Lymphoma versus Carcinoma and Other Collaborations

David Mason started his research career at a time when lymphoma diagnosis was based primarily on cellular morphology, clinical symptoms and special cytochemical stains using formalin fixed tissue sections. There were occasions, however, where the morphology was unhelpful, such as in the case of anaplastic or poorly differentiated tumours, where a distinction between lymphoma and a non-haematopoietic tumour was often problematical. Accurate diagnosis became even more important with the developments in the clinical staging of lymphoma and the availability of more effective treatments. One way forward to improve diagnosis was to use immunohistochemistry to study the antigens expressed by the tumor cells.

Canine pyoderma: mecA persists autogenous bacterin formulation from meticillin-resistant Staphylococcus pseudintermedius (MRSP) and S. aureus (MRSA)

Objective Autogenous Staphylococcus pseudintermedius bacterins can reduce prescribing of antimicrobials in the management of canine recurrent pyoderma. However, increasing prevalence of meticillin-resistant, mecA-positive S. pseudintermedius (MRSP) raises concern over dispersal of mecA through bacterin therapy. We investigated the presence and integrity of mecA in bacterin formulations after manufacturing. Material and methods Twenty clinical isolates (12 MRSP, 7 MR-S. aureus, 1 meticillin-susceptible SP) were investigated. Pellets from overnight growth were washed 3 times with 0.5 % phenol saline, followed by addition of 0.1 ml 10 % formal-saline to 10 ml phenol-saline. Sterility was confirmed, and DNA extracted using both a standard genomic extraction kit and one recommended for formalin-fixed tissue samples (FFPE). The presence of mecA was determined after PCR and its integrity examined in 5 randomly selected samples after sequencing. Results In all bacterins from meticillin-resistant isolates, mecA was detected following FFPE extraction; products aligned fully to a reported mecA sequence. After standard DNA extraction, mecA was seen in 16/19 samples. Conclusion Persistence of mecA in MRSP bacterins suggests that dispersal of this important resistance mediator through therapy may be possible. While the ability of skin bacteria to uptake naked DNA remains unclear, it seems prudent to only formulate autogenous bacterins from mecA-negative S. pseudintermedius to avoid unnecessary spread of mecA.

A novel herpes-like virus inducing branchial lesions in a tiger shark (Galeocerdo cuvier)

A juvenile, male tiger shark ( Galeocerdo cuvier) developed illness after capture in Florida waters and was euthanized. Gross lesions included mild skin abrasions, hepatic atrophy, and coelomic fluid. Histologically, gills contained multifocal lamellar epithelial cell necrosis and thromboses. Scattered gill and esophageal epithelial cells had large, basophilic, intracytoplasmic, and intranuclear inclusions. Ultrastructurally, lamellar epithelial cells contained arrays of intracytoplasmic viral particles and scattered intranuclear nucleocapsids. Capsulated virions were 148 ± 11 nm with an 84 ± 8 nm icosahedral nucleocapsid and an electron-dense core. Next-generation sequencing, quantitative polymerase chain reaction, and in situ hybridization performed on formalin-fixed tissue confirmed a herpes-like viral infection. The viral polymerase shared 24% to 31% protein homology with other alloherpesviruses of fish, indicating a divergent virus. This report documents the pathologic findings associated with a molecularly confirmed novel herpes-like virus in an elasmobranch.

The Development of Novel Primer Sets to Specifically Amplify Each of the Five Different Deltapapillomaviruses That Cause Neoplasia after Cross-Species Infection

Bovine papillomavirus (BPV) types 1 and 2 are recognized as the main cause of equine sarcoids. However, some studies report that up to a quarter of these tumors do not contain detectible BPV1 or BPV2 DNA. The absence of detectible BPV1 or BPV2 in these sarcoids suggests the possible involvement of other papillomavirus types. Currently, five deltapapillomaviruses are recognized to cause mesenchymal neoplasia after cross-species infection. In addition to BPV1 and BPV2, BPV13 has been associated with equine sarcoids in Brazil, BPV14 has been associated with feline sarcoids, and Ovis aries papillomavirus 2 caused a sarcoid-like lesion in a pig. To investigate the cause of equine sarcoids, PCR primers were developed to specifically amplify each of the five different deltapapillomaviruses that have been associated with mesenchymal neoplasia. The specificity of these primers was confirmed using samples of formalin-fixed tissue known to contain each PV type. These primers allow rapid and sensitive detection of deltapapillomavirus DNA in equine sarcoids. As studies have revealed marked regional variability in the cause of equine sarcoids, these primers will be useful to determine the predominant PV type causing sarcoids in a region. Additionally, there is a single report describing mixed infections by BPV1 and BPV2 in equine sarcoids. The specific primer sets are expected to enable more sensitive detection of mixed infections in equine sarcoids. Determining the cause of equine sarcoids is important as vaccines are developed to prevent these common malignant neoplasms.

Do Tissues Fixed in a Non-cross-linking Fixative Require a Dedicated Formalin-free Processor?

We evaluate the consequences of processing alcohol-fixed tissue in a processor previously used for formalin-fixed tissue. Biospecimens fixed in PAXgene Tissue Fixative were cut into three pieces then processed in a flushed tissue processor previously used for formalin-fixed, paraffin-embedded (FFPE) blocks (neutral buffered formalin [NBF]+ve), a formalin-free system (NBF−ve), or left unprocessed. Histomorphology and immunohistochemistry were compared using hematoxylin/eosin staining and antibodies for MLH-1, Ki-67, and CK-7. Nucleic acid was extracted using the PAXgene Tissue RNA/DNA kits and an FFPE RNA extraction kit. RNA integrity was assessed using RNA integrity number (RIN), reverse transcription polymerase chain reaction (RT-PCR) (four amplicons), and quantitative RT-PCR (three genes). For DNA, multiplex PCR, quantitative PCR, DNA integrity number, and gel electrophoresis were used. Compared with NBF−ve, RNA from NBF+ve blocks had 88% lower yield and poorer purity; average RIN reduced from 5.0 to 3.8, amplicon length was 408 base pairs shorter, and Cq numbers were 1.9–2.4 higher. Using the FFPE extraction kit rescued yield and purity, but RIN further declined by 1.1 units. Differences between NBF+ve and NBF−ve in respect of DNA, histomorphology, and immunohistochemistry were either non-existent or small in magnitude. Formalin contamination of a tissue processor and its reagents therefore critically reduce RNA yield and integrity. We discuss the available options users can adopt to ameliorate this problem:

Transcriptome-wide spatial RNA profiling maps the cellular architecture of the developing human neocortex

Spatial genomic technologies can map gene expression in tissues, but provide limited potential for transcriptome-wide discovery approaches and application to fixed tissue samples. Here, we introduce the GeoMX Whole Transcriptome Atlas (WTA), a new technology for transcriptome-wide spatial profiling of tissues with cellular resolution. WTA significantly expands the Digital Spatial Profiling approach to enable in situ hybridisation against 18,190 genes at high-throughput using a sequencing readout. We applied WTA to generate the first spatial transcriptomic map of the fetal human neocortex, validating transcriptome-wide spatial profiling on formalin-fixed tissue material and demonstrating the spatial enrichment of autism gene expression in deep cortical layers. To demonstrate the value of WTA for cell atlasing, we integrated single-cell RNA-sequencing (scRNA-seq) and WTA data to spatially map dozens of neural cell types and showed that WTA can be used to directly measure cell type specific transcriptomes in situ. Moreover, we developed computational tools for background correction of WTA data and accurate integration with scRNA-seq. Our results present WTA as a versatile transcriptome-wide discovery tool for cell atlasing and fixed tissue spatial transcriptomics.

Unraveling Actionable Target Mutations in Formalin Fixed Tissue in Mantle Cell Lymphoma

Introduction: Mantle Cell Lymphoma (MCL) is a B-cell neoplasm with poor prognosis and high relapse frequency. It follows an heterogenous clinical course and, despite clear improvements in survival among young and fit patients, most patients eventually relapse. Recurrent mutations have been previously described, pinpointing alterations in DNA damage pathways, cell survival and cell-cycle regulation. Nevertheless, the current genomic knowledge of the disease cannot fully explain its high clinical variability. The present study aimed at revealing the mutation profile for the development of personalized treatment approaches in a population-based cohort of patients diagnosed with MCL. Material & Methods: We performed sequencing across ~230 lymphomagenesis-related genes on 75 formalin-fixed paraffin-embedded (FFPE) samples from symptomatic patients diagnosed with MCL in Southern Sweden. The panel covered the exonic parts of the chosen genes with capture probes provided by TWIST Bioscience. Sequence alignment was done against the human genome version 37 and variant calling was performed resorting to Sention TNscope. IGV was used for manual validation of each identified mutation. Samples were pre-selected according to DNA quality. Data on patient characteristics, prognostic factors and treatment was extracted from the Swedish Lymphoma Register. Results: MCL tumors were highly heterogenous with respect to the mutational status of the analyzed genes, ranging between 0 and 45 mutated genes in this cohort. ATM was the most commonly mutated gene (49%), followed by KMT2D, NOTCH2, TP53, FAT4, BIRC3, CCND1, UBR5, MEF2B, NOTCH1,SMARCA4, DNAH5,SAMHD1 and SP140 (Figure 1). Our results are in accordance with the previously published analyses, although the frequency of samples with mutated NOTCH2 was higher (18.7%, compared to 3-12.5% of affected tumors in previous studies), and with less samples with mutated NOTCH3 (6.7% of the studied tumors were mutated, vs 12.5% reported by Yang et al., (2018)). We found mutations in DNAH5, PCLO, FAT3 and BRCA1 genes, which have not been previously reported in MCL. Additionally, FAT4 (14.7%) showed recurrent mutations in our cohort, but has only been referred as commonly mutated in MCL before by Zhang et al. (2014). Main affected pathways were NOTCH-related and TP53-related pathways, followed by chromatin remodeling and cell cycle pathways. Due to low numbers of patients with non-classic morphology, no mutations were significantly different comparing classical and non-classical histology, although there was an enrichment of TP53 and NOTCH1 mutations in non-classic cases. It seems that cases with non-classic morphology have stronger driver mutations, which may reflect their aggressiveness. Cases with high proliferation (Ki67 >30%) showed a higher frequency of SMARCA4 (30%, vs 7.7% for low proliferation tumors) and TP53 mutations (36% in highly proliferative tumors vs. 17% in low proliferative tumors). No differences in mutation patterns were found in cases with low vs. high SOX11 expression. Unsurprisingly, TP53 mutations were the strongest independent factor for prognosis among the studied genes. Conclusions: Using the results from a population-based cohort of MCL, we report the feasibility of applying a targeted panel and sequencing technologies on DNA from formalin fixed tissue, as a method applicable in clinical routine in tailoring treatment approaches for MCL. Figure 1. Top 15 genes mutated in the cohort studied. Frequency of mutations on each gene is presented on the right panel and in the upper panel the proportion of affected genes by sample can be seen. Darker blue identifies a missense mutation, light blue corresponds to a frameshift deletion, pink color matches frameshift insertions, orange represent in frame deletions, whereas red in frame insertions. Light green identifies nonsense mutations and dark green stands for different types of mutation affecting that gene in that sample. Figure Disclosures Jerkeman: Roche: Research Funding; Abbvie: Research Funding; Celgene: Research Funding; Gilead: Research Funding; Janssen: Research Funding.

Comparison of methods for quantifying primordial follicles in the mouse ovary

Background Accurate evaluation of primordial follicle numbers in mouse ovaries is an essential endpoint for studies investigating how endogenous and exogenous insults, such as maternal aging and chemotherapy, impact the ovarian reserve. In this study, we compared and contrasted two methods for counting healthy primordial follicles following exposure to cyclophosphamide (75 mg/kg), a well-established model of follicle depletion. The first was the fractionator/optical dissector technique, an unbiased, assumption-free stereological approach for quantification of primordial follicle numbers. While accurate, highly reproducible and sensitive, this method relies on specialist microscopy equipment and software, requires specific fixation, embedding and sectioning parameters to be followed, and is largely a manual process that is tedious and time-consuming. The second method was the more widely used serial section and direct count approach, which is relatively quick and easy. We also compared the impacts of different fixatives, embedding material and section thickness on the overall results for each method. Results Direct counts resulted in primordial follicle numbers that were significantly lower than those obtained by stereology, irrespective of fixation and embedding material. When applied to formalin fixed tissue, the direct count method did not detect differences in follicle numbers between saline and cyclophosphamide treated groups to the same degree of sensitivity as the gold standard stereology method (referred to as the Reference standard). However, when Bouin’s fixative was used, direct counts and stereology were comparable in their ability to detect follicle depletion caused by cyclophosphamide. Conclusions This work indicates that the direct count method can produce similar results to stereology when Bouin’s fixative is used instead of formalin. The findings presented here will assist others to select the most appropriate experimental approach for accurate follicle enumeration, depending on whether the primary objective of the study is to determine absolute primordial follicle numbers or relative differences between groups.

Species differences in the cerebellar distribution of six members of the Kv1 channel subfamily

Comprehensive studies on the distribution of the Kv1 subfamily have been performed in rat (Chung et al., 2001) and gerbil (Chung et al., 2005), but not in mouse or human. We hypothesized that species differences may exist in the localization of these proteins. Two sets of polyclonal antibodies to Kv1.1-6 were used. Immunohistochemistry was performed on archived, formalin-fixed tissue from disease-free human, monkey and mouse cerebellum. Mouse staining corresponded to that described in rat and gerbil, with strong Kv1.1 and Kv1.2 immunoreactivities in the basket cell pinceau at the base of Purkinje cells. Kv1.3, Kv1.4, Kv1.5 and Kv1.6 were predominantly expressed in Purkinje cells. Human and monkey samples showed a similar pattern to mouse for Kv1.1, Kv1.2, Kv1.3 and Kv1.5. However, little or no Purkinje cell staining was seen in the primates with Kv1.4 and Kv1.6, and strong stellate cell expression was noted. All staining was abolished by cognate peptide blocking. Similar distributions were seen with both sets of antibodies. We conclude that there are marked species differences in the distribution of Kv1.4 and Kv1.6 between primates and rodents. Choosing appropriate animal models for studying physiological and disease processes may prove vital for translating research outcomes into clinical applications.

Sequencing of Dna Isolated From Colorectal Sample Fixed in Liquid Formalin : Obstacles and Required Modifications

The necessary modifications in the protocol of general purpose DNA isolation kit to isolate and amplify a target region of genome from colorectal cancer tissues fixed in liquid formalin were made. It is shown that a one hour digestion with proteinase K yields enough DNA from formalin fixed colorectal tissue for subsequent PCR and sequencing. Moreover, using 100% ethanol instead of standard 50% during DNA binding step in the column improves the yield. As DNA fragmentation is unavoidable in formalin fixed tissue, PCR protocol was modified by increasing polymerase concentration to get successful amplification. Following these modifications, two regions of KRAS and BRAF genes were amplified and successfully sequenced from three different patients. These modifications provide a low cost option for Sanger sequencing of DNA isolated from formalin fixed tissue. Dhaka Univ. J. Biol. Sci. 29(2): 165-174, 2020 (July)