Development of an online teaching platform to improve access to postgraduate pathology training in sub-Saharan Africa.

Background: 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. Purpose: 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. Methods: 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. Results: 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. Conclusion: 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.

Lymphoma triage from H&E using AI for improved clinical management.

AIMS: In routine diagnosis of lymphoma, initial non-specialist triage is carried out when the sample is biopsied to determine if referral to specialised haematopathology services is needed. This places a heavy burden on pathology services, causes delays and often results in over-referral of benign cases. We aimed to develop an automated triage system using artificial intelligence (AI) to enable more accurate and rapid referral of cases, thereby addressing these issues. METHODS: A retrospective dataset of H&E-stained whole slide images (WSI) of lymph nodes was taken from Newcastle University Hospital (302 cases) and Manchester Royal Infirmary Hospital (339 cases) with approximately equal representation of the 3 most prevalent lymphoma subtypes: follicular lymphoma, diffuse large B-cell and classic Hodgkin's lymphoma, as well as reactive controls. A subset (80%) of the data was used for training, a further validation subset (10%) for model selection and a final non-overlapping test subset (10%) for clinical evaluation. RESULTS: AI triage achieved multiclass accuracy of 0.828±0.041 and overall accuracy of 0.932±0.024 when discriminating between reactive and malignant cases. Its ability to detect lymphoma was equivalent to that of two haematopathologists (0.925, 0.950) and higher than a non-specialist pathologist (0.75) repeating the same task. To aid explainability, the AI tool also provides uncertainty estimation and attention heatmaps. CONCLUSIONS: Automated triage using AI holds great promise in contributing to the accurate and timely diagnosis of lymphoma, ultimately benefiting patient care and outcomes.

The impact of obesity and bariatric surgery on the immune microenvironment of the endometrium.

BACKGROUND: The incidence of endometrial cancer is rising in parallel with the obesity epidemic. Obesity increases endometrial cancer risk and weight loss is protective, but the underlying mechanisms are incompletely understood. We hypothesise that the immune microenvironment may influence susceptibility to malignant transformation in the endometrium. The aim of this study was to measure the impact of obesity and weight loss on the immunological landscape of the endometrium. METHODS: We conducted a prospective cohort study of women with class III obesity (body mass index, BMI ≥ 40 kg/m2) undergoing bariatric surgery or medically-supervised low-calorie diet. We collected blood and endometrial samples at baseline, and two and 12 months after weight loss intervention. Serum was analysed for inflammatory markers CRP, IL-6 and TNF-α. Multiplex immunofluorescence was used to simultaneously identify cells positive for immune markers CD68, CD56, CD3, CD8, FOXP3 and PD-1 in formalin-fixed paraffin-embedded endometrial tissue sections. Kruskal-Wallis tests were used to determine whether changes in inflammatory and immune biomarkers were associated with weight loss. RESULTS: Forty-three women with matched serum and tissue samples at all three time points were included in the analysis. Their median age and BMI were 44 years and 52 kg/m2, respectively. Weight loss at 12 months was greater in women who received bariatric surgery (n = 37, median 63.3 kg) than low-calorie diet (n = 6, median 12.8 kg). There were significant reductions in serum CRP (p = 3.62 × 10-6, r = 0.570) and IL-6 (p = 0.0003, r = 0.459), but not TNF-α levels, with weight loss. Tissue immune cell densities were unchanged except for CD8+ cells, which increased significantly with weight loss (p = 0.0097, r = -0.323). Tissue CD3+ cell density correlated negatively with systemic IL-6 levels (p = 0.0376; r = -0.318). CONCLUSION: Weight loss is associated with reduced systemic inflammation and a recruitment of protective immune cell types to the endometrium, supporting the concept that immune surveillance may play a role in endometrial cancer prevention.

Immune infiltrate diversity confers a good prognosis in follicular lymphoma.

BACKGROUND: Follicular lymphoma (FL) prognosis is influenced by the composition of the tumour microenvironment. We tested an automated approach to quantitatively assess the phenotypic and spatial immune infiltrate diversity as a prognostic biomarker for FL patients. METHODS: Diagnostic biopsies were collected from 127 FL patients initially treated with rituximab-based therapy (52%), radiotherapy (28%), or active surveillance (20%). Tissue microarrays were constructed and stained using multiplex immunofluorescence (CD4, CD8, FOXP3, CD21, PD-1, CD68, and DAPI). Subsequently, sections underwent automated cell scoring and analysis of spatial interactions, defined as cells co-occurring within 30 µm. Shannon's entropy, a metric describing species biodiversity in ecological habitats, was applied to quantify immune infiltrate diversity of cell types and spatial interactions. Immune infiltrate diversity indices were tested in multivariable Cox regression and Kaplan-Meier analysis for overall (OS) and progression-free survival (PFS). RESULTS: Increased diversity of cell types (HR = 0.19 95% CI 0.06-0.65, p = 0.008) and cell spatial interactions (HR = 0.39, 95% CI 0.20-0.75, p = 0.005) was associated with favourable OS, independent of the Follicular Lymphoma International Prognostic Index. In the rituximab-treated subset, the favourable trend between diversity and PFS did not reach statistical significance. CONCLUSION: Multiplex immunofluorescence and Shannon's entropy can objectively quantify immune infiltrate diversity and generate prognostic information in FL. This automated approach warrants validation in additional FL cohorts, and its applicability as a pre-treatment biomarker to identify high-risk patients should be further explored. The multiplex image dataset generated by this study is shared publicly to encourage further research on the FL microenvironment.

Spatial proximity between T and PD-L1 expressing cells as a prognostic biomarker for oropharyngeal squamous cell carcinoma.

BACKGROUND: Fulfilling the promise of cancer immunotherapy requires novel predictive biomarkers to characterise the host immune microenvironment. Deciphering the complexity of immune cell interactions requires an automated multiplex approach to histological analysis of tumour sections. We tested a new automatic approach to select tissue and quantify the frequencies of cell-cell spatial interactions occurring in the PD1/PD-L1 pathway, hypothesised to reflect immune escape in oropharyngeal squamous cell carcinoma (OPSCC). METHODS: Single sections of diagnostic biopsies from 72 OPSCC patients were stained using multiplex immunofluorescence (CD8, PD1, PD-L1, CD68). Following multispectral scanning and automated regions-of-interest selection, the Hypothesised Interaction Distribution (HID) method quantified spatial proximity between cells. Method applicability was tested by investigating the prognostic significance of co-localised cells (within 30 µm) in patients stratified by HPV status. RESULTS: High frequencies of proximal CD8+ and PD-L1+ (HR 2.95, p = 0.025) and PD1+ and PD-L1+ (HR 2.64, p = 0.042) cells were prognostic for poor overall survival in patients with HPV negative OPSCC (n = 31). CONCLUSION: The HID method can quantify spatial interactions considered to reflect immune escape and generate prognostic information in OPSCC. The new automated approach is ready to test in additional cohorts and its applicability should be explored in research and clinical studies.

Measurement of indicator genes using global complementary DNA (cDNA) amplification, by polyadenylic acid reverse transcriptase polymerase chain reaction (poly A RT-PCR): A feasibility study using paired samples from tissue and ductal juice in patients undergoing pancreatoduodenectomy.

OBJECTIVES: The aim of this study is to compare gene expression profiles in RNA isolated from pancreatic ductal juice with the RNA expression profiles of the same genes from matched intra-operative tissue samples from pancreatic tumours. METHODS: Intra-operative sampling of pancreatic juice and collection of matched tissue samples was undertaken in patients undergoing pancreatoduodenectomy for clinically suspected pancreatic cancer and a precursor lesion, main-duct intraductal papillary mucinous neoplasm. RNA was isolated and Poly A PCR was used to globally amplify the RNA. Real-time polymerase chain reaction (RT-PCR) was used to measure expression levels of 17 genes selected from microarray studies. Spearman's rank correlation test was used to examine the relationship of gene expression between pancreatic juice and tissue. The study was approved by Regional Ethics Committee. RESULTS: Mesothelin (MSLN) showed significant correlation (p < 0.008) in expression levels between paired pancreatic juice and tissue samples in pancreas cancer. In intraductal papillary mucinous neoplasms (IPMN), Matrix Metalloproteinase 7 (MMP7), showed significant correlation (p < 0.01) in the expression levels between paired pancreatic juice and tissue samples. CONCLUSION: This study confirms that RNA analysis of paired pancreatic juice and tissue samples and establishment of cDNA using poly A PCR is technically feasible. Application of the technique to non-invasively obtained pancreatic juice during endoscopic assessment of tumours and the use of gene arrays of cancer indicator genes are the next steps in development of this technique.

HER2 testing of gastro-oesophageal adenocarcinoma: a commentary and guidance document from the Association of Clinical Pathologists Molecular Pathology and Diagnostics Committee.

The use of biologics targeted to the human epidermal growth factor receptor 2 (HER2) protein is the latest addition to the armamentarium used to fight advanced gastric or gastro-oesophageal junction adenocarcinoma. The decision to treat with the biologic trastuzumab is completely dependent on HER2 testing of tumour tissue. In 2017, the College of American Pathologists, American Society for Clinical Pathology and the American Society of Clinical Oncology jointly published guidelines for HER2 testing and clinical decision making in gastro-oesophageal adenocarcinoma. The Association of Clinical Pathologists Molecular Pathology and Diagnostics Committee has issued the following document as a commentary of these guidelines and, in parallel, to provide guidance on HER2 testing in National Health Service pathology departments within the UK. This guidance covers issues related to case selection, preanalytical aspects, analysis and interpretation of such HER2 testing.

Derepression of the Iroquois Homeodomain Transcription Factor Gene IRX3 Confers Differentiation Block in Acute Leukemia.

The Iroquois homeodomain transcription factor gene IRX3 is expressed in the developing nervous system, limb buds, and heart, and transcript levels specify obesity risk in humans. We now report a functional role for IRX3 in human acute leukemia. Although transcript levels are very low in normal human bone marrow cells, high IRX3 expression is found in ∼30% of patients with acute myeloid leukemia (AML), ∼50% with T-acute lymphoblastic leukemia, and ∼20% with B-acute lymphoblastic leukemia, frequently in association with high-level HOXA gene expression. Expression of IRX3 alone was sufficient to immortalize hematopoietic stem and progenitor cells (HSPCs) in myeloid culture and induce lymphoid leukemias in vivo. IRX3 knockdown induced terminal differentiation of AML cells. Combined IRX3 and Hoxa9 expression in murine HSPCs impeded normal T-progenitor differentiation in lymphoid culture and substantially enhanced the morphologic and phenotypic differentiation block of AML in myeloid leukemia transplantation experiments through suppression of a terminal myelomonocytic program. Likewise, in cases of primary human AML, high IRX3 expression is strongly associated with reduced myelomonocytic differentiation. Thus, tissue-inappropriate derepression of IRX3 contributes significantly to the block in differentiation, which is the pathognomonic feature of human acute leukemias.

Primary cilia are increased in number and demonstrate structural abnormalities in human cancer.

AIMS: Primary 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. METHODS: Primary 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. RESULTS: Compared 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). CONCLUSIONS: The 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.

Post-transplant lymphoproliferative disorder in adult renal transplant recipients: survival and prognosis.

Post-transplant lymphoproliferative disorder (PTLD) is a rare, serious complication following solid organ transplantation, with an incidence of 2.6 cases per 1000 patient years. Optimal treatment strategies and risk stratifications specific to kidney transplantation are lacking and PTLD mortality remains high. This study investigated survival and prognosis in 89 cases of PTLD presenting over 44 years at Manchester Royal Infirmary. Patient survival following diagnosis was 72% at 6 months, 67% at 1 year and 54% at 3 years. In multivariate analysis, a poorer 3 year survival was associated with acute kidney injury at diagnosis (p = 0.0001), impaired renal function (p = 0.04), early onset (p = 0.02), T cell disease (p = 0.02) and previous treatment with anti-thymocyte globulin (p = 0.04). The inclusion of graft function adds prognostic value to risk stratification and should be explored further. Strategies to improve survival should include timing and choice of immuno-chemotherapy, preparation for dialysis and aggressive surveillance for sepsis and treatment toxicity.

Automated prognostic pattern detection shows favourable diffuse pattern of FOXP3(+) Tregs in follicular lymphoma.

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.

Increased SYK activity is associated with unfavorable outcome among patients with acute myeloid leukemia.

Recent discoveries have led to the testing of novel targeted therapies for the treatment of acute myeloid leukemia (AML). To better inform the results of clinical trials, there is a need to identify and systematically assess biomarkers of response and pharmacodynamic markers of successful target engagement. Spleen tyrosine kinase (SYK) is a candidate therapeutic target in AML. Small-molecule inhibitors of SYK induce AML differentiation and impair leukemia progression in preclinical studies. However, tools to predict response to SYK inhibition and to routinely evaluate SYK activation in primary patient samples have been lacking. In this study we quantified phosphorylated SYK (P-SYK) in AML cell lines and establish that increasing levels of baseline P-SYK are correlated with an increasing sensitivity to small-molecule inhibitors targeting SYK. In addition, we found that pharmacological inhibition of SYK activity extinguishes P-SYK expression as detected by an immunohistochemical (IHC) test. Quantitative analysis of P-SYK expression by the IHC test in a series of 70 primary bone marrow biopsy specimens revealed a spectrum of P-SYK expression across AML cases and that high P-SYK expression is associated with unfavourable outcome independent of age, cytogenetics, and white blood cell count. This study thus establishes P-SYK as a critical biomarker in AML that identifies tumors sensitive to SYK inhibition, identifies an at-risk patient population, and allows for the monitoring of target inhibition during treatment.

QuantiGene Plex Represents a Promising Diagnostic Tool for Cell-of-Origin Subtyping of Diffuse Large B-Cell Lymphoma.

Emerging therapies targeting the molecularly distinct GCB and non-GCB/ABC subtypes of diffuse large B-cell lymphoma (DLBCL) have created the need to develop an accurate subtyping assay for routine use. We investigated the potential of QuantiGene Plex (QGP)-branched DNA signal amplification assay-for DLBCL subtyping. We performed in silico analysis of public DLBCL datasets to develop and validate a naïve Bayes classifier, and migrated the resulting 21-gene classifier to QGP and real-time quantitative PCR (qPCR) assays. Forty DLBCL formalin-fixed, paraffin-embedded tumors of known subtype (20 per subtype by gene expression profiling of paired fresh-frozen tissues) were reclassified, and results for QGP (on 38/40 for 21/21 targets) and qPCR (on 40/40 samples for 19/21 targets) compared for recapitulation of microarray data and classification accuracy. The 21-gene bayesian classifier achieved mean area under the curve values >0.9 on independent validation. QGP showed a higher correlation with microarray data (mean R(2) = 0.66 ± 0.05 versus 0.34 ± 0.07; P < 0.0001) and classification accuracy (92.1% versus 78.9%). The proportion of validated targets was also higher for QGP (85.7% versus 47.4%). The QGP protocol was rapid and simple to perform, at a cost similar to qPCR. These promising preliminary results strongly support ongoing work to develop a QGP companion diagnostic assay for DLBCL subtyping.

Diagnostic Mutation Profiling and Validation of Non-Small-Cell Lung Cancer Small Biopsy Samples using a High Throughput Platform.

BACKGROUND: A single platform designed for the synchronous screening of multiple mutations can potentially enable molecular profiling in samples of limited tumor tissue. This approach is ideal for the assessment of advanced non-small-cell lung cancer (NSCLC) diagnostic specimens, which often comprise small biopsies. Therefore, we aimed in this study to validate the mass spectrometry-based Sequenom LungCarta panel and MassARRAY platform using DNA extracted from a single 5 µM formalin-fixed paraffin-embedded tissue section. METHODS: Mutations, including those with an equivocal spectrum, detected in 90 cases of NSCLC (72 lung biopsies, 13 metastatic tissue biopsies, three resections, and two cytology samples) were validated by a combination of standard sequencing techniques, immunohistochemical staining for p53 protein, and next-generation sequencing with the TruSight Tumor panel. RESULTS: Fifty-five mutations were diagnosed in 47 cases (52%) in the following genes: TP53 (22), KRAS (15), EGFR (5), MET (3), PIK3CA (3), STK11 (2), NRF-2 (2), EPHA5 (1), EPHA3 (1), and MAP2K1 (1). Of the 90 samples, one failed testing due to poor quality DNA. An additional 7 TP53 mutations were detected by next-generation sequencing, which facilitated the interpretation of p53 immunohistochemistry but required 5 × 10 µM tumor sections per sample tested. CONCLUSIONS: The LungCarta panel is a sensitive method of screening for multiple alterations (214 mutations across 26 genes) and which optimizes the use of limited amounts of tumor DNA isolated from small specimens.

Single-cell analysis of K562 cells: an imatinib-resistant subpopulation is adherent and has upregulated expression of BCR-ABL mRNA and protein.

In chronic myeloid leukemia (CML) cells from different stages of maturation may have differential expression of BCR-ABL at both messenger RNA (mRNA) and protein level. However, the significance of such differential expression to clinical disease behavior is unknown. Using the CML-derived, BCR-ABL expressing cell line, K562, distinct plastic-adherent (K562/Adh) and nonadherent (K562/NonAdh) subpopulations were established and then analyzed both as single cells and as bulk cell populations. BCR-ABL mRNA was upregulated in K562/Adh compared with K562/NonAdh cells in both single cell and bulk population analyses (p < 0.0001). Similarly, phosphorylation of BCR protein was upregulated in K562/Adh, compared with K562/NonAdh cells (63.42% vs. 23.1%; p = 0.007), and these two K562 subpopulations were found to express significantly different microRNA species. Furthermore, treatment with the BCR-ABL tyrosine kinase inhibitor, imatinib, reduced cell viability more rapidly in K562/NonAdh compared with K562/Adh cells (p < 0.005) both at single and bulk cell levels. This discovery of an adherent subpopulation of K562 cells with increased BCR-ABL mRNA, increased phosphorylated BCR protein expression, differential microRNA expression, and increased imatinib resistance suggests that a similar subpopulation of cells can also mediate clinical resistance to imatinib during treatment of patients with CML.

Integrated genomic analysis identifies recurrent mutations and evolution patterns driving the initiation and progression of follicular lymphoma.

Follicular lymphoma is an incurable malignancy, 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.

A statistical framework for analyzing hypothesized interactions between cells imaged using multispectral microscopy and multiple immunohistochemical markers.

BACKGROUND: Multispectral microscopy and multiple staining can be used to identify cells with distinct immunohistochemical (IHC) characteristics. We present here a method called hypothesized interaction distribution (HID) analysis for characterizing the statistical distribution of pair-wise spatial relationships between cells with particular IHC characteristics and apply it to clinical data. MATERIALS AND METHODS: We retrospectively analyzed data from a study of 26 follicular lymphoma patients in which sections were stained for CD20 and YY1. HID analysis, using leave-one-out validation, was used to assign patients to one of two groups. We tested the null hypothesis of no difference in Kaplan-Meier survival curves between the groups. RESULTS: Shannon entropy of HIDs assigned patients to groups that had significantly different survival curves (median survival was 7.70 versus 110 months, P = 0.00750). Hypothesized interactions between pairs of cells positive for both CD20 and YY1 were associated with poor survival. CONCLUSIONS: HID analysis provides quantitative inferences about possible interactions between spatially proximal cells with particular IHC characteristics. In follicular lymphoma, HID analysis was able to distinguish between patients with poor versus good survival, and it may have diagnostic and prognostic utility in this and other diseases.

PLK1 and YY1 interaction in follicular lymphoma is associated with unfavourable outcome.

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.