Distinct clonal identities of B-ALLs arising after lenolidomide therapy for multiple myeloma.

Patients with multiple myeloma (MM) who are treated with lenalidomide rarely develop a secondary B-cell acute lymphoblastic leukemia (B-ALL). The clonal and biological relationship between these sequential malignancies is not yet clear. We identified 17 patients with MM treated with lenalidomide, who subsequently developed B-ALL. Patient samples were evaluated through sequencing, cytogenetics/fluorescence in situ hybridization (FISH), immunohistochemical (IHC) staining, and immunoglobulin heavy chain (IgH) clonality assessment. Samples were assessed for shared mutations and recurrently mutated genes. Through whole exome sequencing and cytogenetics/FISH analysis of 7 paired samples (MM vs matched B-ALL), no mutational overlap between samples was observed. Unique dominant IgH clonotypes between the tumors were observed in 5 paired MM/B-ALL samples. Across all 17 B-ALL samples, 14 (83%) had a TP53 variant detected. Three MM samples with sufficient sequencing depth (>500×) revealed rare cells (average of 0.6% variant allele frequency, or 1.2% of cells) with the same TP53 variant identified in the subsequent B-ALL sample. A lack of mutational overlap between MM and B-ALL samples shows that B-ALL developed as a second malignancy arising from a founding population of cells that likely represented unrelated clonal hematopoiesis caused by a TP53 mutation. The recurrent variants in TP53 in the B-ALL samples suggest a common path for malignant transformation that may be similar to that of TP53-mutant, treatment-related acute myeloid leukemia. The presence of rare cells containing TP53 variants in bone marrow at the initiation of lenalidomide treatment suggests that cellular populations containing TP53 variants expand in the presence of lenalidomide to increase the likelihood of B-ALL development.

Evaluation of PCR cycle threshold values by patient population with the quidel lyra SARS-CoV-2 assay.

The Lyra SARS-CoV-2 assay was the primary method for molecular testing performed at Barnes-Jewish Healthcare System in St. Louis, Missouri during the initial COVID-19 surge from mid-March to late-April 2020. We performed a retrospective analysis of 1,043 positive Lyra SARS-CoV-2 results during these 36 days to investigate associations between cycle threshold (CT)  value and patient characteristics. Total RNA were extracted from NP or OP swabs using either the EasyMag or KingFisher automated extraction systems and quantified with RotorGene Q (Qiagen) or Applied Biosystems 7500 Fast Dx thermocyclers respectively. Notably, we found lower a significant median lower CT for samples tested on the KingFisher-ABI 7500 fastDX (KF/ABI) system compared to the EasyMag/RotorGene (EM/RGQ) platform. Since 77.5% of our tests were ran on the EM/RGQ pipeline we then perform additional analysis on these values and found that C T values in outpatient care settings compared to samples obtained in the emergency department or inpatient had significantly lower C T values. These collective findings suggests a difference in viral load amongst various patient populations.

Lenalidomide demonstrates clinical activity in anaplastic lymphoma kinase-positive large B-cell lymphoma.

Anaplastic lymphoma kinase-positive large B-cell lymphoma (ALK+ LBCL) is known to be a rare and aggressive form of lymphoma that relapses quickly after both conventional chemotherapy and more targeted therapy. Lenalidomide is an immunomodulator that has shown safety and efficacy in multiple myeloma and is also approved for use in several types of lymphoma. In the case described here, the patient had a significant partial response to lenalidomide, which has not previously been described in this type of lymphoma. Given how aggressive and difficult to treat ALK+ LBCL is, further research is warranted to more completely elucidate the mechanism of action of lenalidomide in ALK+ LBCL and its role in treatment.

Napsin A is a highly sensitive marker for nephrogenic adenoma: an immunohistochemical study with a specificity test in genitourinary tumors.

Nephrogenic adenomas are uncommon benign lesions that are typically cytologically bland, but degenerative and reactive changes may make it difficult to distinguish these lesions from malignant entities, such as urothelial carcinoma and prostatic adenocarcinoma. In this study, we explored whether napsin A, a sensitive marker for lung adenocarcinoma, may also have a role in distinguishing nephrogenic adenoma from other genitourinary lesions. Immunohistochemically, napsin A was expressed in all 43 nephrogenic adenomas (bladder: 38, prostatic urethra: 4, and ureter: 1; mean positive tumor cells: 72%, median: 80%, range: 15-100%) and showed regional variability in its expression pattern with a bias toward surface architectures (flat, papillary) compared with stromal architectures (tubular/glandular, microcystic). We also compared napsin A with other markers including PAX8, GATA3, p63, and 34BE12. Although napsin A matched PAX8 in terms of its sensitivity for nephrogenic adenoma (100%), napsin A stained a lower percentage of tumor cells than PAX8 (72% vs 99%, respectively, P = 1.0 × 10-5). P63 was negative in all nephrogenic adenomas, whereas GATA3 showed variable staining in 25 cases (58%). All 43 nephrogenic adenomas showed variable 34BE12 staining. Finally, we profiled napsin A expression among 401 genitourinary tumors on tissue microarrays (n = 308) and full tissue blocks (N = 93) and observed napsin A positivity in 37 tumors (9%), which included urothelial carcinomas with the glandular/microcystic component differentiation (in the glandular/microcystic component in 4/6), bladder adenocarcinomas (primary: 4/4 and metastatic: 3/3), urinary tract clear-cell carcinomas (primary: 8/9, metastatic uterine primary: 1/1), and some renal tumors (17/174). All 81 pure urothelial carcinomas and 53 prostatic acinar adenocarcinomas were negative for napsin A. Our study indicates that napsin A is a highly sensitive marker for nephrogenic adenoma and can serve as a useful addition in immunohistochemical panels seeking to distinguish it from pure urothelial carcinoma and prostatic acinar adenocarcinoma but not clear-cell carcinoma or urothelial carcinoma with glandular differentiation.

Tumor mutation burden and checkpoint immunotherapy markers in primary and metastatic synovial sarcoma.

Synovial sarcoma (SS) is a soft-tissue malignancy that most often affects patients aged between 15 and 40 years, and the prognosis for patients with metastatic disease is generally poor. This study was performed to evaluate checkpoint blockade immunotherapy markers in SS, including tumor mutational burden (TMB), DNA mismatch repair (MMR) status, and PDL-1 (programmed cell death ligand 1), PD1 (programmed cell death 1), and CD8 expression by normal-tumor paired whole-exome sequencing (WES) and immunohistochemistry (IHC). Outcomes evaluated included event-free and overall survival. Twenty one (21) FISH (Fluorescence In Situ Hybridization)-confirmed SS cases (11 F, 10 M) were studied, with age ranging from 8 to 89 years at diagnosis and follow-up ranging from 1 to 16 years. TMB (n = 16) ranged from 0.83 to 212/Mb (median, 1.7). Only one case showed a high TMB of 212/Mb and missense variants of MMR genes in the primary tumor, while the other 15 cases had a low TMB of less than 5/Mb. IHC was performed on all 21 tumor samples for PD-L1, PD1, CD8, and MMR proteins. PD-L1 membranous staining was detected in 3 of 21 cases (14.3%), ranging from 1 to 5% for tumor proportion score and 1-10 for combined positive score. PD1 was detected in 15 of 21 cases (71.4%), ranging from 1 to 25/HPF (high power field) (median, 2). CD8 stain was seen in all cases, ranging from 2 to 60/HPF (median, 5). PD1 staining results correlated with CD8 staining results (P < 0.0001). No correlation of TMB or IHC markers was found with survival. No fixed pattern of TMB or IHCs between primary and metastatic tumors was observed; there was no correlation between TMB or IHCs and age, location, or diagnosis subtype. All of the cases tested showed retained expression of MMR proteins. The results show that for SS, a tumor with strong driver translocation, most cases have a low TMB, but occasionally a high TMB may be present, as observed in 1 of the 16 (6.25%) cases. The demonstration of a subgroup of SS cases with high TMB might explain the 10% response rate to checkpoint immunotherapy observed in clinical trials in patients with SS.

Clinical and Pathological Features of a Newborn With Compound Heterozygous ANKS6 Variants.

We report a term female infant born to nonconsanguineous parents who presented with renal failure at birth, hypothyroidism, cholestasis, and progressive cardiac dysfunction. Multigene next-generation sequencing panels for cholestasis, cardiomyopathy, and cystic renal disease did not reveal a unifying diagnosis. Whole exome sequencing revealed compound heterozygous pathogenic variants in ANKS6 (Ankyrin Repeat and Sterile Alpha Motif Domain Containing 6), which encodes a protein that interacts with other proteins of the Inv compartment of cilium (NEK8, NPHP2/INVS, and NPHP3). ANKS6 has been shown to be important for early renal development and cardiac looping in animal models. Autopsy revealed cystic renal dysplasia and cardiomyocyte hypertrophy, disarray, and focal necrosis. Liver histology revealed cholestasis and centrilobular necrosis, which was likely a result of progressive cardiac failure. This is the first report of compound heterozygous variants in ANKS6 leading to a nephronopthisis-related ciliopathy-like phenotype. We conclude that pathogenic variants in ANKS6 may present early in life with severe renal and cardiac failure, similar to subjects with variants in genes encoding other proteins in the Inv compartment of the cilium.

Reactivity with the EpCAM-specific antibodies MOC-31 and Ber-Ep4 in plasma cell neoplasms: a potential diagnostic pitfall in cytology samples.

OBJECTIVE: Epithelial cell adhesion molecule (EpCAM) is a protein expressed on surfaces of healthy epithelia, and is overexpressed in dysplasias and carcinomas. Immunohistochemistry (IHC) utilizing antibodies that react with EpCAM, such as MOC-31 and Ber-EP4, distinguish reactive mesothelial cells from carcinomas in serous effusions. IHC is crucial in effusions with singly dispersed atypical cells, a scenario with a broad differential, including hematopoietic malignancies. Plasma cell neoplasms (PCN) are the second most common hematopoietic malignancy, manifesting as multiple myeloma or plasmacytoma, with 6% of cases developing serous cavity involvement. Most PCNs are readily recognizable; however, variants that deviate from the classic cytomorphology risk erroneous diagnosis. This study demonstrates EpCAM expression in a subset of PCNs, highlighting a potential diagnostic pitfall in serous effusion cytology. METHODS: A 10-year retrospective search for cytology specimens with a diagnosis of PCN was performed. All cases demonstrating CD138/CD38 and monoclonal immunoglobulin expression, and adequately cellular cell block were included. IHC analysis for MOC-31 and Ber-EP4 was performed using Ventana Benchmark Ultra. Scoring was performed as follows: total IHC score equals the positive proportion (0 = no positive tumor cells; 1 = <1%; 2 = 1-10%; 3 =11-33%; 4 = 34-66%; 5 = 67-100%) plus staining intensity (0, no staining; 1, weak; 2, moderate; 3, strong). A score > 4 was considered positive. RESULTS: 2 of 28 (7%) PCNs demonstrated positivity for MOC-31 and Ber-Ep4. CONCLUSION: A subset of PCNs in cytology samples show positivity for MOC-31 and Ber-EP4 which could result in misinterpretation as carcinoma.

Tumor mutation burden, DNA mismatch repair status and checkpoint immunotherapy markers in primary and relapsed malignant rhabdoid tumors.

INTRODUCTION: Malignant rhabdoid tumor (MRT) is a rare, aggressive pediatric tumor of nuclear lineage. It is mainly characterized by germline or somatic SMARCB1 (INI1) driver mutations. To characterize the potential for immunotherapy in untreated and treated MRT, current study investigated tumor mutational burden (TMB) and other biomarkers in MRT. MATERIAL AND METHODS: Normal-tumor paired whole exome sequencing (WES) and/or immunohistochemistry (IHC) of DNA mismatch repair (MMR) proteins, PD-L1, PD-1 and CD8 were performed in 16 cases, some with both primary and relapsed tumor. RESULTS: Five cases subjected to WES demonstrated germline SMARCB1 (INI1) mutations. TMB was 0.7-1.07/Mb in 4 of the 5 primary untreated tumors, and 33.81/Mb in one case with pathogenic MMR, POLD, and POLE mutations. Ten cases tested for MMR status by IHC showed retained nuclear expression of the proteins. Eight of the 16 cases (8/16, 50%) showed membranous expression of PD-L1 in 10-70% of tumor cells (tumor proportion score, TPS). Nine cases (9/16, 56.3%) showed high (>2/HPF) tumor infiltrating lymphocytes with PD-1 staining ranging 10-60%, correlating with tumor PD-L1 staining (p < 0.0001). Between post-treatment metastatic tumors and the pre-treatment primary tumors, TMB was similar while PD-L1 TPS was similar or lower. CONCLUSION: MRT has a low TMB. Nonetheless, because a subset of MRT cases have a PD-L1 TPS greater than the cutoff for checkpoint therapy in other malignancies, the utility of immune checkpoint inhibitors should be studied in this patient population.

HER2 Basolateral versus Circumferential IHC Expression Is Dependent on Polarity and Differentiation of Epithelial Cells in Gastric/GE Adenocarcinoma.

AIM: Antigenic expression in epithelial cells can be heterogeneous which may pose a problem in immunohistochemical (IHC) analysis of tumor markers, in particular, predictive markers like HER2. Studies have shown that epithelial cells have distinct apical and basolateral domains which are separated by tight junctions. The cell membrane in these two domains has a different composition of macromolecules and hence can have variable antigen expression on immunohistochemistry. In our study, we aimed to investigate this phenomenon of basolateral versus circumferential IHC staining of HER2 in gastric/GE adenocarcinoma. METHODS: We selected 45 cases of gastric/GE adenocarcinoma and evaluated equal number of specimens (15 each) showing well-differentiated, moderately differentiated, and poorly differentiated morphology. All cases had 3+ HER2 score as per CAP guidelines. HER2-membrane staining pattern in all specimens was analyzed. RESULTS: Cases with well-differentiated morphology showed only basolateral or lateral membrane staining in most cases. Poorly differentiated adenocarcinoma samples showed circumferential staining (both basolateral and luminal) in all cases with highly significant p value. Mixed staining pattern was observed in moderately differentiated cases. Diffuse expression of E-cadherin in well-differentiated adenocarcinoma and loss in poorly differentiated tumors were also statistically significant. CONCLUSION: These findings suggest that HER2 in gastric epithelium has a polarized distribution which is maintained by the fence function of tight junctions. With progression to high grade cancer, the glandular structural differentiation in gastric mucosa is lost, along with disruption of tight junctions. This leads to loss of cell polarity and migration of antigens across the membrane.

Knowledge and Perception Regarding Autism among Primary School Teachers: A Cross-sectional Survey from Pakistan, South Asia.

BACKGROUND: Early detection and intervention seem to improve development in autistic children, and teachers form an important part of their early social environment. OBJECTIVES: The objective of this study was to assess baseline knowledge and misconceptions regarding autism among school teachers and evaluate factors influencing their knowledge. MATERIALS AND METHODS: This is a cross-sectional survey enrolling primary school teachers using a self-administered questionnaire. RESULTS: Seventy-three teachers (mean age of 34 years, 66% females) responded. Gaps in awareness and knowledge were found. About 52 (71.2%) teachers identified themselves as having some knowledge about autism, with 23 (44.2%) among this group understanding autism as a neurological/mental disorder. The majority (73.1%) believe that special education is a helpful intervention. The only significant factor that influenced knowledge among teachers was attendance of behavioral classes (P = 0.01). CONCLUSION: Results suggest that teachers have an inadequate understanding of autism due to several misconceptions. This calls for increased education of teachers with regard to autism and other childhood disorders.