensus criteria between Japanese (n = 413) and white (n = 708) patients after first allogeneic hematopoietic cell transplantation. Analysis was stratified according to bone marrow transplantation (BMT) or peripheral blood stem cell transplantation (PBSCT). Japanese patients, compared with white patients, had a similar incidence of late acute GVHD (BMT, 19% vs 16%; PBSCT, 19% vs 16%) but experienced more frequent liver late acute GVHD as defined by transaminase elevation (BMT, 79% vs 8%; PBSCT, 92% vs 33%) and less frequent gastrointestinal late acute GVHD (BMT, 11% vs 58%; PBSCT, 20% vs 68%). Japanese patients were more likely to discontinue systemic immunosuppression after late acute GVHD than white patients (hazard ratio, 3.68; 95% confidence interval, 1.96-6.94; P < .001). Japanese patients, compared with white patients, had a lower incidence of chronic GVHD (BMT, 15% vs 30% [P = .002]; PBSCT, 37% vs 45% [P < .001]) and experienced more frequent chronic GVHD of the mouth, eyes, and liver and less frequent gastrointestinal chronic GVHD. The duration of immunosuppressive treatment of NIH chronic GVHD was similar between the races. These differences could not be entirely attributed to practice variation between the centers. This study shows that the incidence, affected sites, severity, and clinical outcomes of late acute GVHD and NIH chronic GVHD differ between Japanese and white patients.

Measurement of the α1-proteinase inhibitor (α1-antitrypsin) of common marmoset and intestinal protein loss in wasting syndrome.

Although wasting marmoset syndrome (WMS) is one of the biggest problems facing captive marmoset colonies, the mechanisms underlying its pathogenesis remain unclear. In our clinical experience, it is difficult to cure WMS-affected marmosets with severe hypoalbuminemia. Thus, the mechanisms underlying hypoalbuminemia in WMS must be understood. In the present study, we investigated whether intestinal protein loss, a known reason for hypoalbuminemia, occurs in this disease. Fecal α1-proteinase inhibitor (α1-PI, also known as α1-antitrypsin) has been used to diagnose intestinal protein loss in other species. To develop an assay system for this protein, marmoset α1-PI was purified from plasma and antibodies against it were developed using the purified protein. Using the antibodies, a sandwich enzyme-linked immunosorbent assay (ELISA) to measure marmoset α1-PI was developed, and its detection sensitivity for fecal samples was ∼20-fold higher than that of a commercial kit for human α1-PI. From this ELISA, the reference intervals for serum and feces of healthy marmosets were 0.87-1.85 mg/ml and 0.53-395.58 µg/g, respectively. The average concentrations of α1-PI in serum and feces of seven WMS-affected marmosets were 1.17 mg/ml and 1357.58 µg/g, respectively. Although there were no significant differences in the serum concentrations between healthy and WMS-affected marmosets, the fecal concentrations were significantly higher in WMS-affected marmosets than in healthy individuals, suggesting that intestinal protein loss occurs in WMS. Intestinal protein loss of WMS-affected marmosets was significantly attenuated with treatment, suggesting that it is one of the mechanisms involved in the hypoalbuminemia observed in WMS.

Characterization of Late Acute and Chronic Graft-Versus-Host Disease according to the 2014 National Institutes of Health Consensus Criteria in Japanese Patients.

To characterize the incidences and outcomes of late acute (LA) and chronic graft-versus-host disease (GVHD) in East Asians according to the 2014 National Institutes of Health criteria, we retrospectively analyzed 506 consecutive Japanese patients who had a first allogeneic hematopoietic cell transplantation (HCT) at our center between 2006 and 2013. According to manifestations at onset 91 patients (60%) had LA GVHD and 60 (40%) had chronic GVHD. The cumulative incidences of LA and chronic GVHD were 20% and 17%, respectively, at 48 months after HCT. The involved sites at the onset of LA GVHD included the skin (71%), gut (13%), and liver (8%). The cumulative incidences of relapse, nonrelapse mortality (NRM), transition to chronic GVHD, and discontinued systemic treatment were 11%, 6%, 22%, and 46%, respectively, at 48 months after onset of LA GVHD. Cox models showed that prior acute GVHD was associated with NRM, and HCT from a female donor to a male patient, myeloablative conditioning, and low Karnofsky performance status were associated with a longer duration of systemic treatment after LA GVHD. The most frequently involved sites at the onset of chronic GVHD included the mouth (83%), liver (75%), skin (69%), and eyes (62%). Cox models showed that use of antithymocyte globulin in conditioning regimens was associated with a higher risk of discontinued systemic treatment after the onset of chronic GVHD. The cumulative incidences of relapse, NRM, and discontinued systemic treatment were 16%, 11%, and 41%, respectively, at 48 months after the onset of chronic GVHD. Our results suggested several potential differences between Japanese patients and those of other ethnicities. A direct comparison is needed to formally investigate ethnic differences.

Prospective Study of Live Attenuated Vaccines for Patients with Nephrotic Syndrome Receiving Immunosuppressive Agents.

OBJECTIVE: To conduct a prospective study to evaluate the immunogenicity and safety of live attenuated vaccines in patients with nephrotic syndrome receiving immunosuppressive agents. STUDY DESIGN: Patients with nephrotic syndrome receiving immunosuppressive agents with negative or borderline antibody titers (virus-specific IgG levels <4.0) against measles, rubella, varicella, and/or mumps fulfilling the criteria of cellular and humoral immunity were enrolled. Virus-specific IgG levels were measured using an enzyme immunoassay. The primary endpoint was the seroconversion rate (ie, achievement of virus-specific IgG levels ≥4.0) at 2 months after vaccination. Virus-specific IgG levels at 1 year, breakthrough infections (wild-type infections), and adverse events were also evaluated. RESULTS: A total of 116 vaccinations were administered to 60 patients. Seroconversion rates were 95.7% for measles, 100% for rubella, 61.9% for varicella, and 40.0% for mumps. More patients with a borderline antibody titer before vaccination achieved seroconversion than those with negative antibody titer, with statistical significance after varicella and mumps vaccination. The rate of patients who maintained seropositivity at 1 year after vaccination was 83.3% for measles, 94.1% for rubella, 76.7% for varicella, and 20.0% for mumps. No patient experienced breakthrough infection. No serious adverse events, including vaccine-associated infection, were observed. CONCLUSION: Immunization with live attenuated vaccines may be immunogenic and is apparently safe in our cohort of patients with nephrotic syndrome receiving immunosuppressive agents if their cellular and humoral immunologic measures are within clinically acceptable levels. TRIAL REGISTRATION: UMIN-CTR UMIN 000007710.

Serum free IgE guided dose reduction of omalizumab: a case report.

BACKGROUND: Omalizumab is a human IgG1 antibody against IgE used as a therapy for sever asthmatic patients with asthma. According to the guidelines of the Global Initiative for Asthma, omalizumab is an add-on drug at treatment step 5 that is used for severe asthma patients who are allergic to perennial allergens. The effects of omalizumab for severe asthma therapy have been validated in multiple clinical studies. However, the long-term effects of omalizumab on IgE production and possibility of resetting of administration dose of omalizumab remain unknown. CASE PRESENTATION: The serum total and free IgE levels were measured over time in a 63-year-old female patient with allergic asthma who was administered 375 mg omalizumab biweekly for 36 months. Her symptoms did not worsen and clinical course remained favorable after reducing the dose to 375 mg per month. The serum free IgE levels temporarily increased following a dose reduction of omalizumab. The serum free IgE trough level temporarily increased at 4 weeks after capable to reduce the dosage; however, thereafter, the serum free IgE level decreased to desired levels (below 30 ng/mL). CONCLUSIONS: The present case shows the possibility of reducing the dose following the long-term use of omalizumab. Considering the high medical cost of omalizumab, the dose reduction may be a viable option. It may be useful to measure the serum free IgE level to appropriately identify patients in whom the dose can be reduced, and to carefully monitor the clinical course.

Combined genetic analyses can achieve efficient diagnostic yields for subjects with Alagille syndrome and incomplete Alagille syndrome.

AIM: We evaluated combined genetic analyses with targeted next-generation sequencing (NGS), multiplex ligation probe amplification (MLPA) of Jagged1 (JAG1) genes and microarray comparative genomic hybridisation (CGH) in subjects with Alagille syndrome, incomplete clinical features of Alagille syndrome and biliary atresia. METHODS: Subjects recruited from April 2013 to December 2015 underwent a targeted NGS analysis, including JAG1 and Notch homolog 2 (NOTCH2). If no mutations were detected in JAG1 or NOTCH2, or if copy number variations were suggested by the NGS analysis, we performed an MLPA analysis of JAG1. We also performed a microarray CGH analysis with whole-exon deletion detected by the MLPA analysis. RESULTS: We analysed 30 subjects with Alagille syndrome, nine with incomplete Alagille syndrome and 17 with biliary atresia and detected pathogenic mutations in JAG1 or NOTCH2 in 24/30 subjects with Alagille syndrome and in 4/9 subjects with incomplete Alagille syndrome. No pathogenic mutations were detected in subjects with biliary atresia. The frequency of JAG1 mutations was as follows: single nucleotide variants (51.9%), small insertion or deletion (29.6%) and gross deletion (18.5%). CONCLUSION: Combined genetic analyses achieved efficient diagnostic yields for subjects with Alagille syndrome and incomplete Alagille syndrome.