Extrachromosomal circular DNA landscape of breast cancer with lymph node metastasis.

Breast cancer (BC) is a complex disease with diverse manifestations, often resulting in lymph node metastasis (LNM) and impacting patient prognosis. Extrachromosomal circular DNA (eccDNA) has emerged as a key player in tumorigenesis, yet its contribution to BC LNM remains elusive. Here, we examined primary tumors and matched LNM tissues from 19 BC patients using the Circle-Seq method. We identified a median count of 44,682 eccDNA in primary tumor tissues and 38,057 in their paired LNM tissues. Furthermore, a ladder-like size distribution is observed in both primary tumor and LNM tissues. Meanwhile, similar repeat sequence distribution and GC content are identified from both primary tissue and LNM tissues. Finally, we found that eccDNA from both groups are flanked with palindromic trinucleotide motifs. These observations indicate that eccDNA of primary tumor and LNM tissues are from similar chromosomal origins. However, a subset of miRNA-associated eccDNA displayed selective enrichment in metastatic lesions, such as miR-6730 and miR-548AA1 genes. This observation implicates the function of miRNA-related eccDNA in the metastatic cascade. Our study uncovers the potential significance of these unique eccDNA molecules, shedding light on their role in cancer metastasis.

Prediction of tumor origin in cancers of unknown primary origin with cytology-based deep learning.

Cancer of unknown primary (CUP) site poses diagnostic challenges due to its elusive nature. Many cases of CUP manifest as pleural and peritoneal serous effusions. Leveraging cytological images from 57,220 cases at four tertiary hospitals, we developed a deep-learning method for tumor origin differentiation using cytological histology (TORCH) that can identify malignancy and predict tumor origin in both hydrothorax and ascites. We examined its performance on three internal (n = 12,799) and two external (n = 14,538) testing sets. In both internal and external testing sets, TORCH achieved area under the receiver operating curve values ranging from 0.953 to 0.991 for cancer diagnosis and 0.953 to 0.979 for tumor origin localization. TORCH accurately predicted primary tumor origins, with a top-1 accuracy of 82.6% and top-3 accuracy of 98.9%. Compared with results derived from pathologists, TORCH showed better prediction efficacy (1.677 versus 1.265, P < 0.001), enhancing junior pathologists' diagnostic scores significantly (1.326 versus 1.101, P < 0.001). Patients with CUP whose initial treatment protocol was concordant with TORCH-predicted origins had better overall survival than those who were administrated discordant treatment (27 versus 17 months, P = 0.006). Our study underscores the potential of TORCH as a valuable ancillary tool in clinical practice, although further validation in randomized trials is warranted.

Pure red cell aplasia occurring during ibrutinib therapy for chronic lymphocytic leukemia.

INTRODUCTION: Chronic lymphocytic leukemia (CLL) has long been known for its complications related to immune deregulation, of which autoimmune cytopenias (AIC) were frequently reported. Ibrutinib has dramatically changed the overall prognosis of patients with CLL. However, whether ibrutinib can induce or aggravate AIC in CLL patients is still disputable. Here we report a CLL patient with pure red cell aplasia (PRCA) occurring during ibrutinib treatment and review available data to discuss the possible role of ibrutinib in developing AIC. CASE REPORT: A 70-year-old female was diagnosed with CLL with indications to initiate ibrutinib treatment given progressive bulky disease. She was admitted for advanced fatigue on the 14th day of ibrutinib monotherapy. A complete blood count revealed severe anemia of hemoglobin (Hb) 37 g/L and a meager reticulocyte count. After excluding other conditions that could cause anemia, PRCA was diagnosed as a complication of CLL. MANAGEMENT AND OUTCOME: Ibrutinib was discontinued on the day of admission. At the same time, the patient received prednisone and intravenous immunoglobulin (IVIg). Five days later, the Hb did not improve. Cyclosporine A (CsA) was added; IVIg was discontinued, and prednisone was tapered. Ten days later, the Hb had risen to 92 g/L with a high reticulocyte count of 0.279 × 1012/L. The CLL treatment restarted with Zanbrutinib in combination with a low dose of prednisone and CsA. Her CLL was in partial remission by the latest follow-up with an average Hb count. DISCUSSION: Our case demonstrates a need to evaluate the risk of developing AIC before initiating ibrutinib. For patients with high-risk factors for AIC episodes, the transient addition of other immunosuppressive therapies should be taken into consideration.

Clinical characteristics and survival outcomes in patients with primary ovarian carcinoid: A historical cohort study.

INTRODUCTION: Primary ovarian carcinoids are extremely rare ovarian tumors, and there is limited data available on their clinical characteristics and survival outcomes. MATERIAL AND METHODS: We conducted a historical cohort study of 56 patients to investigate their clinical characteristics. The overall survival, disease-specific survival, recurrence-free survival, and potential prognostic factors of these patients were also evaluated. RESULTS: The median age of these patients was 42.0 years (range: 20-71). The average mass and carcinoid size was 7.3 and 0.4 cm, respectively. Elevated tumor marker levels and ascites were observed in 15 and 10 patients, respectively. In 98.2% of the patients, tumors were confined to the ovary, while only one had metastatic disease. Surgery was the mainstay therapy: 37.5% of the patients underwent unilateral salpingo-oophorectomy, 25.0% underwent hysterectomy with bilateral salpingo-oophorectomy, 21.4% underwent ovarian cystectomy, 10.7% underwent comprehensive staging surgery, and 5.4% underwent bilateral salpingo-oophorectomy. Appendectomy and lymphadenectomy were performed in eight and five patients, respectively, but none showed tumor involvement. Chemotherapy was the only adjuvant treatment utilized, and was administered in four patients. Pathological analysis showed that strumal carcinoid was the most predominant subtype, occurring in 66.1% of the patients. The Ki-67 index was reported in 39 patients, 30 of which had an index of no more than 3%, with a maximum of only 5%. Only one relapse occurred after the initial treatment, and that patient experienced recurrences on two occasions, maintaining stable disease after surgery and octreotide therapy. After a median follow-up of 3.6 years, 96.4% of the patients achieved no evidence of disease, while 3.6% were alive with the disease. The 5-year recurrence-free survival rate was 97.9% and no death occurred. No risk factors for recurrence-free survival, overall survival, or disease-specific survival were identified. CONCLUSIONS: The Ki-67 indices were extremely low and prognoses were excellent in patients with primary ovarian carcinoids. Conservative surgery, especially unilateral salpingo-oophorectomy, is preferred. Individualized adjuvant therapy may be considered for patients with metastatic diseases.

Clinical characteristics and survival outcomes in patients with ovarian strumal carcinoid.

BACKGROUND: Ovarian strumal carcinoid is an extremely rare ovarian malignant tumor with limited data on clinical characteristics and survival outcomes. METHODS: A retrospective study of 119 patients was conducted, including 98 cases identified from literature review, and their clinical characteristics were investigated. The overall survival (OS), disease-specific survival (DSS), recurrence-free survival (RFS), and potential prognostic factors of these patients were also evaluated. RESULTS: Lesions of 115 cases were confined to the ovarian while four patients presented with extra-ovarian disease upon initial diagnosis. Surgical treatment options performed in this cohort varied, 5.0% received ovarian cystectomy, 36.1% received unilateral salpingo-oophorectomy (USO), 7.6% received bilateral salpingo-oophorectomy (BSO), 42.0% received hysterectomy with BSO, and 8.4% underwent debulking surgery. Moreover, one patient did not undergo any surgery. No postoperative adjuvant therapy was administered in 89.9% patients, while 7.6% and 2.5% received adjuvant radiotherapy and chemotherapy, of which two patients received combined radiation and chemotherapy. At the final follow-up, 89.1% patients showed no evidence of the disease, and 5.0% were alive with the disease. Only seven deaths occurred, with two attributed to the tumor. The 5-year, 10-year, and 20-year OS rates were 96.0%, 85.0%, and 85.0%, respectively, with a 15-year recurrence rate of 4.4%. The 5-year and 20-year DSS rate were 98.5% and 95.9%. Multivariate Cox regression showed age ≥ 55 years was the only risk factor associated with the OS (P = 0.014, OR 7.988; 95% CI 1.519 - 42.004). However, the univariate and multivariate Cox regression showed no potential risk factor for RFS and DSS. CONCLUSION: Patients with ovarian strumal carcinoid have an excellent prognosis irrespective of the surgical option. Conservative surgery especially USO with individualized adjuvant therapy is recommended.

Investigation of the Relationship between Chronic Endometritis Manifestations under Hysteroscope and CD138 Expression.

Methods: This study was performed to retrospectively analyze clinical cases of 523 CE patients from January 2018 to June 2020 that were confirmed by hysteroscopy before in vitro fertilization. Based on manifestations of CE under hysteroscopy, the cases were divided into three cohorts, i.e., hyperemia cohort where the patients had diffuse endometrium hyperemia; endometrial micropolyp cohort, and endometrial stroma edema and hyperplasia cohort. Small amount of endometrial tissue was collected from the patients, and CD138 IHC examination was performed. According to the results of CD138 IHC, positive patients were given antibiotic treatment (doxycycline 100 mg BID orally for 14 days), and hysteroscopy was performed again after treatment to check the efficacy of antibiotics. Results: In the comparison of overall status for all patient cohorts, infertility type, BMI, bFSH, bLH, bP, bT, PRL, AMH, and CA125 were varied markedly across all cohorts (P < 0.05), with predominant incidences of polycystic ovary syndrome (PCOS) peaking within hyperemia cohort. Incidence/diagnostic rate for CD138 within hyperemia cohort was 10.06%, which was lower than the 63.16% in micropolyp cohort and 74% in edema and hyperplasia cohort (P < 0.05). No major variation existed within CD138 across micropolyp cohort/edema and hyperplasia cohort (P > 0.05). After CD138-positive CE patients were treated with antibiotics, the effective rate (0/16) within hyperemia cohort was lower than micropolyp cohort (73.61%, 53/72) and edema and hyperplasia cohort (83.24%, 154/185) (P < 0.05). The effective rate across micropolyp cohort/edema and hyperplasia cohort was not significantly different (P > 0.05). Conclusion: Cases of diffuse endometrial hyperemia under hysteroscope had the lowest positive rate of CD138, and the effect of antibiotic treatment on these patients was poor. The positive rate of CD138 in patients with endometrial micropolyps and endometrial stroma edema and hyperplasia under hysteroscope was high, and the effect of antibiotic treatment was better.

Developing a routine lab test for absolute quantification of HER2 in FFPE breast cancer tissues using Quantitative Dot Blot (QDB) method.

Developing immunoassay for absolute quantitation of protein biomarkers in Formalin Fixed Paraffin Embedded (FFPE) samples promises improved objectivity, consistency and accuracy in daily clinical practice. The feasibility of Quantitative Dot Blot (QDB) method for this purpose was explored in this study. We were able to measure HER2 protein levels using 0.5 µg/sample total protein lysate extracted from 2 × 5 µm FFPE slices absolutely and quantitatively using QDB method in 332 breast cancer FFPE samples. HER2 levels measured using two clinically validated antibodies for immunohistochemistry respectively were highly correlated (r = 0.963). We also achieved area under the curve (AUC) at 0.9998 ± 0.0002 (p < 0.0001, n = 224) with IHC analysis, and 0.9942 ± 0.0031 (p < 0.0001, n = 319) with combined results from IHC and Fluorescence in situ hybridization (FISH) analyses when analyzed with Receiver Operative Characteristics analysis (ROC) respectively. When the results were converted dichotomously with optimized cutoffs from ROC analyses, we achieved 99.5% concordance with IHC; and 96.9% with combined results from both IHC and FISH analyses. Therefore, we were able to demonstrate QDB method as the first immunoassay platform for absolute quantitation of protein biomarkers in FFPE samples to meet the need of daily clinical practice, especially for local laboratories or laboratories in developing countries.

Signet Ring B Cell Lymphoma: A Potential Diagnostic Pitfall.

Signet ring B cell lymphoma is an unusual non-Hodgkin lymphoma. It is similar to signet ring cell carcinoma and liposarcoma in morphology which should be distinguished. We treated a 63-year-old male patient who suffered from abdominal pain for two months. Multiple enlarged lymph nodes were found in the retroperitoneum by CT scan. The needle biopsy showed neoplastic cells distributed uniformly with clear cytoplasm and the nucleus squeezed to the side mimicking the appearance of signet ring in morphology. By special staining, the neoplastic cells were positive for CD45, Vimentin, Bcl-2 and CD20 but negative for AE1/AE3, S-100, CD3, EMA, CD5, CD10, Bcl-6, MUM1 , Kappa, Lambda and PAS . Ki67 proliferation index was much more than 80%. Based on the histological characters, a diagnosis of signet ring B cell lymphoma was made. Although the patient received six courses of R-CHOP therapy, he died of tumor recurrence at the 34th month after diagnosis.

AnkG hemizygous mice present cognitive impairment and elevated anxiety/depressive-like traits associated with decreased expression of GABA receptors and postsynaptic density protein.

Recent genome-wide association studies (GWAS) of patient populations and genetic linkage assessments have demonstrated that the ankyrin-G (AnkG) gene is involved in neuropsychiatric disorders, including bipolar disorder, schizophrenia, and Alzheimer's disease, but it remains unclear how the genetic variants of AnkG contribute to neuropsychiatric disorders. Here, we generated AnkG hemizygous mice using the gene trapping approach. Homozygous AnkG was embryonically lethal. Western blotting and real-time polymerase chain reaction (qPCR) assessments of wild type (WT) and AnkG +/- mutant mice demonstrated a 50% reduction of ANKG levels, at the gene and protein levels, in AnkG hemizygous mice. In behavioral tests, AnkG hemizygous mice exhibited elevated anxiety- and depression-like traits, as well as cognitive impairment. Moreover, the expression levels of cognitive-related proteins (including metabotropic glutamate receptor subtype-1, brain-derived neurotrophic factor, postsynaptic density-95, GABA-B receptor, and GABA-A receptor alpha-1) were significantly decreased (P < 0.05), suggesting a possible role for AnkG in cognition. It is possible that the loss of AnkG in the brain disrupts the excitation/inhibition balance of neurotransmitters, hindering the synaptic plasticity of neurons, and consequently leading to abnormal behavioral symptoms. Therefore, AnkG possibly contributes to neuroprotection and normal brain function, and may constitute a new target for treating neuropsychiatric diseases, especially cognitive dysfunction.

Histone deacetylase-2 is involved in stress-induced cognitive impairment via histone deacetylation and PI3K/AKT signaling pathway modification.

Exposure to chronic stress upregulates blood glucocorticoid levels and impairs cognition via diverse epigenetic mechanisms, such as histone deacetylation. Histone deacetylation can lead to transcriptional silencing of many proteins involved in cognition and may also cause learning and memory dysfunction. Histone deacetylase­2 (HDAC2) has been demonstrated to epigenetically block cognition via a reduction in the histone acetylation level; however, it is unknown whether HDAC2 is involved in the cognitive decline induced by chronic stress. To the best of authors' knowledge, this is the first study to demonstrate that the stress hormone corticosteroid upregulate HDAC2 protein levels in neuro­2a cells and cause cell injuries. HDAC2 knockdown resulted in a significant amelioration of the pathological changes in N2a cells via the upregulation of histone acetylation and modifications in the phosphoinositide 3­kinase/protein kinase B signaling pathway. In addition, the HDAC2 protein levels were upregulated in 12­month­old female C57BL/6J mice under chronic stress in vivo. Taken together, these findings suggested that HDAC2 may be an important negative regulator involved in chronic stress­induced cognitive impairment.

Histopathological and immunological changes during the acute and recovery phase in Henoch-Schönlein purpura rabbit model.

Henoch-Schönlein purpura (HSP) is a systemic vasculitis mediated by autologous immune complex. Animal models of HSP are scarce. Here, we describe the characteristics of HSP rabbit model in the acute and recovery phase. First, we constructed the HSP rabbit models, and then assessed immunologic indicators of models by enzyme-linked immunosorbent assay and immunoturbidimetry. Histomorphological characteristics were analyzed by haematoxylin-eosin, immunofluorescence and special staining. In the acute stage (24 h) after antigen challenge, the model group rabbits featured skin ecchymosis and abnormal laboratory examination results. Three weeks following the allergic reaction, purple spots improved markedly, and edema and blood seeping decreased, but obvious inflammation was present in the skin, kidneys, joints, gastrointestinal, lung and liver. Serological results of CD4, CD/CD8, IL-2, IL-4, and TNF-α, IgA, IgG, TropI, Alb and T were still abnormal. IgA and C3 expressed in skin and kidney and eosinophils expressed in skin and lungs were increased. The rabbit model can mimic human HSP lesions in symptoms, pathology, and immunology and may provide valuable insight into the pathogenesis of HSP and serve as a tool for future therapeutic development targeting HSP.

[Change of Ankyrin G Promoter Activity Following Treatment with Sodium Valproate at Different Concentrations].

OBJECTIVE: To investigate whether sodium valproate (VPA) directly regulates the activity of Ankyrin G(AnkG) promoter in vitro. METHODS: The mouse AnkG promoter sequence was identified by comparing both human and mouse AnkG promoter sequences. The promoter was amplified from C57BL/6 mouse genome DNA and cloned into pGL3 Luciferase reporter vector. The Luciferase activity was detected in N2a and 293T cells and then treated with 0,0.5, and 1 mmol/L VPA for 12 h. The transcription activity of AnkG promoter in cells and the activity of VPA-treated Luciferase reporter vector in cells were detected using dual Luciferase reporter assay. RESULTS: The AnkG promoter clone and its expression vector were successfully established, as confirmed by enzyme digestion and sequencing. The AnkG promoter showed high transcription activity in both N2a and 293T cells. The Luciferase activity was significantly induced following 0.5 mmol/L VPA treatment in both N2a and 293T cells. CONCLUSIONS VPA can up-regulate the AnkG expression via directly increasing its transcription activity. Thus, the in vivo AnkG expression may be directly regulated by the VPA at transcriptional level.

Hematologic and immunological characteristics of Henoch-Schönlein purpura in rat and rabbit models induced with ovalbumin based on type III hypersensitivity.

Henoch-Schönlein purpura (HSP) is a common systemic vasculitis in children. Animal models of HSP are needed to better understand the mechanism of HSP. Here, we investigated hematologic and immunologic profiles in HSP rat and rabbit models. Models were established with ovalbumin (OVA) based on type III hypersensitivity. During the acute phase, the models exhibited varying degrees of cutaneous purpura, joint inflammatory response, gastrointestinal bleeding, glomerular capsule protein exudation, vascular dilatation, and increased IgA expression and immune complex deposition. Twenty four hours after antigen challenge, compared with the controls, the models showed a significantly increased white blood cell count and granulocytes count and percentage, decreased number and percentage of lymphocytes, no change in platelet concentration, significantly increased serum IL-4 and TNF-α levels, and decreased CD4(+) T cell, CD4/CD8 ratio, and C3 and C4 levels. Compared with the hematologic and immunologic profiles in pediatric HSP patients, the rat and rabbit HSP models can mimic pediatric HSP characteristics. Our studies provide two useful animal models for further investigations of the pathogenesis, diagnosis, drug screening and treatment of HSP.

Tubastatin A/ACY-1215 improves cognition in Alzheimer's disease transgenic mice.

Histone deacetylase 6 (HDAC6) is currently being discussed as a promising therapeutic target for the treatment of Alzheimer's disease (AD). Mounting evidence indicates that increased HDAC6 expression may contribute to AD-associated neurodegeneration, although beneficial effects have also been identified. In the present study, we tested the potential of two selective HDAC6 inhibitors, tubastatin A and ACY-1215, to rescue cognitive deficits in a mouse model of AD. We found that both tubastatin A and ACY-1215 alleviated behavioral deficits, altered amyloid-ß (Aß) load, and reduced tau hyperphosphorylation in AD mice without obvious adverse effects. Our data suggested that tubastatin A and ACY-1215 not only promoted tubulin acetylation, but also reduced production and facilitated autophagic clearance of Aß and hyperphosphorylated tau. Further, the decreased hyperphosphorylated tau and increased tubulin acetylation may account for the improved microtubule stability in AD mice after tubastatin A/ACY-1215 treatment. These preclinical results support the detrimental role of HDAC6 in AD, and offer prospective approaches for using tubastatin A/ACY-1215 as potential therapeutic strategy for AD.