SIOP PNET5 MB Trial: History and Concept of a Molecularly Stratified Clinical Trial of Risk-Adapted Therapies for Standard-Risk Medulloblastoma

Background. SIOP PNET5 MB was initiated in 2014 as the first European trial using clinical, histological, and molecular parameters to stratify treatments for children and adolescents with standard-risk medulloblastoma. Methods. Stratification by upfront assessment of molecular parameters requires the timely submission of adequate tumour tissue. In the standard-risk phase-III cohort, defined by the absence of high-risk criteria (M0, R0), pathological (non-LCA), and molecular biomarkers (MYCN amplification in SHH–MB or MYC amplification), a randomized intensification by carboplatin concomitant with radiotherapy is investigated. In the LR stratum for localized WNT-activated medulloblastoma and age <16 years, a reduction of craniospinal radiotherapy dose to 18 Gy and a reduced maintenance chemotherapy are investigated. Two additional strata (WNT-HR, SHH-TP53) were implemented during the trial. Results. SIOP PNET5 MB is actively recruiting. The availability of adequate tumour tissue for upfront real-time biological assessments to assess inclusion criteria has proven feasible. Conclusion. SIOP PNET5 MB has demonstrated that implementation of biological parameters for stratification is feasible in a prospective multicentre setting, and may improve risk-adapted treatment. Comprehensive research studies may allow assessment of additional parameters, e.g., novel medulloblastoma subtypes, and identification and validation of biomarkers for the further refinement of risk-adapted treatment in the future.

LUMOS - Low and Intermediate Grade Glioma Umbrella Study of Molecular Guided TherapieS at relapse: Protocol for a pilot study

IntroductionGrades 2 and 3 gliomas (G2/3 gliomas), when combined, are the second largest group of malignant brain tumours in adults. The outcomes for G2/3 gliomas at progression approach the dismal outcomes for glioblastoma (GBM), yet there is a paucity of trials for Australian patients with relapsed G2/3 gliomas compared with patients with GBM. LUMOS will be a pilot umbrella study for patients with relapsed G2/3 gliomas that aims to match patients to targeted therapies based on molecular screening with contemporaneous tumour tissue. Participants in whom no actionable or no druggable mutation is found, or in whom the matching drug is not available, will form a comparator arm and receive standard of care chemotherapy. The objective of the LUMOS trial is to assess the feasibility of this approach in a multicentre study across five sites in Australia, with a view to establishing a national molecular screening platform for patient treatment guided by the mutational analysis of contemporaneous tissue biopsiesMethods and analysisThis study will be a multicentre pilot study enrolling patients with recurrent grade 2/3 gliomas that have previously been treated with radiotherapy and chemotherapy at diagnosis or at first relapse. Contemporaneous tumour tissue at the time of first relapse, defined as tissue obtained within 6 months of relapse and without subsequent intervening therapy, will be obtained from patients. Molecular screening will be performed by targeted next-generation sequencing at the reference laboratory (PathWest, Perth, Australia). RNA and DNA will be extracted from representative formalin-fixed paraffin embedded tissue scrolls or microdissected from sections on glass slides tissue sections following a review of the histology by pathologists. Extracted nucleic acid will be quantified by Qubit Fluorometric Quantitation (Thermo Fisher Scientific). Library preparation and targeted capture will be performed using the TruSight Tumor 170 (TST170) kit and samples sequenced on NextSeq 550 (Illumina) using NextSeq V.2.5 hi output reagents, according to the manufacturer’s instructions. Data analysis will be performed using the Illumina BaseSpace TST170 app v1.02 and a custom tertiary pipeline, implemented within the Clinical Genomics Workspace software platform from PierianDx (also refer to section 3.2). Primary outcomes for the study will be the number of patients enrolled and the number of patients who complete molecular screening. Secondary outcomes will include the proportion of screened patients enrolled; proportion of patients who complete molecular screening; the turn-around time of molecular screening; and the value of a brain tumour specific multi-disciplinary tumour board, called the molecular tumour advisory panel as measured by the proportion of patients in whom the treatment recommendation was refined compared with the recommendations from the automated bioinformatics platform of the reference laboratory testing.Ethics and disseminationThe study was approved by the lead Human Research Ethics Committee of the Sydney Local Health District: Protocol No. X19-0383. The study will be conducted in accordance with the principles of the Declaration of Helsinki 2013, guidelines for Good Clinical Practice and the National Health and Medical Research Council National Statement on Ethical Conduct in Human Research (2007, updated 2018 and as amended periodically). Results will be disseminated using a range of media channels including newsletters, social media, scientific conferences and peer-reviewed publications.Trial registration numberACTRN12620000087954; Pre-results.

O-OGC03 Real-time tracking and classification of tumour and non-tumour tissue in upper gastrointestinal cancer specimens using diffuse reflectance spectroscopy

Background Diffuse reflectance spectroscopy (DRS) is a technique that allows discrimination of normal and abnormal tissue based on spectral data. It is a promising technique for cancer margin assessment. However, application in a clinical setting is limited by the inability of DRS to mark the tissue that has been scanned and its lack of continuous real-time spectral measurements. This aim of this study was to develop a real-time tracking system to enable localisation of the tip of a handheld DRS probe to aid classification of tumour and non-tumour tissue. Methods A coloured marker was attached to the DRS fibre probe and was detected using colour segmentation. A Kalman filter was used to estimate the probe’s tip position during scanning of the tissue specimen. In this way, the system was robust to partial occlusion allowing real-time detection and tracking. Supervised classification algorithms were used for the discrimination between tumour and non-tumour tissue, and evaluated in terms of overall accuracy, sensitivity, specificity, and the area under the curve (AUC). A live augmented view with all the tracked and classified optical biopsy sites were presented, providing visual feedback to the surgeons. Results A green coloured marker was successfully used to track the DRS probe. The measured root mean square error of probe tip tracking was 1.18±0.58mm and 1.05±0.28mm for the X and Y directions, respectively, whilst the maximum measured error was 1.76mm. Overall, 47 distinct sets of tumour and non-tumour tissue data were recorded through real-time tracking of ex vivo oesophageal and gastric tissue. The overall diagnostic accuracy of the system to classify tumour and non-tumour tissue in real-time was 94% for stomach and 96% for the oesophagus. Conclusions We have been able to successfully develop a real-time tracking system for a DRS probe when used on stomach and oesophageal tissue for tumour detection, and the accuracy derived demonstrates the strength and clinical value of the technique. The method allows real-time tracking and classification with short data acquisition time to aid margin assessment in cancer resection surgery.

Deciphering tumour tissue organization by 3D electron microscopy and machine learning

Despite recent progress in the characterization of tumour components, the tri-dimensional (3D) organization of this pathological tissue and the parameters determining its internal architecture remain elusive. Here, we analysed the spatial organization of patient-derived xenograft tissues generated from hepatoblastoma, the most frequent childhood liver tumour, by serial block-face scanning electron microscopy using an integrated workflow combining 3D imaging, manual and machine learning-based semi-automatic segmentations, mathematics and infographics. By digitally reconstituting an entire hepatoblastoma sample with a blood capillary, a bile canaliculus-like structure, hundreds of tumour cells and their main organelles (e.g. cytoplasm, nucleus, mitochondria), we report unique 3D ultrastructural data about the organization of tumour tissue. We found that the size of hepatoblastoma cells correlates with the size of their nucleus, cytoplasm and mitochondrial mass. We also found anatomical connections between the blood capillary and the planar alignment and size of tumour cells in their 3D milieu. Finally, a set of tumour cells polarized in the direction of a hot spot corresponding to a bile canaliculus-like structure. In conclusion, this pilot study allowed the identification of bioarchitectural parameters that shape the internal and spatial organization of tumours, thus paving the way for future investigations in the emerging onconanotomy field.

Electron microscopic and pathological changes of lung cancer after intratumoral injection of sodium bicarbonate

Background: Lung cancer can be treated with surgery, chemotherapy, radiation therapy, targeted therapy and palliative care. Palliative therapy is applied for inoperable lung cancer as it induces tumour necrosis. PH of tumour tissue is acidic; application of sodium bicarbonate (SB) into lung cancer locally via bronchoscopy can change its core pH, which may lead to tumour destruction. We aimed to study the ultrastructural characteristics of lung cancer and to assess the destructive effects of sodium bicarbonate 8.4% local injection on tumour tissue integrity by light and electron microscopies. Methods: This study was conducted on 21 patients with central bronchial carcinoma diagnosed according to WHO classification 2015. Three bronchoscopic biopsies were taken; two biopsies before and one after injection of sodium bicarbonate 8.4% solution of 20 ml via transbronchial needle. All biopsies were examined by both light and electron microscopes. The first biopsy was examined to diagnose the tumour morphologically with and without immunostaining. Second and third biopsies were taken before and after SB 8.4% injection to compare pathological changes in tumour tissue integrity as well as cellular ultra-structures. Different lung cancer pathological types were included in the study. Results: Tumour tissue integrity and pathological changes were examined in biopsies before and after injection of sodium bicarbonate 8.4%. Extensive necrosis in all cell types of lung cancer was seen after injection of SB; this important finding was delineated by both light and electron microscopies. Conclusion: Preliminary ultrastructural study of small biopsy of lung tumor has a complementary role to both morphological and immunohistochemical studies. Local injection of sodium bicarbonate into lung cancer induces extensive necrosis that may reflect its important therapeutic role in lung cancer.

Optimization of PLGA nanoparticles for delivery of Novel anticancer CK-10 peptide

The main objective of this project was to formulate novel amphiphilic PLGA nanoparticles having better physicochemical properties for the delivery of the novel peptide (CK-10) to be used for targeting the cancerous/tumour tissue. Double emulsion/Solvent evaporation and novel microfluidic techniques were used to formulate the nanoparticles. Loading efficiency and in-vitro release were measured by a modified Lowry assay. Size and zeta potential were characterized by dynamic light scattering, tuneable pore resistive sensing, and laser obscuration time. Images were scanned by scanning, transmission electron microscopes, and laser obscuration time. Stability was checked by high-performance liquid chromatography and capillary zone electrophoresis. Water absorption and its associated changes in the physicochemical properties were measured by various titration techniques. PLGA/Poloxomer nanoparticles had the highest peptide loading efficiency by 56.13 % for the novel microfluidic technique as well as the highest in-vitro release and water absorption values. It also had the smallest size with the lowest PDI (208.90 nm, 0.11) which are vital parameters for targeting cancer/tumour tissue. The successful development of better physicochemical properties for the CK-10 loaded PLGA nanoparticles could improve the RAN blocking by CK-10.

Association of BRCA1 and BRCA2 genes in arsenic-induced urinary bladder carcinoma

Objective: Study was performed to determine content of arsenic in urinary bladder tumour tissue and association of BRCA1 and BRCA2 protein expression with urinary bladder carcinoma development. Materials and methods: This study was performed in a tertiary care hospital of Eastern India. Post-operative tumour tissue was analysed for arsenic content as well as BRCA1 and BRCA2 expression. Statistical analysis was done and association between stage, grade and BRCA1 and BRCA2 expression with arsenic level in tumour tissue was done. Results: Total 50 patients were included in study. Out of which 26 were arsenic positive as well as 24 were arsenic negative. Maximum patients in arsenic positive group were from arsenic endemic zones of West Bengal, India. There was significant correlation between higher stage and grade of tumour and arsenic positivity. BRCA1 correlation was significant with arsenic positive group whereas BRCA2 correlation was not significant with arsenic positive group. Conclusion: Bladder carcinomas are more common in arsenic endemic zones of our country. This association can help in future to develop drugs which act on selected mutation of genes such as BRCA1, especially in arsenic-associated bladder cancers.

Evaluation of Molecular Positive Margins Using Surrogate P53 and Retinoblastoma Protein Expression and Correlation with Surgical Outcomes in Oral Squamous Cell Carcinoma

BACKGROUND p53 mutation and Rb over-expression have been extensively studied in oral squamous cell carcinoma (OSCC) but their clinical relevance with respect to excision margins is still controversial. The purpose of the study was to determine the expression of molecular markers (p53& Rb) for predicting early locoregional recurrence in oral cancer. METHODS Histopathological specimens of 93 patients of oral cavity squamous cell carcinoma were subjected to p53 mutation and Rb protein testing in tumour and at the closest negative margin on H &E using immunohistochemistry. The expression of p53 and Rb in tumour tissue and at excision margin was correlated with clinicopathologic parameters recurrence and survival over a 2 year follow up period. RESULTS p53 mutation expression in tumour tissue was associated with increased recurrence (22.5 % versus 11.3 % P = 0.13) and mortality (17.5 % versus 5.6 % P = 0.056). p53 expression at margins is also associated with higher recurrence and mortality. Rb overexpression in tumour tissue is not significantly associated with recurrence (15 % and 16.4 %). Rb overexpression at margins had higher recurrence (40 %; P = 0.627) and higher mortality (60 %) in comparison to Rb negative cases (16.4 %versus 6.8 % respectively). CONCLUSIONS Clinical and routine histopathological assessments of margins remain the standard method of prognosticating and planning adjuvant treatment. Determination of molecular positive margins using p53 & Rb in oral cancer may aid in identifying patients at high risk of development of recurrence despite negative pathological margins. KEYWORDS Rb, Margin, P53, Recurrence.

SP7.1.6 Using Diffuse Reflectance Spectroscopy (DRS) to Identify Tumour and Non-tumour Tissue in Upper Gastrointestinal Specimens

Aim Cancers of the upper gastrointestinal (GI) tract remain a major contributor to the global cancer risk. Surgery aims to completely resect tumour with clear margins, whilst preserving as much surrounding tissue. Diffuse reflectance spectroscopy (DRS) is a novel technique that presents a promising advancement in cancer diagnosis. We have developed a novel DRS system with tracking capability. Our aim is to classify tumour and non-tumour GI tissue in real-time using this device to aid intra-operative analysis of resection margins. Method An ex-vivo study was undertaken in which data was collected from consecutive patients undergoing upper GI cancer resection surgery between August 2020- January 2021. A hand-held DRS probe and tracking system was used on normal and cancerous tissue to obtain spectral information. After acquisition of all spectra, a classification system using histopathology results was created. A user interface was developed using Python 3.6 and Qt5. A support vector machine was used to classify the results. Results The data included 4974 normal spectra and 2108 tumour spectra. The overall accuracy of the DRS probe in differentiating normal versus tumour tissue was 88.08% for the stomach (sensitivity 84.8%, specificity 89.3%), and 91.42% for the oesophagus (sensitivity 76.3%, specificity 98.9%). Conclusion We have developed a successful DRS system with tracking capability, able to process thousands of spectra in a small timeframe, which can be used in real-time to distinguish tumour and non-tumour tissue. This can be used for intra-operative decision-making during upper GI cancer surgery to help select the best resection plane.