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Summary of Patients and Clinical Data.

Supplementary Figure 1. Unsupervised Clustering on CCA samples. Supplementary Figure 2. Alterations Found in CCA Clusters. Supplementary Figure 3. MAP2K4 Homozygous Deletions and ERBB2 Amplifications. Supplementary Figure 4. Alterations Related to Structural Variations Found in CCAs. Supplementary Figure 5. FIREFLY Analysis of Pathways Systematically Dysregulated by Somatic Promoter Mutations that Alter Transcription Factor Binding. Supplementary Figure 6. Epigenetic Clusters and Mutation Signatures.

Structural Variations across 71 WGS CCAs.

Summary of CCA Alterations.

Further details of methods, in addition to the supplementary figure and table legends.

Alterations in CCA Clusters.

Coverage Statistics and List of Genes for Targeted Sequencing.

PURPOSE Precision oncology has transformed the management of advanced cancers through implementation of advanced molecular profiling technologies to identify increasingly defined subsets of patients and match them to appropriate therapy. We report outcomes of a prospective molecular profiling study in a high-volume Asian tertiary cancer center. PATIENTS AND METHODS Patients with advanced cancer were enrolled onto a prospective protocol for genomic profiling, the Individualized Molecular Profiling for Allocation to Clinical Trials Singapore study, at the National Cancer Center Singapore. Primary objective was to identify molecular biomarkers in patient's tumors for allocation to clinical trials. The study commenced in February 2012 and is ongoing, with the results of all patients who underwent multiplex next-generation sequencing (NGS) testing until December 2018 presented here. The results were discussed at a molecular tumor board where recommendations for allocation to biomarker-directed trials or targeted therapies were made. RESULTS One thousand fifteen patients were enrolled with a median age of 58 years (range 20-83 years). Most common tumor types were lung adenocarcinoma (26%), colorectal cancer (15%), and breast cancer (12%). A total of 1,064 NGS assays were performed, on fresh tumor tissue for 369 (35%) and archival tumor tissue for 687 (65%) assays. TP53 (39%) alterations were most common, followed by EGFR (21%), KRAS (14%), and PIK3CA (10%). Of 405 NGS assays with potentially actionable alterations, 111 (27%) were allocated to a clinical trial after molecular tumor board and 20 (4.9%) were enrolled on a molecularly matched clinical trial. Gene fusions were detected in 23 of 311 (7%) patients tested, including rare fusions in new tumor types and known fusions in rare tumors. CONCLUSION Individualized Molecular Profiling for Allocation to Clinical Trials Singapore demonstrates the feasibility of a prospective broad molecular profiling program in an Asian tertiary cancer center, with the ability to develop and adapt to a dynamic landscape of precision oncology.

The recently introduced, highly sensitive and specific SS18-SSX immunohistochemistry (IHC) is an attractive alternative to SS18 fluorescence in situ hybridization (FISH) testing in synovial sarcoma (SS). However, little is known about how SS18-SSX IHC correlates with SS18 FISH. We correlated the SS18 FISH results of SS from 36 patients with SS18-SSX IHC. Twenty-six tumours had a classic break-apart FISH pattern (1 fused, 1 red and 1 green signal) and all stained positive for the IHC. Ten had an atypical (non-classic) FISH pattern of which 5 stained positive for the IHC. Four of these (including two with novel atypical SS18 FISH patterns) were confirmed to harbour the SS18-SSX fusion on targeted RNA sequencing, while one had classic features of a biphasic SS. The remaining 5 tumours stained negative for the IHC. One had a TPM3-NTRK1 fusion, and one had no fusion, while the remaining three had insufficient tissue/RNA for sequencing. The sensitivity of the IHC was 91% (after excluding the 2 cases with confirmed absence of SS18-SSX fusion). Twenty histologic mimics of SS also stained negative for the IHC (100% specificity). Our study shows that the SS18-SSX IHC is more specific than SS18 FISH in diagnosing SS, especially in cases with atypical FISH patterns. It correlates well with RNA sequencing result and has the potential to replace SS18 FISH testing. A positive IHC result supports the diagnosis of SS, while a tumour with atypical FISH pattern and negative IHC result should undergo further molecular testing.

The detection of chromosomal abnormalities is important in the diagnosis, prognosis and disease monitoring in plasma cell neoplasia (PCN). However, the gold standard diagnostic techniques of conventional cytogenetics (CC) and fluorescence in situ hybridization (FISH) are hampered by culture difficulties and probe availability. Cytogenomic microarray (CMA), however, is able to surmount such limitations and generate a comprehensive genomic profile with the implementation of plasma cell (PC) enrichment. In this study, we examined 89 bone marrow specimens with CC and FISH without PC enrichment, 35 of which were examined with CMA after PC enrichment. Results revealed that after PC enrichment, CMA was able to detect chromosomal abnormalities in 34 of 35 specimens tested (97.1%), compared to 21 and 32 specimens (60% and 91.4%, respectively) achieved by CC and FISH, respectively, which were similar to the abnormality detection rates among all 89 specimens (59.5% by CC and 92.1% by FISH). In addition, as the only technique capable of detecting copy neutral loss of heterozygosity (CN-LOH) and chromothripsis, CMA appears to be the most powerful tool in risk stratification as it successfully re-stratified 9 (25.7%) and 12 (34.3%) specimens from standard risk (determined by CC and FISH, respectively) to high risk. Based on the encouraging data presented by our study and others, we conclude that implementation of CMA with PC enrichment is of great value in routine clinical workup in achieving a more complete genetic profile of patients with PCN.

PURPOSE Cholangiocarcinoma (CCA) remains a disease with poor prognosis and limited therapeutic options. Identification of driver genetic alterations may lead to the discovery of more effective targeted therapies. CCAs harboring FGFR2 fusions have recently demonstrated promising responses to FGFR inhibitors, highlighting their potential relevance as predictive biomarkers. CCA incidence is high in the northeast of Thailand and its neighboring countries because of chronic infection with the liver fluke Opisthorchis viverrini (Ov). However, there are currently no available data on the prevalence of FGFR alterations in fluke-associated CCA in endemic countries. MATERIALS AND METHODS In this study, we performed anchored multiplex polymerase chain reaction target enrichment RNA sequencing of FGFR1-3, validated by fluorescence in situ hybridization and Sanger sequencing, in 121 Ov-associated and 95 non–Ov-associated CCA tumors. RESULTS Compared with non–fluke-associated CCA (11/95; 11.6%), FGFR2 fusions were significantly less common in fluke-associated CCA (1/121; 0.8%; P = .0006). All FGFR fusions were detected exclusively in intrahepatic CCAs and were mutually exclusive with KRAS/ERBB2/BRAF/FGFR mutations, pointing to their potential roles as oncogenic drivers. CONCLUSION FGFR2 fusions are rare in fluke-associated CCA, underscoring how distinct etiologies may affect molecular landscapes in tumors and highlighting the need to discover other actionable genomic alterations in endemic fluke-associated CCA.

Objectives There is an expanding list of therapeutically relevant biomarkers for non-small cell lung cancer (NSCLC), and molecular profiling at diagnosis is paramount. Tissue attrition in scaling traditional single biomarker assays from small biopsies is an increasingly encountered problem. We sought to compare the performance of targeted next-generation sequencing (NGS) panels with traditional assays and correlate the mutational landscape with PD-L1 status in Singaporean patients. Materials and methods We identified consecutive patients diagnosed between Jan 2016 to Sep 2017 with residual tissue after standard molecular testing. Tissue samples were tested using a targeted NGS panel for DNA alterations (29 selected genes including BRAF, EGFR, ERBB2 and TP53) and an RNA fusion panel (ALK, ROS1 and RET). PD-L1 immunohistochemistry was also performed. A cost-effectiveness analysis of NGS compared to standard molecular testing was conducted. Results A total of 174 samples were evaluated: PD-L1 (n = 169), NGS DNA panel (n = 173) and RNA fusion (n = 119) testing. Median age was 68 years, 53 % were male, 58 % were never smokers, 85 % were Chinese, 66 % had stage IV disease and 95 % had adenocarcinoma histology. In patients profiled with NGS on DNA, EGFR (56 %), KRAS (14 %), BRAF (2 %) and ERBB2 (1 %) mutations were found. RNA fusion testing revealed fusions in ALK (6 %), RET (3 %) and ROS1 (1 %). Cost-effectiveness analysis demonstrated that compared to sequential testing in EGFR negative patients, upfront NGS testing would result in an additional 1 % of patients with actionable alterations for targeted therapy being identified without significant increases in testing cost or turnaround time. Conclusions This study demonstrates that even in an EGFR mutant predominant population, upfront NGS represents a feasible, cost-effective method of diagnostic molecular profiling compared with sequential testing strategies. Our results support the implementation of diagnostic NGS in non-squamous NSCLC in Asia to allow patients access to the most appropriate personalized therapy.

The study aimed to evaluate the impact of the updated 2018 American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines on Human Epidermal Growth Factor Receptor 2 (HER2) testing in invasive breast cancer compared with previous 2013 guidelines. Between Jan 2014 and May 2020, 3364 consecutive invasive breast carcinomas with concurrent HER2 immunohistochemistry (IHC) and fluorescence in-situ hybridization (FISH) results were retrospectively reviewed for HER2 status. Both 2013 and 2018 testing criteria were applied to establish the HER2 status. The testing algorithms involved testing of invasive breast carcinomas by IHC, with equivocal results being reflexed to FISH assays. Concordance rate improved from 92.7% to 94.1% in the non-equivocal IHC cases with the 2018 guidelines. Comparing 2013 versus 2018 criteria, HER2 non-amplified cases increased significantly from 73.7% (n = 2478) to 76.8% (n = 2585), HER2 amplified cases remained similar from 23.4% (n = 789) to 23.2% (n = 779) while equivocal cases decreased from 2.9% (n = 97) to 0% with the new guidelines. Thus, 107 cases (3.2%) were reclassified from HER2 equivocal (n = 97) and amplified (n = 10) to non-amplified with the updated 2018 guidelines. Under the 2018 criteria, a total of 259 cases (7.7%) belonged to the uncommon categories (groups 2 to 4), with group 3 being the most frequent (4.6%), followed by group 4 (2.9%) and group 2 (0.2%). Implementation of 2018 guidelines resulted in a significant increase in HER2 non-amplified cases, mainly due to the abolishment of the equivocal FISH group. This has helped resolve the clinical practice dilemma by providing a more definitive HER2 gene status.

PURPOSE/OBJECTIVE(S) Nasopharyngeal carcinoma (NPC) is a distinct head and neck cancer that is associated with Epstein-Barr virus (EBV). Its histomorphology is characterized by epithelial carcinoma that is admixed with immune cells. This cellular pattern concurs with whole exome sequencing (WES) profiles of NPC that reveal several recurrent mutations involving NFKs and MHC-class genes, which are linked to poor outcomes. There is little information on the transcriptomic landscape of NPC. Here, we performed whole transcriptome sequencing (RNAseq) of a prospectively recruited cohort of NPC patients who were treated at a single institution. MATERIALS/METHODS Patients with biopsy-proven NPC, staged using the UICC/AJCC 8th ed, were prospectively recruited under an institutional approved protocol. All formalin-fixed paraffin-embedded specimens were centrally reviewed. Specimens with tumor cellularity of > 70% were macrodissected and Ranse (Illumina Trusses, CA) was performed. All patients underwent radiotherapy (RT); concurrent (CRT) and/or induction (IC) or adjuvant chemotherapy (AC) was performed for patients with advanced NPC. Primary clinical endpoint was disease-free survival (DFS). RESULTS Of 1,040 patients recruited, 145 patients had specimens that were successfully profiled by Ranse. Median age of this sub-cohort was 54.2 (IQR: 45.2-61.1) y; median follow-up was 4.8 (IQR: 3.3-8.1) y. Stage distribution was as such: 10 (6.9%) for Stage 1, 36 (24.8%) for Stage 2, 47 (32.4%) for Stage 3 and 41 (28.3%) for Stage 4A; 7 (4.8%) for Stage 4B; 4 (2.8%) were unclassified. 77 (53.1%) and 55 (37.9%) had EBV DNA titers of < 4,000 copies/ml and ≥4,000 copies/ml, respectively (13 [9.0%] missing). In terms of treatment received, 42 (29.0%) underwent RT alone; 50 (34.5%) and 41 (28.3%) received CRT and IC/AC+CRT; 12 (8.3%) had other treatment combinations. Unsupervised hierarchical clustering using expression of the top 1,000 most variable genes across the cohort revealed three distinct clusters that were characterized by differential expression of immune-related genes; incidentally, the cluster of patients with low immune score were enriched for recurrences, while the intermediate/high immune score groups had comparable prognoses. Subsequently, we performed gene-selection based on the low and intermediate/high immune score clusters and identified 511 genes that were significantly differentially expressed between them. We further clustered these genes to obtain a representative 5-gene GC that was prognostic for DFS (HR 3.28 [95% CI: 1.36-7.9]) on multivariable analyses, adjusted for T- and N-status and EBV DNA. Our GC was also prognostic for DFS in a validation dataset (N = 88, Zhang et al., 2017); HR 2.1 (95% CI: 0.73-6). CONCLUSION Herein, we developed and validated an immune-based prognostic GC for NPC. Our findings underscore the importance of the immune landscape in EBV-positive NPC.

Umbilical cord blood (UCB) transplants in adults have slower hematopoietic recovery compared to bone marrow (BM) or peripheral blood (PB) stem cells mainly due to low number of total nucleated cells and hematopoietic stem and progenitor cells (HSPC). As such in this study, we aimed to perform ex vivo expansion of UCB HSPC from non-enriched mononucleated cells (MNC) using novel azole-based small molecules. Freshly-thawed UCB–MNC were cultured in expansion medium supplemented with small molecules and basal cytokine cocktail. The effects of the expansion protocol were measured based on in vitro and in vivo assays. The proprietary library of >50 small molecules were developed using structure-activity-relationship studies of SB203580, a known p38-MAPK inhibitor. A particular analog, C7, resulted in 1,554.1 ± 27.8-fold increase of absolute viable CD45+CD34+CD38–CD45RA– progenitors which was at least 3.7-fold higher than control cultures (p 600-fold expansion of CD34+/CD90+/CD49f+ rare HSPCs coupled with significant (p

Background Brush cytology, the conventional method to diagnose cholangiocarcinoma, has been plagued by low diagnostic sensitivity and false-negative results. We aim to study the clinical utility of fluorescence in situ hybridization (FISH) in enhancing identification of malignant biliary strictures. Methods Brush cytologic specimens collected from endoscopic retrograde cholangiopancreatography (ERCP) for biliary strictures in a tertiary hospital in Singapore from March 2013 to July 2015 were examined by FISH technique using UroVysion probe set in this study. Results 30 patients were chosen with 5 patients having multiple FISH done due to indeterminate results. The diagnoses for biliary strictures were 13 (43.3%) cholangiocarcinomas, 7 (23.3%) pancreatic cancers, 7 (23.3%) benign biliary strictures and 3 (10%) primary sclerosing cholangitis. Conventional brush cytology had sensitivity of 53.8% with specificity of 82.4%. FISH had sensitivity of 30.8% with specificity of 100%. When FISH results were interpreted in cases with negative or atypical brush cytology, 2 patients had positive FISH results and cholangiocarcinomas. Based on this pilot study, FISH increased sensitivity of brush cytology in detection of cholangiocarcinoma from 53.8% to 69.2% while preserving specificity of 82.4%. Conclusion Compared with conventional cytology with low sensitivity, FISH may help to increase sensitivity on top of brush cytology while maintaining high specificity.

Clear cell sarcoma of the kidney (CCSK) is a rare paediatric renal malignant tumour. The majority of CCSKs have internal tandem duplications (ITDs) of the BCOR gene, whereas a minority have the YWHAE-NUTM2 gene fusion. A third 'double-negative' (DN) category comprises CCSKs with neither BCOR ITDs nor YWHAE-NUTM2 fusion. The aim of this study was to characterise 11 histologically diagnosed CCSKs immunohistochemically (with CCND1, BCOR and CCNB3 stains) and genetically.By next-generation sequencing, 10 cases (90.9%) had BCOR exon 15 ITDs, with positive BCOR immunoreactivity being found in four (36%) or eight (72%) cases, depending on the antibody clone. By reverse transcription polymerase chain reaction, none had the YWHAE-NUTM2 fusion. The DN case had a BCOR-CCNB3 fusion and strong nuclear CCNB3 and BCOR immunoreactivity. Quantitative polymerase chain reaction showed markedly elevated BCOR expression in this case, whereas BCOR ITD cases had lower levels of elevated BCOR expression.The majority of the CCSKs in our cohort had BCOR ITDs, and none had the YWHAE-NUTM2 fusion. We verified the strong, diffuse cyclin D1 (CCND1) immunoreactivity in CCSKs described in recent reports. BCOR immunoreactivity was not consistently positive in all CCSKs with BCOR ITDs, and therefore cannot be used as a diagnostic immunohistochemical stain to identify BCOR ITD cases. The DN case was a BCOR-CCNB3 fusion sarcoma. BCOR-CCNB3 sarcoma is typically a primary bone sarcoma affecting male adolescents, and this is the first report of it presenting in a kidney of a young child as a CCSK. The full spectrum of DN CCSKs awaits more comprehensive characterisation.

Introduction Conventional cytogenetics (CC) is important in diagnosis, therapy, monitoring of post-transplant bone marrow, and prognosis assessment of acute lymphoblastic leukemia (ALL). However, due to the nature of ALL, CC often encounters difficulties of complex karyotype, poor chromosome morphology, low mitotic index, or normal cells dividing only. In contrast, chromosomal microarray analysis (CMA) showed a specificity >99% and a sensitivity of 100% in chronic lymphocytic leukemia (CLL) patients. Here, we report our experience with CMA on adult ALL patients. Methods Thirty-three bone marrow/blood samples from ALL patients (aged 18-79 years, median 44) at diagnosis/relapse, analyzed by CC and/or fluorescence in situ hybridization (FISH), were recruited. Chromosomal microarray analysis results were compared with CC. Fluorescence in situ hybridization analysis, if available, was applied when there was a discrepancy. Results Copy-neutral loss-of-heterozygosity (CN-LOH) was found in 8 cases (24.2%). Only CN-LOH at 9p was recurrent (3 cases, 9.1%). Copy number alterations (CNAs) were detected in 6 of 9 cases (66.7%) with normal karyotypes, in 3 of 5 cases (60.0%) with sole "balanced" translocations, and in 18 of 19 cases (94.7%) with complex karyotypes. Common CNAs involved CDKN2A/2B (30.3%), IKZF1 (27.3%), PAX5 (9.1%), RB1 (9.1%), BTG1 (6.7%), and ETV6 (6.7%), which regulate cell cycle, B lymphopoiesis, or act as tumor suppressors in ALL. Copy number alteration detection rate by CMA was 81.8% (27 of 33 cases) as compared to 57.6% (19 of 33 cases) by CC. Conclusion Incorporation of CMA as a routine clinical test at the time of diagnosis/relapse, in conjunction with CC and/or FISH, is highly recommended.

Purpose Mesenchymal epithelial transition factor ( MET) activation has been implicated as an oncogenic driver in epidermal growth factor receptor ( EGFR)–mutant non–small-cell lung cancer (NSCLC) and can mediate primary and secondary resistance to EGFR tyrosine kinase inhibitors (TKI). High copy number thresholds have been suggested to enrich for response to MET inhibitors. We examined the clinical relevance of MET copy number gain (CNG) in the setting of treatment-naive metastatic EGFR-mutant–positive NSCLC. Patients and Methods MET fluorescence in situ hybridization was performed in 200 consecutive patients identified as metastatic treatment-naïve EGFR-mutant–positive. We defined MET-high as CNG greater than or equal to 5, with an additional criterion of MET/centromeric portion of chromosome 7 ratiο greater than or equal to 2 for amplification. Time-to-treatment failure (TTF) to EGFR TKI in patients identified as MET-high and -low was estimated by Kaplan-Meier method and compared using log-rank test. Multiregion single-nucleotide polymorphism array analysis was performed on 13 early-stage resected EGFR-mutant–positive NSCLC across 59 sectors to investigate intratumoral heterogeneity of MET CNG. Results Fifty-two (26%) of 200 patients in the metastatic cohort were MET-high at diagnosis; 46 (23%) had polysomy and six (3%) had amplification. Median TTF was 12.2 months (95% CI, 5.7 to 22.6 months) versus 13.1 months (95% CI, 10.6 to 15.0 months) for MET-high and -low, respectively ( P = .566), with no significant difference in response rate regardless of copy number thresholds. Loss of MET was observed in three of six patients identified as MET-high who underwent postprogression biopsies, which is consistent with marked intratumoral heterogeneity in MET CNG observed in early-stage tumors. Suboptimal response (TTF, 1.0 to 6.4 months) to EGFR TKI was observed in patients with coexisting MET amplification (five [3.2%] of 154). Conclusion Although up to 26% of TKI-naïve EGFR-mutant–positive NSCLC harbor high MET CNG by fluorescence in situ hybridization, this did not significantly affect response to TKI, except in patients identified as MET-amplified. Our data underscore the limitations of adopting arbitrary copy number thresholds and the need for cross-assay validation to define therapeutically tractable MET pathway dysregulation in EGFR-mutant–positive NSCLC.

Anaplastic lymphoma kinase (ALK) gene rearrangement in non-small cell lung cancer (NSCLC) is routinely evaluated by fluorescent in-situ hybridization (FISH) testing on biopsy tissues. Testing can be challenging however, when suitable tissue samples are unavailable. We examined the relevance of circulating tumor cells (CTC) as a surrogate for biopsy-based FISH testing. We assessed paired tumor and CTC samples from patients with ALK rearranged lung cancer (n = 14), ALK-negative lung cancer (n = 12), and healthy controls (n = 5) to derive discriminant CTC counts, and to compare ALK rearrangement patterns. Blood samples were enriched for CTCs to be used for ALK FISH testing. ALK-positive CTCs counts were higher in ALK-positive NSCLC patients (3-15 cells/1.88 mL of blood) compared with ALK-negative NSCLC patients and healthy donors (0-2 cells/1.88 mL of blood). The latter range was validated as the 'false positive' cutoff for ALK FISH testing of CTCs. ALK FISH signal patterns observed on tumor biopsies were recapitulated in CTCs in all cases. Sequential CTC counts in an index case of lung cancer with no evaluable tumor tissue treated with crizotinib showed six, three and eleven ALK-positive CTCs per 1.88 mL blood at baseline, partial response and post-progression time points, respectively. Furthermore, ALK FISH rearrangement suggestive of gene copy number increase was observed in CTCs following progression. Recapitulation of ALK rearrangement patterns in the tumor on CTCs, suggested that CTCs might be used to complement tissue-based ALK testing in NSCLC to guide ALK-targeted therapy when suitable tissue biopsy samples are unavailable for testing.

Perivascular epithelioid cell tumor (PEComa) is an uncommon tumor which presents with epithelioid and spindled cell morphology and is immunoreactive for myogenic and melanocytic markers. Recently, a subset of PEComas has been reported to harbor

Umbilical cord blood (UCB) transplants in adults have slower hematopoietic recovery compared to bone marrow (BM) or peripheral blood (PB) stem cells mainly due to low number of total nucleated cells and hematopoietic stem and progenitor cells (HSPC). As such in this study, we aimed to perform ex vivo expansion of UCB HSPC from non‐enriched mononucleated cells (MNC) using novel azole‐based small molecules. Freshly‐thawed UCB–MNC were cultured in expansion medium supplemented with small molecules and basal cytokine cocktail. The effects of the expansion protocol were measured based on in vitro and in vivo assays. The proprietary library of >50 small molecules were developed using structure‐activity‐relationship studies of SB203580, a known p38‐MAPK inhibitor. A particular analog, C7, resulted in 1,554.1 ± 27.8‐fold increase of absolute viable CD45+CD34+CD38–CD45RA– progenitors which was at least 3.7‐fold higher than control cultures (p 600‐fold expansion of CD34+/CD90+/CD49f+ rare HSPCs coupled with significant (p

Tumor recurrence remains the main reason for breast cancer-associated mortality, and there are unmet clinical demands for the discovery of new biomarkers and development of treatment solutions to benefit patients with breast cancer at high risk of recurrence. Here we report the identification of chromosomal copy-number amplification at 1q21.3 that is enriched in subpopulations of breast cancer cells bearing characteristics of tumor-initiating cells (TICs) and that strongly associates with breast cancer recurrence. Amplification is present in ∼10-30% of primary tumors but in more than 70% of recurrent tumors, regardless of breast cancer subtype. Detection of amplification in cell-free DNA (cfDNA) from blood is strongly associated with early relapse in patients with breast cancer and could also be used to track the emergence of tumor resistance to chemotherapy. We further show that 1q21.3-encoded S100 calcium-binding protein (S100A) family members, mainly S100A7, S100A8, and S100A9 (S100A7/8/9), and IL-1 receptor-associated kinase 1 (IRAK1) establish a reciprocal feedback loop driving tumorsphere growth. Notably, this functional circuitry can be disrupted by the small-molecule kinase inhibitor pacritinib, leading to preferential impairment of the growth of 1q21.3-amplified breast tumors. Our study uncovers the 1q21.3-directed S100A7/8/9-IRAK1 feedback loop as a crucial component of breast cancer recurrence, serving as both a trackable biomarker and an actionable therapeutic target for breast cancer.

Brush cytology, the conventional method to diagnose cholangiocarcinoma, has been plagued by low diagnostic sensitivity and false-negative results. This paper aims to study the clinical utility of fluorescence in situ hybridization (FISH) in enhancing identification of malignant biliary strictures.Brush cytologic specimens collected from endoscopic retrograde cholangiopancreatography for biliary strictures in a tertiary hospital in Singapore from March 2013 to July 2015 were examined by FISH technique using UroVysion probe set in this study.Thirty patients were chosen with five patients having multiple FISH performed due to indeterminate results. The diagnoses for biliary strictures were 13 (43.3%) cholangiocarcinomas, seven (23.3%) pancreatic cancers, seven (23.3%) benign biliary strictures, and three (10%) primary sclerosing cholangitis. Conventional brush cytology had sensitivity of 53.8% with specificity of 82.4%. FISH had sensitivity of 30.8% with specificity of 100%. When FISH results were interpreted in cases with negative or atypical brush cytology, two patients had positive FISH results and cholangiocarcinomas. Based on this pilot study, FISH increased sensitivity of brush cytology in detection of cholangiocarcinoma from 53.8% to 69.2% while preserving specificity of 82.4%.Compared with conventional cytology with low sensitivity, FISH may help to increase sensitivity on top of brush cytology while maintaining high specificity.

Formalin-fixed paraffin-embedded (FFPE) tissues are typically the specimens available for FISH analysis of solid tissues, particularly of tumor specimens. Occasionally, tissue cores constructed as tissue microarrays from several patients are presented for simultaneous evaluation. FFPE sections can also be prepared from cell blocks derived from cell suspensions. The interphase fluorescence in situ hybridization assay employs specific nucleic acid sequences (probes) that target complementary sequences of interest to detect gains or losses of genes/gene loci or a fusion gene within the tissue. In this chapter, we describe the protocols utilized in our laboratory and include slide deparaffinization, pretreatment, protease treatment, hybridization, washing, and counterstaining. This protocol can be applied to all of the earlier FFPE preparations. In general, the assay takes 3 consecutive days to complete, although a more rapid assay can be performed.

Much effort has been made to stratify multiple myeloma patients for targeted therapy. However, responses have been varied and improved patient stratifications are needed. Forty-five diagnostic samples from multiple myeloma patients (median age 65 years) were stratified cytogenetically as 15 having non-hyperdiploidy, 20 having hyperdiploidy and 10 having a normal karyotype. Fluorescence in situ hybridization (FISH) assays with FGFR3/IGH, CCND1/IGH, IGH/MAF, RB1 and TP53 probes on bone marrow samples showed that IGH rearrangements were the most common abnormality in the non-hyperdiploid group but these were also found among hyperdiploid patients and patients with normal cytogenetics. Of these, FGFR3/IGH rearrangements were most frequent. Deletion of RB1/monosomy 13 was the most common genetic abnormality across the three groups and was significantly higher among non-hyperdiploid compared to hyperdiploid patients. On the other hand, the study recorded a low incidence of TP53 deletion/monosomy 17. The FGFR3/IGH fusion was frequently seen with RB1 deletion/monosomy 13. FISH with 1p36/1q21 and 6q21/15q22 probes showed that amplification of 15q22 was seen in all of the hyperdiploid patients while amplification of 1q21, Amp(1q21), characterized non-hyperdiploid patients. In contrast, deletions of 1p36 and 6q21 were very rare events. Amp(1q21), FGFR3/IGH fusion, RB1 deletion/monosomy 13, and even TP53 deletion/monosomy 17 were seen in some hyperdiploid patients, suggesting that they have a less than favorable prognosis and require closer monitoring.

ContextHuman epidermal growth factor receptor 2 (HER2/neu) amplification is used as a predictive marker for trastuzumab treatment in breast cancer. Both immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) testing algorithms have been based on the 2007 American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines. In late 2013, the guidelines were updated with new scoring criteria.Objective—To assess the impact of the revised ASCO/CAP recommendations on both IHC and FISH results by using the dual-color HER2/neu and centromeric FISH probes.DesignRetrospective analysis of 590 invasive carcinomas with concurrent IHC and dual-color HER2/neu and centromeric 17 (CEP17) FISH results, based on 2007 ASCO/CAP guidelines, was conducted from July 2011 to June 2013. With the revised guidelines, patients were recategorized and concordance rates between the 2 assays were recalculated.Results—Overall concordance rates for FISH and IHC decreased from 94.9% to 93.8% with reclassification. Negative FISH cases decreased from 79.1% to 69.3%. However, equivocal FISH cases were significantly increased from 0.7% to 9.5%, leading to more retesting. Both positive IHC and FISH cases were also noted to be increased, leading to more patients being eligible for trastuzumab treatment, especially those patients with concurrent HER2/neu and CEP17 polysomy. Approximately 1% of patients with initial FISH negative results were reclassified as having positive results when both the ratios and average copy number of HER2/neu were considered under the revised guidelines.ConclusionsThe revised 2013 ASCO/CAP guidelines can potentially lead to more patients being eligible for trastuzumab therapy but additional retesting is to be expected owing to an increased number of equivocal FISH cases.

Introduction: High workload volumes in a Cytogenetics laboratory can lead to long result turn-around times (TAT). This study aimed to improve laboratory efficiency by adopting Lean Management System initiatives to increase productivity through the elimination of wastes. This study examined if the prerequisite 20-cell analysis was sufficient for a conclusive result or if additional cell workup was necessary to ascertain the presence of a previous chromosome abnormality among cases on follow-up, or when a single abnormal cell was encountered during the analysis to determine the presence of a clone. Materials and Methods: The karyotype results of cases that had additional workup were retrieved from among 8040 bone marrow cases of various haematological disorders performed between June 2003 and June 2008. Results: Of 8040 cases analysed, 2915 cases (36.3%) had additional cell workup. Only 49 cases (1.7%) led to the establishment of a clone. The majority of these cases could have been resolved without the additional workup, especially if fluorescence in situ hybridization (FISH) or polymerase chain reaction (PCR)-based assays had been utilised. Conclusion: This study shows that the additional workup procedure is redundant. The time saved by discontinuing the workup procedure can be used to analyse other cases, leading to increased laboratory efficiency and a faster TAT without compromise to patient care. The practice of additional workup over and above the 20-cell analysis should be dispensed with as little benefit was derived for the amount of additional manpower expended. FISH or PCR-based assays should be utilised to elucidate a case further. Key words: Clone, Fluorescence in situ hybridization, Haematological disorders, Karyotyping, Laboratory efficiency

3612Background: Brain metastases (BM) from colorectal cancer (CRC) are a rare but increasing event as patients survive longer. While human epidermal growth factor receptor 2 (HER2) gene amplificati...

Introduction: Holoprosencephaly (HPE) is an uncommon congenital failure of forebrain development. Although the aetiology is heterogeneous, chromosomal abnormalities or a monogenic defect are the major causes, accounting for about 40% to 50% of HPE cases. At least 7 genes have been positively implicated, including SHH, ZIC2, SIX3, TGIF, PTCH1, GLI2, and TDGF1. Clinical Picture: Twelve antenatally- and 1 postnatally-diagnosed cases are presented in this study. These comprised 6 amniotic fluid, 3 chorionic villus, 2 fetal blood, 1 peripheral blood, and 1 product of conception. Outcome: The total chromosome abnormality rate was 92.3%, comprising predominantly trisomy 13 (66.7%). There was 1 case of trisomy 18, and 3 cases of structural abnormalities, including del13q, del18p, and add4q. Conclusion: Despite the poor outcome of an antenatally-diagnosed HPE and the likely decision by parents to opt for a termination of pregnancy, karyotyping and/or genetic studies should be performed to determine if a specific familial genetic or chromosomal abnormality is the cause. At the very least, a detailed chromosome analysis should be carried out on the affected individual. If the result of highresolution karyotyping is normal, Fluorescence in situ hybridisation (FISH) and/or syndromespecific testing or isolated holoprosencephaly genetic testing may be performed. This information can be useful in making a prognosis and predicting the risk of recurrence. Key words: Chromosomes, Genes, Karyotyping, Trisomies

Monosomy 21 is a rare acquired karyotypic abnormality associated with myeloid disorders. Occurrence of loss of one chromosome 21 in the background of trisomy 21 in Down syndrome, resulting in the pseudo-normalization of trisomy 21, is a novel finding. The case is described of a patient with Down syndrome who acquired such a genetic abnormality as a result of myelodysplastic syndrome.

We describe a patient with acute promyelocytic leukemia (APL) and the karyotype 46,XX,i(17)(q10) with PML-RARA fusion gene detected by fluorescence in situ hybridization (FISH) and nested reverse transcriptase-polymerase chain reaction (RT-PCR). FISH using dual-color translocation probes for PML (promyelocytic leukemia) and RARA (retinoic acid receptor-alpha) showed fusion signal for PML-RARA on both arms of i(17q). The patient attained complete remission (CR) with all-trans retinoic acid treatment and became PML-RARA negative. One year later, while PML-RARA negative on FISH and RT-PCR, the patient presented with thrombocytopenia. Bone marrow examination suggested an acute monoblastic leukemia (AML-M5a) including the karyotype 46,XX,t(8;16) (p11.2;p13.3),inv(11)(p15q22 approximately q23)[11]/47,idem,+i(8)(q10)[9]. She is currently in CR. The occurrence of therapy related acute leukemia after successful therapy for APL is an emerging problem.

The NG2 chondroitin sulfate proteoglycan-expressing neural cells (NG2 cells) have originally been considered as oligodendrocyte progenitor cells (OPCs). However, recent findings on their diverse functions and lineage heterogeneity demonstrated that the NG2 cells contain various sub-populations whose concrete features and therapeutic potential yet remained elucidated. In the present study, we characterized a Nestin(+) 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP)(+) NG2(+) subpopulation from embryonic rat cerebral cortex. The Nestin(+) CNP(+) NG2(+) cells exhibited remarkable progenitor characteristics. Having been immortalized by human telomerase reverse transcriptase (hTERT), the life span of Nestin(+) CNP(+) NG2(+) cells was extended to 230 population doublings (PDs). With immortalized NG2 cells, we demonstrated their differentiation capacities to oligodendrocytes, astrocytes and neurons. Furthermore, transplanted into injured spinal cord of a mouse model, they were able to promote remyelination and neuronal regeneration, thereby enhancing the functional recovery. Our findings suggest that the Nestin(+) CNP(+) NG2(+) progenitor cells could be a good alternative cell source of cell therapy for neurological disorders.

The present study was designed to compare abnormality detection rates using DSP30 + IL2 and 12-O-Tetradecanoylphorbol-13-acetate (TPA) in Asian patients with B-CLL. Hematological specimens from 47 patients (29 newly diagnosed, 18 relapsed) were established as 72 h-DSP30 + IL2 and TPA cultures. Standard methods were employed to identify clonal aberrations by conventional cytogenetics (CC). The B-CLL fluorescence in situ hybridization (FISH) panel comprised ATM, CEP12, D13S25, and TP53 probes. DSP30 + IL2 cultures had a higher chromosomal abnormality detection rate (67 %) compared to TPA (44 %, p0.001). The mean number of analyzable metaphases and abnormal metaphases per slide was also higher (p0.005, p0.001, respectively). Culture success rate, percentage of complex karyotype, and percentage of non-clonal abnormal cell were not significantly different (p0.05). Thirteen cases with abnormalities were found exclusively in DSP30 + IL2 cultures compared to one found solely in TPA cultures. DSP30 + IL2 cultures were comparable to the FISH panel in detecting 11q-, +12 and 17p- but not 13q-. It also has a predilection for 11q- bearing leukemic cells compared to TPA. FISH had a higher abnormality detection rate (84.1 %) compared to CC (66.0 %) with borderline significance (p = 0.051), albeit limited by its coverage. In conclusion, DSP30 + IL2 showed a higher abnormality detection rate. However, FISH is indispensable to circumvent low mitotic indices and detect subtle abnormalities.

Phyllodes tumours of the breast are uncommon fibroepithelial neoplasms which pose management challenges due to difficulties in accurate prediction of clinical behaviour, as histological assessment has its limitations. Molecular studies have improved the understanding of these rare tumours but such findings are scant. We aimed to investigate genetic aberrations in phyllodes tumours stratified according to clinical behaviour, to identify potential genes contributing to disease progression. Twenty phyllodes tumours were separated into prognostically distinct categories depending on whether they had recurred/metastasized within the follow-up period. DNA extracted from FFPE materials was subjected to Affymetrix OncoScan™ FFPE Express molecular inversion probe microarray platform for analysis of copy number changes and mutational status. Results were cross validated with Sanger sequencing, FISH and immunohistochemistry. A higher number of chromosomal aberrations were observed in cases which recurred/metastasized, with median events of 19 compared to 3.5 in cases which did not recur/metastasize. High-level amplification and homozygous deletions were detected exclusively in the former group. Regions of high-level amplification included MDM4 (1q32.1), RAF1 (3p25), EGFR (7p12) and PDZD2 (5p13.3). EGFR amplification was confirmed on FISH and accompanied by intense EGFR immunostaining. Regions of homozygous deletion included CDKN2A (9p21) and MACROD2 (20p12.1). Homozygous deletion of 9p21 which involved CDKN2A was accompanied by loss of protein expression. No mutations were identified in all samples. These findings provide insights into identifying target genes and pathways exploited by phyllodes tumours, which would aid future development of individualised therapy.

The relationship between gestational age and frequency of fetal cells in the maternal blood was studied in order to determine the optimal time for cell recovery. The immunomagnetic colloid system was used to enrich nucleated erythrocytes (NRBCs) and trophoblasts from 20 ml maternal blood samples obtained between 9 and 35 weeks' gestation (n=41). Nested polymerase chain reaction (PCR) for the Y chromosome of enriched NRBCs and trophoblasts showed decreasing negative predictive values with increasing gestational age. The sensitivity and the overall frequency for correct fetal gender diagnosis were the lowest in the third trimester. Fluorescence in situ hybridisation (FISH) using XY DNA-specific probes was used to determine the fetal gender of the trophoblast-enriched fraction. The fetal origin of enriched NRBCs was determined using simultaneous immunophenotyping for fetal hemoglobin and FISH with XY probes. The mean number and mean percentage purity for both fetal trophoblasts and NRBCs showed decreasing values with increasing gestational age. However, statistical analysis showed no relationship between gestational age and frequency of fetal cells even though more fetal cells tend to exist during the first trimester. Nevertheless, the first trimester appears to offer the most optimal time for fetal cell recovery from maternal blood for the purpose of prenatal diagnosis.

The aim of this study was to isolate fetal trophoblasts and nucleated erythrocytes from maternal blood using the immunomagnetic colloid system. About 25 ml of maternal blood was collected from pregnant women between of 14 and 20 weeks gestation. Nucleated erythrocytes (NRBCs) were isolated from 5 ml of maternal blood and a nested polymerase chain reaction for the Y chromosome was used to determine fetal origin. The sensitivity of the fetal gender diagnosis was 80% and the specificity was 86%. Both fetal trophoblasts and NRBCs were isolated from the remaining 20 ml of maternal blood. The fetal gender of the trophoblast-enriched fraction was determined using fluorescence in situ hybridisation (FISH) with dual-colour XY-specific DNA probes. XY-specific signals were observed in 0.38% of cells sorted from all pregnant women carrying male fetuses (n = 10). Simultaneous immunophenotyping for the fetal haemoglobin and FISH using XY probes were used to evaluate the fetal origin of cells enriched with anti-CD71. The mean percentage of male fetal erythroblasts was 0.24% and the number of fetal erythroblasts was estimated to be about 672 in 20 ml of maternal blood. The number of fetal erythroblasts detected in our study was greater than that detected by most other separation techniques. Our study shows that it would be feasible to use the immunomagnetic colloid system for the isolation of both trophoblasts and NRBCs from the same maternal blood sample with relatively good efficiency.

Much is known about the cytogenetic lesions that characterize multiple myeloma (MM) patients from the USA, Europe, and East Asia. However, little has been published about the disease among Southeast Asians. The aim of this study was to determine the chromosomal abnormalities of MM patients in our Singapore population.Forty-five newly-diagnosed, morphologically confirmed patients comprising 18 males and 27 females, aged 46 - 84 years (median 65 years) were investigated by karyotyping and fluorescence in situ hybridization (FISH). FISH employing standard panel probes and 1p36/1q21 and 6q21/15q22 probes was performed on diagnostic bone marrow samples.Thirty-four cases (75.6%) had karyotypic abnormalities. Including FISH, a total detection rate of 91.1% was attained. Numerical and complex structural aberrations were common to both hyperdiploid and non-hyperdiploid patients. Numerical gains of several recurring chromosomes were frequent among hyperdiploid patients while structural rearrangements of several chromosomes including 8q24.1 and 14q32 characterized non-hyperdiploid patients. With FISH, immunoglobulin heavy chain (IGH) gene rearrangements, especially fibroblast growth factor receptor 3 (FGFR3)/IGH and RB1 deletion/monosomy 13 were the most common abnormalities (43.4%). Amplification 1q21 was 10 times more frequent (42.5%) than del(1p36) and del(6q21).We have successfully reported the comprehensive cytogenetic profiling of a cohort of newly-diagnosed myeloma patients in our population. This study indicates that the genetic and cytogenetic abnormalities, and their frequencies, in our study group are generally similar to other populations.

Background: HER2-positive tumors are often associated with poor prognosis, chemo-resistances and some patients eventually develop refractory disease during HER2 targeted therapy. While different mechanisms of trastuzumab resistance are being identified in recent years, contribution of confounding factors such as inherent genomic heterogeneity, equivocal HER2 amplifications and presence of chromosome 17 polysomy has been less understood thus far. In this pilot study, we aim to examine HER2 heterogeneity in CTCs obtained from breast cancer patients in the Asian population setting. Methods: CTCs were enriched from blood samples using a label-free spiral microfluidics-based ClearCell® FX system. A total of 26 samples were collected from patients diagnosed with HER2 positive (17/26) and HER2 negative (9/26) breast cancer. The enriched CTCs were analyzed using conventional diagnostic modalities (fluorescence in-situ hybridisation (FISH) and immunocytochemistry) to examine HER2 status. Concordance rate between CTCs and matched primary tumor was evaluated. Results: HER2-positive CTCs were successfully identified in 14 out of 17 HER2+ patients (82.4%); HER2 gene amplification and chromosome 17 polysomy were observed in 10(58.8%) and 13(76.5%) patients respectively. HER2+ CTC counts ranged from 2 to 30 cells from 7.5ml blood (median: 4 HER2+ CTCs/7.5ml). HER2 amplification was not observed in any of the 9 patients with HER2-negative tumors, though 5 out of 9 patients (55.6%) were identified with CTCs harbouring gain in chromosome 17 (median: 2 HER2+ CTCs/7.5ml). A “false positive” cut-off of more than 2 cells/7.5ml blood were established using receiver operating characteristic (ROC) curve analysis and a concordance rate of approximately 70% between paired tumor tissue and CTC among the 26 patients. Immunofluorescence labelling of CTCs with cytokeratin, CD45 and HER2 antibodies further revealed heterogeneity in HER2 expression on CTCs. Conclusion: CTCs capture the heterogeneity of breast cancer, and could potentially overcome limitations of tissue biopsy which are site specific. HER2- patients, as confirmed by tissue biopsy, with HER2+ CTCs pose interesting questions while determining treatment regime. Citation Format: Man Chun Leong, Tse Hui Lim, Chye Ling Tan, Yong Wei Chua, Elaine Hsuen Lim, Kiley Wei Jen Loh, Guek Eng Lee, Rebecca Dent, Raymond Chee Hui Ng, Andrew Wu, Wan-Teck Lim, Alvin Soon-Tiong Lim, Yoon-Sim Yap. Elucidating HER2 molecular heterogeneity of circulating tumor cells among breast cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5008.