Whole Genome Sequence of a Treponema pallidum Strain From a Formalin-Fixed, Paraffin-Embedded Fine Needle Aspirate of a Cervical Lymph Node.

ABSTRACT: A patient with unilateral cervical lymphadenopathy suspicious for malignancy underwent a fine needle aspiration. Histology demonstrated mixed inflammatory infiltrates with abundant spirochetes. Sufficient spirochete DNA was extracted from paraffin-embedded tissue sections to obtain the near-complete genome sequence of a macrolide-resistant strain belonging to the SS14 omega strain of Treponema pallidum .

Orbital MALT lymphoma with amyloid deposition.

MALT lymphoma is a commonly encountered orbital tumor, and primary amyloidosis is frequently found to be an independent orbital lesion. Orbital MALT lymphoma with associated amyloid deposition is considered rare, with only 12 cases previously published. We describe a 33-year-old man, the youngest patient reported to-date, with a mass in the superonasal quadrant of the right anterior orbit. Pathology demonstrated extranodal marginal zone lymphoma in mucosa-associated lymphoid tissue with associated amyloid deposition. Systemic work-up revealed no other site of either lymphoma or amyloidosis. The patient underwent local irradiation and subsequent surgical resection of the residual mass. Persistent lymphoma was found and treated with rituximab.

Evaluation of CD43 expression in non-hematopoietic malignancies.

OBJECTIVES: CD43 is normally expressed only on the surface of leukocytes, and is considered a sensitive and specific marker for hematologic malignancies. As such, it may have diagnostic utility in confirming hematolymphoid lineage in cases that are negative for CD45. Aberrant CD43 expression has been described in non-hematopoietic tumors, although literature data on this topic is variable and sometimes contradictory. To clarify and expand on existing literature findings, we evaluated CD43 expression by immunohistochemistry (IHC) in a large cohort (307) of non-hematopoietic neoplasms, including poorly differentiated malignancies. METHODS: 17 tissue microarrays and sections from 19 individual cases were stained with CD43 (clone DF-T1) monoclonal antibody. The proportion of positive cells, stain localization (nuclear, cytoplasmic or membranous), and intensity (compared to internal leukocyte controls) were recorded in all cases. RESULTS: There were 98/307 (32%) positive cases, that showed focal weak nuclear staining in 1-25% of cells, including 23/25 (92%) pancreatic ductal adenocarcinomas; 31/34 (91%) breast invasive ductal carcinomas; 13/15 (87%) papillary thyroid carcinomas; 3/4 (75%) follicular thyroid carcinomas; 6/15 (40%) renal cell carcinomas; 9/28 (32%) lung adenocarcinomas; 1/13 (8%) lung squamous cell carcinomas (SCCs); 2/8 (25%) prostate adenocarcinomas; 8/62 (13%) colon adenocarcinomas; and 2/21 (10%) neuroendocrine neoplasms. None of the positive cases demonstrated strong, membranous CD43 expression comparable to that seen in background mature lymphocytes or segmented neutrophils. Negative cases included 11 cervical SCCs, 12 cervical adenocarcinomas, 19 urothelial carcinomas, 10 lung small cell carcinomas, 11 sarcomas, and 19 poorly differentiated carcinomas from various tissue sites. CONCLUSIONS: In our cohort, most non-hematopoietic neoplasms are negative for CD43 expression, with a subset showing focal, weak nuclear positivity. This data indicates that uniform and strong membranous staining appears to be specific to hematopoietic neoplasms.

Proliferation centers in bone marrows involved by chronic lymphocytic leukemia/small lymphocytic lymphoma: a clinicopathologic analysis.

OBJECTIVES: Proliferation centers (PCs) are a characteristic finding in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) lymph nodes, and their presence and extent in this site are not currently felt to be related to clinical course. In contrast, detailed clinicopathologic analyses of bone marrow (BM) PCs have not been previously reported. METHODS: The PCs in 88 CLL/SLL BMs from 45 patients (pts) were graded (0-4) and were correlated with other morphologic, immunophenotypic, cytogenetic, and laboratory features. RESULTS: Proliferation centers were present in 69 BMs (78%) from 32 pts (71%) and were distinct/prominent (grades 2-4) in 21 pts (47%), with the latter more commonly found in follow-up BMs (1/7 diagnostic BMs vs 49/81 follow-up BMs; P=.04). When present, PCs were most commonly graded as distinct nodules easily visible on ×10. No relationships were identified between PCs and any complete blood count parameter, serum lactate dehydrogenase or IgG levels, degree or pattern of BM involvement, blood morphology, CD38 and FMC7 expression by flow cytometry, or fluorescence in situ hybridization results, when the first encountered BM was considered for each patient. CONCLUSIONS: This represents the first detailed analysis of PCs in CLL/SLL BMs. In our tertiary center, PCs were seen frequently, in approximately three-fourths of cases. There were no statistical associations identified between PCs and cytogenetic, immunophenotypic, or other laboratory and morphologic findings.

Utility of targeted gene sequencing to differentiate myeloid malignancies from other cytopenic conditions.

The National Heart, Lung, and Blood Institute-funded National MDS Natural History Study (NCT02775383) is a prospective cohort study enrolling patients with cytopenia with suspected myelodysplastic syndromes (MDS) to evaluate factors associated with disease. Here, we sequenced 53 genes in bone marrow samples harvested from 1298 patients diagnosed with myeloid malignancy, including MDS and non-MDS myeloid malignancy or alternative marrow conditions with cytopenia based on concordance between independent histopathologic reviews (local, centralized, and tertiary to adjudicate disagreements when needed). We developed a novel 2-stage diagnostic classifier based on mutational profiles in 18 of 53 sequenced genes that were sufficient to best predict a diagnosis of myeloid malignancy and among those with a predicted myeloid malignancy, predict whether they had MDS. The classifier achieved a positive predictive value (PPV) of 0.84 and negative predictive value (NPV) of 0.8 with an area under the receiver operating characteristic curve (AUROC) of 0.85 when classifying patients as having myeloid vs no myeloid malignancy based on variant allele frequencies (VAFs) in 17 genes and a PPV of 0.71 and NPV of 0.64 with an AUROC of 0.73 when classifying patients as having MDS vs non-MDS malignancy based on VAFs in 10 genes. We next assessed how this approach could complement histopathology to improve diagnostic accuracy. For 99 of 139 (71%) patients (PPV of 0.83 and NPV of 0.65) with local and centralized histopathologic disagreement in myeloid vs no myeloid malignancy, the classifier-predicted diagnosis agreed with the tertiary pathology review (considered the internal gold standard).

Discordant pathologic diagnoses of myelodysplastic neoplasms and their implications for registries and therapies.

Myelodysplastic neoplasms (MDS) are a collection of hematopoietic disorders with widely variable prognoses and treatment options. Accurate pathologic diagnoses present challenges because of interobserver variability in interpreting morphology and quantifying dysplasia. We compared local clinical site diagnoses with central, adjudicated review from 918 participants enrolled in the ongoing National Heart, Lung, and Blood Institute National MDS Natural History Study, a prospective observational cohort study of participants with suspected MDS or MDS/myeloproliferative neoplasms (MPNs). Locally, 264 (29%) were diagnosed as having MDS, 15 (2%) MDS/MPN overlap, 62 (7%) idiopathic cytopenia of undetermined significance (ICUS), 0 (0%) acute myeloid leukemia (AML) with <30% blasts, and 577 (63%) as other. Approximately one-third of cases were reclassified after central review, with 266 (29%) diagnosed as MDS, 45 (5%) MDS/MPN overlap, 49 (5%) ICUS, 15 (2%) AML with <30%, and 543 (59%) as other. Site miscoding errors accounted for more than half (53%) of the local misdiagnoses, leaving a true misdiagnosis rate of 15% overall, 21% for MDS. Therapies were reported in 37% of patients, including 43% of patients with MDS, 49% of patients with MDS/MPN, and 86% of patients with AML with <30% blasts. Treatment rates were lower (25%) in cases with true discordance in diagnosis compared with those for whom local and central diagnoses agreed (40%), and receipt of inappropriate therapy occurred in 7% of misdiagnosed cases. Discordant diagnoses were frequent, which has implications for the accuracy of study-related and national registries and can lead to inappropriate therapy. This trial was registered at www.clinicaltrials.gov as #NCT05074550.

How I Diagnose Mature T-Cell Proliferations by Flow Cytometry.

OBJECTIVES: Mature T-cell neoplasms are a challenging area of diagnostic hematopathology. Flow cytometry has emerged as a useful technique for T-cell assessment. METHODS: We discuss the application of flow cytometry to the evaluation of mature T-cell proliferations, to include illustrative cases, theoretical framework, detailed review of normal and reactive T-cell subsets, and examination of diagnostic pitfalls. RESULTS: Immunophenotypic aberrancy can be construed as a direct expression of the neoplastic phenotype, in contrast to clonal expansion, which is seen in reactive and neoplastic T-cell proliferations. Major and minor T-cell subsets show characteristic patterns of antigen expression. Reactive states can manifest expansions of normal minor subsets and also show alterations of antigen expression on certain populations. However, some patterns of antigen expression are either never or very rarely encountered in reactive T cells. Flow cytometric tools are now available to directly assess clonality in specific T-cell populations. Technical and biological pitfalls may complicate the interpretation of T-cell flow cytometry. CONCLUSIONS: Flow cytometry is a very useful tool in the diagnostic armamentarium for the assessment of mature T-cell proliferations, but it must be interpreted based on a thorough knowledge of the T-cell immune response, as well as an awareness of clinical context.

Clinicopathologic analysis of the impact of CD23 expression in plasma cell myeloma with t(11;14)(q13;q32).

A recent study has shown that 10% of plasma cell myelomas (PCMs) express CD23 and that expression is associated with abnormalities of chromosome 11, mainly t(11;14)(q13;q32); however, only 40% of t(11;14)(+) PCMs express CD23. Because these results were generated in a limited patient cohort and because the clinical relevance of CD23 expression in PCMs with t(11;14)(q13;q32) has not been fully characterized, we addressed this question in a large series of patients with t(11;14)(+) PCM. Forty-two bone marrow biopsies from patients with t(11;14)(+) PCM were evaluated for CD23 expression by immunohistochemistry. CD23 expression was correlated with laboratory and clinical data and outcome after autologous stem cell transplantation, including event-free survival and overall survival (OS). Plasma cell myelomas with t(11;14)(q13;q32) were frequently CD20(+) (46.4%) and CD56(-) (53.8%) and had a nonhyperdiploid karyotype (97.6%) with frequent 13q deletion (33.3%). Of 42 cases, 19 (45.2%) expressed CD23. CD23(+) PCMs were more likely to present with platelet counts less than 150 × 10(3)/µL (100% vs 50%, P = .006). There were no significant differences in other laboratory or presenting clinical data. The median event-free survival in patients treated with autologous stem cell transplantation (n = 29) was similar regardless of CD23 status, whereas the median OS (all patients) was longer in CD23(-) than in CD23(+) PCMs: not reached vs 3365 days (P = .08). Our findings suggest that patients with t(11;14)(+)/CD23(+) PCM present with lower platelet counts and may have a shorter OS than those with t(11;14)(+)/CD23(-) PCM.

Laboratory Workup of Lymphoma in Adults.

OBJECTIVES: The diagnostic workup of lymphoma continues to evolve rapidly as experience and discovery lead to the addition of new clinicopathologic entities and techniques to differentiate them. The optimal clinically effective, efficient, and cost-effective approach to diagnosis that is safe for patients can be elusive, in both community-based and academic practice. Studies suggest that there is variation in practice in both settings. THE AIM OF THIS REVIEW IS TO: develop an evidence-based guideline for the preanalytic phase of testing, focusing on specimen requirements for the diagnostic evaluation of lymphoma. METHODS: The American Society for Clinical Pathology, the College of American Pathologists, and the American Society of Hematology convened a panel of experts in the laboratory workup of lymphoma to develop evidence-based recommendations. The panel conducted a systematic review of the literature to address key questions. Using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach, recommendations were derived based on the available evidence, the strength of that evidence, and key judgments as defined in the GRADE Evidence to Decision framework. RESULTS: Thirteen guideline statements were established to optimize specimen selection, ancillary diagnostic testing, and appropriate follow-up for safe and accurate diagnosis of indolent and aggressive lymphoma. CONCLUSIONS: Primary diagnosis and classification of lymphoma can be achieved with a variety of specimens. Application of the recommendations can guide decisions about specimen suitability, diagnostic capabilities, and correct utilization of ancillary testing. Disease prevalence in patient populations, availability of ancillary testing, and diagnostic goals should be incorporated into algorithms tailored to each practice environment.

Laboratory Workup of Lymphoma in Adults: Guideline From the American Society for Clinical Pathology and the College of American Pathologists.

CONTEXT.­: The diagnostic workup of lymphoma continues to evolve rapidly as experience and discovery led to the addition of new clinicopathologic entities and techniques to differentiate them. The optimal clinically effective, efficient, and cost-effective approach to diagnosis that is safe for patients can be elusive, in both community-based and academic practice. Studies suggest that there is variation in practice in both settings. OBJECTIVE.­: To develop an evidence-based guideline for the preanalytic phase of testing, focusing on specimen requirements for the diagnostic evaluation of lymphoma. DESIGN.­: The American Society for Clinical Pathology, the College of American Pathologists, and the American Society of Hematology convened a panel of experts in the laboratory workup of lymphoma to develop evidence-based recommendations. The panel conducted a systematic review of literature to address key questions. Using the Grading of Recommendations Assessment, Development, and Evaluation approach, recommendations were derived based on the available evidence, strength of that evidence, and key judgements as defined in the Grading of Recommendations Assessment, Development, and Evaluation Evidence to Decision framework. RESULTS.­: Thirteen guideline statements were established to optimize specimen selection, ancillary diagnostic testing, and appropriate follow-up for safe and accurate diagnosis of indolent and aggressive lymphoma. CONCLUSIONS.­: Primary diagnosis and classification of lymphoma can be achieved with a variety of specimens. Application of the recommendations can guide decisions on specimen suitability, diagnostic capabilities, and correct use of ancillary testing. Disease prevalence in patient populations, availability of ancillary testing, and diagnostic goals should be incorporated into algorithms tailored to each practice environment.

Constrained chromatin accessibility in PU.1-mutated agammaglobulinemia patients.

The pioneer transcription factor (TF) PU.1 controls hematopoietic cell fate by decompacting stem cell heterochromatin and allowing nonpioneer TFs to enter otherwise inaccessible genomic sites. PU.1 deficiency fatally arrests lymphopoiesis and myelopoiesis in mice, but human congenital PU.1 disorders have not previously been described. We studied six unrelated agammaglobulinemic patients, each harboring a heterozygous mutation (four de novo, two unphased) of SPI1, the gene encoding PU.1. Affected patients lacked circulating B cells and possessed few conventional dendritic cells. Introducing disease-similar SPI1 mutations into human hematopoietic stem and progenitor cells impaired early in vitro B cell and myeloid cell differentiation. Patient SPI1 mutations encoded destabilized PU.1 proteins unable to nuclear localize or bind target DNA. In PU.1-haploinsufficient pro-B cell lines, euchromatin was less accessible to nonpioneer TFs critical for B cell development, and gene expression patterns associated with the pro- to pre-B cell transition were undermined. Our findings molecularly describe a novel form of agammaglobulinemia and underscore PU.1's critical, dose-dependent role as a hematopoietic euchromatin gatekeeper.

Persistent localized bone marrow aplasia after radiotherapy with preserved peripheral counts: a study of 8 cases.

Localized, radiation (XRT)-induced sternal bone marrow (BM) aplasia was described in early studies in the radiation oncology literature; however, no pathologic studies have examined in detail this phenomenon in random iliac crest biopsies and its relationship to overall hematopoiesis. We retrospectively reviewed aplastic iliac crest BMs with discrepant peripheral blood (PB) counts after localized pelvic XRT. BM aplasia was defined as 5% or less cellularity in an adequate biopsy and/or hypocellular particles on aspirate smears. Discrepant PB counts were defined as either within or higher than normal limits or mild cytopenias. Eight patients with BM aplasia and discrepant PB counts were identified; each had received localized XRT to the sacrum, lumbar spine, or pelvis. Aplastic BMs showed replacement by mature fat and/or virtually acellular spicules. One case showed focal reticulin fibrosis. Mild cytopenias were seen in 6 cases and normal or increased counts in one case each. Aplastic BMs were observed 5 to 43 months after XRT. A myeloproliferative neoplasm was diagnosed in one case based on PB findings and JAK-2 mutation, despite BM aplasia. In one case, a right-sided aplastic BM, diagnosed 8 months after XRT, was followed 14 months later by a normocellular right aspirate and aplastic left BM biopsy. Prolonged, localized BM sterilization may be seen as a result of XRT to the iliac crest for several years. In the setting of preserved PB counts, this is not likely representative of overall hematopoiesis and serves as a potential diagnostic pitfall. Regeneration of hematopoietic activity at exposed sites may be possible.

Carcinocythemia: A rare entity becoming more common? A 3-year, single institution series of seven cases and literature review.

INTRODUCTION: Carcinocythemia is a rare phenomenon defined as morphologically identifiable, circulating tumor cells in the peripheral blood. No modern case series of carcinocythemia exists in the literature. METHODS: Blood smears from carcinocythemia patients were reviewed and associated clinicopathologic findings described and compared to the literature. When available, bone marrows were examined. RESULTS: We identified 7 carcinocythemia cases over 3 years at our institution in 5 females and 2 males with a median age of 57 and compare them to 26 case reports in the literature (19 females, 10 males; median age 57). The primary neoplasms were carcinomas of breast (3 cases), lung, non-small cell (2 cases), prostate (1), and 1 case of unknown primary. Circulating tumor cells were associated with fragmentation hemolysis (2 cases), asplenic RBC changes (3 cases), and myeloid antigen expression by flow cytometry (2 cases) and were most commonly found at the feathered edge of the slide (6 cases) as single cells or in clusters. CONCLUSIONS: This represents the largest series of carcinocythemia reported. The identification of 7 cases at one institution over a 3-year period suggests carcinocythemia may becoming more common. Raising awareness of this entity and its associated clinicopathologic findings may help avoid diagnostic pitfalls in blood smear examinations and may guide timely clinical management.

Treatment of diffuse large B-cell lymphoma of the liver with yttrium-90 microsphere embolization.

BACKGROUND: A 41-year-old male with a 4-year history of chronic hepatitis C presented with a 1-month history of abdominal pain, fatigue, weight loss, and night sweats. INVESTIGATIONS: Laboratory examinations, chest, abdomen, and pelvic CT scans, PET-CT scans, ultrasound-guided needle biopsies of liver lesions, bone-marrow biopsy, flow cytometry, and immunohistochemical staining for B-cell markers including CD20. DIAGNOSIS: Chemoresistant diffuse large B-cell lymphoma, with gradual loss of CD20 antigen expression. MANAGEMENT: Embolization of hepatic tumors using yttrium-90 microspheres (Therasphere, Theragenics Corporation, Buford, GA).

Long-term remission with imatinib mesylate in Philadelphia chromosome-positive AML presenting as primary extramedullary myeloid sarcoma.

In this case report, we describe the first account in the literature of a patient who presented with extramedullary myeloid sarcomas (EMS) and Ph+ AML without leukemic manifestations. EMS are rare, destructive, extramedullary tumor masses that consist of immature leukemia cells. These tumors can occur anywhere in the body and have to be differentiated from lymphoma, carcinoma or infectious processes. We report a previously healthy 61-year-old male who presented with progressive left-sided hip pain. Magnetic resonance imaging (MRI) of the left hip joint revealed an extensive lytic lesion with pathological fracture of the femur. The lesion was confirmed by biopsy to be EMS with bcr-abl rearrangement in tumor cell nuclei by fluorescence in situ hybridization (FISH). Laboratory tests showed normal metabolic and complete blood counts with normal differential. Peripheral blood RT-PCR for bcr-abl was negative. Bone marrow examination revealed a normocellular with progressive trilineage hematopoiesis and no evidence of increased blast count. On the basis of available literature for the treatment of EMS, our patient received standard AML induction chemotherapy consisting of idarabucin and cytarabine. After cytogenetics studies of the bone marrow revealed bcr-abl rearrangement, we discontinued further chemotherapy and managed the patient with imatinib mesylate 400mg p.o.q. day. Follow-up at time of manuscript preparation, the patient remains in complete cytogenetic remission (CCR) for 22 months. To the best of our knowledge, this is the first case report of Ph+ AML presenting as a primary EMS without leukemic manifestations.

Iron deficiency anemia, beta-thalassemia minor, and anemia of chronic disease: a morphologic reappraisal.

We observed increased numbers of an infrequently referenced poikilocyte, the prekeratocyte, in iron deficiency anemia (IDA) compared with beta-thalassemia minor and anemia of chronic disease (ACD) and, therefore, chose to quantify these cells and other morphologic features in these anemias. Prekeratocytes were observed in 31 (78%) of 40 IDAs vs 11 (37%) of 30 beta-thalassemias (P = .001) and 5 (13%) of 40 ACDs (P < .001) and averaged 0.78 per 1,000 RBCs in IDA vs 0.21 in beta-thalassemia (P < .001) and 0.075 in ACD (P < .001). Pencil cells also were more commonly seen and more numerous in IDAs than in beta-thalassemia or ACD. Target cells were present in most IDAs and thalassemia and in similar numbers. Basophilic stippling was seen in only 5 (17%) of the beta-thalassemias. Our results lend quantitative support to prekeratocytes and pencil cells as morphologic features favoring the diagnosis of IDA but fail to support the diagnostic usefulness of target cells and basophilic stippling in discriminating IDA and beta-thalassemia minor.

Gene expression analysis of gallium-resistant and gallium-sensitive lymphoma cells reveals a role for metal-responsive transcription factor-1, metallothionein-2A, and zinc transporter-1 in modulating the antineoplastic activity of gallium nitrate.

Several clinical trials have shown gallium nitrate to be an active agent in the treatment of lymphoma. Whereas gallium is known to target cellular iron homeostasis, the basis for lymphoma cell resistance to gallium is not known. Understanding mechanisms of resistance may suggest strategies to enhance the clinical efficacy of gallium. In the present study, we used a focused DNA microarray to compare the expression of genes related to metal metabolism in gallium-resistant and gallium-sensitive lymphoma cell lines developed by us. Gallium-resistant cells were found to display a marked increase in gene expression for metallothionein-2A and the zinc transporter ZnT-1. Cells exposed to gallium nitrate displayed an increase in the binding of metal-responsive transcription factor-1 to metal response element sequences involved in the transcriptional regulation of metallothionein and ZnT-1 genes. Gallium nitrate induced metallothionein-2A and ZnT-1 expression in cells. A role for metallothionein in modulating the antineoplastic activity of gallium was confirmed by showing that the induction of metallothionein expression by zinc provided partial protection against the cytotoxicity of gallium and by showing that the level of endogenous metallothionein in lymphoma cell lines correlated with their sensitivity to gallium nitrate. Immunohistochemical staining of lymphomatous tissues revealed metallothionein protein to be variably expressed in different lymphomas. Our studies show for the first time that gallium acts on pathways related to zinc metabolism and that metal-responsive transcription factor-1 activity and metallothionein expression contribute to the development of gallium drug resistance. Furthermore, the endogenous level of metallothionein in lymphoma may be an important determinant of clinical response to gallium nitrate.