Fibrin microthrombi in bladder urothelium after SARS-CoV-2 infection: Case report.

A 45-year-old male with diabetes, hypertension and hyperlipidemia was referred to urology due to persistent symptoms of urinary frequency, urgency, nocturia, erectile dysfunction, and constant pain localized to the bladder, pelvis, and perineal area, 3-4 months after SARS-CoV-2 infection. A bladder biopsy showed urothelial mucosa and submucosa with hemorrhage and fibrin microthrombi in blood vessels. Hydrodistention of the bladder and pelvic floor physical therapy resolved symptoms, though bladder and pain symptoms returned upon reinfection with SARS-CoV-2. Urinalysis revealed elevated urinary interleukin-8, which may indicate localized bladder inflammation.

Focal Segmental Glomerulosclerosis (FSGS) Progressing to Collapsing Glomerulopathy in Renal Transplant Recipients With and Without COVID-19 Infection.

BACKGROUND: Collapsing glomerulopathy (CGN) secondary to HIV or COVID-19 infection mainly occurs in patients of African American descent due to APOL-1 gene mutations, but CGN is occasionally reported in white patients. CGNs are rarely reported in renal transplant biopsies and their association with idiopathic focal segmental glomerulosclerosis (FSGS) is unclear. METHODS AND RESULTS: Patient #1 was a 48-year-old Caucasian white man who had a renal transplant 8 years ago and was recently diagnosed with COVID-19 infection. Two weeks post infection, his serum creatinine (SCr) increased to 2.01 mg/dL from a baseline of 1.40 mg/dL, and he developed concomitant nephrotic range proteinuria. The first renal transplant biopsy showed FSGS. Four weeks later, his sCr level increased to 2.65 mg/dL with worsening proteinuria, and a second renal transplant biopsy revealed CGN. Patient #2 was a 32-year-old African American man whose native renal biopsy revealed primary FSGS. He received a renal transplant with initial post-transplant sCr level at 1.17 mg/dL. Four months later, his sCr and protein-to-creatinine ratio began to rise. Sequential biopsies revealed that the patient had developed recurrent FSGS, which progressed to show features of CGN. The CGN was further confirmed in his transplant kidney graft at autopsy later. CONCLUSIONS: This is the first case report of CGN in a white renal recipient with COVID-19 infection. The pathologic presentations of FSGS progressing to collapsing FSGS in our 2 renal transplant recipients suggest that FSGS and GGN may share a common pathophysiologic mechanism of podocytopathy.

Clinical Significance of PTEN Deletion, Mutation, and Loss of PTEN Expression in De Novo Diffuse Large B-Cell Lymphoma.

PTEN loss has been associated with poorer prognosis in many solid tumors. However, such investigation in lymphomas is limited. In this study, PTEN cytoplasmic and nuclear expression, PTEN gene deletion, and PTEN mutations were evaluated in two independent cohorts of diffuse large B-cell lymphoma (DLBCL). Cytoplasmic PTEN expression was found in approximately 67% of total 747 DLBCL cases, more frequently in the activated B-cell-like subtype. Nuclear PTEN expression was less frequent and at lower levels, which significantly correlated with higher PTEN mRNA expression. Remarkably, loss of PTEN protein expression was associated with poorer survival only in DLBCL with AKT hyperactivation. In contrast, high PTEN expression was associated with Myc expression and poorer survival in cases without abnormal AKT activation. Genetic and epigenetic mechanisms for loss of PTEN expression were investigated. PTEN deletions (mostly heterozygous) were detected in 11.3% of DLBCL, and showed opposite prognostic effects in patients with AKT hyperactivation and in MYC rearranged DLBCL patients. PTEN mutations, detected in 10.6% of patients, were associated with upregulation of genes involved in central nervous system function, metabolism, and AKT/mTOR signaling regulation. Loss of PTEN cytoplasmic expression was also associated with TP53 mutations, higher PTEN-targeting microRNA expression, and lower PD-L1 expression. Remarkably, low PTEN mRNA expression was associated with down-regulation of a group of genes involved in immune responses and B-cell development/differentiation, and poorer survival in DLBCL independent of AKT activation. Collectively, multi-levels of PTEN abnormalities and dysregulation may play important roles in PTEN expression and loss, and that loss of PTEN tumor-suppressor function contributes to the poor survival of DLBCL patients with AKT hyperactivation.

AKT Hyperactivation and the Potential of AKT-Targeted Therapy in Diffuse Large B-Cell Lymphoma.

AKT signaling is important for proliferation and survival of tumor cells. The clinical significance of AKT activation in diffuse large B-cell lymphoma (DLBCL) is not well analyzed. Here, we assessed expression of phosphorylated AKT (p-AKT) in 522 DLBCL patients. We found that high levels of p-AKT nuclear expression, observed in 24.3% of the study cohort, were associated with significantly worse progression-free survival and Myc and Bcl-2 overexpression. However, multivariate analysis indicated that AKT hyperactivation was not an independent factor. miRNA profiling analysis demonstrated that 63 miRNAs directly or indirectly related to the phosphatidylinositol 3-kinase/AKT/mechanistic target of rapamycin pathway were differentially expressed between DLBCLs with high and low p-AKT nuclear expression. We further targeted AKT signaling using a highly selective AKT inhibitor MK-2206 in 26 representative DLBCL cell lines and delineated signaling alterations using a reverse-phase protein array. MK-2206 treatment inhibited lymphoma cell viability, and MK-2206 sensitivity correlated with AKT activation status in DLBCL cells. On MK-2206 treatment, p-AKT levels and downstream targets of AKT signaling were significantly decreased, likely because of the decreased feedback repression; Rictor and phosphatidylinositol 3-kinase expression and other compensatory pathways were also induced. This study demonstrates the clinical and therapeutic implications of AKT hyperactivation in DLBCL and suggests that AKT inhibitors need to be combined with other targeted agents for DLBCL to achieve optimal clinical efficacy.

An exploratory clinical trial of bortezomib in patients with lower risk myelodysplastic syndromes.

Myelodysplastic syndromes (MDSs) are characterized by ineffective hematopoiesis and an increased risk of transformation. Few effective therapies are available for lower risk MDS patients, especially after the failure of hypomethylating agents. MDS progenitor cells are dependent on the nuclear factor-κB (NF-κB) for survival, which makes it an attractive therapeutic target. As a proteosomal inhibitor, bortezomib is thought to have inhibitory activity against NF-κB. We designed a proof-of-principle study of subcutaneous (SC) bortezomib in lower risk MDS patients with evidence of NF-κB activation in their bone marrow. Fifteen patients were treated, their median age was 71 (range 56-87), 33% were low and 67% int-1 by IPSS, median number of prior therapies was 2, all patients were transfusion dependent. Baseline median pp65 percentage was 31% and 11 patients had evidence of ring sideroblasts (RS). SC bortezomib was safe, well tolerated with no excess toxicity. Three patients out of the 15 (20%) had evidence of response with hematologic improvement (HI-E). Bortezomib caused a decrease in pp65 levels in 7 out of 13 evaluable patients (54%, P = .025). Of interest, unexpectedly, we observed a significant decrease in RS in 7 out of 10 (70%) evaluable patients during treatment. In conclusion, this study suggests that NF-κB activation, measured by pp65 levels, may be a useful biomarker in MDS. Bortezomib is safe in this patient population but has modest clinical activity. The role of the proteasome in the genesis of RS needs further study.

Assessment of CD37 B-cell antigen and cell of origin significantly improves risk prediction in diffuse large B-cell lymphoma.

CD37 (tetraspanin TSPAN26) is a B-cell surface antigen widely expressed on mature B cells. CD37 is involved in immune regulation and tumor suppression but its function has not been fully elucidated. We assessed CD37 expression in de novo diffuse large B-cell lymphoma (DLBCL), and investigated its clinical and biologic significance in 773 patients treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) and 231 patients treated with CHOP. We found that CD37 loss (CD37-) in ∼60% of DLBCL patients showed significantly decreased survival after R-CHOP treatment, independent of the International Prognostic Index (IPI), germinal center B-cell-like (GCB)/activated B-cell-like (ABC) cell of origin, nodal/extranodal primary origin, and the prognostic factors associated with CD37-, including TP53 mutation, NF-κBhigh, Mychigh, phosphorylated STAT3high, survivinhigh, p63-, and BCL6 translocation. CD37 positivity predicted superior survival, abolishing the prognostic impact of high IPI and above biomarkers in GCB-DLBCL but not in ABC-DLBCL. Combining risk scores for CD37- status and ABC cell of origin with the IPI, defined as molecularly adjusted IPI for R-CHOP (M-IPI-R), or IPI plus immunohistochemistry (IHC; IPI+IHC) for CD37, Myc, and Bcl-2, significantly improved risk prediction over IPI alone. Gene expression profiling suggested that decreased CD20 and increased PD-1 levels in CD37- DLBCL, ICOSLG upregulation in CD37+ GCB-DLBCL, and CD37 functions during R-CHOP treatment underlie the pivotal role of CD37 status in clinical outcomes. In conclusion, CD37 is a critical determinant of R-CHOP outcome in DLBCL especially in GCB-DLBCL, representing its importance for optimal rituximab action and sustained immune responses. The combined molecular and clinical prognostic indices, M-IPI-R and IPI+IHC, have remarkable predictive values in R-CHOP-treated DLBCL.

COLD-PCR: Applications and Advantages.

Co-amplification at lower denaturation temperature-based polymerase chain reaction (COLD-PCR) is a single-step amplification method that results in the enhancement of both known and unknown minority alleles during PCR, irrespective of mutation type and position. This method is based on exploitation of the critical temperature, Tc, at which mutation-containing DNA is preferentially melted over wild type. COLD-PCR can be a good strategy for mutation detection in specimens with high nonneoplastic cell content, small specimens in which neoplastic cells are difficult to micro-dissect and therefore enrich, and whenever a mutation is suspected to be present but is undetectable using conventional PCR and sequencing methods. We describe in this chapter our COLD-PCR-based pyrosequencing method for KRAS mutation detection in various clinical samples using DNA extracted from either fresh or fixed paraffin-embedded tissue specimens.

Tetraspanin CD37 protects against the development of B cell lymphoma.

Worldwide, B cell non-Hodgkin lymphoma is the most common hematological malignancy and represents a substantial clinical problem. The molecular events that lead to B cell lymphoma are only partially defined. Here, we have provided evidence that deficiency of tetraspanin superfamily member CD37, which is important for B cell function, induces the development of B cell lymphoma. Mice lacking CD37 developed germinal center-derived B cell lymphoma in lymph nodes and spleens with a higher incidence than Bcl2 transgenic mice. We discovered that CD37 interacts with suppressor of cytokine signaling 3 (SOCS3); therefore, absence of CD37 drives tumor development through constitutive activation of the IL-6 signaling pathway. Moreover, animals deficient for both Cd37 and Il6 were fully protected against lymphoma development, confirming the involvement of the IL-6 pathway in driving tumorigenesis. Loss of CD37 on neoplastic cells in patients with diffuse large B cell lymphoma (DLBCL) directly correlated with activation of the IL-6 signaling pathway and with worse progression-free and overall survival. Together, this study identifies CD37 as a tumor suppressor that directly protects against B cell lymphomagenesis and provides a strong rationale for blocking the IL-6 pathway in patients with CD37- B cell malignancies as a possible therapeutic intervention.

Clinical validation of a multipurpose assay for detection and genotyping of CALR mutations in myeloproliferative neoplasms.

OBJECTIVES: To develop a polymerase chain reaction (PCR)-based approach to detect CALR mutations in myeloproliferative neoplasms (MPNs) in a clinical laboratory. METHODS: DNA was extracted from bone marrow aspirate samples of 67 JAK2 wild-type MPNs (22 with matched peripheral blood), 54 cases of unclassifiable myelodysplastic syndrome/MPN, and 16 cases of atypical chronic myeloid leukemia. We used genomic DNA to detect somatic mutations in exon 9 of CALR and PCR with fluorescently labeled and M13-tagged primers and subjected the products to capillary electrophoresis (CE) followed by Sanger sequencing. Detailed assay performance characteristics were established. RESULTS: We identified CALR mutations in 19 (28.4%) of 67 JAK2-negative MPNs, including 14 type I (52-base pair [bp] deletion), four type II (5-bp insertions), and one type III (18-bp deletion). All mutations were confirmed by Sanger sequencing. Sensitivity studies showed 2.5% and 5% mutation detection levels by CE and Sanger sequencing, respectively, with high reproducibility. CONCLUSIONS: This assay allows for rapid, convenient screening for CALR mutations in MPNs, thereby reducing the number of cases that require assessment by Sanger sequencing, reducing labor and improving turnaround time.

Prognostic impact of acquisition of cytogenetic abnormalities during the course of chronic myelomonocytic leukemia.

Karyotypic abnormalities are detected in 20-40% of chronic myelomonocytic leukemia (CMML) patients at initial diagnosis and have been shown to correlate with patients' outcome. The significance of acquisition of cytogenetic abnormalities (ACA) during the course of CMML, however, is largely unknown. In a cohort of 314 CMML patients, karyotypic abnormalities were detected in 106 (34%) patients at the time of diagnosis; and ACA were detected in 80 (25%) patients after a median interval of 17 months (range, 2-117 months). The most frequently observed ACA were a complex karyotype, followed by +21, -7/del(7q), del(20q), i(17q), and -17/del(17p). ACA appeared to occur more frequently in patients with a normal or lower risk karyotype. Progression to AML was seen in 44 of 80 (55%) patients with ACA versus 67 of 234 (29%) patients without ACA (P < 0.0001). Presence of ACA predicted an inferior leukemia-free survival (LFS) by univariate (P = 0.0435) and multivariate analysis (HR = 1.892, P = 0.006). While acquisition of a complex karyotype was positively correlated with AML progression (P = 0.0086), del(20q) was associated with a stable disease (P = 0.0198). We conclude that ACA occur in ∼20-30% of CMML patients during the course of disease, and are significantly associated with AML progression and a shorter LFS. Karyotypic abnormalities, either present at diagnosis or acquired during the course of disease, have prognostic implication in CMML patients.

Clinical and biological significance of de novo CD5+ diffuse large B-cell lymphoma in Western countries.

CD5 is a pan-T-cell surface marker and is rarely expressed in diffuse large B-cell lymphoma (DLBCL). Large-scale studies of de novo CD5+ DLBCL are lacking in Western countries. In this study by the DLBCL Rituximab-CHOP Consortium, CD5 was expressed in 5.5% of 879 DLBCL patients from Western countries. CD5+ DLBCL was associated with higher frequencies of >1 ECOG performance status, bone marrow involvement, central nervous system relapse, activated B-cell-like subtype, Bcl-2 overexpression, and STAT3 and NF-κB activation, whereas rarely expressed single-stranded DNA-binding protein 2 (SSBP2), CD30 or had MYC mutations. With standard R-CHOP chemotherapy, CD5+ DLBCL patients had significantly worse overall survival (median, 25.3 months vs. not reached, P< .0001) and progression-free survival (median, 21.3 vs. 85.8 months, P< .0001) than CD5- DLBCL patients, which was independent of Bcl-2, STAT3, NF-κB and the International Prognostic Index. Interestingly, SSBP2 expression abolished the prognostic significance of CD5 expression, suggesting a tumor-suppressor role of SSBP2 for CD5 signaling. Gene-expression profiling demonstrated that B-cell receptor signaling dysfunction and microenvironment alterations are the important mechanisms underlying the clinical impact of CD5 expression. This study shows the distinctive clinical and biological features of CD5+ DLBCL patients in Western countries and underscores important pathways with therapeutic implications.

Hotspot mutation panel testing reveals clonal evolution in a study of 265 paired primary and metastatic tumors.

PURPOSE: We used a clinical next-generation sequencing (NGS) hotspot mutation panel to investigate clonal evolution in paired primary and metastatic tumors. EXPERIMENTAL DESIGN: A total of 265 primary and metastatic tumor pairs were sequenced using a 46-gene cancer mutation panel capable of detecting one or more single-nucleotide variants as well as small insertions/deletions. Mutations were tabulated together with tumor type and percentage, mutational variant frequency, time interval between onset of primary tumor and metastasis, and neoadjuvant therapy status. RESULTS: Of note, 227 of 265 (85.7%) tumor metastasis pairs showed identical mutation calls. Of the tumor pairs with identical mutation calls, 160 (60.4%) possessed defining somatic mutation signatures and 67 (25.3%) did not exhibit any somatic mutations. There were 38 (14.3%) cases that showed at least one novel mutation call between the primary and metastasis. Metastases were almost two times more likely to show novel mutations (n = 20, 7.5%) than primary tumors (n = 12, 4.5%). TP53 was the most common additionally mutated gene in metastatic lesions, followed by PIK3CA and SMAD4. PIK3CA mutations were more often associated with metastasis in colon carcinoma samples. CONCLUSIONS: Clinical NGS hotspot panels can be useful in analyzing clonal evolution within tumors as well as in determining subclonal mutations that can expand in future metastases. PIK3CA, SMAD4, and TP53 are most often involved in clonal divergence, providing potential targets that may help guide the clinical management of tumor progression or metastases.

Comparison of next-generation sequencing mutation profiling with BRAF and IDH1 mutation-specific immunohistochemistry.

Mutation-specific antibodies for BRAF V600E and IDH1 R132H offer convenient immunohistochemical (IHC) assays to detect these mutations in tumors. Previous studies using these antibodies have shown high sensitivity and specificity, but use in routine diagnosis with qualitative assessment has not been well studied. In this retrospective study, we reviewed BRAF and IDH1 mutation-specific IHC results compared with separately obtained clinical next-generation sequencing results. For 67 tumors with combined IDH1 IHC and mutation data, IHC was unequivocally reported as positive or negative in all cases. Sensitivity of IHC for IDH1 R132H was 98% and specificity was 100% compared with mutation status. Four IHC-negative samples showed non-R132H IDH1 mutations including R132C, R132G, and P127T. For 128 tumors with combined BRAF IHC and mutation data, IHC was positive in 33, negative in 82, and equivocal in 13 tumors. The sensitivity of IHC was 97% and specificity was 99% when including only unequivocally positive or negative results. If equivocal IHC cases were included in the analysis as negative, sensitivity fell to 81%. If equivocal cases were classified as positive, specificity dropped to 91%. Eight IHC-negative samples showed non-V600E BRAF mutations including V600K, N581I, V600M, and K601E. We conclude that IHC for BRAF V600E and IDH1 R132H is relatively sensitive and specific, but there is a discordance rate that is not trivial. In addition, a significant proportion of patients harbor BRAF non-V600E or IDH1 non-R132H mutations not detectable by IHC, potentially limiting utility of IHC screening for BRAF and IDH1 mutations.

Flow cytometric immunophenotypic analysis of systemic mastocytosis involving bone marrow.

CONTEXT: Mast cells of systemic mastocytosis (SM) have aberrant immunophenotypes that are useful for their detection by flow cytometry immunophenotyping. OBJECTIVES: To assess the usefulness of CD2, CD25, and other antigens for establishing the diagnosis of SM in bone marrow using flow cytometry immunophenotyping. DESIGN: We studied 50 bone marrow aspirates of patients with SM using flow cytometry immunophenotyping. The bone marrow aspirates were stained with antibodies specific for CD2, CD25, CD35, CD59, CD63, and CD69. For the detection of CD2 and CD25, antibodies conjugated with phycoerythrin (PE) or fluorescein isothiocyanate (FITC) were compared. CD45-PerCP and CD117-APC were used for gating. Data were acquired on FACS Calibur cytometers and analyzed using CellQuest software. RESULTS: CD2 and CD25 were positive in 41 of 50 (82%) and 45 of 50 (90%) SM cases, respectively. For CD2, the PE-conjugated antibody yielded better sensitivity than the FITC-conjugated antibody (31 of 40 [78%] versus 28 of 40 [70%]). For CD25, PE-conjugated and FITC-conjugated antibodies showed similar detection sensitivity, although the intensity of expression was brighter with CD25-PE. Compared with immunohistochemistry, flow cytometry immunophenotyping was superior for detecting CD2 (14 of 23 [61%] versus 9 of 23 [39%]). Other antigens frequently overexpressed in SM were CD35 (43 of 50 [86%]), CD59 (46 of 50 [92%]), CD63 (43 of 49 [88%]), and CD69 (39 of 48 [81%]). CONCLUSIONS: Flow cytometry immunophenotyping is a rapid and sensitive technique for characterizing mast cells in bone marrow aspirate specimens. The use of PE-conjugated antibodies for CD2 and CD25 improves the detection rate (CD2) or facilitates analysis (CD25); therefore, PE-conjugated antibodies are suggested. Antibodies reactive with CD35, CD59, CD63, and CD69 are also helpful in detecting SM in bone marrow.

Pathobiology of hemophilic synovitis I: overexpression of mdm2 oncogene.

Hemophilia is a genetic disease caused by a deficiency of blood coagulation factor VIII or IX. Bleeding into joints is the most frequent manifestation of hemophilia. Hemarthrosis results in an inflammatory and proliferative disorder termed hemophilic synovitis (HS). In time, a debilitating, crippling arthritis, hemophilic arthropathy, develops. Although the clinical sequence of events from joint bleeding to synovitis to arthropathy is well documented, the component or components in blood and the molecular changes responsible for hemophilic synovitis are not known. Iron has long been suspected to be the culprit but direct evidence has been lacking. Previously, we showed that iron increased human synovial cell proliferation and induced c-myc expression. Here we show that bleeding into a joint in vivo and iron in vitro result in increased expression of the p53-binding protein, mdm2. Iron induced the expression of mdm2 by normal human synovial cells approximately 8-fold. In a murine model of human hemophilia A, hemarthrosis resulted in pathologic changes observed in human hemophilic synovitis and a marked increase in synovial cell proliferation. Iron, in vitro, induced the expression of mdm2. The molecular changes induced by iron in the blood may be the basis of the increase in cell proliferation and the development of hemophilic synovitis.