Flow Cytometric Characteristics of Extrathymic Thymocytes in Adenoid Tissue: A Case Report and Comparison to Normal Thymus and Thymoma.

BACKGROUND: Normal thymocyte precursors in secondary lymphoid organs have previously been described. It is important to recognize normal thymocyte precursors by flow cytometry to differentiate them from T-cell lymphoblastic leukemia. METHODS: A 3-year-old boy status 2 years postallogenic cardiac transplant underwent adenoidectomy to exclude post-transplant lymphoproliferative disorder. Microscopic, immunohistochemical, and flow cytometry analyses of the adenoid were performed. RESULTS: By flow cytometry, a population of CD45+(dim)/CD7+(bright)/CD3- cells were observed at 1.0% of lymphocytes. These cells expressed CD10, partial CD34 and exhibited acquisition of CD4 followed by CD8. Within the brighter CD45+ lymphocytes, a population of CD3-/CD4+/CD8+ thymocytes and a similarly sized population of CD4+/CD8+ cells exhibiting acquisition of low-density CD3 were identified. By immunostaining, clusters of TdT+/CD1a+/CD4+/CD8+ T-cells were identified in the interfollicular areas. Compared to normal thymus, thymocytes in the adenoid tissue lacked the classic CD4xCD8 winged differentiation profile but showed a normal early precursor pattern. CONCLUSIONS: Thymocytes in adenoid show a similar differentiation pattern to thymus and thymoma. However, the classic winged pattern of common thymocyte differentiation may not be readily apparent in thymocytes differentiating outside of the thymus. Recognition of the early thymocyte precursor antigen acquisition profile can be crucial to correct interpretation. © 2017 International Clinical Cytometry Society.

Flow cytometric characteristics of extrathymic thymocytes in adenoid tissue: A case report and comparison to normal thymus and thymoma.

BACKGROUND: Normal thymocyte precursors in secondary lymphoid organs have previously been described. It is important to recognize normal thymocyte precursors by flow cytometry to differentiate them from T-cell lymphoblastic leukemia. METHODS: A 3-year-old boy status 2 years post-allogenic cardiac transplant underwent adenoidectomy to exclude post-transplant lymphoproliferative disorder. Microscopic, immunohistochemical, and flow cytometry analyses of the adenoid were performed. RESULTS: By flow cytometry, a population of CD45+(dim)/CD7+(bright)/CD3- cells were observed at 1.0% of lymphocytes. These cells expressed CD10, partial CD34, and exhibited acquisition of CD4 followed by CD8. Within the brighter CD45+ lymphocytes, a population of CD3-/CD4+/CD8+ thymocytes and a similarly sized population of CD4+/CD8+ cells exhibiting acquisition of low-density CD3 were identified. By immunostaining, clusters of TdT+/CD1a+/CD4+/CD8+ T-cells were identified in the interfollicular areas. Compared to normal thymus, thymocytes in the adenoid tissue lacked the classic CD4xCD8 winged differentiation profile but showed a normal early precursor pattern. CONCLUSIONS: Thymocytes in adenoid show a similar differentiation pattern to thymus and thymoma. However, the classic winged pattern of common thymocyte differentiation may not be readily apparent in thymocytes differentiating outside of the thymus. Recognition of the early thymocyte precursor antigen acquisition profile can be crucial to correct interpretation. © 2017 International Clinical Cytometry Society.

Synchronous or Metachronous Hairy Cell Leukemia and Chronic Lymphocytic Leukemia: A Case Series and Literature Review.

INTRODUCTION: Concurrent hairy cell leukemia (HCL) and chronic lymphocytic leukemia (CLL) is rare; management is inadequately described in the literature. METHODS: Retrospective chart review and clinical follow-up. RESULTS: Five patients are described. The first patient developed synchronous HCL and CLL and was treated with rituximab for 13 months with HCL in remission and stable CLL. The second patient developed HCL and was treated with cladribine. His disease recurred 7 years later which was retreated with cladribine. Seven years later, he developed asymptomatic CLL. The third patient developed CLL, managed expectantly, then developed HCL 10 months later, and was treated with cladribine. Although his HCL went into remission, there was a slow redevelopment of CLL for which expectant management was done. The fourth patient developed concurrent CLL and HCL, received cladribine, with subsequent development of worsening abdominal lymphadenopathy and was lost to follow-up. The last patient developed concurrent HCL and CLL and was also diagnosed with lung adenocarcinoma; this patient was also lost to follow-up. CONCLUSION: The development of concurrent HCL and CLL may indicate a common origin. Patients with HCL may subsequently develop CLL, thus mimicking a relapse of HCL. Therapy requires individualized approach including watchful waiting in asymptomatic cases. Rituximab may be useful to treat both disorders simultaneously.

Flow cytometric analysis of lymphoid enhancer-binding factor 1 in diagnosis of chronic lymphocytic leukemia/small lymphocytic lymphoma.

OBJECTIVES: Nuclear overexpression of lymphoid enhancer-binding factor 1 (LEF1) assessed by immunohistochemistry has been shown to be highly associated with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) among small B-cell lymphomas. The purpose of this study was to evaluate the utility of flow cytometric analysis of LEF1 in the diagnosis of CLL/SLL. METHODS: Normal peripheral blood was used to validate the test. Flow cytometric analysis of LEF1 was performed in 64 patient samples qualitatively and quantitatively by comparing the staining intensity and the ratios of the median fluorescence intensities (MFIs) of LEF1 in B cells of interest to the internal reference cell populations. The results were correlated with the pathologic diagnosis. RESULTS: Proper sample processing ensured sufficient separation of positive LEF1 staining in T cells from negative staining in normal B and natural killer (NK) cells. Qualitative analysis of patient samples showed that all 25 cases of CLL/SLL but none of the other small B-cell lymphomas were positive for LEF1. Using a B/NK MFI ratio of 1.5 and B/T MFI ratio of 0.45 separated CLL/SLL cases from non-CLL lymphomas. CONCLUSIONS: Flow cytometric analysis of LEF1 is sufficient to differentiate CLL/SLL from other small B-cell lymphomas and may serve as a useful tool in the diagnosis of CLL/SLL.

Significance of isolated vasculitis in the gynecological tract: what clinicians do with the pathologic diagnosis of vasculitis?

Vasculitides includes a heterogeneous group of disorders with the common histologic findings of vascular wall inflammation. Systemic or localized disease (eg, renal vasculitis) has serious consequences. The incidence of isolated gynecologic vasculitis diagnosed on pathology specimens and its significance is little known. We performed a 20 year retrospective review including 53 cases with vasculitis diagnosis affecting the female genital tract identified in pathology reports. None had prior symptoms or were diagnosed with generalized vasculitis, while one patient had prior diagnosis of fibromyalgia. Most patients presented with abnormal bleeding and were treated for conditions unrelated to vasculitis. The different types of vasculitis were: predominantly lymphocytic (nonspecific) 30 cases, necrotizing 17 cases and granulomatous 6 cases. Only 2 patients had additional serologic tests. None of the patients with isolated gynecologic vasculitis received corticosteroids or additional treatment related to the vasculitis. None of the patients developed systemic vasculitis at follow-up (2 months-19.5 years; mean, 5.5 years). Isolated gynecologic vasculitis diagnosed on pathology slides is rarely associated with systemic vasculitis. Potential isolated gynecologic vasculitis causes include: previous surgical interventions and vascular inflammation secondary to local neoplasm. In almost all cases, clinicians did not perform a thorough laboratory analysis to exclude systemic vasculitis and therapy was not required in any case, suggesting minimal clinical significance.

Evaluation of bioabsorbable seamguard for staple line reinforcement in stapled rectal anastomoses.

INTRODUCTION: The concept of staple line reinforcement is a growing area of interest. This study evaluated the feasibility and effect of using bioabsorbable Seamguard (BSG) to bolster end-to-end stapled rectal anastomoses in a porcine model. METHODS: Eleven female 45-kg Yucatan domestic pigs were used. Each animal served as its own control by creating a BSG and nonreinforced anastomosis using a 29-mm end-to-end anastomotic stapling device. Reinforced anastomoses were randomized to proximal and distal positions along the rectum. Each staple line reinforcement agent consisted of adding BSG to the stapling device according to the manufacturer's instructions. Barium enemas were then performed and the 2 anastomotic sites harvested. Each anastomosis underwent burst testing. The internal diameter of each anastomosis was measured and underwent pathologic review. RESULTS: Bolstered anastomoses offered no strength advantage as burst pressures were no different as compared with unbolstered anastomoses. There was also no difference in anastomotic internal or external diameters. Only 1 stapled anastomosis burst during testing and none in the bolstered group. On histological analysis, there was a significant increase in inflammatory infiltrate in the bolstered group as compared with the stapled group (P = .041), with a higher incidence of lymphocytes (P = .047) and giant cells (P = .037). There was no difference in mucosal loss at the anastomotic site, neovascularization, fibroblast presence, extent of fibrosis, muscle layer disruption, percentage of anastomosis replaced by collagen, and elastin deposition. CONCLUSIONS: The routine use of BSG bolsters in stapled rectal anastomoses is safe and results in equivalent anastomotic strength as traditional stapled anastomoses.

Use of classic and novel immunohistochemical markers in the diagnosis of cutaneous myeloid sarcoma.

Cutaneous myeloid sarcoma is often challenging to diagnose based solely upon histopathological features. Although immunohistochemistry can aid in its diagnosis, specific markers have not been clearly identified. We evaluated the utility of immunohistochemical markers in 57 cutaneous myeloid sarcoma cases. In addition to classical markers (CD117, CD163, CD34, myeloperoxidase and lysozyme), we used CD33 and CD14, recently described markers in paraffin-embedded tissue samples, and Kruppel-like factor 4 (KLF-4), a novel monocytic marker. Our results show that lysozyme was expressed in 91%, CD33 in 60%, myeloperoxidase in 54%, CD34 in 39% and CD117 in 36% of cases. An antibody panel that included lysozyme, CD117 and CD33 identified all cases. The monocytic markers CD14, KLF-4 and CD163 were expressed in 60, 58 and 40% of all cases, respectively. CD14 and KLF-4 expression was significantly more common in cases with monocytic differentiation. CD14 is the single most sensitive and specific marker for monocytic differentiation (79 and 80%). Although KLF-4 in isolation is relatively insensitive (50 and 87%), it enhances sensitivity in detecting monocytic cutaneous myeloid sarcoma when combined with CD14. Our results indicate that in addition to classical immunohistochemical markers, targeted use of newer antibodies, including CD33, CD14 and KLF-4 is useful in the diagnosis of cutaneous myeloid sarcoma and in the detection of monocytic differentiation.

Combined core needle biopsy and fine-needle aspiration with ancillary studies correlate highly with traditional techniques in the diagnosis of nodal-based lymphoma.

Core needle biopsy (CNB) and fine-needle aspiration (FNA) are increasingly replacing excisional lymph node biopsy in the diagnosis of lymphomas. However, evaluation of CNB and FNA remains challenging owing to limited architectural information and the more detailed subclassification of lymphomas required by the WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Our study is the largest study to assess diagnostic accuracy of CNB and FNA in conjunction with ancillary studies. We analyzed 263 cases and a diagnosis was established in 237, of which 193 were completely subclassified. In cases in which excisional biopsy was available as a reference for comparison, CNB and FNA had a sensitivity of 96.5%, a specificity of 100%, a positive predictive value of 100%, and a negative predictive value of 90%. CNB and FNA with ancillary studies represent a viable alternative in the diagnosis of lymphoma, as long as the number and size of cores for morphologic studies are not compromised.

JAZF1 and JJAZ1 gene fusion in primary extrauterine endometrial stromal sarcoma.

Endometrial stromal sarcoma predominantly occurs as a primary tumor of the uterus. The most common cytogenetic abnormality in these tumors is t(7;17)(p15;q21), which occurs in 33% to 80% of cases and results in a JAZF1-JJAZ1 gene fusion. Rare cases of primary extrauterine endometrial stromal sarcoma have been reported, but it remains uncertain whether the genetic features of uterine endometrial stromal sarcoma are also characteristic of extrauterine tumors. The present study evaluates the prevalence of the t(7;17)(p15;q21) and JAZF1-JJAZ1 gene fusion in a series of 6 cases of primary extrauterine endometrial stromal sarcoma. Conventional nested reverse transcriptase-polymerase chain reaction was performed using primers complementary to sense and antisense JAZF1 and JJAZ1 sequences. Interphase fluorescence in situ hybridization was performed to detect t(7;17)(p15;q21) using a break-apart strategy for both JAZF1 and JJAZ1. In one of the 6 extrauterine endometrial stromal sarcoma cases, JAZF1-JJAZ1 fusion transcripts were detected by reverse transcriptase-polymerase chain reaction. The same case showed evidence of both JAZF1 and JJAZ1 rearrangements by interphase fluorescence in situ hybridization. The remaining 5 cases were negative for the t(7;17)(p15;q21) by both reverse transcriptase-polymerase chain reaction and fluorescence in situ hybridization analysis. These findings demonstrate that the t(7;17)(p15;q21) and associated JAZF1-JJAZ1 fusion transcripts are present in only a subset of primary extrauterine endometrial stromal sarcoma. Although molecular testing for the t(7;17)(p15;q21) and associated gene fusion may be useful for confirming primary extrauterine endometrial stromal sarcoma, the low prevalence of the genetic aberration limits the clinical utility of the testing.

TDP-43 in differential diagnosis of motor neuron disorders.

Motor neuron disorders are clinically and pathologically heterogeneous. They can be classified into those that affect primarily upper motor neurons, lower motor neurons or both. The most common disorder to affect both upper and lower motor neurons is amyotrophic lateral sclerosis (ALS). Some forms of motor neuron disease (MND) affect primarily motor neurons in the spinal cord or brainstem, while others affect motor neurons at all levels of the neuraxis. A number of genetic loci have been identified for the various motor neuron disorders. Several of the MND genes encode for proteins important for cytoskeletal stability and axoplasmic transport. Despite these genetic advances, the relationship of the various motor neuron disorders to each other is unclear. Except for rare familial forms of ALS associated with mutations in superoxide dismutase type 1 (SOD1), which are associated with neuronal inclusions that contain SOD1, specific molecular or cellular markers that differentiate ALS from other motor neuron disorders have not been available. Recently, the TAR DNA binding protein 43 (TDP-43) has been shown to be present in neuronal inclusions in ALS, and it has been suggested that TDP-43 may be a specific marker for ALS. This pilot study aimed to determine the value of TDP-43 in the differential diagnosis of MND. Immunohistochemistry for TDP-43 was used to detect neuronal inclusions in the medulla of disorders affecting upper motor neurons, lower motor neurons or both. Medullary motor neuron pathology also was assessed in frontotemporal lobar degeneration (FTLD) that had no evidence of MND. TDP-43 immunoreactivity was detected in the hypoglossal nucleus in all cases of ALS, all cases of FTLD-MND and some of cases of primary lateral sclerosis (PLS). It was not detected in FTLD-PLS. Surprisingly, sparse TDP-43 immunoreactivity was detected in motor neurons in about 10% of FTLD that did not have clinical or pathologic features of MND. The results suggest that TDP-43 immunoreactivity is useful in differentiating FTLD-MND and ALS from other disorders associated with upper or lower motor neuron pathology. It also reveals subclinical MND in a subset of cases of FTLD without clinical or pathologic evidence of MND.

TDP-43 immunoreactivity in hippocampal sclerosis and Alzheimer's disease.

OBJECTIVE: This study aimed to determine the frequency of frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U) in the setting of hippocampal sclerosis (HpScl) and Alzheimer's disease (AD) using immunohistochemistry for TAR DNA binding protein 43 (TDP-43), a putative marker for FTLD-U. METHODS: Initially, 21 cases of HpScl associated with a variety of other pathological processes and 74 cases of AD were screened for FTLD-U with TDP-43 immunohistochemistry. A confirmation study was performed on 93 additional AD cases. Specificity of TDP-43 antibodies was assessed using double-immunolabeling confocal microscopy, immunoelectron microscopy, and biochemistry. RESULTS: TDP-43 immunoreactivity was detected in 71% of HpScl and 23% of AD cases. Double immunostaining of AD cases for TDP-43 and phospho-tau showed that the TDP-43-immunoreactive inclusions were usually distinct from neurofibrillary tangles. At the ultrastructural level, TDP-43 immunoreactivity in AD was associated with granular and filamentous cytosolic material and only occasionally associated with tau filaments. Western blots of AD cases showed a band that migrated at a higher molecular weight than normal TDP-43 that was not present in AD cases without TDP-43 immunoreactivity. INTERPRETATION: These results suggest that as many as 20% of AD cases and more than 70% of HpScl cases have pathology similar to that found in FTLD-U. Whether this represents concomitant FTLD-U or is analogous to colocalization of alpha-synuclein and tau in AD, reflecting a propensity for codeposition of abnormal protein conformers, remains to be determined.

Hippocampal sclerosis dementia differs from hippocampal sclerosis in frontal lobe degeneration.

Hippocampal sclerosis (HS) is characterized by selective neuronal loss and gliosis in CA1 and the subiculum and has been associated with several disorders, including Alzheimer's disease, frontotemporal lobar degeneration with ubiquitin immunoreactive inclusions (FTLD-U), vascular dementia and some tauopathies. In some cases, HS is not associated with other degenerative pathologies. Such cases are sometimes referred to as HS dementia (HSD). Differences between HSD and HS in the setting of FTLD-U have not been systematically investigated. To this end, eight cases of HSD and ten cases of HS associated with FTLD-U were studied with Nissl and periodic acid-Schiff stains to assess neuronal loss and corpora amylacea, respectively. Sections were immunostained with antibodies to glial fibrillary acidic protein, HLA-DR and synaptophysin and immunoreactivity was measured with image analysis in CA1 and the subiculum of each case. Additionally, sections were immunostained with antibodies to 4-R tau to determine the presence of argyrophilic grains. HSD was different from HS associated with FTLD-U. Specifically, it was more common in the elderly, and it was associated with more marked neuronal and synaptic loss and with greater reactive gliosis. Corpora amylacea tended to be more frequent in HSD than in FTLD-U, but there was no difference in frequency of argyrophilic grains.