Meiosis is a conserved yet evolutionarily varied process underpinning sexual reproduction in eukaryotes. In the malaria parasite Plasmodium, meiosis is unconventional: it occurs immediately after fertilisation (post-zygotic) and must be coordinated with the transformation of the zygote into a motile ookinete. The mechanisms synchronising these meiotic and morphogenetic programmes remain unknow. Here, we identify the Plasmodium berghei NIMA-related kinase, NEK4 as a key regulator that couples meiotic initiation with zygote morphogenesis. Using ultrastructure expansion microscopy, we show that NEK4 accumulates at the microtubule-organising centre (MTOC) and the apical polar complex (APC) shortly after fertilisation, preceding the assembly of perinuclear and cortical microtubules. We reveal that Plasmodium zygotes undergo a nuclear migration driven by the MTOC, analogous to the meiotic nuclear movement in fission yeast. Deletion of nek4 results in complete developmental arrest: MTOC duplication and microtubule formation are blocked, chromatin remains uncondensed, and nuclear migration and cell polarity fail to establish. Transcriptomic and phosphoproteomic analyses reveal that NEK4 absence causes a collapse in transcriptional and phosphoregulatory networks governing meiosis and cytoskeletal organisation, leading to reduced expression and phosphorylation of important players, including HOP1, REC8, and AP2-O. These findings establish NEK4 as a key regulator driving meiotic entry and zygote maturation.

Mitosis in Plasmodium spp., the causative agent of malaria, is fundamentally different from model eukaryotes, proceeding via a bipartite microtubule organising centre (MTOC) and lacking canonical regulators such as Polo kinases. During schizogony, asynchronous nuclear replication produces a multinucleate schizont, while rapid male gametogony generates an octaploid nucleus before gamete formation. Here, we identify Aurora-related kinase 1 (ARK1) as a key component of inner MTOC and spindle formation, controlling kinetochore dynamics and driving mitotic progression. Conditional ARK1 depletion disrupts spindle biogenesis, kinetochore segregation, karyokinesis and cytokinesis in both stages, and affects parasite transmission. Interactome analysis shows that ARK1 forms the catalytic core of a non-canonical chromosomal passenger complex (CPC) containing two highly divergent inner centromere proteins (INCENPs), which we term INCENP-A and INCENP-B, and lacking the canonical chromatin-targeting subunits Survivin and Borealin. Comparative genomics suggests that apicomplexan INCENPs arose through recurrent lineage-specific duplications, reflecting an evolutionary rewiring of CPC architecture in this eukaryotic lineage. Together, these findings reveal key adaptations in Plasmodium mitosis involving ARK1 and its INCENP scaffolds, and identify the ARK1-INCENP interface as a potential multistage target for antimalarial intervention.

Sexual development and male gamete formation of the malaria parasite in the mosquito midgut are initiated by rapid endomitosis in the activated male gametocyte. This process is highly regulated by protein phosphorylation, specifically by three divergent male-specific protein kinases (PKs): CDPK4, SRPK1, and MAP2. Here, we localise each PK during male gamete formation using live-cell imaging, identify their putative interacting partners by immunoprecipitation, and determine the morphological consequences of their absence using ultrastructure expansion and transmission electron microscopy. Each PK has a distinct location in either the nuclear or the cytoplasmic compartment. Protein interaction studies revealed that CDPK4 and MAP2 interact with key drivers of rapid DNA replication, whereas SRPK1 is involved in RNA translation. The absence of each PK results in severe defects in either microtubule-organising centre organisation, kinetochore segregation, or axoneme formation. This study reveals the crucial role of these PKs during endomitosis in formation of the flagellated male gamete and uncovers some of their interacting partners that may drive this process.

Malaria, a vector borne disease, is a major global health and socioeconomic problem caused by the apicomplexan protozoan parasite Plasmodium. The parasite alternates between mosquito vector and vertebrate host, with meiosis in the mosquito and proliferative mitotic cell division in both hosts. In the canonical eukaryotic model, cell division is either by open or closed mitosis and karyokinesis is followed by cytokinesis; whereas in Plasmodium closed mitosis is not directly accompanied by concomitant cell division. Key molecular players and regulatory mechanisms of this process have been identified, but the pivotal role of certain protein complexes and the post-translational modifications that modulate their actions are still to be deciphered. Here, we discuss recent evidence for the function of known proteins in Plasmodium cell division and processes that are potential novel targets for therapeutic intervention. We also identify key questions to open new and exciting research to understand divergent Plasmodium cell division.

This May marked the 20th anniversary of the BioMalPar conference at the European Molecular Biology Laboratory (EMBL) in Heidelberg, Germany. The meeting originated as part of a European network of excellence but has evolved to a world-leading conference on the biology and pathology of malaria parasites. Ally Olotu (Ifakara Health Institute, Tanzania) kicked off the conference this year with a keynote presentation on malaria prevention, emphasizing current and emerging vaccine strategies. Another highlight was the presentation of the BioMalPar Lifetime Achievement Award to Andy Waters (University of Glasgow, UK). In his speech, Andy not only reminisced about his distinguished academic career in fundamental Plasmodium research but stressed the importance of supporting such research in endemic communities. These and many other talks showcased the significant progress that has been made in the field and the challenges that remain in eradicating this devastating disease. In this TrendsTalk, we invite both early-career and established Plasmodium researchers to highlight the excellent work presented over six '2005-themed' sessions at BioMalPar XX.

PURPOSE:Here, we report the sensitivity of a personalized, tumor-informed circulating tumor DNA (ctDNA) assay (Signatera) for detection of molecular relapse during long-term follow-up of patients with breast cancer. METHODS:A total of 156 patients with primary breast cancer were monitored clinically for up to 12 years after surgery and adjuvant chemotherapy. Semiannual blood samples were prospectively collected, and analyzed retrospectively to detect residual disease by ultradeep sequencing using ctDNA assays, developed from primary tumor whole-exome sequencing data. RESULTS:Personalized Signatera assays detected ctDNA ahead of clinical or radiologic relapse in 30 of the 34 patients who relapsed (patient-level sensitivity of 88.2%). Relapse was predicted with a lead interval of up to 38 months (median, 10.5 months; range, 0-38 months), and ctDNA positivity was associated with shorter relapse-free survival (P < .0001) and overall survival (P < .0001). All relapsing triple-negative patients (n = 7/23) had a ctDNA-positive test within a median of 8 months (range, 0-19 months), while the 16 nonrelapsed patients with triple-negative breast cancer remained ctDNA-negative during a median follow-up of 58 months (range, 8-99 months). The four patients who had negative tests before relapse all had hormone receptor-positive (HR+) disease and conversely, five of the 122 nonrelapsed patients (all HR+) had an occasional positive test. CONCLUSION:Serial postoperative ctDNA assessment has strong prognostic value, provides a potential window for earlier therapeutic intervention, and may enable more effective monitoring than current clinical tests such as cancer antigen 15-3. Our study provides evidence that those with serially negative ctDNA tests have superior clinical outcomes, providing reassurance to patients with breast cancer. For select cases with HR+ disease, decisions about treatment management might require serial monitoring despite the ctDNA-positive result.

Background: Breast mass is one of the main symptoms of breast cancer. Effective and accurate detection of breast masses at an early stage would be of great value for clinical breast cancer analysis. Methods: We developed a novel mass detection framework named GFNet. The GFNet is comprised of three modules, including patch extraction, feature extraction, and mass detection. The developed breast mass detection framework is of high robustness and generality that can be self-adapted to images collected by different imaging devices. The patch-based detection is deployed to improve performance. A novel feature extraction technique based on gradient field convergence features (GFCF) is proposed to enhance the information of breast mass and, therefore, provide useful information for the following patch extraction module. A novel false positives reduction method is designed by combining the texture and morphological features in breast mass patch. This is the first attempt at fusing morphological and texture features for breast mass false positive reduction. Results: Compared to other state-of-the-art methods, the proposed GFNet showed the best performance on CBIS-DDSM and INbreast with an accuracy of 0.90 at 2.91 false positive per image (FPI) and 0.99 at only 0.97 FPI, respectively. Conclusions: The GFNet is an effective tool for detecting breast mass.

Amongst all types of cancer, breast cancer has become one of the most common cancers in the UK threatening millions of people's health. Early detection of breast cancer plays a key role in timely treatment for morbidity reduction. Compared to biopsy, which takes tissues from the lesion for further analysis, image-based methods are less time-consuming and pain-free though they are hampered by lower accuracy due to high false positivity rates. Nevertheless, mammography has become a standard screening method due to its high efficiency and low cost with promising performance. Breast mass, as the most palpable symptom of breast cancer, has received wide attention from the community. As a result, the past decades have witnessed the speeding development of computer-aided systems that are aimed at providing radiologists with useful tools for breast mass analysis based on mammograms. However, the main issues of these systems include low accuracy and require enough computational power on a large scale of datasets. To solve these issues, we developed a novel breast mass classification system called DF-dRVFL. On the public dataset DDSM with more than 3500 images, our best model based on deep random vector functional link network showed promising results through five-cross validation with an averaged AUC of 0.93 and an average accuracy of 81.71%. Compared to sole deep learning based methods, average accuracy has increased by 0.38. Compared with the state-of-the-art methods, our method showed better performance considering the number of images for evaluation and the overall accuracy.

To assess their roles in breast cancer diagnostics, we aimed to compare plasma cell-free DNA (cfDNA) levels with the circulating metabolome in a large breast screening cohort of women recalled for mammography, including healthy women and women with mammographically detected breast diseases, ductal carcinoma in situ and invasive breast cancer: the Breast Screening and Monitoring Study (BSMS). In 999 women, plasma was analyzed by nuclear magnetic resonance (NMR) and Ultra-Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS) and then processed to isolate and quantify total cfDNA. NMR and UPLC-MS results were compared with data for 186 healthy women derived from the AIRWAVE cohort. Results showed no significant differences between groups for all metabolites, whereas invasive cancers had significantly higher plasma cfDNA levels than all other groups. When stratified the supervised OPLS-DA analysis and total cfDNA concentration showed high discrimination accuracy between invasive cancers and the disease/medication-free subjects. Furthermore, comparison of OPLS-DA data for invasive breast cancers with the AIRWAVE cohort showed similar discrimination between breast cancers and healthy controls. This is the first report of agreement between metabolomics and plasma cfDNA levels for discriminating breast cancer from healthy subjects in a true screening population. It also emphasizes the importance of sample standardization. Follow on studies will involve analysis of candidate features in a larger validation series as well as comparing results with serial plasma samples taken at the next routine screening mammography appointment. The findings here help establish the role of plasma analysis in the diagnosis of breast cancer in a large real-world cohort.

Meiosis is sexual cell division, a process in eukaryotes whereby haploid gametes are produced. Compared to canonical model eukaryotes, meiosis in apicomplexan parasites appears to diverge from the process with respect to the molecular mechanisms involved; the biology of Plasmodium meiosis, and its regulation by means of post-translational modification, are largely unexplored. Here, we discuss the impact of technological advances in cell biology, evolutionary bioinformatics, and genome-wide functional studies on our understanding of meiosis in the Apicomplexa. These parasites, including Plasmodium falciparum, Toxoplasma gondii, and Eimeria spp., have significant socioeconomic impact on human and animal health. Understanding this key stage during the parasite's life cycle may well reveal attractive targets for therapeutic intervention.

Mechanisms of cell division are remarkably diverse, suggesting the underlying molecular networks among eukaryotes differ extensively. The Aurora family of kinases orchestrates the process of chromosome segregation and cytokinesis during cell division through precise spatiotemporal regulation of their catalytic activities by distinct scaffolds. Plasmodium spp., the causative agents of malaria, are unicellular eukaryotes that have three divergent aurora-related kinases (ARKs) and lack most canonical scaffolds/activators. The parasite uses unconventional modes of chromosome segregation during endomitosis and meiosis in sexual transmission stages within mosquito host. This includes a rapid threefold genome replication from 1N to 8N with successive cycles of closed mitosis, spindle formation and chromosome segregation within eight minutes (termed male gametogony). Kinome studies had previously suggested likely essential functions for all three Plasmodium ARKs during asexual mitotic cycles; however, little is known about their location, function, or their scaffolding molecules during unconventional sexual proliferative stages. Using a combination of super-resolution microscopy, mass spectrometry, and live-cell fluorescence imaging, we set out to investigate the role of the atypical Aurora paralog ARK2 to proliferative sexual stages using rodent malaria model Plasmodium berghei . We find that ARK2 primarily localises to the spindle apparatus in the vicinity of kinetochores during both mitosis and meiosis. Interactomics and co-localisation studies reveal a unique ARK2 scaffold at the spindle including the microtubule plus end-binding protein EB1, lacking conserved Aurora scaffold proteins. Gene function studies indicate complementary functions of ARK2 and EB1 in driving endomitotic divisions and thereby parasite transmission. Our discovery of a novel Aurora kinase spindle scaffold underlines the emerging flexibility of molecular networks to rewire and drive unconventional mechanisms of chromosome segregation in the malaria parasite Plasmodium .

Supplementary data, figures and tables Supplementary Fig. 1. Patient clinical timelines and samples taken. Supplementary Fig. 2. Total cfDNA levels track with CTC counts and CA15-3 in patient CTCM167. Supplementary Table 1: Summary of individual CTCs and WBCs, and respective pools isolated by DEPArray for NGS analysis Supplementary Table 2: Details of PCR amplicons for the custom 30 amplicon NGS panel Supplementary Table 3: Details of PCR amplicons for the Ampli1 Custom Cancer NGS panel showing confirmed mutations in cfDNA and CTCs Supplementary Table 4: Primer sequences of the ESR1 screening assays Supplementary Table 5: Patient details and variables for correlation and survival analyses Supplementary Table 6: Spearman's correlation of each circulating biomarker Supplementary Table 7: Sequencing metrics per panel for each sample Supplementary Table 8: Allelic dropout rate (%) for individual CTCs, WBCs and sample pools by patient Supplementary Table 9: Private mutations detected in single CTCs and WBCs Supplementary Table 10: AF and coverage for each mutation in patient CTCM155 Supplementary Table 11: AF and coverage for each mutation in patient CTCM138 Supplementary Table 12: AF and coverage for each variant in patient CTCM105 Supplementary Table 13: AF and coverage for each mutation in patient CTCM292

As an important imaging modality, mammography is considered to be the global gold standard for early detection of breast cancer. Computer-Aided (CAD) systems have played a crucial role in facilitating quicker diagnostic procedures, which otherwise could take weeks if only radiologists were involved. In some of these CAD systems, breast pectoral segmentation is required for breast region partition from breast pectoral muscle for specific analysis tasks. Therefore, accurate and efficient breast pectoral muscle segmentation frameworks are in high demand. Here, we proposed a novel deep learning framework, which we code-named PeMNet, for breast pectoral muscle segmentation in mammography images. In the proposed PeMNet, we integrated a novel attention module called the Global Channel Attention Module (GCAM), which can effectively improve the segmentation performance of Deeplabv3+ using minimal parameter overheads. In GCAM, channel attention maps (CAMs) are first extracted by concatenating feature maps after paralleled global average pooling and global maximum pooling operation. CAMs are then refined and scaled up by multi-layer perceptron (MLP) for elementwise multiplication with CAMs in next feature level. By iteratively repeating this procedure, the global CAMs (GCAMs) are then formed and multiplied elementwise with final feature maps to lead to final segmentation. By doing so, CAMs in early stages of a deep convolution network can be effectively passed on to later stages of the network and therefore leads to better information usage. The experiments on a merged dataset derived from two datasets, INbreast and OPTIMAM, showed that PeMNet greatly outperformed state-of-the-art methods by achieving an IoU of 97.46%, global pixel accuracy of 99.48%, Dice similarity coefficient of 96.30%, and Jaccard of 93.33%, respectively.

562 Background: Up to 30% of patients with breast cancer relapse after primary treatment. There are no sensitive or reliable tests to monitor these patients and detect distant metastases before overt recurrence. Here, we demonstrate the use of personalized circulating tumour DNA (ctDNA) profiling performed postoperatively, postadjuvantly, and serially for detection of recurrence in breast cancer. Methods: Patients with primary breast cancer (n=188) were recruited following surgery and adjuvant therapy and were followed-up for up to 10 years with semi-annual blood sampling for ctDNA analysis. Patients (n=29) with insufficient residual tumour for whole exome sequencing (WES) were excluded from the analysis. Tumour WES profiles were generated for 159 patients; samples from 2 patients failed WES QC requirements and a personalised ctDNA panel could not be generated for 1 patient. In 156 patients, plasma samples (n=1141) were retrospectively tested for the presence of ctDNA using personalized Signatera assays (mPCR-NGS) targeting up to 16 somatic single nucleotide variants selected from primary tumour WES. Results: Plasma ctDNA was detected ahead of clinical or radiologic relapse in 30 of the 34 relapsed patients (sensitivity of 88%). Metastatic relapse was predicted with a lead interval of up to 2 years (median: 10 months, range: 0-39 months); median lead intervals for HR+/HER2- were 15 (2 - 39); for HR-/HER2+ 6 (0.5 – 12) for HR+/HER2+ 8 (5 – 14) and 9 (0-20) for TNBC. Patients with a positive ctDNA test had poorer relapse-free-survival (RFS) (HR=47.5; 95% CI 18.5-161.4; p <0.001) from surgery and all four breast cancer subgroups showed a similarly reduced RFS. Overall survival was also significantly reduced for patients who were ctDNA positive (HR=84.15; 95%CI 16.43-1538; p <0.001). The number of variants, mean VAF and MTM/mL varied between patients, with significantly higher values at the time closest to relapse than in the first ctDNA positive sample (p =0.0002). Among the 4 relapsed patients not detected in the study all were HR+/HER2-, 1 had a local recurrence, 2 had bone recurrence (1 with axillary LN involvement) and 1 had cancer cells in pleural fluid. Of the remaining 122 patients, only 5 developed ctDNA-positivity, all with low VAF, none of them have relapsed by the follow-up census date (31 December 2021). However, follow-up for some of these patients limits definitive assessment. Lastly, 4 patients developed a second primary cancer (2 breast, 2 lung) all of whom were ctDNA-negative. Conclusions: This study demonstrates that serial post-operative ctDNA analysis has strong prognostic value. More importantly, earlier detection of metastatic disease provides a possible window for therapeutic intervention, while repeated negative ctDNA tests can provide reassurance to patients. Future interventional studies may assess the clinical utility of ctDNA-based risk-stratification.

Background We report copy-number profiling by low-pass WGS (LP-WGS) in individual circulating tumour cells (CTCs) for guiding treatment in patients with metastatic breast cancer (MBC), comparing CTC results with mutations detected in circulating tumour DNA (ctDNA) in the same blood samples. Methods Across 10 patients with MBC who were progressing at the time of blood sampling and that had >20 CTCs detected by CellSearch ® , 63 single cells (50 CTCs and 13 WBCs) and 16 cell pools (8 CTC pools and 8 WBC pools) were recovered from peripheral blood by CellSearch ® /DEPArray™ and sequenced with Ampli1 LowPass technology (Menarini Silicon Biosystems). Copy-number aberrations were identified using the MSBiosuite software platform, and results were compared with mutations detected in matched plasma cfDNA analysed by targeted next-generation sequencing using the Oncomine™ Breast cfDNA Assay (Thermo Fisher). Results LP-WGS data demonstrated copy-number gains/losses in individual CTCs in regions including FGFR1, JAK2 and CDK6 in five patients, ERBB2 amplification in two HER2-negative patients and BRCA loss in two patients. Seven of eight matched plasmas also had mutations in ctDNA in PIK3CA, TP53, ESR1 and KRAS genes with mutant allele frequencies (MAF) ranging from 0.05 to 33.11%. Combining results from paired CTCs and ctDNA, clinically actionable targets were identified in all ten patients. Conclusion This combined analysis of CTCs and ctDNA may offer a new approach for monitoring of disease progression and to direct therapy in patients with advanced MBC, at a time when they are coming towards the end of other treatment options.

Chemotherapy-refractory diffuse large B-cell lymphoma (DLBCL) remains a significant clinical problem. The ability to predict patients likely to have poor outcomes with conventional therapies may facilitate rational use of alternative, targeted treatment approaches before fulminant relapse. The utility of circulating tumor DNA (ctDNA) in DLBCL is currently being investigated. Initial studies have reported high levels of sensitivity for detection of residual disease, outperforming imaging techniques such as 18FDG-PET/CT scans.1 For example, Alizadeh et al recently demonstrated the feasibility of CAncer Personalized Profiling by deep Sequencing (CAPP-Seq) approach in DLBCL; the mean elapsed time between the first ctDNA-positive time point and radiological relapse was 188 days.2 Kurtz et al detected pretreatment ctDNA in 98% of patients with DLBCL receiving treatment with frontline or salvage immunochemotherapy.3 Early and major molecular responses after 1 and 2 cycles of treatment, resulted in superior outcome at 24 months. However, only 9% and 23% of patients within these validation sets had stage I and II disease, respectively. Limitations to the use of ctDNA to detect early stage malignancy have been identified; for example, in lung, where low-volume disease cannot be reliably detected using mutation profiles in ctDNA.4 Furthermore, some specific subtypes of disease (lung adenocarcinoma) do not shed detectable ctDNA into the peripheral blood.5 Primary cutaneous diffuse large B-cell lymphoma - leg type (PCDLBCL-LT) is a rare but distinct form of aggressive B-cell lymphoma, which is often resistant to therapy and associated with a poor prognosis.6 We have studied a case of chemotherapy and radiotherapy-refractory PCDLBCL-LT that remained localized to the lower leg for 13 years before systemic dissemination. We evaluated sequential plasma-derived ctDNA samples over 3 years during 3 different treatment modalities but were only able to detect ctDNA transiently, when the patient was taking the Bruton’s tyrosine kinase inhibitor, ibrutinib, and not during 2 episodes of systemic relapse. A previously well 78-year-old gentleman was diagnosed with PCDLBCL-LT in 2006 involving the left lower leg. He received 4 cycles of systemic immunochemotherapy (R-CHOP) followed by involved-field radiotherapy (40 Gy, 15 fractions) with rapid and complete response. The patient subsequently relapsed in 2010 with left leg cutaneous involvement only, adjacent to the previously treated sites, and received additional treatment with 4 cycles of R-CHOP and radiotherapy (40 Gy in 15 fractions), again attaining clinical response. Two further relapses in 2011 and 2012, again in the left leg were treated with radiotherapy alone (4 Gy, single fraction). The patient remained disease free for 4 years before experiencing a further localized cutaneous relapse, refractory to rituximab, gemcitabine, cyclophosphamide, vincristine, and prednisolone (R-GVCP). He commenced venetoclax in January 2017. A rapid and complete metabolic response was obtained,7 before he relapsed in January 2019 (Figure 1). The patient then received 3 weeks of ibrutinib but experienced further rapid clinical progression. In April 2019, he commenced treatment on a phase 1 trial with a bispecific antibody, obtaining a complete metabolic response after 2 cycles; however, he progressed once again in January 2020.Figure 1.: Mutational and phylogenetic analysis of tumor biopsies at points of relapse on venetoclax and Ibrutinib. (A) Timeline of clinical course. The patient received single agent venetoclax at a dose of 400 and 800 mg OD on alternating days from January 2017 until January 2019 (violet timeline). An excisional biopsy was during treatment with venetoclax (biopsy 1); biopsy 1 mutational data are shown in Suppl. Table S1. Following relapse, he was treated with single agent ibrutinib at a dose of 480 mg bd for a period of 3 weeks (pale blue timeline) but progressed rapidly with enlarging left inguinal lymph nodes (biopsy 2, WES). He then received treatment with a T cell–engaging bispecific antibody (orange timeline) and once again entered a 18FDG-PET/CT scan negative complete remission only to relapse 8 months after completion of therapy. (B) Clonal evolution in PCDLBCL-LT; clonal frequencies inferred from WES. Figure 2B1—analysis of biopsy 1 showed 2 clonal populations C1 (38%) and C2 (55%) with a small population of residual normal cells (7%). Figure 2B3 in contrast showing analysis of biopsy 2 with no detectable C2 cells and only 4.5% of C1. Two new subclones C3 and C4 comprising 63.5% and 20.5% of all cells were detected. Figure 2B2 shows possible driver mutations in the inferred subclones and percentages of cells carrying the different mutations, with further expansion of clonal mutations observed in Suppl. Figure S3. (C) Immunohistochemistry staining showing maintained BCL2 expression in biopsy 2 following 2 years venetoclax therapy. WES = whole exome sequencing.Tumor DNA from freshly excised biopsy tissue and constitutional DNA from buffy coat were extracted using the DNeasy Blood and Tissue kit (Qiagen). Cell-free DNA (cfDNA) was isolated from blood plasma using the QIAamp Circulating Nucleic Acid Kit (Qiagen). Samples were quantified and quality tested using Qubit fluorometer (Thermofisher and Agilent Tapestation (Santa Clara, California, United States), respectively. Whole exome sequencing (WES) of tumors was carried out using hybridization capture-based Agilent Sure Select all exon target enrichment on Illumina to Novoseq 6000 by Novogene, to a minimum of 100× after deduplication. Targeted deep sequencing of plasma was performed following the Roche SeqCap-EZ workflow with KAPA HyperPrep library generation, captured with a custom 80 gene panel, sequenced on an Illumina Novaseq to a minimum of 1000× deduplicated coverage. Variant detection in tumor DNA and plasma was performed using the genome analysis toolkit v4 Mutect2 caller against germline DNA derived from buffy coat.8 Clonal phylogeny between tumor samples were analyzed in “R”9 with package “CLOE.”10 Serial monitoring of plasma by ARID1A c.4381C>T fs variant and NOTCH2 c.7198C>T fs variant mutant TaqMan assays was performed using the BIORAD Q×200 digital droplet polymerase chain reaction (PCR) system (BIORAD).11 WES of excision biopsies sampled at each episode of progression throughout the study was performed (Figure 1A). There were no activating mutations in the B-cell receptor (BCR) signaling pathway (MYD88, CD79A/B, or CARD11), as commonly reported in PCDLBCL-LT.12–14 However, a previously unreported G29W mutation within the ATP binding site of the MAP4KI/HPK1 kinase was identified. From modeling using crystal structures, this mutation would impair MAP4K1 activity by reducing ATP binding. MAP4K1 has been shown to be a critical regulator of BCR signaling.15 Other truncal, potential genetic driver aberrations included ARID1A (COSM4031017), NOTCH2 (COSM36210), and FBXO28 missense mutations, as well as biallelic deletion of RB1 and CDKN2A/B with monoallelic loss of TP53 with the remaining TP53 allele retaining germline sequences. Clonal phylogeny across biopsy samples prevenetoclax and following progression on ibrutinib treatment is summarized in Figure 1B. Interestingly, 2 subclones were detected by WES before starting venetoclax, C1 and C2 (Figure 1B.1). Subclone C1 evolved resulting in the derivation of 2 new subclones C3 and C4, representing the acquisition of 34 previously undetected mutations (Figure 1B.2, B3). No BCL2 mutations nor new mutations involved in regulation of apoptosis were detected, and levels of BCL2 expression were maintained on immunohistochemistry despite treatment with venetoclax for 2 years (Figure 1C). Following relapse on venetoclax,7 to enhance detection of residual disease, we analyzed sequential ctDNA samples. Serial plasma samples were analyzed by ddPCR over a 3-year period for the ARID1A p.R1461* and NOTCH2 p.R2400* nonsense variants, present in all biopsy samples, which are likely truncal mutations (Figure 2); 1 plasma sample was additionally assessed by CAPP-Seq during treatment on venetoclax (Figure 2B). Before and after starting venetoclax, no ctDNA harboring the ARID1A nor NOTCH2 mutations could be detected by ddPCR or CAPP-Seq. This remained the case after the disease had spread to the left inguinal nodal region before starting ibrutinib (420 mg OD, Figure 2A.1). Surprisingly, higher levels of ctDNA (600 HHGE/ml plasma) were readily detectable 1 week later (Figure 2B). There were no detectable B cells in the peripheral blood by flow cytometry before starting ibrutinib and no detectable egress of tumor cells into the peripheral blood as assessed by sequential full blood counts (data not shown). Ibrutinib therapy was discontinued after 3 weeks due to rapid clinical progression. After 4 weeks washout and reassessment, the patient was started on experimental therapy (Figures 1A and 2B). By this time, levels of the ARID1A and NOTCH2 mutations in ctDNA had fallen considerably in the face of disease progression, and again became undetectable following treatment on trial, remaining undetectable at subsequent relapse in January 2020 (data not shown).Figure 2.: Serial analysis of ctDNA by ddPCR and CAPP-Seq through treatment with Venetoclax, Ibrutinib and bispecific antibody. (A) 18FDG-PET/CT scans taken at relapse following (1) venetoclax, (2) ibrutinib, and (3) bispecific /PD-L1 antibody therapy showing lumbar and lower leg regions. (B) Timeline in weeks of 7 plasma samples, analyzed for ARID1A c.4381C>T variant and NOTCH2 c.7198C>T variant by ddPCR assay throughout treatment, quantified as human haploid genomic equivalents per mL of plasma (HHGE/mL). Arrows showing points of 18FGD-PET/CT scans, dotted-lines marking treatment intervals, solid line indicating timepoint of biopsy A, and red star indicating additional plasma sample analyzed by CAPP-Seq during treatment on venetoclax. (C) Droplet frequency plots corresponding to (B) detected with ARID1A assay (C1, Black) and NOTCH2 assay (C2, Green) of 7 serial plasmas, with additional biopsy A positive and HGD negative controls. Pink line indicating amplitude thresholds. Mutant droplets indicated in blue; negative droplets gray. (D) IGV plot of processed bam files for CAPP-Seq of biopsy A (blue) and plasma (pink) indicated in this figure, showing stacked and sorted reads for the ARID1A c.4381C>T variant (D1, red stacked reads) and NOTCH2 c.7198C>T variant (D2, green stacked reads). Plasma samples obtained 4 months after biopsy A showing ARID1A c.4381 site and NOTCH2 c.7198 site with no mutations and biopsy A harboring the ARID1A c.4381C>T fs variant and NOTCH2 c.7198C>T fs variant. HGD = human genomic DNA; IGV = Integrated genome viewer.Monitoring of ctDNA levels in DLBCL may have a transformational role in predicting response and relapse. However, whether this is universally the case, across all biological subtypes and stages of DLBCL is not clear. From the example of solid tumors, specific subtypes of disease may not be associated with detectable levels of ctDNA and datasets studied in DLBCL include only small numbers presenting with early stage disease. In this case of PCDLBCL-LT, no ctDNA was detectable throughout the patients’ clinical course including following 2 systemic relapses. For reasons that are unclear, ctDNA was only detectable, while the patient was taking ibrutinib; whether this is a reproducible feature in other patients with DLBCL receiving Bruton’s tyrosine kinase inhibitor is not known. Other methods of disease monitoring may be necessary for some DLBCL subtypes and relying on a singular mutational approach to monitor low level disease may be ineffective. ACKNOWLEDGMENTS We thank our patient for his kind perseverance and the staff of the Hope Clinical Trials Facility for their kind help. We thank Dr Paresh Sewpaul (Janssen Pharmaceuticals) for his kind help in obtaining compassionate use ibrutinib for this case. This study was approved by the University Hospitals of Leicester NHS Trust Research and Development (UK; 06/Q2501/122). Written informed consent from the patient was obtained for publication purposes. AUTHOR CONTRIBUTIONS CST designed research, performed research, analyzed data, and wrote the article. RS and ANMA contributed data. HSW, DSG, YG, MJA, GSS, SPNJ, and MJSD wrote the article and supervised the study. DISCLOSURES The authors have no conflicts of interest to disclose SOURCES OF FUNDING This work was supported by Cancer Research UK in conjunction with the UK Department of Health on an Experimental Cancer Medicine Centre grant [C10604/A25151] and grants from Hope Against Cancer, CRUK, Leicester Haematology Research Fund and the Scott-Waudby Charitable Trust.

Mutations in the estrogen receptor (ESR1) gene are common in ER-positive breast cancer patients who progress on endocrine therapies. Most mutations localise to just three residues at, or near, the C-terminal helix 12 of the hormone binding domain, at leucine-536, tyrosine-537 and aspartate-538. To investigate these mutations, we have used CRISPR-Cas9 mediated genome engineering to generate a comprehensive set of isogenic mutant breast cancer cell lines. Our results confirm that L536R, Y537C, Y537N, Y537S and D538G mutations confer estrogen-independent growth in breast cancer cells. Growth assays show mutation-specific reductions in sensitivities to drugs representing three classes of clinical anti-estrogens. These differential mutation- and drug-selectivity profiles have implications for treatment choices following clinical emergence of ER mutations. Our results further suggest that mutant expression levels may be determinants of the degree of resistance to some anti-estrogens. Differential gene expression analysis demonstrates up-regulation of estrogen-responsive genes, as expected, but also reveals that enrichment for interferon-regulated gene expression is a common feature of all mutations. Finally, a new gene signature developed from the gene expression profiles in ER mutant cells predicts clinical response in breast cancer patients with ER mutations.

The malaria parasite life cycle alternates between two hosts: a vertebrate and the female Anopheles mosquito vector. Cell division, proliferation, and invasion are essential for parasite development, transmission, and survival. Most research has focused on Plasmodium development in the vertebrate, which causes disease; however, knowledge of malaria parasite development in the mosquito (the sexual and transmission stages) is now rapidly accumulating, gathered largely through investigation of the rodent malaria model, with Plasmodium berghei. In this review, we discuss the seminal genome-wide screens that have uncovered key regulators of cell proliferation, invasion, and transmission during Plasmodium sexual development. Our focus is on the roles of transcription factors, reversible protein phosphorylation, and molecular motors. We also emphasize the still-unanswered important questions around key pathways in cell division during the vector transmission stages and how they may be targeted in future studies.

Background : Endometrial cancer (EC) is the commonest gynaecologic malignancy in many countries and its incidence is rising. Although excellent prognosis is associated with early stage disease, response to systemic treatment for metastatic or recurrent EC is often low and treatment options are limited. Aims-Methods : The aim of the study was to propose improved targeted drug treatments suitable for subsequent testing in pre-clinical models of EC. Cell proliferation assay (MTS) was used to assess viability of EC cell lines following treatment with drug inhibitors and Western blotting to explore the effect of inhibitors in molecular pathways. Results : We identified that CUDC-907, a PI3K and HDAC inhibitor, was the most effective monotherapy treatment of a panel of drugs screened in EC cells. Moreover, several combination treatments showed synergism in EC cell lines, with the most efficacious being CUDC-907 combined with the MEK inhibitor PD0325901. This indicates that simultaneous inhibition of two main oncogenic pathways, PI3K and MEK, could improve drug sensitivity in EC. Conclusions : In summary, we propose a range of targeted inhibitory drugs, alone or in combination, showing in vitro efficacy in endometrial cancer cells, which could provide novel therapeutic strategies for advanced EC.