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Jessica Okosun, Csaba Bödör and colleagues performed whole-genome or whole-exome sequencing on 10 follicular lymphoma and transformed follicular lymphoma pairs, followed by deep sequencing of 28 target genes in an additional 122 cases. They identify recurrent mutations in linker histone genes and genes involved in JAK-STAT signaling, NF-κB signaling and B cell development. Follicular lymphoma is an incurable malignancy 1 , with transformation to an aggressive subtype representing a critical event during disease progression. Here we performed whole-genome or whole-exome sequencing on 10 follicular lymphoma–transformed follicular lymphoma pairs followed by deep sequencing of 28 genes in an extension cohort, and we report the key events and evolutionary processes governing tumor initiation and transformation. Tumor evolution occurred through either a 'rich' or 'sparse' ancestral common progenitor clone (CPC). We identified recurrent mutations in linker histone, JAK-STAT signaling, NF-κB signaling and B cell developmental genes. Longitudinal analyses identified early driver mutations in chromatin regulator genes ( CREBBP , EZH2 and KMT2D (MLL2)), whereas mutations in EBF1 and regulators of NF-κB signaling ( MYD88 and TNFAIP3 ) were gained at transformation. Collectively, this study provides new insights into the genetic basis of follicular lymphoma and the clonal dynamics of transformation and suggests that personalizing therapies to target key genetic alterations in the CPC represents an attractive therapeutic strategy.

AimsPrimary cilia play an important role in the regulation of cell signalling pathways and are thought to have a role in cancer but have seldom been studied in human cancer samples.MethodsPrimary cilia were visualised by dual immunofluorescence for anti-CROCC (ciliary rootlet coiled-coil) and anti-tubulin in a range of human cancers (including carcinomas of stomach, pancreas, prostate, lung and colon, lobular and ductal breast cancers and follicular lymphoma) and in matched normal tissue (stomach, pancreas, lung, large and small intestines, breast and reactive lymph nodes) samples using a tissue microarray; their frequency, association with proliferation, was measured by Ki-67 staining and their structure was analysed.ResultsCompared with normal tissues, primary cilia frequency was significantly elevated in adenocarcinoma of the lung (2.75% vs 1.85%, p=0.016), adenocarcinoma of the colon (3.80% vs 2.43%, respectively, p=0.017), follicular lymphoma (1.18% vs 0.83%, p=0.003) and pancreatic adenocarcinoma (7.00% vs 5.26%, p=0.002); there was no statistically significant difference compared with normal control tissue for gastric and prostatic adenocarcinomas or for lobular and ductal breast cancers. Additionally, structural abnormalities of primary cilia were identified in cancer tissues, including elongation of the axoneme, multiple basal bodies and branching of the axoneme. Ki-67 scores ranged from 0.7% to 78.4% and showed no statistically significant correlation with primary cilia frequency across all tissues (p=0.1501).ConclusionsThe results show upregulation of primary cilia and the presence of structural defects in a wide range of human cancer tissue samples demonstrating association of dysregulation of primary cilia with human cancer.

Resource barriers to the provision of accessible training in cancer diagnosis in lower- and middle-income countries (LMICs) limit the potential of African health systems. Long-term provision via teaching visits from senior pathologists and trainee foreign placements is unsustainable due to the prohibitive costs of travel and subsistence. Emerging eLearning methods would allow pathologists to be trained by experts in a cheaper, more efficient, and more scalable way. This study aimed to develop an online teaching platform, starting with hematopathology, for trainee pathologists in sub-Saharan Africa, initially in Nairobi, Kenya, and Lusaka, Zambia. Course materials were prepared for both Canvas and the Zoom eLearning platforms using digitally scanned slides of lymph nodes and bone marrow trephines. Initial in-person visits were made to each site to establish trainee rapport and maximize engagement, evaluate different methods and course content, and obtain feedback to develop the project. The knowledge of trainees before and after course completion was used to measure initial effectiveness. Online teaching with the preferred platform is to be continued for 1 year before re-evaluation for long-term effectiveness. Canvas was selected as the preferred delivery platform as it is freely available and has good functionality to support all required tasks. Face-to-face teaching was considered optimal to establish the initial rapport necessary to maximize subsequent engagement with online teaching. Challenges have included sub-optimal internet speeds and connections and scheduling issues. Weekly online hematopathology teaching sessions using live image capture microscope sessions, Zoom, and Canvas have been delivered to students in Kenya and Zambia, with good attendance and interaction in case discussions. Our team has successfully designed and delivered an online training program in hematopathology to trainee pathologists in Kenya and Zambia, which has been ongoing for over a year. This project is now being scaled to other sub-Saharan countries and other sub-specialties.

This report identifies upregulation of HGF as an autocrine growth pathway in several subsets of AML. Ligand-dependent activation of MET represents a new oncogenic stimulus, and the dynamic regulation of HGF can overcome the effects of MET inhibition. These results suggest that combination treatments may be needed to disrupt this autocrine signaling loop and quell the growth of AML. Although the treatment of acute myeloid leukemia (AML) has improved substantially in the past three decades, more than half of all patients develop disease that is refractory to intensive chemotherapy 1 , 2 . Functional genomics approaches offer a means to discover specific molecules mediating the aberrant growth and survival of cancer cells 3 , 4 , 5 , 6 , 7 , 8 . Thus, using a loss-of-function RNA interference genomic screen, we identified the aberrant expression of hepatocyte growth factor (HGF) as a crucial element in AML pathogenesis. We found HGF expression leading to autocrine activation of its receptor tyrosine kinase, MET, in nearly half of the AML cell lines and clinical samples we studied. Genetic depletion of HGF or MET potently inhibited the growth and survival of HGF-expressing AML cells. However, leukemic cells treated with the specific MET kinase inhibitor crizotinib developed resistance resulting from compensatory upregulation of HGF expression, leading to the restoration of MET signaling. In cases of AML where MET is coactivated with other tyrosine kinases, such as fibroblast growth factor receptor 1 (FGFR1) 9 , concomitant inhibition of FGFR1 and MET blocked this compensatory HGF upregulation, resulting in sustained logarithmic cell killing both in vitro and in xenograft models in vivo . Our results show a widespread dependence of AML cells on autocrine activation of MET, as well as the key role of compensatory upregulation of HGF expression in maintaining leukemogenic signaling by this receptor. We anticipate that these findings will lead to the design of additional strategies to block adaptive cellular responses that drive compensatory ligand expression as an essential component of the targeted inhibition of oncogenic receptors in human cancers.

Background: Histopathological prognostication relies on morphological pattern recognition, but as numbers of biomarkers increase, human prognostic pattern-recognition ability decreases. Follicular lymphoma (FL) has a variable outcome, partly determined by FOXP3 Tregs. We have developed an automated method, hypothesised interaction distribution (HID) analysis, to analyse spatial patterns of multiple biomarkers which we have applied to tumour-infiltrating lymphocytes in FL. Methods: A tissue microarray of 40 patient samples was used in triplex immunohistochemistry for FOXP3, CD3 and CD69, and multispectral imaging used to determine the numbers and locations of CD3 + , FOXP3/CD3 + and CD69/CD3 + T cells. HID analysis was used to identify associations between cellular pattern and outcome. Results: Higher numbers of CD3 + ( P =0.0001), FOXP3/CD3 + ( P =0.0031) and CD69/CD3 + ( P =0.0006) cells were favourable. Cross-validated HID analysis of cell pattern identified patient subgroups with statistically significantly different survival (35.5 vs 142 months, P =0.00255), a more diffuse pattern associated with favourable outcome and an aggregated pattern with unfavourable outcome. Conclusions: A diffuse pattern of FOXP3 and CD69 positivity was favourable, demonstrating ability of HID analysis to automatically identify prognostic cellular patterns. It is applicable to large numbers of biomarkers, representing an unsupervised, automated method for identification of undiscovered prognostic cellular patterns in cancer tissue samples.

AimsFollicular lymphoma is the second most common type of non-Hodgkin's lymphoma worldwide. The majority of patients diagnosed as having follicular lymphoma have an indolent form of the disease, but a subset of patients have aggressive disease with a shorter survival interval. Optimal treatment stratification requires a distinction between these two groups, though there are presently few prognostic biomarkers available. The transcription factor YY1 has been shown to play an important role in cancer biology. The authors have previously reported a correlation of Yin Yang 1 (YY1) mRNA levels with survival in FL. This study aimed to validate these findings at the protein level.MethodsQuantification of YY1 protein was carried out on 26 FL biopsy samples using quantum dot labelled immunohistochemistry. Ki-67 percentage, grade, YY1 protein levels and T cell and macrophage markers were used in a multivariable analysis for survival in 26 cases of FL.ResultsExpression levels of YY1 protein were significantly increased in patients alive in comparison with those dead after follow-up (p≤0.025). Kaplan–Meier analysis showed association of higher expression levels of YY1 with longer survival (p≤0.01) (hazard ratio 3.33, 95% CI 1.26 to 8.85). The multivariable analysis identified YY1 protein level as the strongest predictor of outcome (p≤0.018), with none of the other markers being significantly associated with outcome.ConclusionThese results support the prognostic utility of YY1 in FL, indicating potential as a clinical biomarker.

Aims Ying Yang 1 (YY1) is a transcription factor involved in both proliferation and apoptosis. It is prognostic in follicular lymphoma (FL), increased protein levels being associated with favourable outcome. PLK1 is a critical regulator of mitosis, playing a role in spindle formation and in regulation of the G2/M cell cycle checkpoint. PLK1 phosphorylates YY1 at the G2/M checkpoint with activation of YY1 and resultant progression from G2 into mitosis. Methods This study aims to investigate possible molecular coexpression and interaction of YY1 with PLK1 in FL using Duolink II in situ proximity ligation assay (PLA) in 51 FL samples in a tissue microarray. Results Positive PLA signals were present at variable frequency and Kaplan-Meier analysis showed association of signal frequency above the median with unfavourable outcome (p=0.0270). PLA signals were localised to the nuclear edge, with only one signal per cell, suggesting PLK1 and YY1 coexpression at the centrosome. In a minority of cells, two very close PLA signals were present in a single cell, and occasionally, there was a strong ring of semi-confluent fluorescent PLA signals round the nucleus of non-dividing cells, while rarely events were observed in the cytoplasm surrounding dividing cells. Conclusions The results confirm association of YY1 and PLK1 with outcome in FL and suggest coexpression at the centrosome. Given the reported interaction of YY1 with PLK1 at the centriole and promotion of cell division at the G2/M checkpoint, the results would concord with the known association of higher proliferation with poor outcome in FL.

Primary NHLs of the uterus and cervix are rare, comprising only 0.54%-0.64% of all extranodal NHLs, most occurring in the cervix. 1 Marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT) of the uterus is extremely rare, with only seven cases reported in the literature. 2-8 We report a rare case of an extranodal marginal zone B cell lymphoma arising from the MALT tissue of the endometrium. The histological differential diagnosis of extranodal marginal zone lymphomas of the uterus includes other haematological malignancies, endometrial stromal sarcoma and florid benign reactive lymphoid proliferations seen in inflammatory conditions such as follicular endometritis.

BackgroundMultiplexed immunohistochemistry (IHC) has the potential to improve conventional IHC staining allowing for analysis of multiple cell phenotypes while maintaining spatial context. Automated multispectral image analysis and computer-based cell recognition make the process more attainable, but stringent validation of multiplex IHC is still required. Pertinently, multiplex IHC allows for the characterisation and enumeration of immune cell densities in the tumour micro-environment, of particular importance for analysis of tumour-infiltrating immune cells which require multiple co-localised markers for their identification. However, issues of antibody blocking, cross-reactivity and masking have caused concern that the results of multiplex staining may not accurately reflect those of single-plex staining. The Opal workflow from PerkinElmer uses a heat-induced epitope retrieval step between each antibody detected which aims to obviate these potential problems and enable single-species antibody use. In this study we systematically validated multiplex staining for a range of immune cell markers against single-plex staining for each marker to determine the accuracy of the multiplex method.MethodsValidation of multiplex IHC was undertaken using a multi-tissue TMA stained in multiplex for CD3, CD4, CD56 and CD20 in a 4-marker validation and for CD3, CD4, CD8, CD20 and FOXP3 in a 5-marker validation. The TMA was composed of 72 cores including normal lung, pancreas, breast, prostate and stomach and malignant prostate, lung and colon. Spearman correlations measured agreement of immune cell populations with those of singly stained serial sections on a per-core basis. Single-stain/single-stain comparisons of corresponding immune cell populations provided baseline variation of immune cells in subsequent sections.ResultsAll validation comparisons showed a highly significant correlation (P < 0.0001) with strong correlation was observed between cores for the majority of multiplex stains. Specifically, for the 4-plex experiment, a high degree of correlation was observed for CD3, CD8 and CD20 with R2 of 0.835, 0.950 and 0.870 respectively (P= < 0.001); CD56 showed a lower degree of correlation between the multiplex and single stains with an R2 of 0.584 (P < 0.0001). For the 5-plex experiment, a high degree of correlation was observed for high degree of correlation for CD3, CD8 and CD20 with R2 of 0.87, 0.95 and 0.87 respectively (P < 0.0001). FOXP3 showed a poor correlation of 0.74 and CD4 showed a poor correlation of 0.58 (P < 0.0001).ConclusionMultiplex IHC is comparable to single stain IHC preserving precious samples and reagents while enhancing the information gained.