NIMG-72. QUANTIFYING INTRA-TUMOR MULTI-GENE HETEROGENEITY OF GBM FROM MRI USING A DATA-INCLUSIVE MACHINE LEARNING ALGORITHM

Intra-tumor genetic heterogeneity is an important cause of treatment failure of GBM. Using MRI and image-localized biopsies, it is possible to train machine learning (ML) models to predict regional genetic status. However, biopsy samples are limited, making it difficult to train a robust ML model. We proposed a data-inclusive model called Weakly Supervised Ordinal Support Vector Machines (WSO-SVM) which leverages the vast amount of MRI data outside the sparsely sampled biopsy regions to augment the biopsy samples to improve ML accuracy. Our study included a unique dataset of 104 image-localized biopsies with spatially matched multiparametric MRI from 30 untreated Glioblastoma (GBM) patients. Each biopsy sample went through genetic sequencing analysis and our study focused on two GBM hallmark genes, EGFR and PDGFRA. For each gene, a biopsy sample was labeled as “altered” if the copy number of this gene was amplified or a mutation was found, and “non-altered” otherwise. From the localized region of six MRI contrasts from T1gd, T2w, diffusion and perfusion imaging, over 300 texture features were extracted. To account for biopsy sample location uncertainty, six neighboring regions of the biopsy sample including four neighbors two pixels away from the biopsy location and two neighbors on adjacent slices were also included in model training. WSO-SVM achieved 0.83 accuracy, 0.77 sensitivity, and 0.86 specificity for classifying EGFR; 0.77 accuracy, 0.74 sensitivity, and 0.79 specificity for classifying PDGFRA, based on 10-fold cross validation. Furthermore, using the trained models, we generated regional EGFR and PDGFRA alteration maps for each patient within the enhancing and non-enhancing tumoral areas. On average we found a greater proportion of enhancing tumor with co-alteration than non-enhancing tumor, while this trend was reversed when considering only one altered gene. The ratio of EGFR vs PDGFRA alterations was higher in non-enhancing tumor than enhancing tumor.

P–572 Purifying selection for aneuploidy cells in mosaicism embryo at post-implantation stage

Study question Why low ratio mosaicism embryos develop to normal karyotype babies? Summary answer Our in vitro implantation assay clarified purifying selection for aneuploid cells in post implantation embryos. What is known already There are some reports about healthy live birth after transfer of mosaic embryos, which was reported for the first time from Italy in 2015. It is also reported that the abnormal cell is screened with the mouse in the embryo development, and only a normal cell contributes to the development. But it has not been examined in human. Study design, size, duration To clarify the change of aneuploid cells and mitochondrial activity in human embryo, we biopsied several parts from one blastocyst and examined karyotype. After in vitro implantation assay for biopsied embryos, we compared the karyotype of biopsy sample with that of cultured cell mass. Participants/materials, setting, methods Under the ethical review of Yokohama City University and informed consent with patients, we collected human surplus blastocysts those are donated after successful clinical treatment or discarded because of poor development grade. We biopsied multiple parts from one blastocyst and cultured the biopsied embryos, and extracted whole DNA from the biopsy samples and cultured embryos. Karyotyping by next generation sequencing were performed. Main results and the role of chance We analyzed 34 samples from 11 embryos, including 25 biopsy sample from 11 embryos and 9 cell mass from 7 cultured embryos. In the karyotype tracking results, even though biopsy sample analysis before the culture were uniformed aneuploid or chromosome mosaic, the developing embryo cell mass had normal karyotype. In one embryo as an example, among the three biopsied extra trophectoderm samples from that, two of them were mosaic, and one of them had uniformed chromosome 21 trisomy and chromosome 16 mosaic monosomy. But the embryo formed multiple cell mass in implantation assay. We examined karyotype of three cell mass, and the result from all were normal karyotype. We suggested that the chromosome aberration cells were screened in the human embryo development, and when the function was not carried out the embryo stopped the development. Limitations, reasons for caution Because of small number of samples available, we need more samples for a more accurate evaluation. Furthermore, we cannot evaluate the absolute mechanism that cells with chromosome aberration decreases. Wider implications of the findings: Conventional PGT-A techniques are based on uniformed embryos developing hypothesized past time. As showed in some clinical reports, PGT-A can reduce of spontaneous abortion and chance of embryo transfer. Thinking about aneuploid cell purifying system in embryo development, effectiveness of PGT-A should be more questionable for infertility treatment. Trial registration number A200326004

Rheumatological features of Whipple disease

Whipple disease (WD) is a rare infectious systemic disease. Rheumatologists are at the frontline of WD diagnosis due to the early rheumatological manifestations. An early diagnosis is crucial, as usual anti-rheumatic drugs, especially TNF inhibitors, may worsen the disease course. We conducted a retrospective multicentre national study from January 2010 to April 2020 to better characterize the rheumatological features of WD. Classic WD (CWD) was defined by positive periodic acid-Schiff (PAS) staining of a small-bowel biopsy sample, and non-CWD (NCWD) was defined by negative PAS staining of a small-bowel biopsy sample but at least one positive Tropheryma whipplei (TW) polymerase chain reaction (PCR) for a digestive or extradigestive specimen. Sixty-eight patients were enrolled, including 11 CWD patients. Twenty patients (30%) received TNF inhibitors during the WD course, with inefficacy or symptom worsening. More digestive symptoms and systemic biological features were observed in CWD patients than in NCWD patients, but both patient groups had similar outcomes, especially concerning the response to antibiotics and relapse rate. Stool and saliva TW PCR sensitivity were both 100% for CWD and 75% for NCWD and 89% and 60% for small-bowel biopsy sample PCR, respectively. WD encountered in rheumatology units has many presentations, which might result from different pathophysiologies that are dependent on host immunity. Given the heterogeneous presentations and the presence of chronic carriage, multiple TW PCR tests on samples from specific rheumatological sites when possible should be performed, but samples from nonspecific digestive and extradigestive sites also have great value.

A135 ASSESSING THE HISTOLOGICAL QUALITY OF ENDOSCOPIC BIOPSY SAMPLES OBTAINED USING NOVEL MULTIBITE FORCEPS FROM A PORCINE GASTRIC SPECIMEN

Background Tissue sampling is often limited to acquisition of one to two biopsy samples during a single pass. The ability to obtain more than two biopsies during a single pass can improve diagnostic yield however is potentially limited by poor specimen quality and loss of specimens. The multibite forceps used in this study have a unique geometry with the ability to store up to six biopsy samples taken consecutively with easy removal of the samples when shaken in solution. If multiple biopsies can be taken during a single pass with preserved specimen quality then we can reduce procedure time, improve efficiency and sensitivity of biopsies. Aims To evaluate the histological quality of the first biopsy sample compared to the last (sixth) biopsy sample acquired consecutively with the multibite forcep during a single act. Methods A porcine stomach was coloured with surgical dye to create six separate segments. An experienced endoscopist used single use disposable MultiCROC multi-sampling biopsy forceps to acquire six consecutive biopsies. Biopsies were manually separated into the order of which they were acquired (biopsy one through six) and each sample was placed in formalin solution. A total of 35 sets of 6 biopsies were obtained producing a total of 210 samples. Samples were randomized and two independent pathologists who were blinded to the biopsy order assessed the histological quality of specimens. Specimens were evaluated for presence of full thickness mucosa, absence of fragmentation, crush artifact and diagnostic utility. Each pathologist then scored each specimen and the mean scores were used to compare the histological quality of the first biopsy vs. the sixth biopsy for each set. Results Our preliminary results include 12 of the 35 sets of biopsies. Using a paired sample t test, there was no significant difference between the mean score given to biopsy one and biopsy six for all twelve pairs [3.62 (SD 1.46) vs. 3.67 (SD 1.15), correlation factor=0.498 and p=.10). There was no significant difference between the first and sixth biopsy when comparing the presence of full thickness mucosa [0.59 (SD 0.49) vs. 0.59 (SD 0.44), p=.086], absence of fragmentation [0.50 (SD 0.50) vs. 0.73 (SD 0.34), p=0.06], absence of crush artifact [0.96 (SD 0.15) vs. 0.91 (SD 0.30), p=0.77), and specimen size [1.64 (SD 0.92) vs. 1.70 (SD 0.65), p=0.56). Conclusions No significant differences were found between the histological quality of the first biopsy and the sixth biopsy. Additional parameters such as specimen size, full thickness mucosa, absence of fragmentation and absence of crush artifact revealed no significant differences between the first and sixth biopsy. This preliminary data thus far shows that there is no difference between the histological quality when multiple biopsies are retrieved consecutively. Funding Agencies NoneNone

High efficiency rare sperm separation from biopsy samples in an inertial focusing device

Immotile and rare sperm isolation from a complex cell background is an essential process for infertility treatment. The traditional sperm collection process from a biopsy sample requires long, tedious searches,...

A Case Report of Isolated Pigmentosus Atrophic Lichen Planus on the Supramaxillary Area: A New Variant ？

Background: Lichen planus pigmentosus (LPP) and atrophic lichen planus (ALP) are two rare subtypes of Lichen Planus（LP）, the former is characterized by epidermal atrophy and the latter by over pigmentation. LP with both of the above manifestations has few reports and there is a lack of treatment experience. Case presentation: We herein reported a 22-year old girl with a complaint of a sharply edged brown plaque on the supramaxillary area with pain and itchy for 6 months. The skin biopsy sample from the forehead revealed thinning of epidermal ridges, liquefaction degeneration of basal cells, loss of cuticular process, lichenoid lymphocytic infiltration and incontinence of pigment as well as numerous melanophages. It was diagnosed as atrophic Lichen planus pigmentosus (ALPP), and the plaque was completely cleared after 10 months of Alternating topical corticosteroids or calcineurin inhibitors. Discussion and Conclusions: ALPP might be an independent variant of LP or LPP that causes significant epidermal atrophy in the degenerative phase. This case revealed the special type of LP and provided a clinical reference for the treatment.

Analytical Validation and Performance of a 7-gene Next-generation Sequencing Panel in Uveal Melanoma

Background: A 15-gene expression profiling (GEP) test is widely used for prognostication of metastatic risk in uveal melanoma (UM) patients. Because the amount of tumor tissue that can be safely obtained by biopsy from UM is limited, it is critical to obtain as much individualized genomic information as possible from each biopsy sample. Mutational profiling of UM tumors using next generation sequencing (NGS) in combination with GEP allows for analysis of both DNA and RNA from a single tumor sample, offers additional prognostic value, and can potentially inform therapy selection. This study evaluated the analytical performance of a targeted custom NGS panel for mutational profiling of the seven genes known to be commonly mutated in primary UM.Methods: 105 primary UM samples were analyzed, including 37 formalin-fixed paraffin embedded (FFPE) specimens and 68 fine needle aspiration biopsy (FNAB) specimens obtained with a 25- or 27-gauge needle. Sequencing was performed on the Ion GeneStudio S5 platform to an average read depth of greater than 500X per region of interest in a clinical laboratory accredited by the College of American Pathologists (CAP) and certified under the Clinical Laboratory Improvement Amendments (CLIA).Results: The 7-gene panel assay achieved a positive percent agreement (PPA) of 100% for detection of both single nucleotide variants (SNVs) and insertions/deletions (INDELs), with a technical positive predictive value (TPPV) of 99.4% and 100%, respectively. Intra-assay and inter-assay concordance studies confirmed the reproducibility and repeatability of the assay. The limit of detection was determined to be 5% variant allele frequency (VAF) for both SNVs and INDELs, with a minimum DNA input requirement of 1.5ng for FNAB and 5ng for FFPE samples.Conclusions: The 7-gene panel is a robust, highly accurate NGS test that can be successfully performed, along with GEP, from a single small gauge needle biopsy sample.