Clinical utility of next-generation sequencing in the diagnosis of hereditary haemolytic anaemias.

Hereditary haemolytic anaemias are genetically and phenotypically heterogeneous disorders characterized by increased red cell destruction, with consequences ranging from innocuous to severe life-threatening anaemia. Diagnostic laboratories endeavour to assist clinicians reach the exact patient diagnosis by using tests principally based on morphological and biochemical techniques. However, these routine studies may be inconclusive, particularly in newborn infants and when transfusions have recently been administered. Large numbers and size of the potentially involved genes also impose a practical challenge for molecular diagnosis using routine sequencing approaches. To overcome these diagnostic shortcomings, we have utilized next-generation sequencing to provide a high-throughput, highly sensitive assay. We developed a panel interrogating 28 genes encoding cytoskeletal proteins and enzymes with sequencing coverage of the coding regions, splice site junctions, deep intronic and regulatory regions. We then evaluated 19 samples, including infants with unexplained extreme hyperbilirubinaemia and patients with transfusion-dependent haemolytic anaemia. Where possible, inheritance patterns of pathogenic mutations were determined by sequencing of immediate relatives. We conclude that this next-generation sequencing panel could be a cost-effective approach to molecular diagnosis of hereditary haemolytic anaemia, especially when the family history is uninformative or when routine laboratory testing fails to identify the causative haemolytic process.

Overdiagnosis of high-grade dysplasia in Barrett's esophagus: a multicenter, international study.

Numerous histological mimics of high-grade dysplasia in Barrett's esophagus predispose to overdiagnosis and potential serious mismanagement, including unnecessary esophagectomy. This study investigates the prevalence and sources of this problem. Biopsies from 485 patients diagnosed with Barrett's high-grade dysplasia were screened for a multi-institutional, international Barrett's endoscopic ablation trial. Screening included review of the original diagnostic slides and an additional protocol endoscopy with an extensive biopsy sampling. Observer variability by the study pathologists was assessed through two blinded diagnostic rounds on 437 biopsies from 26 random study endoscopies. Study diagnostic reassessments revealed significantly lower rates of high-grade dysplasia. Only 248 patients (51%) were confirmed to have high-grade dysplasia. The remaining patients had inflamed gastric cardia without Barrett's (n=18; 7%), Barrett's without dysplasia (n=35; 15%), indefinite change (n=61; 26%), low-grade dysplasia (n=79; 33%), adenocarcinoma (n=43; 18%), and other (n=1; <1%), yielding an alarming total of 194 or 40% of patients who were overdiagnosed with Barrett's high-grade dysplasia. Study pathologists achieved a high-level agreement (90% three-way inter-observer agreement per biopsy, Kappa value 0.77) for high-grade dysplasia. Confounding factors promoting overdiagnosis included Barrett's inflammatory atypia (n=182), atypia limited to the basal metaplastic glands (n=147), imprecise criteria for low grade neoplasia (n=102), tangential sectioning artifact (n=59), and reactive gastric cardiac mucosa (n=38). A total of 194 patients (40%) were overdiagnosed with Barrett's high-grade dysplasia, as affirmed by the extensive screening process and high-level study pathologist agreement. The multiple diagnostic pitfalls uncovered should help raise pathologists' awareness of this problem and improve diagnostic accuracy.

Optimized immunohistochemical panel to differentiate myeloid sarcoma from blastic plasmacytoid dendritic cell neoplasm.

Myeloid sarcoma (MS) and blastic plasmacytoid dendritic cell neoplasm (BPDCN) can be difficult to distinguish morphologically, even with the use of extensive immunohistochemical studies. Three new research markers, myxovirus A (MxA), CLA/CD162, and CD303/BDCA-2, have been reported to be positive in BPDCN, but their clinical utility has never been tested. We compared these markers to other antibodies that have been used traditionally to distinguish MS from BPDCN to assess the utility of these newer antibodies in differential diagnosis. Formalin-fixed, paraffin-embedded tissue sections of 23 MS and 17 BPDCN cases were assessed using immunohistochemical analysis for CD4, CD14, CD33, CD43, CD56, CD68, CD123, CD163, myeloperoxidase, lysozyme, terminal deoxynucleotidyl transferase (TdT), T-cell leukemia 1 (TCL-1), MxA, cutaneous lymphocyte-associated antigen (CLA)/CD162, and blood dendritic cell antigen 2 (BDCA2)/CD303. We identified antibodies with a high predictive value of ≥ 90% and used these markers to develop an approach to classification using specific staining criteria. Diagnostic classification criteria were based on staining patterns of one or more of the seven markers. BPDCN was associated with positive staining for CD56, TdT, or TCL1, or negative staining for lysozyme. MS was associated with positive staining for lysozyme or myeloperoxidase, or negative staining for CD56, CD123, myxovirus, or TCL1. The immunohistochemical staining patterns observed using a panel that includes MPO, CD56, CD123, TCL1, TdT, and MxA, are predictive of MS or BPDCN. In this study, neither CD162 nor CD303 had good predictive value in distinguishing MS from BPDCN.

Myelodysplastic Neoplasms (MDS) with Ring Sideroblasts or SF3B1 Mutations: The Improved Clinical Utility of World Health Organization and International Consensus Classification 2022 Definitions, a Single-Centre Retrospective Chart Review.

Myelodysplastic neoplasms (MDS) with ring sideroblasts (RS) are diagnosed via bone marrow aspiration in the presence of either (i) ≥15% RS or (ii) 5-14% RS and an SF3B1 mutation. In the MEDALIST trial and in an interim analysis of the COMMANDS trial, lower-risk MDS-RS patients had decreased transfusion dependency with luspatercept treatment. A total of 6817 patients with suspected hematologic malignancies underwent molecular testing using a next-generation-sequencing-based genetic assay and 395 MDS patients, seen at our centre from 1 January 2018 to 31 May 2023, were reviewed. Of these, we identified 39 evaluable patients as having lower-risk MDS with SF3B1 mutations: there were 20 (51.3%) males and 19 (48.7%) females, with a median age of 77 years (range of 57 to 92). Nineteen (48.7%) patients had an isolated SF3B1 mutation with a mean variant allele frequency of 35.2% +/- 8.1%, ranging from 7.4% to 46.0%. There were 29 (74.4%) patients with ≥15% RS, 6 (15.4%) with 5 to 14% RS, one (2.6%) with 1% RS, and 3 (7.7%) with no RS. Our study suggests that a quarter of patients would be missed based on the morphologic criterion of only using RS greater than 15% and supports the revised 2022 definitions of the World Health Organization (WHO) and International Consensus Classification (ICC), which shift toward molecularly defined subtypes of MDS and appropriate testing.

Pericardial Extramedullary Hematopoiesis Associated with Metastatic Adenocarcinoma of Gastrointestinal or Pancreaticobiliary Origin: A Case Report.

Extramedullary hematopoiesis (EMH) is a rare complication of solid tumor malignancies. We describe the first case of a patient who developed EMH in the pericardium secondary to metastatic gastrointestinal or pancreaticobiliary cancer. A 58-year-old man presented with recurrent episodes of fatigue and shortness of breath and was treated with thoracocentesis and pericardiocentesis for pleural and pericardial effusions, respectively. Owing to a markedly elevated alkaline phosphatase, a bone scan was performed and demonstrated diffuse sclerotic lesions. Evaluation of pleural effusion diagnosed metastatic adenocarcinoma, and cytospin morphology of the pericardial fluid demonstrated EMH. While EMH secondary to solid tumors is commonly suggested to be due to cytokine signaling, we propose the mechanism of EMH in this patient was due to extensive disruption of bone marrow hematopoiesis, similar to what is seen in myeloproliferative neoplasms.

Extramedullary plasmacytoma of the uterine cervix arising in an asymptomatic 46-year-old female.

An extramedullary plasmacytoma is a rare type of plasma cell tumour that can be found in soft tissues throughout the body. The most common location for an extramedullary plasmacytoma is in the head and neck region. Few case reports have previously documented patients with an extramedullary plasmacytoma within the female genital tract. We report a case of a healthy and asymptomatic 46-year-old female who presented to Colposcopy Clinic with a finding of low-grade squamous intraepithelial lesion seen on a routine Pap smear. She was found to have a cervical polyp that was excised. Pathology revealed diffuse sheets of atypical plasma cells with lambda light chain restriction. She was referred to Hematology for extensive work-up as the pathology finding was concerning for a plasma cell neoplasm. Staging investigations, including bone marrow biopsy, skeletal survey, whole body PET-CT scan, serum protein electrophoresis, and serum free light chain testing, were all negative. Surgical resection with a hysterectomy was recommended as the most appropriate course of management. The treatment approach is consistent with guidelines outlined in the literature, whereby extramedullary plasmacytomas, which arise outside of the head and neck region and have clear margins, should undergo surgical resection. Extramedullary plasmacytomas carry a risk of progressing to systemic disease, such as multiple myeloma, making it crucial that these patients be followed with routine surveillance to achieve the most optimal long term survival outcome.

Radiation management of a late thoracic metastasis from an intracranial solitary fibrous tumour.

Solitary fibrous tumours (SFTs) are a rare soft tissue sarcoma. We present a case of a male patient with an SFT of the right posterior fossa, with a late metastasis to the right lung and chest wall identified 18 years later.A small number of late metastases of SFTs have previously been reported. Metastases are typically managed surgically, although there is limited evidence suggesting that radiotherapy may be effective for primary SFTs.In this case, the patient declined treatment for his metastasised cancer for 5 years. He then only agreed to radiation treatment without surgery, which uniquely resulted in excellent symptom relief and durable local control. This case illustrates the importance of further research on the role of radiation in managing SFTs, the value of long-term follow-up and the necessity of exploring barriers to care.This case also highlights issues regarding barriers to care related to late diagnoses of recurrence in rare tumours. In this case, at the time of recurrence the original tissue blocks were not available for review. The patient had moved to a different province where his former records were not easily accessible, and the original tissue blocks had been discarded. In that jurisdiction, laboratories must keep cytology slides for 5 years, histopathology slides for 10 years and paraffin blocks for 2 years. This contributed to a misdiagnosis of the recurrence as an Ewing sarcoma, resulting in the patient initially declining treatment at the time of disease recurrence, and leading to a long-standing mistrust of his physicians which impacted his decision-making.

Serous carcinoma of the fallopian tube presenting as axillary lymphadenopathy.

Serous carcinoma originating in the fallopian tube usually presents at an advanced stage with extensive pelvic disease. Palpable axillary lymphadenopathy as the initial presentation of primary fallopian tube cancer without extensive extratubal spread in the pelvis is very uncommon. We report a case of a woman with a high-grade serous carcinoma of fallopian tube origin whose initial clinical presentation was palpable axillary lymphadenopathy. On histopathologic evaluation of her primary tumor, only minimal extension to the ipsilateral ovary was identified, with no other pelvic involvement. No additional supradiaphragmatic involvement was identified on imaging. Although the primary route of spread of tubal cancer is primarily through the direct exfoliation of the cells onto the adjacent surfaces in the peritoneal cavity, less commonly, lymphatic spread can result in distant metastasis, preceding intraperitoneal extension.

Prevalence of heparin in samples submitted for lupus anticoagulant testing.

INTRODUCTION: Lupus anticoagulant (LA) testing can be affected by the presence of anticoagulant medications such as heparin, and current guidelines recommend caution when performing and interpreting LA testing for patients who are receiving anticoagulation treatment. We searched our reference laboratory database to determine the prevalence of heparin in samples submitted for LA testing. METHODS: We reviewed 18,676 LA reflexive testing panels. Heparin was identified by partial thromboplastin time (before and after heparin neutralization), thrombin time, and reptilase time results. Samples containing heparin were subclassified (not significant, significant) according to the degree of thrombin time prolongation. RESULTS: Of the panels, 1909 panels (10%) were LA positive. We found that 2011 samples (11%) contained some heparin and that 616 samples contained a significant amount of heparin (3% of all samples and 31% of samples containing heparin). LA-positive results were obtained for 80 (13%) of these samples, which represented 4% of the samples containing heparin and 0.4% of all samples. CONCLUSION: LA-testing guidelines recommend that samples not contain anticoagulant medications. Despite these recommendations, our data show that a significant proportion (11%) of these samples contain heparin. We conclude that LA-testing algorithms should use methods to identify and neutralize heparin and that laboratories should provide education regarding appropriate sample collection for LA testing.

Evaluation of an open-source machine-learning tool to quantify bone marrow plasma cells.

AIMS: The objective of this study was to develop and validate an open-source digital pathology tool, QuPath, to automatically quantify CD138-positive bone marrow plasma cells (BMPCs). METHODS: We analysed CD138-scanned slides in QuPath. In the initial training phase, manual positive and negative cell counts were performed in representative areas of 10 bone marrow biopsies. Values from the manual counts were used to fine-tune parameters to detect BMPCs, using the positive cell detection and neural network (NN) classifier functions. In the testing phase, whole-slide images in an additional 40 cases were analysed. Output from the NN classifier was compared with two pathologist's estimates of BMPC percentage. RESULTS: The training set included manual counts ranging from 2403 to 17 287 cells per slide, with a median BMPC percentage of 13% (range: 3.1%-80.7%). In the testing phase, the quantification of plasma cells by image analysis correlated well with manual counting, particularly when restricted to BMPC percentages of <30% (Pearson's r=0.96, p<0.001). Concordance between the NN classifier and the pathologist whole-slide estimates was similarly good, with an intraclass correlation of 0.83 and a weighted kappa for the NN classifier of 0.80 with the first rater and 0.90 with the second rater. This was similar to the weighted kappa between the two human raters (0.81). CONCLUSIONS: This represents a validated digital pathology tool to assist in automatically and reliably counting BMPC percentage on CD138-stained slides with an acceptable error rate.

Contrast-Enhanced Mammography in the Diagnosis of Breast Angiosarcoma.

A 60-year-old female presented for further assessment of a new right breast lump (November 2020). She had a history of a stage I (T1bN0M0) right breast invasive mammary carcinoma, grade 2 (score 7/9) with receptors ER/PR-negative, HER2/neu-positive, diagnosed four years prior to her current presentation. At that time, she was treated with a right breast lumpectomy and local radiation. Breast assessment with contrast-enhanced mammography showed new skin thickening with associated enhancement within the palpable region. Histology of subsequent ultrasound-guided biopsy found radiation-induced breast angiosarcoma. Breast angiosarcoma is a rare entity that represents less than 1% of all breast cancers. To our knowledge, this is the first case describing the imaging findings of breast angiosarcoma on contrast-enhanced mammography.

Gastrointestinal Histoplasmosis Mimicking Crohn's Disease.

Disseminated histoplasmosis is a life-threatening disease usually seen in immunocompromised patients living in endemic areas. We present an apparently immunocompetent patient with gastrointestinal histoplasmosis who was initially diagnosed with biopsy-proven Crohn's disease. Following discontinuation of anti-inflammatory drugs and institution of antifungal therapy, his gastrointestinal illness completely improved. Specific fungal staining should be routinely included in histopathologic assessment of tissue specimens diagnosed as Crohn's disease.

Recurrence of achondrogenesis type 2 in sibs: Additional evidence for germline mosaicism.

Achondrogenesis Type II (ACG2) is a lethal skeletal disorder caused by a dominant mutation in the type II collagen gene (COL2A1). Familial cases have been reported, suggesting both germline and somatic mosaicism. We report on two pregnancies from the same couple with gross, radiologic, and microscopic findings of ACG2. Molecular analysis of the second infant demonstrated heterozygosity for a c.2303G > A transition (p.Gly768Asp) in exon 33 of the COL2A1 gene. Although this mutation could not be proven by molecular studies in the first infant, identical findings in two affected pregnancies support germline mosaicism as the cause of ACG2 in this family.

EBV-positive T/NK-associated lymphoproliferative disorders of childhood: A complete autopsy report.

INTRODUCTION: Epstein-Barr Virus (EBV)-associated systemic T-cell lymphoproliferative disorder of childhood is a rare but severe manifestation of chronic EBV infection. Despite several case reports characterizing this rare hematological neoplasm, the literature describes extensive heterogeneity in the presentation of this disease. CASE PRESENTATION: Here we present a complete autopsy of a 16-year-old girl who ultimately succumbed to EBV-associated systemic T-cell lymphoproliferative disorder of childhood. Her clinical presentation demonstrated a non-specific pharyngitis with positive mono spot test, evolving into fulminant multi-organ failure, disseminated intravascular coagulopathy, sepsis, and ultimately death. CONCLUSIONS: Post-mortem findings included extensive hemorrhage, and infiltration of the liver, spleen, lymph nodes and bone marrow with neoplastic T-cells. There was extensive hemophagocytic lymphohistiocytosis (HLH) within these organs, suggesting overlap between the EBV-associated systemic T-cell lymphoproliferative disorder of childhood and EBV-associated HLH. We hope these findings provide a more comprehensive overview of several possible manifestations of EBV-associated systemic T-cell lymphoproliferative disorder of childhood.

Metastatic cervical adenocarcinoma to the breast: A case report and literature review.

Breast metastases from gynecologic cancers are rare. Cervical cancer most commonly metastasizes to the lung, liver or bone. When cervical cancer metastasizes to the breast, the presentation is usually a solitary breast mass; rarely, however, breast metastases can mimic inflammatory breast cancer. We present a case of metastatic cervical adenocarcinoma presenting clinically as inflammatory breast cancer and review the relevant literature.

Iatrogenic immunodeficiency-associated lymphoproliferative disorders in transplant and nontransplant settings.

Iatrogenic immunodeficiency-associated lymphoproliferative disorders comprise a group of lymphoid neoplasms that are associated with an immunosuppressed state, either in the posttransplant period, or during the treatment of various autoimmune and rheumatologic disorders by immunomodulatory medications. Their morphologies vary widely but are generally classified according to the lymphomas that they most closely resemble. This group is strongly associated with infections by the Epstein-Barr virus as a result of impaired immune function in the immunosuppressed state. Although further classification may become necessary in the coming years, they are distinguished from lymphomas in immunocompetent hosts because reduction or cessation of immunosuppressive or immunomodulatory therapy can result in complete clinical remission.

Improved harmonization of eosin-5-maleimide binding test across different instruments and age groups.

BACKGROUND: The eosin-5'maleimide (EMA) binding test has been studied extensively for the detection of hereditary spherocytosis (HS). Its performance characteristics have been compared to NaCl-based or glycerol lysis-based red cell osmotic fragility tests and cryohemolysis. HS samples are also better identified when both mean channel fluorescence (MCF) of EMA relative to controls and the coefficient of variation (CV) are analyzed. METHODS: We looked at 65 normal controls including 30 adults 25-65 years old and 35 newborns and 12 HS cases. In addition to the MCF and the CV, we used a side scatter (SSC) vs. EMA fluorescence gate or "footprint" to depict where normal erythrocytes should appear. Erythrocytes that have reduced band 3 protein appear outside of the footprint. RESULTS: In our study, newborn data did not cluster with the samples from working age individuals. The MCF and the CVs of normal newborns were higher than normal adult group. However, the footprint data of normal samples relative to their controls was around 99.5% for each group, because the footprint was moved to fit the pattern of the normal. CONCLUSIONS: The inclusion of footprint parameter will help in better standardization as well as implementation of this test across different age groups as well as different instruments. © 2015 International Clinical Cytometry Society.