Incorporation of ultrasound-guided core biopsy with flow cytometry to assist the diagnosis of cervical lymphoma.

PURPOSE: The main purpose of surgery for cervical lymphoma is only for tissue sampling. To establish a patient-friendly diagnostic approach, we investigated the feasibility of ultrasound-guided core biopsy with flow cytometry in the patients with suspected cervical lymphoma. METHODS: We prospectively recruited patients with suspected cervical lymphoma from Nov 2017 till Jan 2021 in a referral medical center and performed retrospective interpretation of the prospectively acquired data. Ultrasound-guided core biopsy as the tissue sampling approach for the targeted lesions was performed in all patients. The ultrasound-guided core biopsy samples were analyzed by immunohistochemical stains and flow cytometry. The sample quality and the rate of definite and decisive diagnosis obtained by ultrasound-guided core biopsy alone and ultrasound-guided core biopsy with flow cytometry were evaluated. RESULTS: Total 81 consecutive patients were recruited for analysis. All ultrasound-guided core biopsy samples were qualified for analysis of pathology and flow cytometry. Pathologically, the diagnoses were definite and compatible with their flow cytometry results in 70 patients (86.42%). Either newly-diagnosed or recurrent cervical lymphoma/lymphoproliferative disorders with histologic transformation could be diagnosed by ultrasound-guided core biopsy with flow cytometry. Nine of the 11 patients with pathologically indefinite diagnosis became clinically decisive when flow cytometry was incorporated into the process, which improved the rate of decisive diagnosis to 98.77% (Odds ratio [95% CI]: 6.21 [1.28, 58.96]). CONCLUSION: Ultrasound-guided core biopsy combined with flow cytometry is suggested to serve as the first-line and patient-friendly diagnostic approach for the patients with suspected cervical lymphoma.

Polatuzumab vedotin-based salvage immunochemotherapy as third-line or beyond treatment for patients with diffuse large B-cell lymphoma: a real-world experience.

Polatuzumab vedotin (PoV) has recently shown promising activity when combined with rituximab-bendamustine (BR) in patients with relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL). However, few studies have described the prognostic factors predicting response. Here, we aimed to evaluate the efficacy and safety profile of PoV-based chemotherapy, including regimens other than BR, as third-line or beyond treatment for patients with R/R DLBCL and to explore prognostic factors. Overall, 40 patients, including 37 with de novo and 3 with transformed DLBCL, were enrolled. The overall response rate was 52.5%, and 25% and 27.5% of patients showed a complete response and partial response, respectively. With a median follow-up of 18.8 months, the median overall survival (OS) of the total cohort was 8.5 months, and that of those receiving subsequent hematopoietic stem cell transplantation (HSCT) was 24 months. Low/intermediate risk according to the revised International Prognostic Index score at diagnosis and before PoV treatment predicted better OS. Furthermore, a normal lactate dehydrogenase level and an absolute lymphocyte count/absolute monocyte count ratio > 1.5 were favorable OS prognostic factors. The most common adverse event was cytopenia, with 42.5% of patients developing febrile neutropenia. Grade 1-3 peripheral neuropathy associated with PoV was reported in 25% of patients and resolved in most patients after the cessation of treatment. In summary, we demonstrated that PoV combined with either BR or other intensive chemotherapy is an effective and well-tolerated salvage option for patients with R/R DLBCL. Subsequent HSCT has the potential to further improve survival outcomes in this high-risk population. Clinicaltrials.gov number: NCT05006534.

Investigating early progression of Hodgkin lymphoma in a two-center analysis.

BACKGROUND/PURPOSE: The early progression of disease (POD) of Hodgkin lymphoma (HL) leads to a poor prognosis. To identify risk factors for early POD, this retrospective two-center cohort analysis was conducted. METHODS: Medical records of HL patients between 1998 and 2020 from two referral centers were reviewed. RESULTS: Two-hundred and sixty-nine patients were analyzed. The distribution of early vs. advanced stages was 51.1 vs. 48.9%, respectively. The 5-year progression free survival (PFS) was 63%, and the overall survival (OS) was 87% with a median follow-up of 52.0 months. The complete remission (CR) rate was 85.7%. Disease progression or relapsed disease occurred in 33.9% (n = 85) of patients while 17.0% of this cohort had early POD within 12 months of induction therapy. Patients with early POD had a worse median OS than those without (p < 0.001). Failure to achieve post-induction CR and high international prognostic score (IPS, 3-7) were independent risk factors for early POD. Compared with chemotherapy alone, consolidative radiotherapy after induction chemotherapy was associated with a lower risk of early POD (21.3% vs. 6.2%, p = 0.006). CONCLUSION: High IPS was an independent risk factor for early POD, which was less observed in those with consolidative radiotherapy.

A draft map of the human proteome.

The availability of human genome sequence has transformed biomedical research over the past decade. However, an equivalent map for the human proteome with direct measurements of proteins and peptides does not exist yet. Here we present a draft map of the human proteome using high-resolution Fourier-transform mass spectrometry. In-depth proteomic profiling of 30 histologically normal human samples, including 17 adult tissues, 7 fetal tissues and 6 purified primary haematopoietic cells, resulted in identification of proteins encoded by 17,294 genes accounting for approximately 84% of the total annotated protein-coding genes in humans. A unique and comprehensive strategy for proteogenomic analysis enabled us to discover a number of novel protein-coding regions, which includes translated pseudogenes, non-coding RNAs and upstream open reading frames. This large human proteome catalogue (available as an interactive web-based resource at http://www.humanproteomemap.org) will complement available human genome and transcriptome data to accelerate biomedical research in health and disease.

Phosphoproteomic Profiling Reveals Epstein-Barr Virus Protein Kinase Integration of DNA Damage Response and Mitotic Signaling.

Epstein-Barr virus (EBV) is etiologically linked to infectious mononucleosis and several human cancers. EBV encodes a conserved protein kinase BGLF4 that plays a key role in the viral life cycle. To provide new insight into the host proteins regulated by BGLF4, we utilized stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics to compare site-specific phosphorylation in BGLF4-expressing Akata B cells. Our analysis revealed BGLF4-mediated hyperphosphorylation of 3,046 unique sites corresponding to 1,328 proteins. Frequency analysis of these phosphosites revealed a proline-rich motif signature downstream of BGLF4, indicating a broader substrate recognition for BGLF4 than its cellular ortholog cyclin-dependent kinase 1 (CDK1). Further, motif analysis of the hyperphosphorylated sites revealed enrichment in ATM, ATR and Aurora kinase substrates while functional analyses revealed significant enrichment of pathways related to the DNA damage response (DDR), mitosis and cell cycle. Phosphorylation of proteins associated with the mitotic spindle assembly checkpoint (SAC) indicated checkpoint activation, an event that inactivates the anaphase promoting complex/cyclosome, APC/C. Furthermore, we demonstrated that BGLF4 binds to and directly phosphorylates the key cellular proteins PP1, MPS1 and CDC20 that lie upstream of SAC activation and APC/C inhibition. Consistent with APC/C inactivation, we found that BGLF4 stabilizes the expression of many known APC/C substrates. We also noted hyperphosphorylation of 22 proteins associated the nuclear pore complex, which may contribute to nuclear pore disassembly and SAC activation. A drug that inhibits mitotic checkpoint activation also suppressed the accumulation of extracellular EBV virus. Taken together, our data reveal that, in addition to the DDR, manipulation of mitotic kinase signaling and SAC activation are mechanisms associated with lytic EBV replication. All MS data have been deposited in the ProteomeXchange with identifier PXD002411 (http://proteomecentral.proteomexchange.org/dataset/PXD002411).

Annotation of the zebrafish genome through an integrated transcriptomic and proteomic analysis.

Accurate annotation of protein-coding genes is one of the primary tasks upon the completion of whole genome sequencing of any organism. In this study, we used an integrated transcriptomic and proteomic strategy to validate and improve the existing zebrafish genome annotation. We undertook high-resolution mass-spectrometry-based proteomic profiling of 10 adult organs, whole adult fish body, and two developmental stages of zebrafish (SAT line), in addition to transcriptomic profiling of six organs. More than 7,000 proteins were identified from proteomic analyses, and ∼ 69,000 high-confidence transcripts were assembled from the RNA sequencing data. Approximately 15% of the transcripts mapped to intergenic regions, the majority of which are likely long non-coding RNAs. These high-quality transcriptomic and proteomic data were used to manually reannotate the zebrafish genome. We report the identification of 157 novel protein-coding genes. In addition, our data led to modification of existing gene structures including novel exons, changes in exon coordinates, changes in frame of translation, translation in annotated UTRs, and joining of genes. Finally, we discovered four instances of genome assembly errors that were supported by both proteomic and transcriptomic data. Our study shows how an integrative analysis of the transcriptome and the proteome can extend our understanding of even well-annotated genomes.

Ablation of Dicer leads to widespread perturbation of signaling pathways.

Dicer is an essential ribonuclease involved in the biogenesis of miRNAs. Previous studies have reported downregulation of Dicer in multiple cancers including hepatocellular carcinoma. To identify signaling pathways that are altered upon Dicer depletion, we carried out quantitative phosphotyrosine profiling of liver tissue from Dicer knockout mice. We employed antibody-based enrichment of phosphotyrosine containing peptides coupled with SILAC spike-in approach for quantitation. High resolution mass spectrometry-based analysis identified 349 phosphotyrosine peptides corresponding to 306 unique phosphosites of which 75 were hyperphosphorylated and 78 were hypophosphorylated. Several receptor tyrosine kinases including MET, PDGF receptor alpha, Insulin-like growth factor 1 and Insulin receptor as well as non-receptor tyrosine kinases such as Src family kinases were found to be hyperphosphorylated upon depletion of Dicer. In addition, signaling molecules such as IRS-2 and STAT3 were hyperphosphorylated. Activation of these signaling pathways has been implicated previously in various types of cancers. Interestingly, we observed hypophosphorylation of molecules including focal adhesion kinase and paxillin. Our study profiles the perturbed signaling pathways in response to dysregulated miRNAs resulting from depletion of Dicer. Our findings warrant further studies to investigate oncogenic effects of downregulation of Dicer in cancers.

TSLP signaling network revealed by SILAC-based phosphoproteomics.

Thymic stromal lymphopoietin (TSLP) is a cytokine that plays diverse roles in the regulation of immune responses. TSLP requires a heterodimeric receptor complex consisting of IL-7 receptor α subunit and its unique TSLP receptor (gene symbol CRLF2) to transmit signals in cells. Abnormal TSLP signaling (e.g. overexpression of TSLP or its unique receptor TSLPR) contributes to the development of a number of diseases including asthma and leukemia. However, a detailed understanding of the signaling pathways activated by TSLP remains elusive. In this study, we performed a global quantitative phosphoproteomic analysis of the TSLP signaling network using stable isotope labeling by amino acids in cell culture. By employing titanium dioxide in addition to antiphosphotyrosine antibodies as enrichment methods, we identified 4164 phosphopeptides on 1670 phosphoproteins. Using stable isotope labeling by amino acids in cell culture-based quantitation, we determined that the phosphorylation status of 226 proteins was modulated by TSLP stimulation. Our analysis identified activation of several members of the Src and Tec families of kinases including Btk, Lyn, and Tec by TSLP for the first time. In addition, we report TSLP-induced phosphorylation of protein phosphatases such as Ptpn6 (SHP-1) and Ptpn11 (Shp2), which has also not been reported previously. Co-immunoprecipitation assays showed that Shp2 binds to the adapter protein Gab2 in a TSLP-dependent manner. This is the first demonstration of an inducible protein complex in TSLP signaling. A kinase inhibitor screen revealed that pharmacological inhibition of PI-3 kinase, Jak family kinases, Src family kinases or Btk suppressed TSLP-dependent cellular proliferation making them candidate therapeutic targets in diseases resulting from aberrant TSLP signaling. Our study is the first phosphoproteomic analysis of the TSLP signaling pathway that greatly expands our understanding of TSLP signaling and provides novel therapeutic targets for TSLP/TSLPR-associated diseases in humans.

Unmet Needs in the First-Line Treatment of Diffuse Large B-cell Lymphoma: Expert Recommendations From the Asia-Pacific Region With a Focus on the Challenging Subtypes.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma, accounting for around 30-60% of all cases. The management of DLBCL in Asia has several unmet needs due to the diversity of the population, the heterogeneity of local clinical guidelines for DLBCL and the wide disparity in resources and healthcare systems across different regions. Rituximab combined with cyclophosphamide, doxorubicin, vincristine, and prednisone (RCHOP) is widely recognized as the standard first-line treatment for DLBCL; however, alternative regimens are required to improve patient outcomes in challenging subtypes, such as patients with high International Prognostic Index scores, old/frail patients, and patients with double-hit and double-expressor DLBCL or concurrent central nervous system disease. This review article draws from the expertise of practicing hematologists/oncologists in the region, with the aim of integrating data from current scientific evidence to address the unmet needs and unique socioeconomic challenges faced by challenging high risk patient groups in the Asia-Pacific region.

Clinical manifestations and risk factors for COVID-19 and its severity in patients with hematological malignancies.

BACKGROUND: Patients with hematological malignancies (HM) were at a high risk of developing severe disease from coronavirus disease 2019 (COVID-19). We aimed to assess the clinical outcome of COVID-19 in hospitalized patients with HM. METHODS: Adult patients with HM who were hospitalized with a laboratory-confirmed COVID-19 between May, 2021 and November, 2022 were retrospectively identified. Primary outcome was respiratory failure requiring mechanical ventilation or mortality within 60 days after hospitalization. We also analyzed associated factors for de-isolation (defined as defervescence with a consecutive serial cycle threshold value > 30) within 28 days. RESULTS: Of 152 eligible patients, 22 (14.5%) developed respiratory failure or mortality in 60 days. Factors associated with developing respiratory failure that required mechanical ventilation or mortality included receipt of allogeneic hematopoietic stem-cell transplantation (allo-HSCT) (adjusted hazards ratio [aHR], 5.10; 95% confidence interval [CI], 1.64-15.85), type 2 diabetes mellitus (aHR, 2.47; 95% CI, 1.04-5.90), lymphopenia at admission (aHR, 6.85; 95% CI, 2.45-19.15), and receiving <2 doses of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines (aHR, 3.00; 95% CI, 1.19-7.60). Ninety-nine (65.1%) patients were de-isolated in 28 days, against which two hazardous factors were identified: receipt of B-cell depletion therapies within one year prior to COVID-19 (aHR, 0.55, 95% CI, 0.35-0.87) and lymphopenia upon admission (aHR, 0.65; 95% CI, 0.43-1.00). CONCLUSION: We found a high rate of respiratory failure and mortality among patients with HM who contracted the SARS-CoV-2. Factors associated with developing respiratory failure or mortality in 60 days included receipt of allo-HSCT, type 2 diabetes mellitus and lymphopenia upon admission. Having received ≥2 doses of vaccination conferred protection against clinical progression.

Proteomics for understanding miRNA biology.

MicroRNAs (miRNAs) are small noncoding RNAs that play important roles in posttranscriptional regulation of gene expression. Mature miRNAs associate with the RNA interference silencing complex to repress mRNA translation and/or degrade mRNA transcripts. Mass spectrometry-based proteomics has enabled identification of several core components of the canonical miRNA processing pathway and their posttranslational modifications which are pivotal in miRNA regulatory mechanisms. The use of quantitative proteomic strategies has also emerged as a key technique for experimental identification of miRNA targets by allowing direct determination of proteins whose levels are altered because of translational suppression. This review focuses on the role of proteomics and labeling strategies to understand miRNA biology.

Regulation of lipid metabolism by Dicer revealed through SILAC mice.

Dicer is a ribonuclease whose major role is to generate mature microRNAs, although additional functions have been proposed. Deletion of Dicer leads to embryonic lethality in mice. To study the role of Dicer in adults, we generated mice in which administration of tamoxifen induces deletion of Dicer. Surprisingly, disruption of Dicer in adult mice induced lipid accumulation in the small intestine. To dissect the underlying mechanisms, we carried out miRNA, mRNA, and proteomic profiling of the small intestine. The proteomic analysis was done using mice metabolically labeled with heavy lysine (SILAC mice) for an in vivo readout. We identified 646 proteins, of which 80 were up-regulated >2-fold and 75 were down-regulated. Consistent with the accumulation of lipids, Dicer disruption caused a marked decrease of microsomal triglyceride transfer protein, long-chain fatty acyl-CoA ligase 5, fatty acid binding protein, and very-long-chain fatty acyl-CoA dehydrogenase, among others. We validated these results using multiple reaction monitoring (MRM) experiments by targeting proteotypic peptides. Our data reveal a previously unappreciated role of Dicer in lipid metabolism. These studies demonstrate that a systems biology approach by integrating mouse models, metabolic labeling, gene expression profiling, and quantitative proteomics can be a powerful tool for understanding complex biological systems.

WT1 mutation in 470 adult patients with acute myeloid leukemia: stability during disease evolution and implication of its incorporation into a survival scoring system.

The impact of WT1 mutations in acute myeloid leukemia (AML) is not completely settled. We aimed to determine the clinical implication of WT1 mutation in 470 de novo non-M3 AML patients and its stability during the clinical course. WT1 mutations were identified in 6.8% of total patients and 8.3% of younger patients with normal karyotype (CN-AML). The WT1 mutation was closely associated with younger age (P < .001), French-American-British M6 subtype (P = .006), and t(7;11)(p15;p15) (P = .003). Multivariate analysis demonstrated that the WT1 mutation was an independent poor prognostic factor for overall survival and relapse-free survival among total patients and the CN-AML group. A scoring system incorporating WT1 mutation, NPM1/FLT3-ITD, CEBPA mutations, and age into survival analysis proved to be very useful to stratify CN-AML patients into different prognostic groups (P < .001). Sequential analyses were performed on 133 patients. WT1 mutations disappeared at complete remission in all WT1-mutated patients studied. At relapse, 3 of the 16 WT1-mutated patients who had paired samples lost the mutation and 2 acquired additional mutations, whereas 3 of 110 WT1-wild patients acquired novel mutations. In conclusion, WT1 mutations are correlated with poor prognosis in AML patients. The mutation status may be changed in some patients during AML progression.

Safety, Feasibility, and Effects of Short-Term Calorie Reduction during Induction Chemotherapy in Patients with Diffuse Large B-Cell Lymphoma: A Pilot Study.

Short-term calorie reduction (SCR) requires individuals to reduce their calorie intake to less than 50% of normal requirements and has shown good tolerance and potential benefits in prior studies addressing gynecological cancer patients. More studies are needed to further confirm its safety, feasibility, and effects in patients with different cancers, including hematological malignancies. This pilot cohort study with a matched-pair comparison group was registered at ClinicalTrails.gov [201810112RIND]. Adult patients diagnosed with advanced-stage diffuse large-B cell lymphoma were recruited (SCR group) and matched with one comparison patient (comparison group), each in a manner blinded to their outcomes. The SCR group undertook at least two cycles of 48 h water fast along with their chemotherapy R-CHOP. Descriptive analysis and generalized estimating equations were used to analyze the data. Six participants completed multiple cycles of SCR and were compared to their six counterparts in the comparison group. The results showed that SCR is safe and feasible in terms of a high compliance rate and stable nutritional status. The SCR was associated with benefits in post-chemotherapy hematological parameters (i.e., erythrocyte [p < 0.001] and lymphocyte counts [p < 0.001]). More randomized controlled trials are needed to validate the effects of SCR on different types of cancer populations.

A Clinicopathological Study of Cytomegalovirus Lymphadenitis and Tonsillitis and Their Association with Epstein-Barr Virus.

INTRODUCTION: Histopathological characteristics of cytomegalovirus (CMV) lymphadenitis have been well described. Rare studies have reported the immune status and clinical features. Clinically, experts believed that CMV lymphadenitis develops in immunocompromised and immunocompetent patients. Infectious mononucleosis (IM)-like syndrome is the most well-known clinical presentation. METHODS: We reviewed archived CMV immunohistochemical stains on lymphoid tissues. The clinicopathological features of CMV-positive cases were studied. RESULTS: For lymph nodes, we detected CMV in 29% (5/17) allogeneic peripheral blood hematopoietic stem cell transplantation (PBSCT) recipients, 29% (4/14) post-autologous PBSCT patients, 13% (6/47) patients treated with intravenous chemotherapy, and 9% (9/96) immunocompetent patients. We detected CMV in 7% (2/24) of tonsils but not in the nasopharynx, tongue base, or spleen specimens. The patients with iatrogenic immunodeficiency ranged from 37 to 76 years old. CMV infections developed a few years after lymphoma treatment (median duration after allogeneic PBSCT, 932 days; after autologous PBSCT, 370 days; and after chemotherapy, 626 days). The most common clinical presentation was neck mass (13/25, 42%), followed by asymptomatic image finding (10/25, 40%). Positron emission tomography/computed tomography (PET/CT) scan showed increased uptake compared to the liver in all patients (11/11, 100%). Of 10 lymphoma patients, 8 (80%) had a Deauville score of 4-5; they accounted for 30% (8/27) of lymphoma patients with false-positive PET/CT scan results. All cases were self-limiting. 96% (23/25) cases had Epstein-Barr virus coinfection, and EBER-positive cells were predominantly in a few germinal centers. CONCLUSIONS: Cytomegalovirus (CMV) lymphadenitis and tonsillitis were subclinical infections, not primary CMV infection with IM-like syndrome. The lymphadenopathy typically developed a few years after lymphoma treatments in the middle-aged and the elderly. The lesions mimicked lymphoma relapse in PET scans. Therefore, recognizing CMV infection in lymphoid tissues is of clinical importance.

Epigenetic modulation of immune synaptic-cytoskeletal networks potentiates γδ T cell-mediated cytotoxicity in lung cancer.

γδ T cells are a distinct subgroup of T cells that bridge the innate and adaptive immune system and can attack cancer cells in an MHC-unrestricted manner. Trials of adoptive γδ T cell transfer in solid tumors have had limited success. Here, we show that DNA methyltransferase inhibitors (DNMTis) upregulate surface molecules on cancer cells related to γδ T cell activation using quantitative surface proteomics. DNMTi treatment of human lung cancer potentiates tumor lysis by ex vivo-expanded Vδ1-enriched γδ T cells. Mechanistically, DNMTi enhances immune synapse formation and mediates cytoskeletal reorganization via coordinated alterations of DNA methylation and chromatin accessibility. Genetic depletion of adhesion molecules or pharmacological inhibition of actin polymerization abolishes the potentiating effect of DNMTi. Clinically, the DNMTi-associated cytoskeleton signature stratifies lung cancer patients prognostically. These results support a combinatorial strategy of DNMTis and γδ T cell-based immunotherapy in lung cancer management.

Feasibility, Process, and Effects of Short-Term Calorie Reduction in Cancer Patients Receiving Chemotherapy: An Integrative Review.

Recent preclinical studies have shown the potential benefits of short-term calorie reduction (SCR) on cancer treatment. In this integrative review, we aimed to identify and synthesize current evidence regarding the feasibility, process, and effects of SCR in cancer patients receiving chemotherapy. PubMed, Cumulative Index to Nursing and Allied Health Literature, Ovid Medline, PsychINFO, and Embase were searched for original research articles using various combinations of Medical Subject Heading terms. Among the 311 articles identified, seven studies met the inclusion criteria. The majority of the reviewed studies were small randomized controlled trials or cohort study with fair quality. The results suggest that SCR is safe and feasible. SCR is typically arranged around the chemotherapy, with the duration ranging from 24 to 96 h. Most studies examined the protective effects of SCR on normal cells during chemotherapy. The evidence supports that SCR had the potential to enhance both the physical and psychological wellbeing of patients during chemotherapy. SCR is a cost-effective intervention with great potential. Future well-controlled studies with sufficient sample sizes are needed to examine the full and long-term effects of SCR and its mechanism of action.