Glioblastoma and the search for non-hypothesis driven combination therapeutics in academia.

Glioblastoma (GBM) remains a cancer of high unmet clinical need. Current standard of care for GBM, consisting of maximal surgical resection, followed by ionisation radiation (IR) plus concomitant and adjuvant temozolomide (TMZ), provides less than 15-month survival benefit. Efforts by conventional drug discovery to improve overall survival have failed to overcome challenges presented by inherent tumor heterogeneity, therapeutic resistance attributed to GBM stem cells, and tumor niches supporting self-renewal. In this review we describe the steps academic researchers are taking to address these limitations in high throughput screening programs to identify novel GBM combinatorial targets. We detail how they are implementing more physiologically relevant phenotypic assays which better recapitulate key areas of disease biology coupled with more focussed libraries of small compounds, such as drug repurposing, target discovery, pharmacologically active and novel, more comprehensive anti-cancer target-annotated compound libraries. Herein, we discuss the rationale for current GBM combination trials and the need for more systematic and transparent strategies for identification, validation and prioritisation of combinations that lead to clinical trials. Finally, we make specific recommendations to the preclinical, small compound screening paradigm that could increase the likelihood of identifying tractable, combinatorial, small molecule inhibitors and better drug targets specific to GBM.

Respiratory Viruses Cause Late Morbidity in Recipients of Hematopoietic Stem Cell Transplantation.

Common respiratory viral infections (CRVIs) frequently complicate hematopoietic stem cell transplantation (HSCT). We conducted a retrospective, single-center, observational cohort study to determine the incidence of CRVI in patients who received an allogeneic (allo) or autologous (auto) HSCT at the Royal Adelaide Hospital between 2009 and 2017. The median follow-up was 8.9 and 4.5 years for auto- and allo-HSCT recipients, respectively. There were 149 CRVI episodes in 74 patients, with rhinovirus being the most commonly isolated virus (n = 81, 47%). The majority of CRVIs (113/149, 75.8%) occurred more than 100 days post-HSCT and 67% were diagnosed in the outpatient setting. There was evidence of lower respiratory tract infection (LRTI) in 45.6% (68/149) of CRVIs. On multivariate logistic regression analysis, coviral infections and cytomegalovirus viremia were independent risk factors for progression of CRVI to LRTI. Ten (6.7%) CRVI episodes resulted in admission to intensive care for ventilatory support and 8 (5.4%) patients died within 30 days of CRVI diagnosis. In our study, 10.4% of HSCT recipients experienced a CRVI post-transplant, primarily causing late morbidity and potentially mortality. Prevention with strict infection control practices, vaccination, and patient education is essential.

Increased EZH2 expression in prostate cancer is associated with metastatic recurrence following external beam radiotherapy.

BACKGROUND: Enhancer of zeste 2 (EZH2) promotes prostate cancer progression. We hypothesized that increased EZH2 expression is associated with postradiotherapy metastatic disease recurrence, and may promote radioresistance. METHODS: EZH2 expression was investigated using immunohistochemistry in diagnostic prostate biopsies of 113 prostate cancer patients treated with radiotherapy with curative intent. Associations between EZH2 expression in malignant and benign tissue in prostate biopsy cores and outcomes were investigated using univariate and multivariate Cox regression analyses. LNCaP and PC3 cell radiosensitivity was investigated using colony formation and γH2AX assays following UNC1999 chemical probe-mediated EZH2 inhibition. RESULTS: While there was no significant association between EZH2 expression and biochemical recurrence following radiotherapy, univariate analysis revealed that prostate cancer cytoplasmic and total EZH2 expression were significantly associated with metastasis development postradiotherapy (P = 0.034 and P = 0.003, respectively). On multivariate analysis, the prostate cancer total EZH2 expression score remained statistically significant (P = 0.003), while cytoplasmic EZH2 expression did not reach statistical significance (P = 0.053). No association was observed between normal adjacent prostate EZH2 expression and biochemical recurrence or metastasis. LNCaP and PC3 cell treatment with UNC1999 reduced histone H3 lysine 27 tri-methylation levels. Irradiation of LNCaP or PC3 cells with a single 2 Gy fraction with UNC1999-mediated EZH2 inhibition resulted in a statistically significant, though modest, reduction in cell colony number for both cell lines. Increased γH2AX foci were observed 24 hours after ionizing irradiation in LNCaP cells, but not in PC3, following UNC1999-mediated EZH2 inhibition vs controls. CONCLUSIONS: Taken together, these results reveal that high pretreatment EZH2 expression in prostate cancer in diagnostic biopsies is associated with an increased risk of postradiotherapy metastatic disease recurrence, but EZH2 function may only at most play a modest role in promoting prostate cancer cell radioresistance.

Clinical Features and Laboratory Findings of Travelers Returning to South Australia with Dengue Virus Infection.

Reported cases of dengue are rising in South Australia (SA) in travellers returning from dengue-endemic regions. We have undertaken a retrospective analysis to identify the clinical and laboratory characteristics of patients returning to SA with suspected dengue virus (DENV) infection. From 488 requests, 49 (10%) were defined by serology as acute dengue, with the majority of patients (75%) testing as non-structural protein 1 (NS1) and/or IgM positive. Dengue was most commonly acquired in Indonesia (42.9%) with clinical features of fever (95%), headache (41%) and myalgia/arthralgia (56%). The presence of rash (36%) and laboratory findings of neutropenia, leukopenia, thrombocytopenia, but not elevated C-reactive protein, were distinct from findings in DENV-seronegative patients. Available dengue seropositive samples were analysed by RT-PCR, with 14/32 (43.8%) positive by a serotype non-specific DENV assay, but 28/32 positive (87.5%) when also assessed by serotype-specific RT-PCR. Serotype analysis revealed the predominance of DENV-1 and DENV-2 and the presence of DENV-3, but not DENV-4 or Zika virus (ZIKV). Thus, dengue in returned travellers in SA presents in a manner consistent with World Health Organization (WHO) definitions, with symptoms, travel history and laboratory results useful in prioritising the likelihood of dengue. This definition will assist the future management in DENV-non-endemic regions, such as SA.

Application of an allele-specific PCR to clinical HIV genotyping samples detects additional K103N mutations in both therapy naïve and experienced patients.

Low-level drug resistance is not detected by routine consensus sequence genotype analysis (CSA) but low levels of specific mutations, such as the non-nucleoside reverse transcriptase inhibitor (NNRTI)-resistant mutation K103N, can be quantitated by allele-specific PCR (ASP). This study has applied an ASP to quantitate low-level K103N in patients presenting for clinical HIV genotyping and assess the correlation with antiretroviral treatment history and outcomes. HIV RNA was extracted from patient plasma and subjected to PCR amplification of the reverse transcriptase (RT) region followed by genotyping by CSA and real-time ASP for K103N. When applied to samples from patients presenting for genotyping, the ASP detects K103N, not K103 nor K103R, but cross-reacts with K103S. ASP identified all samples that were K103N by CSA (10.5%) and an additional 14% by ASP only, representing patients who were therapy naïve and with NNRTI treatment history. ASP detected therapy-acquired K103N at low levels up to 6 years after cessation of NNRTI therapy. In three patients with new HIV diagnosis and K103N detected by ASP only, K103N virus declined rapidly from the circulation but persisted in PBMC DNA at >12 months post-diagnosis. Efavirenz (EFV) combination therapy in three patients with low-level K103N suppressed successfully viral load, although one patient developed failure and CSA-detectable K103N after 15 months of therapy. Thus, analysis of K103N by ASP in conjunction with CSA genotyping provides additional information that reflects K103N transmission and persistence but detection of low-level K103N does not preclude successful EFV-containing combination therapy.

A novel bocavirus associated with acute gastroenteritis in Australian children.

Acute gastroenteritis (AGE) is a common illness affecting all age groups worldwide, causing an estimated three million deaths annually. Viruses such as rotavirus, adenovirus, and caliciviruses are a major cause of AGE, but in many patients a causal agent cannot be found despite extensive diagnostic testing. Proposing that novel viruses are the reason for this diagnostic gap, we used molecular screening to investigate a cluster of undiagnosed cases that were part of a larger case control study into the etiology of pediatric AGE. Degenerate oligonucleotide primed (DOP) PCR was used to non-specifically amplify viral DNA from fecal specimens. The amplified DNA was then cloned and sequenced for analysis. A novel virus was detected. Elucidation and analysis of the genome indicates it is a member of the Bocavirus genus of the Parvovirinae, 23% variant at the nucleotide level from its closest formally recognized relative, the Human Bocavirus (HBoV), and similar to the very recently proposed second species of Bocavirus (HBoV2). Fecal samples collected from case control pairs during 2001 for the AGE study were tested with a bocavirus-specific PCR, and HBoV2 (sequence confirmed) was detected in 32 of 186 cases with AGE (prevalence 17.2%) compared with only 15 controls (8.1%). In this same group of children, HBoV2 prevalence was exceeded only by rotavirus (39.2%) and astrovirus (21.5%) and was more prevalent than norovirus genogroup 2 (13.4%) and adenovirus (4.8%). In a univariate analysis of the matched pairs (McNemar's Test), the odds ratio for the association of AGE with HBoV2 infection was 2.6 (95% confidence interval 1.2-5.7); P = 0.007. During the course of this screening, a second novel bocavirus was detected which we have designated HBoV species 3 (HBoV3). The prevalence of HBoV3 was low (2.7%), and it was not associated with AGE. HBoV2 and HBoV3 are newly discovered bocaviruses, of which HBoV2 is the thirdmost-prevalent virus, after rotavirus and astrovirus, associated with pediatric AGE in this study.

Possible Radiation Sensitisation by Trastuzumab Leading to Radiation-Induced Myelitis.

SUMMARY: BACKGROUND: Trastuzumab is used as adjuvant treatment in patients with HER2-positive breast cancers and has been shown to reduce the chance of recurrence by up to 50%. However, experience with it given with radiotherapy is limited and there is in vitro evidence of a radiosensiti-sation effect. We describe the first case of trastuzumab-associated radiation-induced myelitis. CASE REPORT: This patient received a calculated dose of 28 Gy to the spinal cord when receiving adjuvant radiotherapy to the chest wall and supraclavicular and axillary lymph nodes. This is well below the accepted radiation tolerance of the spinal cord (50-60 Gy) but she developed radiation-induced myelitis of her spinal cord with characteristic magnetic resonance imaging changes. We postulate that trastuzu-mab given concurrently with radiation may have acted as a radiosensitiser and that normal repair mechanisms in the acute stage were affected by trastuzumab blockage of epidermal growth factor receptors, resulting in demy-elination at a lower dose of radiation than normally seen. CONCLUSIONS: Concomitant radiotherapy and adjuvant trastuzumab treatment should be given with caution and consideration made of delaying trastuzumab until after radiotherapy has been completed. As longer-term data become available for patients who received trastuzumab and radiation, it will become clearer whether there is a significant interaction on organs such as the heart and spinal cord in the radiation field.

Outcome analysis of 300 prostate cancer patients treated with neoadjuvant androgen deprivation and hypofractionated radiotherapy.

PURPOSE: Neoadjuvant androgen deprivation and radical radiotherapy is an established treatment for localized prostate carcinoma. This study sought to analyze the outcomes of patients treated with relatively low-dose hypofractionated radiotherapy. METHODS AND MATERIALS: Three hundred patients with T1-T3 prostate cancer were treated between 1996 and 2001. Patients were prescribed 3 months of neoadjuvant androgen deprivation before receiving 5250 cGy in 20 fractions. Patients' case notes and the oncology database were used to retrospectively assess outcomes. Median follow-up was 58 months. RESULTS: Patients presented with prostate cancer with poorer prognostic indicators than that reported in other series. At 5 years, the actuarial cause-specific survival rate was 83.2% and the prostate-specific antigen (PSA) relapse rate was 57.3%. Metastatic disease had developed in 23.4% of patients. PSA relapse continued to occur 5 years from treatment in all prognostic groups. Independent prognostic factors for relapse included treatment near the start of the study period, neoadjuvant oral anti-androgen monotherapy rather than neoadjuvant luteinizing hormone releasing hormone therapy, and diagnosis through transurethral resection of the prostate rather than transrectal ultrasound. CONCLUSION: This is the largest reported series of patients treated with neoadjuvant androgen deprivation and hypofractionated radiotherapy in the United Kingdom. Neoadjuvant hormonal therapy did not appear to adequately compensate for the relatively low effective radiation dose used.

Sensitive detection of RNA viruses associated with gastroenteritis by a hanging-drop single-tube nested reverse transcription-PCR method.

The detection of the human RNA viruses, calicivirus and astrovirus, requires high sensitivity and broad reactivity. A novel single-tube nested reverse transcription-PCR (RT-PCR) method is described here, in which all of the required reagents are included in the one tube; however, those required for the nested amplification are separated in a "hanging drop" in the cap to be introduced by centrifugation after the RT and first-round cDNA amplification steps. Broad reactivity was obtained by using primer cocktails covering the published sequence variation in the primer targets. The method was evaluated with clinical fecal samples from outbreak and sporadic cases. Norwalk-like virus types 1 and 2 and rotavirus were the causal agents in 10 of 12 outbreaks. A viral agent was detected in 44% of 197 samples from sporadic infections in patients presenting to community health centers and a children's hospital. Interestingly, whereas rotavirus was more common than astrovirus in patients presenting to the hospital (33 and 7.6%, respectively), the reverse was true for patients presenting to community health centers (4.2 and 34%, respectively).