Antibodies targeting merozoites are important in protection from malaria. Therefore, merozoite surface proteins are attractive vaccine candidates. There is a need for robust functional assays to ...investigate mechanisms of acquired immunity and vaccine efficacy. To date, the study of merozoite phagocytosis has been confounded by the complexity and variability of in vitro assays.
We have developed a new flow cytometry-based merozoite phagocytosis assay. An optimized merozoite preparation technique produced high yields of merozoites separated from haemozoin. Phagocytosis by the undifferentiated THP-1 monocytic cell line was mediated only by Fc Receptors, and was therefore ideal for studying opsonising antibody responses. The assay showed robust phagocytosis with highly diluted immune sera and strong inter-assay correlation. The assay effectively measured differences in opsonisation-dependent phagocytosis among individuals.
This highly reproducible assay has potential applications in assessing the role of opsonic phagocytosis in naturally acquired immunity and vaccine trials.
Uterine leiomyosarcoma (uLMS) is a rare and aggressive gynaecological malignancy, with individuals with advanced uLMS having a five-year survival of < 10%. Mutations in the homologous recombination ...(HR) DNA repair pathway have been observed in ~ 10% of uLMS cases, with reports of some individuals benefiting from poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi) therapy, which targets this DNA repair defect. In this report, we screened individuals with uLMS, accrued nationally, for mutations in the HR repair pathway and explored new approaches to therapeutic targeting.
A cohort of 58 individuals with uLMS were screened for HR Deficiency (HRD) using whole genome sequencing (WGS), whole exome sequencing (WES) or NGS panel testing. Individuals identified to have HRD uLMS were offered PARPi therapy and clinical outcome details collected. Patient-derived xenografts (PDX) were generated for therapeutic targeting.
All 13 uLMS samples analysed by WGS had a dominant COSMIC mutational signature 3; 11 of these had high genome-wide loss of heterozygosity (LOH) (> 0.2) but only two samples had a CHORD score > 50%, one of which had a homozygous pathogenic alteration in an HR gene (deletion in BRCA2). A further three samples harboured homozygous HRD alterations (all deletions in BRCA2), detected by WES or panel sequencing, with 5/58 (9%) individuals having HRD uLMS. All five individuals gained access to PARPi therapy. Two of three individuals with mature clinical follow up achieved a complete response or durable partial response (PR) with the subsequent addition of platinum to PARPi upon minor progression during initial PR on PARPi. Corresponding PDX responses were most rapid, complete and sustained with the PARP1-specific PARPi, AZD5305, compared with either olaparib alone or olaparib plus cisplatin, even in a paired sample of a BRCA2-deleted PDX, derived following PARPi therapy in the patient, which had developed PARPi-resistance mutations in PRKDC, encoding DNA-PKcs.
Our work demonstrates the value of identifying HRD for therapeutic targeting by PARPi and platinum in individuals with the aggressive rare malignancy, uLMS and suggests that individuals with HRD uLMS should be included in trials of PARP1-specific PARPi.
Further reduction in malaria prevalence and its eventual elimination would be greatly facilitated by the development of biomarkers of exposure and/or acquired immunity to malaria, as well as the ...deployment of effective vaccines against Plasmodium falciparum and Plasmodium vivax. A better understanding of the acquisition of immunity in naturally-exposed populations is essential for the identification of antigens useful as biomarkers, as well as to inform rational vaccine development.
ELISA was used to measure total IgG to a synthetic form of glycosylphosphatidylinositol from P. falciparum (PfGPI) in a cohort of 1-3 years old Papua New Guinea children with well-characterized individual differences in exposure to P. falciparum and P. vivax blood-stage infections. The relationship between IgG levels to PfGPI and measures of recent and past exposure to P. falciparum and P. vivax infections was investigated, as well as the association between antibody levels and prospective risk of clinical malaria over 16 months of follow-up.
Total IgG levels to PfGPI were low in the young children tested. Antibody levels were higher in the presence of P. falciparum or P. vivax infections, but short-lived. High IgG levels were associated with higher risk of P. falciparum malaria (IRR 1.33-1.66, P = 0.008-0.027), suggesting that they are biomarkers of increased exposure to P. falciparum infections. Given the cross-reactive nature of antibodies to PfGPI, high IgG levels were also associated with reduced risk of P. vivax malaria (IRR 0.65-0.67, P = 0.039-0.044), indicating that these antibodies are also markers of acquired immunity to P. vivax.
This study highlights that in young children, IgG to PfGPI might be a useful marker of immune-status to both P. falciparum and P. vivax infections, and potentially useful to help malaria control programs to identify populations at-risk. Further functional studies are necessary to confirm the potential of PfGPI as a target for vaccine development.
Background:
Despite initial response to platinum-based chemotherapy and PARP inhibitor therapy (PARPi), nearly all recurrent high-grade serous ovarian cancer (HGSC) will acquire lethal drug ...resistance; indeed, ~15% of individuals have de novo platinum-refractory disease.
Objectives:
To determine the potential of anti-microtubule agent (AMA) therapy (paclitaxel, vinorelbine and eribulin) in platinum-resistant or refractory (PRR) HGSC by assessing response in patient-derived xenograft (PDX) models of HGSC.
Design and methods:
Of 13 PRR HGSC PDX, six were primary PRR, derived from chemotherapy-naïve samples (one was BRCA2 mutant) and seven were from samples obtained following chemotherapy treatment in the clinic (five were mutant for either BRCA1 or BRCA2 (BRCA1/2), four with prior PARPi exposure), recapitulating the population of individuals with aggressive treatment-resistant HGSC in the clinic. Molecular analyses and in vivo treatment studies were undertaken.
Results:
Seven out of thirteen PRR PDX (54%) were sensitive to treatment with the AMA, eribulin (time to progressive disease (PD) ⩾100 days from the start of treatment) and 11 out of 13 PDX (85%) derived significant benefit from eribulin time to harvest (TTH) for each PDX with p < 0.002. In 5 out of 10 platinum-refractory HGSC PDX (50%) and one out of three platinum-resistant PDX (33%), eribulin was more efficacious than was cisplatin, with longer time to PD and significantly extended TTH (each PDX p < 0.02). Furthermore, four of these models were extremely sensitive to all three AMA tested, maintaining response until the end of the experiment (120d post-treatment start). Despite harbouring secondary BRCA2 mutations, two BRCA2-mutant PDX models derived from heavily pre-treated individuals were sensitive to AMA. PRR HGSC PDX models showing greater sensitivity to AMA had high proliferative indices and oncogene expression. Two PDX models, both with prior chemotherapy and/or PARPi exposure, were refractory to all AMA, one of which harboured the SLC25A40-ABCB1 fusion, known to upregulate drug efflux via MDR1.
Conclusion:
The efficacy observed for eribulin in PRR HGSC PDX was similar to that observed for paclitaxel, which transformed ovarian cancer clinical practice. Eribulin is therefore worthy of further consideration in clinical trials, particularly in ovarian carcinoma with early failure of carboplatin/paclitaxel chemotherapy.
How platelets are produced by megakaryocytes in vivo remains controversial despite more than a century of investigation. Megakaryocytes readily produce proplatelet structures in vitro; however, ...visualization of platelet release from proplatelets in vivo has remained elusive. We show that within the native prenatal and adult environments, the frequency and rate of proplatelet formation is incompatible with the physiological demands of platelet replacement. We resolve this inconsistency by performing in-depth analysis of plasma membrane budding, a cellular process that has previously been dismissed as a source of platelet production. Our studies demonstrate that membrane budding results in the sustained release of platelets directly into the peripheral circulation during both fetal and adult life without induction of cell death or proplatelet formation. In support of this model, we demonstrate that in mice deficient for NF-E2 (the thrombopoietic master regulator), the absence of membrane budding correlates with failure of in vivo platelet production. Accordingly, we propose that membrane budding, rather than proplatelet formation, supplies the majority of the platelet biomass.
We report the discovery of GATA2 as a new myelodysplastic syndrome (MDS)-acute myeloid leukemia (AML) predisposition gene. We found the same, previously unidentified heterozygous c.1061C>T ...(p.Thr354Met) missense mutation in the GATA2 transcription factor gene segregating with the multigenerational transmission of MDS-AML in three families and a GATA2 c.1063_1065delACA (p.Thr355del) mutation at an adjacent codon in a fourth MDS family. The resulting alterations reside within the second zinc finger of GATA2, which mediates DNA-binding and protein-protein interactions. We show differential effects of the mutations on the transactivation of target genes, cellular differentiation, apoptosis and global gene expression. Identification of such predisposing genes to familial forms of MDS and AML is critical for more effective diagnosis and prognosis, counseling, selection of related bone marrow transplant donors and development of therapies.
Abstract
Whilst the majority of Endometrial Cancers (ECs) are common Type 1 endometrioid cancers, accounting for 75-80% of cases, with a good prognosis, Type 2 ECs include high-grade, clinically ...aggressive histologies, with poor response rates to hormonal therapies. Serous endometrial carcinoma is the second most common type, accounting for ~10 percent of cases, most with a p53 abnormality and a lesser proportion with HER2 overexpression/amplification/mutation. Clear cell endometrial carcinoma accounts for <5 % of EC, with the most aggressive cases also having p53 mutations. Mixed histologies and undifferentiated ECs are also aggressive. Carcinosarcoma (ECS) is a rare, aggressive, biphasic carcinoma that accounts for <5 percent of ECs, 90% with p53 abnormalities. Endometrial stromal sarcomas (ESS) can be low-grade or high-grade, or undifferentiated or resemble ovarian sex cord tumors. Uterine leiomyosarcoma (uLMS) are epithelioid or myxoid in type. Adenosarcoma involves a benign epithelial component mixed with a malignant stromal element. Whilst there have been considerable improvements in death rates for all cancers combined over the last 20 years, these improvements have not been seen for most rare cancers (RC). In order to ensure that those diagnosed with RC have access to research and novel therapies, we designed the Walter and Eliza Hall Institute of Medical Research (WEHI) Stafford Fox Rare Cancer Program (SFRCP). The WEHI-SFRCP has streamlined ethics, governance, consenting processes, including remote consent at home anywhere in Australia, and data collection protocols to allow the analysis of data and tissue of any type of RC. We have interconnected clinical and laboratory RC Databases within BioGrid Australia using the online REDCap platform. We have extensive laboratory processes in place for processing of tumour and blood samples, including the generation of PBMCs, DNA, RNA, to generate NGS including WGS; patient-derived xenografts (PDX), organoids, cell lines and other derivatives. We have high grade serous endometrial cancer (33 cases, including 4 PDX with 3 pending); uLMS (32 cases, including 2 PDX with 3 pending); endometrial carcinosarcoma (13 cases, including 3 PDX, 1 pending); rare (other, adenosarcoma, STUMP). We perform NGS testing on cases, depending on tumour purity, including Whole Genome Sequencing from fresh tumour samples; characterize PDX according to current chemotherapy and relevant novel therapeutics; study rare endometrial subtypes in specific projects and also provide information back to patients to guide therapy in the clinic. By integrating data sets with endometrial projects of similar depth we will drive forward the study of rare endometrial cancer subtypes.
Citation Format: Clare L. Scott, Ratana Lim, Amandine Carmagnac, Cassandra Vandenberg, Gayanie Ratnayake, Genevieve Dall, Joshua Tram, Justin Bedo, Jocelyn Penington, Joep Vissers, Sean Grimmond, Matthew Wakefield, Anthony Papenfuss, Holly Barker. Building the infrastructure required to research novel therapies for 35% of women with endometrial cancer who have rare subtypes abstract. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO026.
Abstract
High-grade serous endometrial carcinoma (HGSEC) accounts for just 10% of endometrial cancer (EC) cases but is responsible for at least 40% of all EC-related deaths. It typically arises in ...post-menopausal women, with 70% of patients presenting with stage III or IV disease, does not respond to hormone therapy unlike the less aggressive forms of EC, and has a lower overall survival rate of just 18-27%, which has not improved over the past two decades. The primary treatment for HGSEC is surgery, followed by a combination of standard chemotherapies (platinum and taxane) with or without localised radiotherapy. However, recurrent HGSEC is less responsive to chemotherapy than are other subtypes of EC and even initial responses to chemotherapy are poor. Therefore, there is a great unmet clinical need to find better treatment options for women with this aggressive cancer. Apart from TP53 (mutated in up to 90% of cases), the other most frequently mutated genes in HGSEC are PPP2R1A (31%), PIK3CA (22%), FBXW7 (28%), CHD4 (17%) and BRCA2 (12%). Focal amplifications of the genes MYC, ERBB2, CCNE1, FGFR3 and SOX17 are also common. The presence of ERBB2 amplification and/or HER2 over-expression in around 30% of HGSEC suggests these patients may respond to HER2-targeting drugs, such as trastuzumab. However, only modest benefit has so far been seen for single-agent HER2-targeted therapies (ie trastuzumab or lapatinib) against HGSEC, suggesting resistance mechanisms are present. Another feature of HGSEC that could be exploited therapeutically is homologous recombination deficiency (HRD), which may be targeted with PARP inhibitors (PARPi). It is not clear what proportion of HGSEC are HRD and neither HER2-targeting drugs or PARPi have been approved for the treatment of HGSEC. Due to its rarity and a lack of pre-clinical models, HGSEC has so far been understudied, resulting in a lack of effective treatment options. We currently have 33 HGSEC patients consented to the WEHI-Stafford Fox Rare Cancer Program and have developed pre-clinical models from fresh patient tumour samples received (4 patient-derived xenograft (PDX) models validated, with 3 pending). Preliminary molecular analysis of whole-genome sequencing (5 samples, one of which gave rise to a PDX model), whole-exome sequencing (4 samples), and cancer panel sequencing (3 samples, 2 of which gave rise to PDX models; one harbouring ERBB2 amplification and one harbouring an AKT mutation) data from our HGSEC cohort has been performed. This has identified potential treatment targets, including ERBB2 amplifications and mutations in HR genes. I am using the PDX models for initial in vivo therapeutic characterization studies and to develop organoid models for use in high-throughput drug assays in vitro. This will guide subsequent novel drug combination testing in our PDX models. By combining specific targeted drugs I hope to overcome de novo resistance mechanisms and prevent acquired resistance. Results from this study will guide future decisions about therapeutic strategies to improve survival of women with HGSEC.
Citation Format: Holly E. Barker, Ratana Lim, Amandine Carmagnac, Cassandra Vandenberg, Gayanie Ratnayake, Genevieve Dall, Briony Milesi, Angela Komiti, Emily O'Grady, Joshua Tram, Kym Pham Stewart, Justin Bedo, Jocelyn Penington, Joep Vissers, Sean Grimmond, Matthew Wakefield, Tony Papenfuss, Clare Scott. Identifying effective combinations of targeted therapies, using novel pre-clinical models, to improve treatment options for high-grade serous endometrial cancer abstract. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO037.
Abstract
Uterine sarcomas make up 1-4% of uterine malignancies. Of these 60% are classified as leiomyosarcoma (uLMS). The 5-year survival rate of uLMS is 35-65.2% for tumours that have not spread ...beyond the uterus. However, women are often diagnosed at a late stage due to a lack of screening options by which time the tumour has often spread to adjacent and distant tissues. Current standard of care for uLMS patients is surgical de-bulking followed by adjuvant chemotherapy, but significant improvement in progression free survival and overall survival is not consistently observed. The lack of advances for the treatment and screening of uLMS is due in part to the scarcity of appropriate research resources for this rare disease. Genetic analyses have been performed but on relatively small samples and there are just 14 reported patient-derived xenograft (PDX) models of uLMS in the literature to date. Through the WEHI Stafford Fox Rare Cancer Program, as well as collaborations (facilitated by ANZGOG) throughout the country and internationally, we have access to a large biobank of uLMS tissue. We have received 8 fresh uLMS samples in the laboratory, 2 of which have established PDX lines that were validated as uLMS by our anatomical pathologist. All fresh samples received into the laboratory are snap frozen for whole-genome sequencing as well as viably frozen to enable regeneration of the tissue for future applications. One application is organoid culturing, which allows the tissue to retain its 3D growth properties and is significantly cheaper than growing tissue as a PDX. Organoid culturing also allows for higher throughput of samples in drug screening assays, enabling us to fast-track the selection of drugs for validation in our PDX models. In addition to these fresh samples we also have 23 archival uLMS samples (formalin fixed, paraffin embedded) that can be used for lower coverage genetic analysis, and protein expression by immunohistochemistry. This unique biobank of uLMS tissue is the first of its kind in Australia and with it we will endeavour to gain a comprehensive understanding of this disease. Through our PDX modelling we also have the opportunity to predict resistance to therapy and test emerging therapies in a clinically relevant context. We believe this biobank will provide a critical resource which will ultimately lead to better outcomes for uLMS patients.
Citation Format: Genevieve Dall, Cassandra Vandenberg, Amandine Carmagnac, Ratana Lim, Briony Milesi, Angela Komiti, Emily O'Grady, Joshua Tram, Gayanie Ratnayake, Kym Pham Stewart, Justin Bedo, Jocelyn Penington, Joep Vissers, Inger Olesen, Sean Grimmond, Holly Barker, Tony Papenfuss, Clare Scott. Developing pre-clinical models of uterine leiomyosarcoma abstract. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO021.
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