Comparison of histological delineations of medial temporal lobe cortices by four independent neuroanatomy laboratories.

The medial temporal lobe (MTL) cortex, located adjacent to the hippocampus, is crucial for memory and prone to the accumulation of certain neuropathologies such as Alzheimer's disease neurofibrillary tau tangles. The MTL cortex is composed of several subregions which differ in their functional and cytoarchitectonic features. As neuroanatomical schools rely on different cytoarchitectonic definitions of these subregions, it is unclear to what extent their delineations of MTL cortex subregions overlap. Here, we provide an overview of cytoarchitectonic definitions of the entorhinal and parahippocampal cortices as well as Brodmann areas (BA) 35 and 36, as provided by four neuroanatomists from different laboratories, aiming to identify the rationale for overlapping and diverging delineations. Nissl-stained series were acquired from the temporal lobes of three human specimens (two right and one left hemisphere). Slices (50 µm thick) were prepared perpendicular to the long axis of the hippocampus spanning the entire longitudinal extent of the MTL cortex. Four neuroanatomists annotated MTL cortex subregions on digitized slices spaced 5 mm apart (pixel size 0.4 µm at 20× magnification). Parcellations, terminology, and border placement were compared among neuroanatomists. Cytoarchitectonic features of each subregion are described in detail. Qualitative analysis of the annotations showed higher agreement in the definitions of the entorhinal cortex and BA35, while the definitions of BA36 and the parahippocampal cortex exhibited less overlap among neuroanatomists. The degree of overlap of cytoarchitectonic definitions was partially reflected in the neuroanatomists' agreement on the respective delineations. Lower agreement in annotations was observed in transitional zones between structures where seminal cytoarchitectonic features are expressed less saliently. The results highlight that definitions and parcellations of the MTL cortex differ among neuroanatomical schools and thereby increase understanding of why these differences may arise. This work sets a crucial foundation to further advance anatomically-informed neuroimaging research on the human MTL cortex.

Comparison of histological delineations of medial temporal lobe cortices by four independent neuroanatomy laboratories.

The medial temporal lobe (MTL) cortex, located adjacent to the hippocampus, is crucial for memory and prone to the accumulation of certain neuropathologies such as Alzheimer's disease neurofibrillary tau tangles. The MTL cortex is composed of several subregions which differ in their functional and cytoarchitectonic features. As neuroanatomical schools rely on different cytoarchitectonic definitions of these subregions, it is unclear to what extent their delineations of MTL cortex subregions overlap. Here, we provide an overview of cytoarchitectonic definitions of the cortices that make up the parahippocampal gyrus (entorhinal and parahippocampal cortices) and the adjacent Brodmann areas (BA) 35 and 36, as provided by four neuroanatomists from different laboratories, aiming to identify the rationale for overlapping and diverging delineations. Nissl-stained series were acquired from the temporal lobes of three human specimens (two right and one left hemisphere). Slices (50 µm thick) were prepared perpendicular to the long axis of the hippocampus spanning the entire longitudinal extent of the MTL cortex. Four neuroanatomists annotated MTL cortex subregions on digitized (20X resolution) slices with 5 mm spacing. Parcellations, terminology, and border placement were compared among neuroanatomists. Cytoarchitectonic features of each subregion are described in detail. Qualitative analysis of the annotations showed higher agreement in the definitions of the entorhinal cortex and BA35, while definitions of BA36 and the parahippocampal cortex exhibited less overlap among neuroanatomists. The degree of overlap of cytoarchitectonic definitions was partially reflected in the neuroanatomists' agreement on the respective delineations. Lower agreement in annotations was observed in transitional zones between structures where seminal cytoarchitectonic features are expressed more gradually. The results highlight that definitions and parcellations of the MTL cortex differ among neuroanatomical schools and thereby increase understanding of why these differences may arise. This work sets a crucial foundation to further advance anatomically-informed human neuroimaging research on the MTL cortex.

Neuroanatomical and cellular degeneration associated with a social disorder characterized by new ritualistic belief systems in a TDP-C patient vs. a Pick patient.

Frontotemporal dementia (FTD) is a spectrum of clinically and pathologically heterogenous neurodegenerative dementias. Clinical and anatomical variants of FTD have been described and associated with underlying frontotemporal lobar degeneration (FTLD) pathology, including tauopathies (FTLD-tau) or TDP-43 proteinopathies (FTLD-TDP). FTD patients with predominant degeneration of anterior temporal cortices often develop a language disorder of semantic knowledge loss and/or a social disorder often characterized by compulsive rituals and belief systems corresponding to predominant left or right hemisphere involvement, respectively. The neural substrates of these complex social disorders remain unclear. Here, we present a comparative imaging and postmortem study of two patients, one with FTLD-TDP (subtype C) and one with FTLD-tau (subtype Pick disease), who both developed new rigid belief systems. The FTLD-TDP patient developed a complex set of values centered on positivity and associated with specific physical and behavioral features of pigs, while the FTLD-tau patient developed compulsive, goal-directed behaviors related to general themes of positivity and spirituality. Neuroimaging showed left-predominant temporal atrophy in the FTLD-TDP patient and right-predominant frontotemporal atrophy in the FTLD-tau patient. Consistent with antemortem cortical atrophy, histopathologic examinations revealed severe loss of neurons and myelin predominantly in the anterior temporal lobes of both patients, but the FTLD-tau patient showed more bilateral, dorsolateral involvement featuring greater pathology and loss of projection neurons and deep white matter. These findings highlight that the regions within and connected to anterior temporal lobes may have differential vulnerability to distinct FTLD proteinopathies and serve important roles in human belief systems.

Associations of phosphorylated tau pathology with whole-hemisphere ex vivo morphometry in 7 tesla MRI.

INTRODUCTION: Neurodegenerative disorders are associated with different pathologies that often co-occur but cannot be measured specifically with in vivo methods. METHODS: Thirty-three brain hemispheres from donors with an Alzheimer's disease (AD) spectrum diagnosis underwent T2-weighted magnetic resonance imaging (MRI). Gray matter thickness was paired with histopathology from the closest anatomic region in the contralateral hemisphere. RESULTS: Partial Spearman correlation of phosphorylated tau and cortical thickness with TAR DNA-binding protein 43 (TDP-43) and α-synuclein scores, age, sex, and postmortem interval as covariates showed significant relationships in entorhinal and primary visual cortices, temporal pole, and insular and posterior cingulate gyri. Linear models including Braak stages, TDP-43 and α-synuclein scores, age, sex, and postmortem interval showed significant correlation between Braak stage and thickness in the parahippocampal gyrus, entorhinal cortex, and Broadman area 35. CONCLUSION: We demonstrated an association of measures of AD pathology with tissue loss in several AD regions despite a limited range of pathology in these cases. HIGHLIGHTS: Neurodegenerative disorders are associated with co-occurring pathologies that cannot be measured specifically with in vivo methods. Identification of the topographic patterns of these pathologies in structural magnetic resonance imaging (MRI) may provide probabilistic biomarkers. We demonstrated the correlation of the specific patterns of tissue loss from ex vivo brain MRI with underlying pathologies detected in postmortem brain hemispheres in patients with Alzheimer's disease (AD) spectrum disorders. The results provide insight into the interpretation of in vivo structural MRI studies in patients with AD spectrum disorders.

Menin inhibitor MI-3454 induces remission in MLL1-rearranged and NPM1-mutated models of leukemia.

The protein-protein interaction between menin and mixed lineage leukemia 1 (MLL1) plays a critical role in acute leukemias with translocations of the MLL1 gene or with mutations in the nucleophosmin 1 (NPM1) gene. As a step toward clinical translation of menin-MLL1 inhibitors, we report development of MI-3454, a highly potent and orally bioavailable inhibitor of the menin-MLL1 interaction. MI-3454 profoundly inhibited proliferation and induced differentiation in acute leukemia cells and primary patient samples with MLL1 translocations or NPM1 mutations. When applied as a single agent, MI-3454 induced complete remission or regression of leukemia in mouse models of MLL1-rearranged or NPM1-mutated leukemia, including patient-derived xenograft models, through downregulation of key genes involved in leukemogenesis. We also identified MEIS1 as a potential pharmacodynamic biomarker of treatment response with MI-3454 in leukemia, and demonstrated that this compound is well tolerated and did not impair normal hematopoiesis in mice. Overall, this study demonstrates, for the first time to our knowledge, profound activity of the menin-MLL1 inhibitor as a single agent in clinically relevant PDX models of leukemia. These data provide a strong rationale for clinical translation of MI-3454 or its analogs for leukemia patients with MLL1 rearrangements or NPM1 mutations.

Cytokines increase engraftment of human acute myeloid leukemia cells in immunocompromised mice but not engraftment of human myelodysplastic syndrome cells.

Patient-derived xenotransplantation models of human myeloid diseases including acute myeloid leukemia, myelodysplastic syndromes and myeloproliferative neoplasms are essential for studying the biology of the diseases in pre-clinical studies. However, few studies have used these models for comparative purposes. Previous work has shown that acute myeloid leukemia blasts respond to human hematopoietic cytokines whereas myelodysplastic syndrome cells do not. We compared the engraftment of acute myeloid leukemia cells and myelodysplastic syndrome cells in NSG mice to that in NSG-S mice, which have transgene expression of human cytokines. We observed that only 50% of all primary acute myeloid leukemia samples (n=77) transplanted in NSG mice provided useful levels of engraftment (>0.5% human blasts in bone marrow). In contrast, 82% of primary acute myeloid leukemia samples engrafted in NSG-S mice with higher leukemic burden and shortened survival. Additionally, all of 5 injected samples from patients with myelodysplastic syndrome showed persistent engraftment on week 6; however, engraftment was mostly low (<2%), did not increase over time, and was only transiently affected by the use of NSG-S mice. Co-injection of mesenchymal stem cells did not enhance human myelodysplastic syndrome cell engraftment. Overall, we conclude that engraftment of acute myeloid leukemia samples is more robust compared to that of myelodysplastic syndrome samples and unlike those, acute myeloid leukemia cells respond positively to human cytokines, whereas myelodysplastic syndrome cells demonstrate a general unresponsiveness to them.

Multifluorescence confocal microscopy: application for a quantitative analysis of hemostatic proteins in human venous valves.

Confocal laser scanning microscopy is commonly used to visualize and quantify protein expression. Visualization of the expression of multiple proteins in the same region via multifluorescence allows for the analysis of differential protein expression. The defining step of multifluorescence labeling is the selection of primary antibodies from different host species. In addition, species-appropriate secondary antibodies must also be conjugated to different fluorophores so that each protein can be visualized in separate channels. Quantitative analysis of proteins labeled via multifluorescence can be used to compare relative changes in protein expression. Multifluoresecence labeling and analysis of fluorescence intensity within and among human venous specimens, for example, allowed us to determine that the anticoagulant phenotype of the venous valve is defined not by increased anticoagulant expression, but instead by significantly decreased procoagulant protein expression (Blood 114:1276-1279, 2009 and Histochem Cell Biol 135:141-152, 2011).

Discrimination of 'driver' and 'passenger' HPV in tonsillar carcinomas by the polymerase chain reaction, chromogenic in situ hybridization, and p16(INK4a) immunohistochemistry.

Human papillomavirus (HPV) positive tonsillar squamous cell carcinoma (TSCC) is associated with a favorable clinical outcome. However, the HPV detected in a given tumor may be causal (driver HPV) or an incidental bystander (passenger HPV). There is a need to discriminate these forms of HPV in TSCCs to understand their impact on HPV as a biomarker for use in TSCC patient management. This study has compared the polymerase chain reaction (PCR), chromogenic in situ hybridization (CISH), and p16(INK4a) immunohistochemistry in the assessment of HPV status in TSCC. Archival specimens of TSCC from thirty patients were investigated. HPV was detected by PCR in 25/30 (83.3%) tumors; HPV16 (70.0%) and HPV52 (6.7%) were the most common types. HPV was corroborated by CISH in 22/25 (88.0%) specimens; integrated HPV was implicated by the presence of punctate signals in each of these cases. p16(INK4a) staining was found in 20/22 (90.9%) HPV PCR positive samples; two PCR/CISH HPV positive cases were p16(INK4a) negative and two HPV negative samples were p16(INK4a) positive. These data suggest that a minority of HPV positive TSCCs are positive for passenger HPV and that two or more assays may be required for diagnosing driver HPV status. Further studies are required to exam whether oropharyngeal tumors positive for passenger HPV have a less favorable prognosis than tumors that are driver HPV positive. The clinical significance of TSCCs that test HPV negative/p16(INK4a) positive, PCR and CISH HPV positive/p16 (INK4a) negative, or PCR HPV positive/p16 (INK4a) and CISH negative, also requires further investigation.

Screening for human papillomavirus in basaloid squamous carcinoma: utility of p16(INK4a), CISH, and PCR.

This study compares p16( INK4a) immunohistochemistry (IHC), HPV chromogenic in situ hybridization (ISH), and HPV polymerase chain reaction (PCR) genotyping for detection of HPV infection in basaloid squamous carcinoma (BSCC). A retrospective histopathological analysis of 40 BSCC from a single institution was carried out. p16 IHC, HPV DNA extraction and ISH, and HPV PCR genotyping were performed, and there was excellent agreement between all 3 methods of HPV detection. Analysis of variance yielded no significant differences between the results of the 3 tests ( P = .354) and Pearson product-moment correlation coefficients calculated for each pair of tests demonstrated direct correlation (r = .61 for PCR and IHC, r = .61 for PCR and ISH, and r = 1.00 for ISH and IHC). This supports the use of p16(INK4a) IHC as an initial screening tool for HPV infection in BSCC, while definitive evidence of HPV DNA can be sought subsequently with PCR or CISH.

Cell adhesion molecule 1: a novel risk factor for venous thrombosis.

Protein C (PC) deficiency increases the risk of venous thrombosis (VT) among members of Kindred Vermont II but fails to fully account for the inheritance pattern. A genome scan of the pedigree supported the presence of a prothrombotic gene on chromosome 11q23 (nominal P < .0001), with weaker support on chromosomes 10p12 (P < .0003) and 18p11.2-q11 (P < .0007). Resequencing of 109 genes in the linkage regions identified 5030 variants in a sample of 20 kindred members. Of 16 single nucleotide polymorphisms in 6 genes tested in the larger family set, only single nucleotide polymorphisms in cell adhesion molecule 1 (CADM1) associated with VT. Among the 8 CADM1 single nucleotide polymorphisms genotyped in the complete sample, rs6589488 was most strongly supported (P < .000007), but the association was limited to the PC-deficient subset of the sample (P < .000001). Haplotype analysis narrowed the region containing the causative variant to the coding region of the CADM1 gene. CADM1 gene expression analyzed in blood outgrowth endothelial cells cultured from family members was decreased compared with control subjects, lending phenotypic support to this conclusion. Finally, we have for the first time demonstrated CADM1 in endothelial cells, where it appears to be selectively involved in endothelial cell migration, suggesting a role in endothelial barrier repair.

Valves of the deep venous system: an overlooked risk factor.

Deep venous valves are frequent sites of deep venous thrombosis initiation. However, the possible contribution of the valvular sinus endothelium has received little attention in studies of thrombosis risk. We hypothesized that the endothelium of valve sinus differs from that of vein lumen with up-regulation of anticoagulant and down-regulation of procoagulant activities in response to the local environment. In pursuit of this hypothesis, we quantified endothelial protein C receptor (EPCR), thrombomodulin (TM), and von Willebrand factor (VWF) by immunofluorescence in great saphenous veins harvested at cardiac bypass surgery. We found significantly increased expression of EPCR and TM in the valvular sinus endothelium as opposed to the vein lumenal endothelium, and the opposite pattern with VWF (paired t test for TM and EPCR, each P < .001; for VWF, P = .01). These data support our hypothesis and suggest that variation in valvular sinus thromboresistance may be an important factor in venous thrombogenesis.

Ber-EP4, CK1, CK7 and CK14 are useful markers for basaloid squamous carcinoma: a study of 45 cases.

Diagnosis of basaloid squamous carcinoma (BSCC) currently relies mainly on histological criteria, with variable immunohistochemical results reported in small series. We explored the use of a battery of immunohistochemical stains to elucidate this diagnosis on 45 cases of BSCC. To further elucidate the immunohistochemical profile of BSCC, to explore potential genetic pathways of malignant transformation using proliferation markers, and to investigate a possible link with Human Papillomavirus (HPV). Forty-five cases of BSCC and 34 site-matched cases of squamous cell carcinoma (SCC) were obtained from the archives of the pathology department at our institution. Extensive literature review was undertaken utilizing Medline. Ber-EP4 is a useful diagnostic marker for BSCC, positive in 82% (37/45) of the cases and in 68% (23/34) of SCC. An alternative is the combination of cytokeratins CK14 and CK7, known to be negative, and CK1, known to be positive, which achieves an accuracy of 73% (33/45) in BSCC and 88% (30/34) in SCC. The two diagnostic approaches were in agreement in 66% of the cases; both methods were equally accurate in the divergent cases. Increased expression of the proliferation markers supports the concept that BSCC is a rapidly growing tumor. Results of p16 stains support an etiological link between BSCC and HPV; interestingly, HPV was present significantly more in BSCC (71% (32/45)), than in SCC (59% (20/34)) in this study (P=0.02).

Glypican-3 immunocytochemistry in liver fine-needle aspirates : a novel stain to assist in the differentiation of benign and malignant liver lesions.

BACKGROUND: Glypican-3 (GPC3) is a heparan sulfate proteoglycan which is elevated in the serum of patients with hepatocellular carcinoma (HCC), but not in healthy blood donors, or patients with benign liver disease. GPC3 immunohistochemistry (IHC) is a promising marker of HCC in surgical pathology. This study explores the value of GPC3 expression in liver fine-needle aspirates (FNAs) by immunocytochemistry (ICC), and compares its sensitivity and staining intensity with that of IHC. METHODS: Archival cytologic material in hepatic FNAs from 20 patients with HCC, 20 patients with metastatic tumors, and 20 patients with benign lesions, were studied. Correlating surgical specimens and/or cell blocks were available for GPC-3 IHC in 16 patients with HCC. All slides were stained with GPC3-1G12 antibody with appropriate positive and negative controls. Staining intensity was graded as 0, no staining; 1, weak cytoplasmic staining; 2, moderate cytoplasmic staining; 3, strong cytoplasmic staining with membranous accentuation. Grades 0 and 1 were regarded as negative; grades 2 and 3 were considered positive for GPC3. RESULTS: In the HCC group, positive staining was found in 18/20 (90%) samples. In contrast, GPC3 ICC of 20/20 (100%) metastatic tumors and 20/20 (100%) benign cases displayed negative staining, no cases showing moderate or strong expression. The sensitivity and specificity of GPC3 in HCC ICC were 90% and 100% respectively. The surgical sections and cell blocks of HCC demonstrated positive staining less frequently, in 11/16 (68.8%) cases, with 12/16 (75%) correlation with ICC. CONCLUSIONS: Results indicated positive staining for GPC3 as defined in 90% of liver FNAs from HCC patients. All metastatic tumors and benign aspirates studied were negative for GPC3. ICC was superior to IHC in 25% of cases. This pilot study supports the diagnostic utility of GPC3 in hepatic FNAs to aid in distinction of HCC from metastatic tumors and benign liver lesions.

FISH detection of t(14;18) in follicular lymphoma on Papanicolaou-stained archival cytology slides.

BACKGROUND: The t(14;18)(q32;q21) translocation is present in about 85% of follicular lymphomas (FL) and can be identified using fluorescence in situ hybridization (FISH). In the diagnostic laboratory setting, the cytologic archival material consists of stained slides, and only rarely is material saved for molecular testing. The authors proposed FISH for FL using Papanicolaou-stained archival cytology material as a practical ancillary technique for diagnosing FL. METHODS: Cases included 35 FL, 6 small lymphocytic lymphomas/chronic lymphocytic leukemias (SLL/CLL), 4 mantle cell lymphomas (MCL), 4 marginal zone lymphomas (MZL), 1 lymphoplasmacytic lymphoma (LPL), and 10 reactive lymphoid tissues (RLT). FISH was performed on Papanicolaou-stained archival cytology slides using probes for immunoglobulin heavy chain (IGH) on chromosome 14 and BCL2 on chromosome 18. RESULTS: In all, 25 of 32 (81%) FL cases exhibited the t(14;18) translocation, whereas 7 of 32 (19%) lacked the translocation. No cases of non-FL were positive for t(14;18). This series shows a sensitivity of 81% and specificity of 100% for detecting the t(14;18) translocation as a diagnostic tool in FL. CONCLUSIONS: When performed on Papanicolaou-stained cytology slides, FISH for t(14;18) is relatively sensitive and quite specific for FL. These findings are similar to those reported on other specimens, such as paraffin-embedded tissue and unstained cytology slides. The authors proposed that their technique would allow the pathologist and clinician the flexibility to utilize previously stained fine-needle aspiration slides for FISH evaluation.

Mechanisms of compensatory beta-cell growth in insulin-resistant rats: roles of Akt kinase.

The physiological mechanisms underlying the compensatory growth of beta-cell mass in insulin-resistant states are poorly understood. Using the insulin-resistant Zucker fatty (fa/fa) (ZF) rat and the corresponding Zucker lean control (ZLC) rat, we investigated the factors contributing to the age-/obesity-related enhancement of beta-cell mass. A 3.8-fold beta-cell mass increase was observed in ZF rats as early as 5 weeks of age, an age that precedes severe insulin resistance by several weeks. Closer investigation showed that ZF rat pups were not born with heightened beta-cell mass but developed a modest increase over ZLC rats by 20 days that preceded weight gain or hyperinsulinemia that first developed at 24 days of age. In these ZF pups, an augmented survival potential of beta-cells of ZF pups was observed by enhanced activated (phospho-) Akt, phospho-BAD, and Bcl-2 immunoreactivity in the postweaning period. However, increased beta-cell proliferation in the ZF rats was only detected at 31 days of age, a period preceding massive beta-cell growth. During this phase, we also detected an increase in the numbers of small beta-cell clusters among ducts and acini, increased duct pancreatic/duodenal homeobox-1 (PDX-1) immunoreactivity, and an increase in islet number in the ZF rats suggesting duct- and acini-mediated heightened beta-cell neogenesis. Interestingly, in young ZF rats, specific cells associated with ducts, acini, and islets exhibited an increased frequency of PDX-1+/phospho-Akt+ staining, indicating a potential role for Akt in beta-cell differentiation. Thus, several adaptive mechanisms account for the compensatory growth of beta-cells in ZF rats, a combination of enhanced survival and neogenesis with a transient rise in proliferation before 5 weeks of age, with Akt serving as a potential mediator in these processes.

Dual Y chromosome painting and in situ cell-specific immunofluorescence staining in lung tissue: an improved method of identifying donor marrow cells in lung following bone marrow transplantation.

Several recent studies have demonstrated localization of donor bone marrow-derived cells in recipient lungs following transplantation from male to female mice or patients. Donor cells are identified by detection of the Y chromosome by fluorescence in situ hybridization (FISH). However, protein digestion pretreatments usually required for tissue FISH significantly limit the ability to detect cell type-specific markers by immunohistochemistry. We have used an alternative protein digest approach that entails heating the slides in 10 mM sodium citrate rather than utilizing a protease digestion. This approach preserves cell proteins following FISH, and allows lung tissue to remain intact for subsequent detection of cell-specific markers by immunohistochemistry. We have examined this technique in mouse lungs using a Y chromosome paint and three cell-specific markers, a pan-cytokeratin for epithelial cells, PECAM-1 for endothelial cells, and CD45 for leukocytes. Excellent visualization of both the Y chromosome and cell-specific surface protein markers was obtained on a single slide. This approach will significantly enhance the ability to detect and identify donor bone marrow cells in recipient mouse lungs following male to female transplantation.