Browsing by Author "Knowlton N"

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Accurate machine learning model for human embryo morphokinetic stage detection
(Springer Science+Business Media, LLC, 2025-08-20) Misaghi H; Cree L; Knowlton N
Purpose: The ability to detect, monitor, and precisely time the morphokinetic stages of human pre-implantation embryo development plays a critical role in assessing their viability and potential for successful implantation. Therefore, there is a need for accurate and accessible tools to analyse embryos. This work describes a highly accurate, machine learning model designed to predict 17 morphokinetic stages of pre-implantation human development, an improvement on existing models. This model provides a robust tool for researchers and clinicians, enabling the automation of morphokinetic stage prediction, standardising the process, and reducing subjectivity between clinics. Method: A computer vision model was built on a publicly available dataset for embryo Morphokinetic stage detection. The dataset contained 273,438 labelled images based on Embryoscope/ + © embryo images. The dataset was split 70/10/20 into training/validation/test sets. Two different deep learning architectures were trained and tested, one using EfficientNet-V2-Large and the other using EfficientNet-V2-Large with the addition of fertilisation time as input. A new postprocessing algorithm was developed to reduce noise in the predictions of the deep learning model and detect the exact time of each morphokinetic stage change. Results: The proposed model reached an overall test F1-score of 0.881 and accuracy of 87% across 17 morphokinetic stages on an independent test set. Conclusion: The proposed model shows a 17% accuracy improvement, compared to the best models on the same dataset. Therefore, our model can accurately detect morphokinetic stages in static embryo images as well as detecting the exact timings of stage changes in a complete time-lapse video.
Analysis of HER2-Low Breast Cancer in Aotearoa New Zealand: A Nationwide Retrospective Cohort Study
(MDPI (Basel, Switzerland), 2024-09-20) Lasham A; Ramsaroop R; Wrigley A; Knowlton N; Radisky D; Lambertini M
OBJECTIVES: To perform the first national analysis of demographic and clinicopathological features associated with the HER2 positive, HER2-low, and HER2-zero invasive breast cancers in New Zealand. The study will reveal the proportion of women who may benefit from new HER2-targeted antibody drug conjugate (ADC) therapies. METHODS: Utilising data from Te Rēhita Mate Ūtaetae (Breast Cancer Foundation NZ National Register), the study analysed data from women diagnosed with invasive breast cancer over a 21-year period. The HER2 status of tumours was classified into three categories-HER2-zero, HER2-low, HER2-positive. RESULTS: From 2009-2021, 94% of women underwent HER2 testing, with 14% diagnosed with HER2-positive breast cancer. For advanced-stage disease, 38% of those formerly classified as HER2-negative were reclassified as HER2-low. Including HER2-positive breast cancers, this indicates that 60% of women with advanced breast cancer may potentially benefit from the new HER2-directed ADCs (approximately 120 women per year). CONCLUSIONS: The findings suggest a significant proportion of women with invasive breast cancer in New Zealand could benefit from new HER2-targeted treatments. There is a need to standardise HER2 testing to enhance personalised treatment and improve outcomes.
Breast Cancer Patient Prognosis Is Determined by the Interplay between TP53 Mutation and Alternative Transcript Expression: Insights from TP53 Long Amplicon Digital PCR Assays
(MDPI (Basel, Switzerland), 2021-03-26) Lasham A; Knowlton N; Mehta SY; Braithwaite AW; Print CG
The TP53 gene locus is capable of producing multiple RNA transcripts encoding the different p53 protein isoforms. We recently described multiplex long amplicon droplet digital PCR (ddPCR) assays to quantify seven of eight TP53 reference transcripts in human tumors. Here, we describe a new long amplicon ddPCR assay to quantify expression of the eighth TP53 reference transcript encoding ∆40p53α. We then applied these assays, alongside DNA sequencing of the TP53 gene locus, to tumors from a cohort of New Zealand (NZ) breast cancer patients. We found a high prevalence of mutations at TP53 splice sites in the NZ breast cancer cohort. Mutations at TP53 intron 4 splice sites were associated with overexpression of ∆133TP53 transcripts. Cox proportional hazards survival analysis showed that interplay between TP53 mutation status and expression of TP53 transcript variants was significantly associated with patient outcome, over and above standard clinical and pathological information. In particular, patients with no TP53 mutation and a low ratio of TP53 transcripts t2 to t1, which derive from alternative intron 1 acceptor splice sites, had a remarkably good outcome. We suggest that this type of analysis, integrating mutation and transcript expression, provides a step-change in our understanding of TP53 in cancer.
Limitations of ploidy prediction by time-lapse morphokinetics and artificial-intelligence-based embryo selection algorithms: trisomies fly under the radar
(Elsevier Ltd on behalf of Reproductive Healthcare Ltd, 2025-07-01) Roman IC; Coull G; Knowlton N; Doherty-Eagles T; Ozturk O; Michielsen A; Carty H; Yefremenkova O; Zăhan M
Research question: How does the genetic constitution of embryos impact the accuracy and effectiveness of time-lapse ploidy detection? Design: A retrospective analysis of chromosomal constitution, morphokinetic characteristics and embryo grading was conducted on 1012 embryos, originating from 386 intracytoplasmic sperm injection cycles at a single clinic. Morphokinetic checkpoints of pronuclear fading, cleavage stages and post-cleavage stages – including start of compaction; time to compacted morula; time to start of blastulation; and time to full, expanded and hatching blastocyst – were recorded for all analysed embryos. Morphokinetic profiles of 363 euploid embryos were used as reference to analyse 649 embryos with aneuploidies, according to their level of gain or loss of chromosomal material. Embryo grading was performed using commercially available time-lapse-based algorithmic embryo selection tools. Results: Embryos with loss of genetic material and embryos with multiple aneuploidies exhibited consistent developmental delays that accumulated and became significant at the time of blastulation. In contrast, embryos with chromosomal gains displayed a morphokinetic profile almost identical to that of euploid embryos. Subset analyses suggested that aneuploidies of large chromosomes tend to have a greater impact on the morphokinetic profile. Time-lapse-based algorithmic or artificial intelligence scoring downgraded blastocysts with loss of chromosomal material or with complex aneuploidies, but discrimination potential was not observed between embryos with gain of chromosomes and euploid embryos. Conclusions: Embryos with extra chromosomes show similar morphokinetic patterns to euploid embryos, reducing the effectiveness of non-invasive selection tools. However, embryos with monosomies or multiple aneuploidies experience significant delays in reaching blastulation, and generally score lower on time-lapse selection algorithms.
N-Methyl-D-Aspartate Receptor Hypofunction in Meg-01 Cells Reveals a Role for Intracellular Calcium Homeostasis in Balancing Megakaryocytic-Erythroid Differentiation
(Thieme Gruppe, 2020-04-14) Hearn JI; Green TN; Chopra M; Nursalim YNS; Ladvanszky L; Knowlton N; Blenkiron C; Poulsen RC; Singleton DC; Bohlander SK; Kalev-Zylinska ML
The release of calcium ions (Ca2+) from the endoplasmic reticulum (ER) and related store-operated calcium entry (SOCE) regulate maturation of normal megakaryocytes. The N-methyl-D-aspartate (NMDA) receptor (NMDAR) provides an additional mechanism for Ca2+ influx in megakaryocytic cells, but its role remains unclear. We created a model of NMDAR hypofunction in Meg-01 cells using CRISPR-Cas9 mediated knockout of the GRIN1 gene, which encodes an obligate, GluN1 subunit of the NMDAR. We found that compared with unmodified Meg-01 cells, Meg-01-GRIN1 -/- cells underwent atypical differentiation biased toward erythropoiesis, associated with increased basal ER stress and cell death. Resting cytoplasmic Ca2+ levels were higher in Meg-01-GRIN1 -/- cells, but ER Ca2+ release and SOCE were lower after activation. Lysosome-related organelles accumulated including immature dense granules that may have contributed an alternative source of intracellular Ca2+. Microarray analysis revealed that Meg-01-GRIN1 -/- cells had deregulated expression of transcripts involved in Ca2+ metabolism, together with a shift in the pattern of hematopoietic transcription factors toward erythropoiesis. In keeping with the observed pro-cell death phenotype induced by GRIN1 deletion, memantine (NMDAR inhibitor) increased cytotoxic effects of cytarabine in unmodified Meg-01 cells. In conclusion, NMDARs comprise an integral component of the Ca2+ regulatory network in Meg-01 cells that help balance ER stress and megakaryocytic-erythroid differentiation. We also provide the first evidence that megakaryocytic NMDARs regulate biogenesis of lysosome-related organelles, including dense granules. Our results argue that intracellular Ca2+ homeostasis may be more important for normal megakaryocytic and erythroid differentiation than currently recognized; thus, modulation may offer therapeutic opportunities.
Recurrent loss of heterozygosity correlates with clinical outcome in pancreatic neuroendocrine cancer
(Springer Nature in partnership with the Center of Excellence in Genomic Medicine Research at King Abdulaziz University, 2018-07-20) Lawrence B; Blenkiron C; Parker K; Tsai P; Fitzgerald S; Shields P; Robb T; Yeong ML; Kramer N; James S; Black M; Fan V; Poonawala N; Yap P; Coats E; Woodhouse B; Ramsaroop R; Yozu M; Robinson B; Henare K; Koea J; Johnston P; Carroll R; Connor S; Morrin H; Elston M; Jackson C; Reid P; Windsor J; MacCormick A; Babor R; Bartlett A; Damianovich D; Knowlton N; Grimmond S; Findlay M; Print C
Pancreatic neuroendocrine tumors (pNETs) are uncommon cancers arising from pancreatic islet cells. Here we report the analysis of gene mutation, copy number, and RNA expression of 57 sporadic well-differentiated pNETs. pNET genomes are dominated by aneuploidy, leading to concordant changes in RNA expression at the level of whole chromosomes and chromosome segments. We observed two distinct patterns of somatic pNET aneuploidy that are associated with tumor pathology and patient prognosis. Approximately 26% of the patients in this series had pNETs with genomes characterized by recurrent loss of heterozygosity (LoH) of 10 specific chromosomes, accompanied by bi-allelic MEN1 inactivation and generally poor clinical outcome. Another ~40% of patients had pNETs that lacked this recurrent LoH pattern but had chromosome 11 LoH, bi-allelic MEN1 inactivation, and universally good clinical outcome. The somatic aneuploidy allowed pathogenic germline variants (e.g., ATM) to be expressed unopposed, with RNA expression patterns showing inactivation of downstream tumor suppressor pathways. No prognostic associations were found with tumor morphology, single gene mutation, or expression of RNAs reflecting the activity of immune, differentiation, proliferative or tumor suppressor pathways. In pNETs, single gene mutations appear to be less important than aneuploidy, with MEN1 the only statistically significant recurrently mutated driver gene. In addition, only one pNET in the series had clearly actionable single nucleotide variants (SNVs) (in PTEN and FLCN) confirmed by corroborating RNA expression changes. The two clinically relevant patterns of LoH described here define a novel oncogenic mechanism and a plausible route to genomic precision oncology for this tumor type.
Tumor mutational burden is a determinant of immune-mediated survival in breast cancer
(Taylor and Francis, England, 2018-07-30) Thomas A; Routh ED; Pullikuth A; Jin G; Su J; Chou JW; Hoadley KA; Print C; Knowlton N; Black MA; Demaria S; Wang E; Bedognetti D; Jones WD; Mehta GA; Gatza ML; Perou CM; Page DB; Triozzi P; Miller LD
Mounting evidence supports a role for the immune system in breast cancer outcomes. The ability to distinguish highly immunogenic tumors susceptible to anti-tumor immunity from weakly immunogenic or inherently immune-resistant tumors would guide development of therapeutic strategies in breast cancer. Genomic, transcriptomic and clinical data from The Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) breast cancer cohorts were used to examine statistical associations between tumor mutational burden (TMB) and the survival of patients whose tumors were assigned to previously-described prognostic immune subclasses reflecting favorable, weak or poor immune-infiltrate dispositions (FID, WID or PID, respectively). Tumor immune subclasses were associated with survival in patients with high TMB (TMB-Hi, P < 0.001) but not in those with low TMB (TMB-Lo, P = 0.44). This statistical relationship was confirmed in the METABRIC cohort (TMB-Hi, P = 0.047; TMB-Lo, P = 0.39), and also found to hold true in the more-indolent Luminal A tumor subtype (TMB-Hi, P = 0.011; TMB-Lo, P = 0.91). In TMB-Hi tumors, the FID subclass was associated with prolonged survival independent of tumor stage, molecular subtype, age and treatment. Copy number analysis revealed the reproducible, preferential amplification of chromosome 1q immune-regulatory genes in the PID immune subclass. These findings demonstrate a previously unappreciated role for TMB as a determinant of immune-mediated survival of breast cancer patients and identify candidate immune-regulatory mechanisms associated with immunologically cold tumors. Immune subtyping of breast cancers may offer opportunities for therapeutic stratification.