Rituximab versus tocilizumab in anti-TNF inadequate responder patients with rheumatoid arthritis (R4RA): 16-week outcomes of a stratified, biopsy-driven, multicentre, open-label, phase 4 randomised controlled trial.

BACKGROUND: Although targeted biological treatments have transformed the outlook for patients with rheumatoid arthritis, 40% of patients show poor clinical response, which is mechanistically still unexplained. Because more than 50% of patients with rheumatoid arthritis have low or absent CD20 B cells-the target for rituximab-in the main disease tissue (joint synovium), we hypothesised that, in these patients, the IL-6 receptor inhibitor tocilizumab would be more effective. The aim of this trial was to compare the effect of tocilizumab with rituximab in patients with rheumatoid arthritis who had an inadequate response to anti-tumour necrosis factor (TNF) stratified for synovial B-cell status. METHODS: This study was a 48-week, biopsy-driven, multicentre, open-label, phase 4 randomised controlled trial (rituximab vs tocilizumab in anti-TNF inadequate responder patients with rheumatoid arthritis; R4RA) done in 19 centres across five European countries (the UK, Belgium, Italy, Portugal, and Spain). Patients aged 18 years or older who fulfilled the 2010 American College of Rheumatology and European League Against Rheumatism classification criteria for rheumatoid arthritis and were eligible for treatment with rituximab therapy according to UK National Institute for Health and Care Excellence guidelines were eligible for inclusion in the trial. To inform balanced stratification, following a baseline synovial biopsy, patients were classified histologically as B-cell poor or rich. Patients were then randomly assigned (1:1) centrally in block sizes of six and four to receive two 1000 mg rituximab infusions at an interval of 2 weeks (rituximab group) or 8 mg/kg tocilizumab infusions at 4-week intervals (tocilizumab group). To enhance the accuracy of the stratification of B-cell poor and B-cell rich patients, baseline synovial biopsies from all participants were subjected to RNA sequencing and reclassified by B-cell molecular signature. The study was powered to test the superiority of tocilizumab over rituximab in the B-cell poor population at 16 weeks. The primary endpoint was defined as a 50% improvement in Clinical Disease Activity Index (CDAI50%) from baseline. The trial is registered on the ISRCTN database, ISRCTN97443826, and EudraCT, 2012-002535-28. FINDINGS: Between Feb 28, 2013, and Jan 17, 2019, 164 patients were classified histologically and were randomly assigned to the rituximab group (83 [51%]) or the tocilizumab group (81 [49%]). In patients histologically classified as B-cell poor, there was no statistically significant difference in CDAI50% between the rituximab group (17 [45%] of 38 patients) and the tocilizumab group (23 [56%] of 41 patients; difference 11% [95% CI -11 to 33], p=0·31). However, in the synovial biopsies classified as B-cell poor with RNA sequencing the tocilizumab group had a significantly higher response rate compared with the rituximab group for CDAI50% (rituximab group 12 [36%] of 33 patients vs tocilizumab group 20 [63%] of 32 patients; difference 26% [2 to 50], p=0·035). Occurrence of adverse events (rituximab group 76 [70%] of 108 patients vs tocilizumab group 94 [80%] of 117 patients; difference 10% [-1 to 21) and serious adverse events (rituximab group 8 [7%] of 108 vs tocilizumab group 12 [10%] of 117; difference 3% [-5 to 10]) were not significantly different between treatment groups. INTERPRETATION: The results suggest that RNA sequencing-based stratification of rheumatoid arthritis synovial tissue showed stronger associations with clinical responses compared with histopathological classification. Additionally, for patients with low or absent B-cell lineage expression signature in synovial tissue tocilizumab is more effective than rituximab. Replication of the results and validation of the RNA sequencing-based classification in independent cohorts is required before making treatment recommendations for clinical practice. FUNDING: Efficacy and Mechanism Evaluation programme from the UK National Institute for Health Research.

Radiologically guided percutaneous core needle biopsy of the spleen: a reliable and safe diagnostic procedure for neoplastic and reactive conditions.

RATIONALE: Percutaneous core needle biopsy (CNB) of the spleen is rarely performed, due to concerns about its complications and low diagnostic yield. However, this procedure represents a potentially useful diagnostic tool, especially in patients with splenomegaly and no definitive diagnosis after a clinical and radiological work-up. METHODS AND RESULTS: We report the data on a cohort of 45 radiologically guided percutaneous core needle biopsies of the spleen from 44 patients performed at two centres. Platelet count and prothrombin time were within normal limits in all patients at the time of the procedure. The biopsy was ultrasound-guided in all cases except one, which was guided by computed tomography. An 18G needle was used in 82% of the cases, followed by 16G (10.2%) and 20G (7.8%) needles. The biopsy provided sufficient material for histological examination (including immunohistochemical studies) in 41 cases (91.1%). Haematological malignancies were most commonly diagnosed (52.3%); diffuse large B cell lymphoma (DLBCL) was the most frequent, followed by splenic marginal zone lymphoma (SMZL). For the most recent cases of DLBCL, the CNB provided sufficient material for fluorescence in-situ hybridisation to assess the status of MYC, BCL2 and BCL6. This allowed the identification of a case of high-grade B cell lymphoma with MYC and BCL2 rearrangement. Major complications were not reported; minor complications occurred in three cases (6.7%). CONCLUSIONS: Our data demonstrate that radiologically guided percutaneous CNB should be considered as a valid diagnostic tool, as it provides quick and reliable histological diagnoses avoiding the complications and risks of splenectomy.

Microscopic Observation Drug Susceptibility Assay for Rapid Diagnosis of Lymph Node Tuberculosis and Detection of Drug Resistance.

In this study, 132 patients with lymphadenopathy were investigated. Fifty-two (39.4%) were diagnosed with tuberculosis (TB). The microscopic observation drug susceptibility (MODS) assay provided rapid (13 days), accurate diagnosis (sensitivity, 65.4%) and reliable drug susceptibility testing (DST). Despite its lower sensitivity than that of other methods, its faster results and simultaneous DST are advantageous in resource-poor settings, supporting the incorporation of MODS into diagnostic algorithms for extrapulmonary TB.

United Kingdom immune thrombocytopenia registry: retrospective evaluation of bone marrow fibrosis in adult patients with primary immune thrombocytopenia and correlation with clinical findings.

Fibrosis has been reported in some patients with immune thrombocytopenia (ITP) treated with thrombopoietin receptor agonists (TPO-RA). However, fibrosis has also been reported in patients with various stages of ITP, who were TPO-RA treatment-naïve. In our study, we looked for fibrosis in bone marrow trephine biopsies taken at initial diagnosis from 32 adult patients with ITP. Ten of the 32 evaluated samples (31·25%) showed increased reticulin (Grade 1-2 on Bauermeister scale and Grade 0-1 on the European Consensus scale), which showed a positive correlation with ethnicity (0·3%) but did not correlate with disease severity, any clinical features or co-morbidities.

Applications of machine and deep learning to thyroid cytology and histopathology: a review.

This review synthesises past research into how machine and deep learning can improve the cyto- and histopathology processing pipelines for thyroid cancer diagnosis. The current gold-standard preoperative technique of fine-needle aspiration cytology has high interobserver variability, often returns indeterminate samples and cannot reliably identify some pathologies; histopathology analysis addresses these issues to an extent, but it requires surgical resection of the suspicious lesions so cannot influence preoperative decisions. Motivated by these issues, as well as by the chronic shortage of trained pathologists, much research has been conducted into how artificial intelligence could improve current pipelines and reduce the pressure on clinicians. Many past studies have indicated the significant potential of automated image analysis in classifying thyroid lesions, particularly for those of papillary thyroid carcinoma, but these have generally been retrospective, so questions remain about both the practical efficacy of these automated tools and the realities of integrating them into clinical workflows. Furthermore, the nature of thyroid lesion classification is significantly more nuanced in practice than many current studies have addressed, and this, along with the heterogeneous nature of processing pipelines in different laboratories, means that no solution has proven itself robust enough for clinical adoption. There are, therefore, multiple avenues for future research: examine the practical implementation of these algorithms as pathologist decision-support systems; improve interpretability, which is necessary for developing trust with clinicians and regulators; and investigate multiclassification on diverse multicentre datasets, aiming for methods that demonstrate high performance in a process- and equipment-agnostic manner.

Cutaneous mucinous carcinoma arising in extramammary Paget disease of the perineum.

We present the case of a 74-year-old woman with a 7-year history of an expanding vulval and perianal erythematous plaque, which failed to respond to topical treatments in the community. Biopsy of the affected skin showed typical features of extramammary Paget disease. No underlying associated malignancy was identified. After 2 months of treatment with 5% topical imiquimod, the patient developed a new tender nodule in the perineal region. Histological examination revealed a mucinous carcinoma, which, after careful clinical assessment, was deemed to be a primary cutaneous mucinous carcinoma. This is the second reported case of a primary cutaneous mucinous carcinoma arising on a background of extramammary Paget disease of the vulva and perineum.

Development of a Validated Exam to Assess Pathologist Knowledge of Genomic Oncology.

CONTEXT.­: There is a clear need to educate health professionals in genomic medicine. Pathologists, given their critical role in cancer diagnostics, must understand core concepts in genomic oncology. Although high-quality evaluation is a cornerstone of medical education, to our knowledge a rigorously validated genomic oncology assessment tool has not been published. OBJECTIVE.­: To develop and validate a genomic oncology exam. DESIGN.­: A previously developed exam was updated and validated using 3 approaches: pretesting/posttesting in relation to a live genomic pathology workshop; comparison of scores of individuals at a priori defined knowledge levels; and use of Rasch analysis. This last approach is used in high-stakes testing, such as licensing exams. The exam included both knowledge-based as well as skills-based questions related to the use of online genomics tools. RESULTS.­: There was a significant difference in exam scores preworkshop and postworkshop (37.5% to 75%; P < .001). Individuals at a priori defined beginner, intermediate, and expert levels scored 35%, 58%, and 89%, respectively (P < .001). Rasch analysis demonstrated excellent fit and reliability and led to further exam refinement with the removal of 2 questions deemed unnecessary for assessment. CONCLUSIONS.­: A rigorously validated exam has now been created to assess pathologist genomic oncology knowledge and skills. The exam can be used to assess both individual learners as well as educational interventions. The exam may also be applicable to other specialties involved in genomic-based cancer care.

Immune landscape in Burkitt lymphoma reveals M2-macrophage polarization and correlation between PD-L1 expression and non-canonical EBV latency program.

BACKGROUND: The Tumor Microenviroment (TME) is a complex milieu that is increasingly recognized as a key factor in multiple stages of disease progression and responses to therapy as well as escape from immune surveillance. However, the precise contribution of specific immune effector and immune suppressor components of the TME in Burkitt lymphoma (BL) remains poorly understood. METHODS: In this paper, we applied the computational algorithm CIBERSORT to Gene Expression Profiling (GEP) datasets of 40 BL samples to draw a map of immune and stromal components of TME. Furthermore, by multiple immunohistochemistry (IHC) and multispectral immunofluorescence (IF), we investigated the TME of additional series of 40 BL cases to evaluate the role of the Programmed Death-1 and Programmed Death Ligand-1 (PD-1/PD-L1) immune checkpoint axis. RESULTS: Our results indicate that M2 polarized macrophages are the most prominent TME component in BL. In addition, we investigated the correlation between PD-L1 and latent membrane protein-2A (LMP2A) expression on tumour cells, highlighting a subgroup of BL cases characterized by a non-canonical latency program of EBV with an activated PD-L1 pathway. CONCLUSION: In conclusion, our study analysed the TME in BL and identified a tolerogenic immune signature highlighting new potential therapeutic targets.

Correction to: Immune landscape in Burkitt lymphoma reveals M2-macrophage polarization and correlation between PD-L1 expression and non-canonical EBV latency program.

[This corrects the article DOI: 10.1186/s13027-020-00292-w.].

Epigenetic Regulation of iASPP-p63 Feedback Loop in Cutaneous Squamous Cell Carcinoma.

Keratinocyte skin cancer, comprising cutaneous squamous (cSCC) and basal cell carcinoma, is the most common malignancy in the United Kingdom. P53 is frequently mutated in cSCC. iASPP is a key inhibitor of p53 and NF-κB signaling pathways and has been documented as highly expressed in several types of human cancer. We have previously identified an autoregulatory feedback loop between iASPP and p63, which is critical in epidermal homeostasis. We hypothesized a potential role for dysregulation of this axis in the pathogenesis of keratinocyte malignancies. Immunostaining of 116 cSCC clinical samples revealed increased iASPP and ΔNp63 expression, but also highlighted a significant alteration of iASPP cellular localization, with consequent deregulation of its function. Expression patterns, functionality, and gene and microRNA expression analysis were further investigated in 10 cSCC cell lines. Our data suggest that while direct effects of iASPP and p63 upon each other's expression are maintained in cSCC, epigenetic dysregulation of the feedback loop occurs at the microRNA level by a previously unreported mechanism controlling p63 expression. We demonstrate that this autoregulatory feedback loop controls cell migration in cSCC by blocking epithelial-mesenchymal transition and promoting proliferation, and provides future directions for clinical biomarker and therapeutic target discovery in cutaneous SCC.

Novel markers in pediatric-type follicular lymphoma.

The aim of this study was to review the histopathological, phenotypic, and molecular characteristics of pediatric-type follicular lymphoma (PTFL) and to assess the diagnostic value of novel immunohistochemical markers in distinguishing PTFL from follicular hyperplasia (FH). A total of 13 nodal PTFLs were investigated using immunohistochemistry, fluorescence in situ hybridization (FISH), and PCR and were compared with a further 20 reactive lymph nodes showing FH. Morphologically, PTFL cases exhibited a follicular growth pattern with irregular lymphoid follicles in which the germinal centers were composed of numerous blastoid cells showing a starry-sky appearance. Immunohistochemistry highlighted preserved CD10 (13/13) and BCL6 (13/13) staining, CD20 (13/13) positivity, a K light chain predominance (7/13), and partial BCL2 expression in 6/13 cases (using antibodies 124, E17, and SP66). The germinal center (GC)-associated markers stathmin and LLT-1 were positive in most of the cases (12/13 and 12/13, respectively). Interestingly, FOXP-1 was uniformly positive in PTFL (12/13 cases) in contrast to reactive GCs in FH, where only a few isolated positive cells were observed. FISH revealed no evidence of BCL2, BCL6, or MYC rearrangements in the examined cases. By PCR, clonal immunoglobulin gene rearrangements were detected in 100% of the tested PTFL cases. Our study confirmed the unique morphological and immunophenotypic features of PTFL and suggests that FOXP-1 can represent a novel useful diagnostic marker in the differential diagnosis between PTFL and FH.

The Role of Human Papillomaviruses and Polyomaviruses in BRAF-Inhibitor Induced Cutaneous Squamous Cell Carcinoma and Benign Squamoproliferative Lesions.

Background: Human papillomavirus (HPV) has long been proposed as a cofactor in the pathogenesis of cutaneous squamous cell carcinoma (cSCC). More recently, the striking clinico-pathological features of cSCCs that complicate treatment of metastatic melanoma with inhibitors targeting BRAF mutations (BRAFi) has prompted speculation concerning a pathogenic role for oncogenic viruses. Here, we investigate HPV and human polyomaviruses (HPyV) and correlate with clinical, histologic, and genetic features in BRAFi-associated cSCC. Materials and Methods: Patients receiving BRAFi treatment were recruited at Barts Health NHS Trust. HPV DNA was detected in microdissected frozen samples using reverse line probe technology and degenerate and nested PCR. HPV immunohistochemistry was performed in a subset of samples. Quantitative PCR was performed to determine the presence and viral load of HPyVs with affinity for the skin (HPyV6, HPyV7, HPyV9, MCPyV, and TSPyV). These data were correlated with previous genetic mutational analysis of H, K and NRAS, NOTCH1/2, TP53, CDKN2A, CARD11, CREBBP, TGFBR1/2. Chromosomal aberrations were profiled using single nucleotide polymorphism (SNP) arrays. Results: Forty-five skin lesions from seven patients treated with single agent vemurafenib in 2012-2013 were analyzed: 12 cSCC, 19 viral warts (VW), 2 actinic keratosis (AK), 5 verrucous keratosis/other squamoproliferative (VK/SP) lesions, one melanocytic lesion and 6 normal skin samples. Significant histologic features of viral infection were seen in 10/12 (83%) cSCC. HPV DNA was detected in 18/19 (95%) VW/SP, 9/12 (75%) cSCC, 4/5 (80%) SP, and 3/6 (50%) normal skin samples and in 1/12 cases assessed by immunohistochemistry. HPyV was co-detected in 22/30 (73%) of samples, usually at low viral load, with MCPyV and HPyV7 the most common. SNP arrays confirmed low levels of chromosomal abnormality and there was no significant correlation between HPV or HPyV detection and individual gene mutations or overall mutational burden. Conclusion: Despite supportive clinicopathologic evidence, the role for HPV and HPyV infection in the pathogenesis of BRAFi-induced squamoproliferative lesions remains uncertain. Synergistic oncogenic mechanisms are plausible although speculative. Nonetheless, with the prospect of a significant increase in the adjuvant use of these drugs, further research is justified and may provide insight into the pathogenesis of other BRAFi-associated malignancies.

The Undergraduate Training in Genomics (UTRIG) Initiative: early & active training for physicians in the genomic medicine era.

Genomic medicine is transforming patient care. However, the speed of development has left a knowledge gap between discovery and effective implementation into clinical practice. Since 2010, the Training Residents in Genomics (TRIG) Working Group has found success in building a rigorous genomics curriculum with implementation tools aimed at pathology residents in postgraduate training years 1-4. Based on the TRIG model, the interprofessional Undergraduate Training in Genomics (UTRIG) Working Group was formed. Under the aegis of the Undergraduate Medical Educators Section of the Association of Pathology Chairs and representation from nine additional professional societies, UTRIG's collaborative goal is building medical student genomic literacy through development of a ready-to-use genomics curriculum. Key elements to the UTRIG curriculum are expert consensus-driven objectives, active learning methods, rigorous assessment and integration.

Intracellular TCR-signaling pathway: novel markers for lymphoma diagnosis and potential therapeutic targets.

Despite the immunologic functions of T-cell receptor signaling molecules being extensively investigated, their potential as immunohistochemical markers has been poorly explored. With this background, we evaluated the expression of 5 intracellular proteins-GADS, DOK2, SKAP55, ITK, and PKCα-involved in T-cell receptor signaling in normal and neoplastic hematologic tissue samples, using antibodies raised against fixation-resistant epitopes of the 5 molecules. All 5 antibodies were associated with normal T-cell differentiation. GADS, DOK2, SKAP55, and ITK turned out to be T-cell lineage-specific markers in the setting of lymphoid and myeloid precursor neoplasms but showed differential expression in peripheral T-cell lymphoma (PTCL) subtypes, being detected in PTCL/not otherwise specified (NOS) and angioimmunoblastic T-cell lymphoma but negative in anaplastic large cell lymphoma (ALCL). Peripheral B-cell lymphomas were consistently negative for ITK, with occasional cases showing expression of DOK2 and SKAP55, and a proportion (47%) of hairy cell leukemias were GADS. Notably, PKCα highlighted a defective antigen in both PTCL/NOS (6%) and angioimmunoblastic T-cell lymphoma (10%), mostly negative in ALCL, and was aberrantly expressed in classical Hodgkin lymphoma (65%), Burkitt lymphoma (48%), and plasma cell myeloma (48%). In conclusion, all five molecules evaluated play a role in T-cell differentiation in normal and neoplastic tissues. They can be applied confidently to routine sections contributing primarily to assignment of T-lineage differentiation in the setting of hematopoietic precursor neoplasms (GADS/DOK2/SKAP55/ITK) and for the differential diagnosis between ALCL and PTCL/NOS (GADS/DOK2/SKAP55/ITK) or classical Hodgkin lymphoma (PKCα). Finally, association with specific tumor subtypes may have therapeutic potential.