A novel COVID-19 program, delivering vaccines throughout rural and remote Australia.

Background: The Royal Flying Doctor Service of Australia (RFDS) established a unique SARS-CoV-2 vaccination program for vaccinating Australians that live in rural and remote areas. This paper describes the preparation and response phases of the RFDS response. Methods: This study includes vaccinations conducted by the RFDS from 01 January 2021 until 31 December 2021 when vaccines were mandatory for work and social activities. Prior to each clinic, we conducted community consultation to determine site requirements, patient characteristics, expected vaccination numbers, and community transmission rates. Findings: Ninety-five organizations requested support. The majority (n = 60; 63.2%) came from Aboriginal Community Controlled Health Organizations. Following consultation, 360 communities were approved for support. Actual vaccinations exceeded expectations (n = 70,827 vs. 49,407), with a concordance correlation coefficient of 0.88 (95% CI, 0.83, 0.93). Areas that reported healthcare workforce shortages during the preparation phase had the highest population proportion difference between expected and actual vaccinations. Areas that reported high vaccine hesitancy during the preparation phase had fewer than expected vaccines. There was a noticeable increase in vaccination rates in line with community outbreaks and positive polymerase chain reaction cases [r (41) = 0.35, p = 0.021]. Engagement with community leaders prior to clinic deployment was essential to provide a tailored response based on community expectations.

Colony stimulating factor-1 dependence of paneth cell development in the mouse small intestine.

BACKGROUND & AIMS: Paneth cells (PCs) secrete defensins and antimicrobial enzymes that contribute to innate immunity against pathogen infections within the mucosa of the small intestine. We examined the role of colony stimulating factor-1 (CSF-1) in PC development. METHODS: CSF-1-deficient and CSF-1 receptor (CSF-1R)-deficient mice and administration of neutralizing anti-CSF-1R antibody were used to study the requirement of CSF-1 for the development of epithelial cells of the small intestine. CSF-1 transgenic reporter mice and mice that express only the membrane-spanning, cell-surface CSF-1 isoform were used to investigate regulation by systemic versus local CSF-1. RESULTS: Mice deficient in CSF-1 or CSF-1R had greatly reduced numbers of mature PCs. PCs express the CSF-1R, and administration of anti-CSF-1R antibody to neonatal mice significantly reduced the number of PCs. Analysis of transgenic CSF-1 reporter mice showed that CSF-1-expressing cells are in close proximity to PCs. CSF-1/CSF-1R-deficient mice also had reduced numbers of the proliferating epithelial cell progenitors and lamina propria macrophages. Expression of the membrane-spanning, cell-surface CSF-1 isoform in CSF-1-deficient mice completely rescued the deficiencies of PCs, proliferating progenitors, and lamina propria macrophages. CONCLUSIONS: These results indicate local regulation by CSF-1 of PC development, either directly, in a juxtacrine/paracrine manner, or indirectly, by lamina propria macrophages. Therefore, CSF-1R hyperstimulation could be involved in hyperproliferative disorders of the small intestine, such as Crohn's disease and ulcerative colitis.

Terminal osteoblast differentiation, mediated by runx2 and p27KIP1, is disrupted in osteosarcoma.

The molecular basis for the inverse relationship between differentiation and tumorigenesis is unknown. The function of runx2, a master regulator of osteoblast differentiation belonging to the runt family of tumor suppressor genes, is consistently disrupted in osteosarcoma cell lines. Ectopic expression of runx2 induces p27KIP1, thereby inhibiting the activity of S-phase cyclin complexes and leading to the dephosphorylation of the retinoblastoma tumor suppressor protein (pRb) and a G1 cell cycle arrest. Runx2 physically interacts with the hypophosphorylated form of pRb, a known coactivator of runx2, thereby completing a feed-forward loop in which progressive cell cycle exit promotes increased expression of the osteoblast phenotype. Loss of p27KIP1 perturbs transient and terminal cell cycle exit in osteoblasts. Consistent with the incompatibility of malignant transformation and permanent cell cycle exit, loss of p27KIP1 expression correlates with dedifferentiation in high-grade human osteosarcomas. Physiologic coupling of osteoblast differentiation to cell cycle withdrawal is mediated through runx2 and p27KIP1, and these processes are disrupted in osteosarcoma.

Novel molecular subtypes of serous and endometrioid ovarian cancer linked to clinical outcome.

PURPOSE: The study aim to identify novel molecular subtypes of ovarian cancer by gene expression profiling with linkage to clinical and pathologic features. EXPERIMENTAL DESIGN: Microarray gene expression profiling was done on 285 serous and endometrioid tumors of the ovary, peritoneum, and fallopian tube. K-means clustering was applied to identify robust molecular subtypes. Statistical analysis identified differentially expressed genes, pathways, and gene ontologies. Laser capture microdissection, pathology review, and immunohistochemistry validated the array-based findings. Patient survival within k-means groups was evaluated using Cox proportional hazards models. Class prediction validated k-means groups in an independent dataset. A semisupervised survival analysis of the array data was used to compare against unsupervised clustering results. RESULTS: Optimal clustering of array data identified six molecular subtypes. Two subtypes represented predominantly serous low malignant potential and low-grade endometrioid subtypes, respectively. The remaining four subtypes represented higher grade and advanced stage cancers of serous and endometrioid morphology. A novel subtype of high-grade serous cancers reflected a mesenchymal cell type, characterized by overexpression of N-cadherin and P-cadherin and low expression of differentiation markers, including CA125 and MUC1. A poor prognosis subtype was defined by a reactive stroma gene expression signature, correlating with extensive desmoplasia in such samples. A similar poor prognosis signature could be found using a semisupervised analysis. Each subtype displayed distinct levels and patterns of immune cell infiltration. Class prediction identified similar subtypes in an independent ovarian dataset with similar prognostic trends. CONCLUSION: Gene expression profiling identified molecular subtypes of ovarian cancer of biological and clinical importance.

Patients with multiple myeloma treated with thalidomide: evaluation of clinical parameters, cytokines,angiogenic markers, mast cells and marrow CD57+ cytotoxic T cells as predictors of outcome.

BACKGROUND AND OBJECTIVES: In vitro studies suggest that thalidomide has an immunoregulatory role and alters the marrow microenvironment. We assessed laboratory and clinical parameters in patients with myeloma treated with thalidomide as potential prognostic markers and looked for changes with therapy. DESIGN AND METHODS: Seventy-five patients with relapsed/refractory myeloma received thalidomide in a phase II trial. Serial samples of platelet-poor plasma and bone marrow were tested for angiogenic cytokines including vascular endothelial growth factor (VEGF), marrow microvessel-density (MVD), mast cells and CD57+ cell expression. The effects of these parameters on response rate (RR), progression-free survival (PFS) and overall survival (OS) were analyzed. RESULTS: Elevated baseline VEGF predicted for a superior RR (p=0.018) and PFS. Elevated CD57+ cells also predicted superior PFS (p=0.012). MVD did not predict for RR, PFS or OS, but MVD and VEGF fell significantly in responders. Multivariate analysis identified that inferior OS was associated with age >65 years (p=0.017), raised lactate dehydrogenase (p=0.001), raised hepatocyte growth factor levels (p=0.012) and low pre-treatment CD57+ cells (p<0.001). INTERPRETATION AND CONCLUSIONS: Our findings support the suggestion that thalidomide has anti-angiogenic and immunomodulatory effects in myeloma. The preferred method for assessing angiogenesis is plasma VEGF levels and the assessment of CD57+ cells for patients with myeloma receiving novel immunomodulatory drugs should be further investigated.

Use of molecular tumor characteristics to prioritize mismatch repair gene testing in early-onset colorectal cancer.

PURPOSE: The relationships between mismatch repair (MMR) protein expression, microsatellite instability (MSI), family history, and germline MMR gene mutation status have not been studied on a population basis. METHODS: We studied 131 unselected patients with colorectal cancer diagnosed younger than age 45 years. For the 105 available tumors, MLH1, MSH2, MSH6, and PMS2 protein expression using immunohistochemistry (IHC) and MSI were measured. Germline DNA was screened for hMLH1, hMSH2, hMSH6, and hPMS2 mutations for the following patients: all from families fulfilling the Amsterdam Criteria for hereditary nonpolyposis colorectal cancer (HNPCC); all with tumors that were high MSI, low MSI, or that lacked expression of any MMR protein; and a random sample of 23 with MS-stable tumors expressing all MMR proteins. RESULTS: Germline mutations were found in 18 patients (nine hMLH1, four hMSH2, four hMSH6, and one hPMS2); all tumors exhibited loss of MMR protein expression, all but one were high MSI or low MSI, and nine were from a family fulfilling Amsterdam Criteria. Sensitivities of IHC testing, MSI (high or low), and Amsterdam Criteria for MMR gene mutation were 100%, 94%, and 50%, respectively. Corresponding positive predictive values were 69%, 50%, and 75%. CONCLUSIONS: Tumor IHC analysis of four MMR proteins and MSI testing provide a highly sensitive strategy for identifying MMR gene mutation-carrying, early-onset colorectal cancer patients, half of whom would have been missed using Amsterdam Criteria alone. Tumor-based approaches for triaging early-onset colorectal cancer patients for MMR gene mutation testing, irrespective of family history, appear to be an efficient screening strategy for HNPCC.

Brca1 inactivation induces p27(Kip1)-dependent cell cycle arrest and delayed development in the mouse mammary gland.

One common characteristic of breast cancers arising in carriers of the predisposition gene BRCA1 is a loss of expression of the CDK inhibitor p27(Kip1) (p27), suggesting that p27 interacts epistatically with BRCA1. To investigate this relationship, we examined expression of p27 in mice expressing a dominant negative allele of Brca1 (MMTV-trBr) in the mammary gland. While these mice rarely develop tumors, they showed a 50% increase in p27 protein and a delay in mammary gland development associated with reduced proliferation. In contrast, on a p27 heterozygote background, MMTV-trBrca1 mice showed an increase in S phase cells, and normal mammary development. p27 was the only protein in the cyclin-cyclin-dependent kinase network to show altered expression, suggesting that it may be a central mediator of cell cycle arrest in response to loss of function of BRCA1. Furthermore, in human mammary epithelial MCF7 cells expressing BRCA1-specific RNAi and in the BRCA1-deficient human tumor cell line HCC1937, p27 is elevated at the mRNA level compared to cells expressing wild-type BRCA1. We hypothesize that disruption of BRCA1 induces an increase in p27 that inhibits proliferation. Accordingly, reduction in p27 expression leads to enhancement of cellular proliferation in the absence of BRCA1.

Bone marrow plasma cell microaggregates detected by immunohistology predict earlier relapse in patients with minimal disease after high-dose therapy for myeloma.

Plasma cell microaggregates detected by CD138 immunohistology were demonstrated in 22% of patients achieving morphologic remission 3 months after high-dose therapy for myeloma. Microaggregates were predictive of earlier disease progression, indicating that immunohistology may represent a useful tool in the assessment of minimal disease in patients after high-dose therapy for myeloma.

EGFR blockade with ZD1839 ("Iressa") potentiates the antitumor effects of single and multiple fractions of ionizing radiation in human A431 squamous cell carcinoma. Epidermal growth factor receptor.

PURPOSE: Signaling pathways initiated by the epidermal growth factor receptor (EGFR) play important roles in the response to ionizing radiation. In this study the consequences of inhibiting the EGFR on the response of A431 cells (human vulvar squamous cell carcinoma cells that overexpress EGFR) to radiation, were investigated in vitro and in vivo, using the selective EGFR-tyrosine kinase inhibitor, ZD1839 ("Iressa"). METHODS AND MATERIALS: The effect of ZD1839 on proliferation, apoptosis, and clonogenic survival after radiation was determined in vitro. For in vivo studies, athymic nude mice with established subcutaneous A431 xenografts (approximately 100 mm(3)) were treated with either a single 10 Gy fraction or 4 daily 2.5 Gy fractions of radiation with or without ZD1839 (75 mg/kg/day intraperitoneally for 10 days) to determine effects on tumor growth delay. RESULTS: Treatment of A431 cells with ZD1839 in vitro reduced proliferation, increased apoptosis, and reduced clonogenic survival after radiation. Strikingly greater than additive effects of ZD1839 in combination with radiation on tumor growth delay were observed in vivo after either a single 10 Gy fraction (enhancement ratio: 1.5) or multiple 4 x 2.5 Gy fractions (enhancement ratio: 4). ZD1839 reduced tumor vascularity, as well as levels of vascular endothelial growth factor (VEGF) protein and mRNA induced by stimulation with epidermal growth factor (EGF), suggesting a possible role of inhibition of angiogenesis in the effect. CONCLUSIONS: Inhibiting EGFR-mediated signal transduction cascades with ZD1839 potentiates the antitumor effect of single and multiple fractions of radiation. These data provide preclinical rationale for clinical trials of EGFR inhibitors including ZD1839 in combination with radiation.

Diagnostic application of Epstein-Barr virus-encoded RNA in situ hybridisation.

AIMS: Epstein-Barr virus (EBV) has been implicated in the pathogenesis of nasopharyngeal carcinoma and a range of proliferative lymphoid conditions. In situ hybridisation (ISH) looking for virus-encoded RNA (EBER) transcripts is performed simply using a commercially available probe. We aimed to examine the application of this test in a routine diagnostic setting. METHODS: In total, 26 cases in which EBV ISH was requested for diagnostic purposes were examined. We looked at the indication for testing, the result and its implication for the final diagnosis. RESULTS: Cases were classified into three categories: possible nasopharyngeal carcinoma; possible EBV-related lymphoma; and possible immunodeficiency-associated lymphoproliferative disorder. Six of nine cases of possible nasopharyngeal carcinoma were EBV ISH positive (3/3 primary and 3/6 secondary), confirming the diagnosis. Three of 14 possible lymphoma cases were EBV ISH positive which, along with appropriate ancillary tests, assisted in making the diagnoses of Burkitt's lymphoma, lymphomatoid granulomatosis and extranodal NK/T-cell lymphoma of nasal type. All of three immunodeficiency-associated cases were EBV ISH positive. Two of these were post-transplant lymphoproliferative disorders, monomorphic type. The third case was classified as HIV-related polymorphic lymphoproliferative disorder. CONCLUSIONS: In our experience, EBV ISH is a straightforward and rapid procedure to perform, giving unequivocal results. Used in the appropriate clinicopathological setting it can be a highly useful ancillary diagnostic aid.

Parathyroid hormone-related protein protects against mammary tumor emergence and is associated with monocyte infiltration in ductal carcinoma in situ.

Parathyroid hormone-related protein (PTHrP) is required for mammary gland development and promotes the growth of breast cancer metastases within bone. However, there are conflicting reports of the prognostic significance of its expression in primary breast cancers. To study the role of PTHrP in early breast cancer, the effect of conditional deletion of PTHrP was examined in the context of neu-induced mammary tumorigenesis. Loss of PTHrP resulted in a higher tumor incidence. Transcriptional profiling of the tumors revealed that PTHrP influenced genes relevant to heterotypic cell signaling, including regulators of monocyte recruitment. Immunohistochemical analysis of human breast cancers revealed that PTHrP expression was associated with both HER-2/neu expression and macrophage infiltration in preinvasive ductal carcinoma in situ. The gene expression signature associated with loss of PTHrP expression in vivo correlated with poorer outcome in human breast cancer. Together, these data indicate that loss of PTHrP accelerates mammary tumorigenesis possibly by a non-cell-autonomous tumor suppressor pathway.

Wnt inhibitory factor 1 is epigenetically silenced in human osteosarcoma, and targeted disruption accelerates osteosarcomagenesis in mice.

Wnt signaling increases bone mass by stimulating osteoblast lineage commitment and expansion and forms the basis for novel anabolic therapeutic strategies being developed for osteoporosis. These strategies include derepression of Wnt signaling by targeting secreted Wnt pathway antagonists, such as sclerostin. However, such therapies are associated with safety concerns regarding an increased risk of osteosarcoma, the most common primary malignancy of bone. Here, we analyzed 5 human osteosarcoma cell lines in a high-throughput screen for epigenetically silenced tumor suppressor genes and identified Wnt inhibitory factor 1 (WIF1), which encodes an endogenous secreted Wnt pathway antagonist, as a candidate tumor suppressor gene. In vitro, WIF1 suppressed beta-catenin levels in human osteosarcoma cell lines, induced differentiation of human and mouse primary osteoblasts, and suppressed the growth of mouse and human osteosarcoma cell lines. Wif1 was highly expressed in the developing and mature mouse skeleton, and, although it was dispensable for normal development, targeted deletion of mouse Wif1 accelerated development of radiation-induced osteosarcomas in vivo. In primary human osteosarcomas, silencing of WIF1 by promoter hypermethylation was associated with loss of differentiation, increased beta-catenin levels, and increased proliferation. These data lead us to suggest that derepression of Wnt signaling by targeting secreted Wnt antagonists in osteoblasts may increase susceptibility to osteosarcoma.

The Lewis-Y carbohydrate antigen is expressed by many human tumors and can serve as a target for genetically redirected T cells despite the presence of soluble antigen in serum.

In this study we aimed to determine the suitability of the Lewis-Y carbohydrate antigen as a target for immunotherapy using genetically redirected T cells. Using the 3S193 monoclonal antibody and immunohistochemistry, Lewis-Y was found to be expressed on a range of tumors including 42% squamous cell lung carcinoma, 80% lung adenocarcinoma, 25% ovarian carcinoma, and 25% colorectal adenocarcinoma. Expression levels varied from low to intense on between 1% and 90% of tumor cells. Lewis- was also found in soluble form in sera from both normal donors and cancer patients using a newly developed enzyme-linked immunosorbent assay. Serum levels in patients was often less than 1 ng/mL, similar to normal donors, but approximately 30% of patients had soluble Lewis-Y levels exceeding 1 ng/mL and up to 9 ng/mL. Lewis-Y-specific human T cells were generated by genetic modification with a chimeric receptor encoding a single-chain humanized antibody linked to the T-cell signaling molecules, T-cell receptor-zeta, and CD28. T cells responded against the Lewis-Y antigen by cytokine secretion and cytolysis in response to tumor cells. Importantly, the T-cell response was not inhibited by patient serum containing soluble Lewis-Y. This study demonstrates that Lewis-Y is expressed on a large number of tumors and Lewis-Y-specific T cells can retain antitumor function in the presence of patient serum, indicating that this antigen is a suitable target for this form of therapy.

No evidence of clonal somatic genetic alterations in cancer-associated fibroblasts from human breast and ovarian carcinomas.

There is increasing evidence showing that the stromal cells surrounding cancer epithelial cells, rather than being passive bystanders, might have a role in modifying tumor outgrowth. The molecular basis of this aspect of carcinoma etiology is controversial. Some studies have reported a high frequency of genetic aberrations in carcinoma-associated fibroblasts (CAFs), whereas other studies have reported very low or zero mutation rates. Resolution of this contentious area is of critical importance in terms of understanding both the basic biology of cancer as well as the potential clinical implications of CAF somatic alterations. We undertook genome-wide copy number and loss of heterozygosity (LOH) analysis of CAFs derived from breast and ovarian carcinomas using a 500K SNP array platform. Our data show conclusively that LOH and copy number alterations are extremely rare in CAFs and cannot be the basis of the carcinoma-promoting phenotypes of breast and ovarian CAFs.

Distinct clinical and pathological features are associated with the BRAF(T1799A(V600E)) mutation in primary melanoma.

The BRAF(T1799A) mutation encodes BRAF(V600E) that leads to activation of the mitogen-activated protein kinase pathway. This study aimed to assess the clinico-pathological features of primary invasive melanomas containing the BRAF(T1799A) mutation. Patients (n=251) with invasive primary melanomas from Australia were interviewed and examined with respect to their melanoma characteristics and risk factors. Independent review of pathology, allele-specific PCR for the BRAF(T1799A) mutation, immunohistochemical staining with Ki67, and phospho-histone-H3 (PH3) were performed. The BRAF(T1799A) mutation was found in 112 (45%) of the primary melanomas. Associations with the BRAF(T1799A) mutation (P<0.05) were as follows: low tumor thickness (odds ratio (OR)=3.3); low mitotic rate (OR=2.0); low Ki67 score (OR=5.0); low PH3 score (OR=3.3); superficial spreading melanoma (OR=10.0); pigmented melanoma (OR=3.7); a lack of history of solar keratoses (OR=2.7); a location on the trunk (OR=3.4) or extremity (OR=2.0); a high level of self-reported childhood sun exposure (OR=2.0); < or =50 years of age (OR=2.5); and fewer freckles (OR=2.5). We conclude that the BRAF(T1799A) mutation has associations with host phenotype, tumor location, and pigmentation. Although implicated in the control of the cell cycle, the BRAF(T1799A) mutation is associated with a lower rate of tumor proliferation.

Parathyroid hormone-related protein production in the lamprey Geotria australis: developmental and evolutionary perspectives.

This study explored the distribution of parathyroid hormone-related protein (PTHrP) and its mRNA in tissues of the lamprey Geotria australis, a representative of one of the two surviving groups of an early and jawless stage in vertebrate evolution. For this purpose, antibodies to N-terminal and mid-molecule human PTHrP were used to determine the locations of the antigen. Sites of mRNA production were demonstrated by in situ hybridisation with a digoxigenin-labelled riboprobe to exon VI of the human PTHrP gene. The results revealed that antigen and its mRNA were widely distributed among similar sites of tissue localisation to those described for mammalian and avian species. However, some novel sites of localisation, such as in the gill and notochord, were also found. Some differences in PTHrP localisation were noted among individuals at different intervals of the life cycle, indicating that the distributions of PTHrP, and possibly its roles, change with the stage of development in this species. The widespread tissue distribution in G. australis implies diverse physiological roles for this protein. The presence of PTHrP in the lamprey, a representative of a group of vertebrates, which apparently evolved over 540 million years ago, strongly suggests that it is a protein of ancient origin. In addition, the successful use of antibodies and probes based on the human sequence in the lamprey also provides evidence that the PTHrP molecule may have been conserved from lampreys through to humans.

Molecular profiling of giant cell tumor of bone and the osteoclastic localization of ligand for receptor activator of nuclear factor kappaB.

Giant cell tumor of bone (GCT) is a generally benign, osteolytic neoplasm comprising stromal cells and osteoclast-like giant cells. The osteoclastic cells, which cause bony destruction, are thought to be recruited from normal monocytic pre-osteoclasts by stromal cell expression of the ligand for receptor activator of nuclear factor kappaB (RANKL). This model forms the foundation for clinical trials in GCTs of novel cancer therapeutics targeting RANKL. Using expression profiling, we identified both osteoblast and osteoclast signatures within GCTs, including key regulators of osteoclast differentiation and function such as RANKL, a C-type lectin, osteoprotegerin, and the wnt inhibitor SFRP4. After ex vivo generation of stromal- and osteoclast-enriched cultures, we unexpectedly found that RANKL mRNA and protein were more highly expressed in osteoclasts than in stromal cells, as determined by expression profiling, flow cytometry, immunohistochemistry, and reverse transcriptase-polymerase chain reaction. The expression patterns of molecules implicated in signaling between stromal cells and monocytic osteoclast precursors were analyzed in both primary and fractionated GCTs. Finally, using array-based comparative genomic hybridization, neither GCTs nor the derived stromal cells demonstrated significant genomic gains or losses. These data raise questions regarding the role of RANKL in GCTs that may be relevant to the development of molecularly targeted therapeutics for this disease.