Choon Ong

Introduction: Natural-killer/T-cell lymphoma (NKTL) is a rare subset of non-Hodgkin's lymphoma that is more prevalent in Asia than in the West. We report a case of two brothers who were diagnosed with NKTL and died of their disease at 23 and 37 years old respectively, and further investigations performed to determine the underlying genetic basis to their disease. Methods Both patients were recruited into the Singapore lymphoma study where retrieval of their clinical data and biospecimens had been approved by the Singhealth instititutional review board. Their clinical courses are described using information from electronic medical records. For the older brother we extracted tumor DNA from a patient-derived xenograft (PDX), and DNA from a buccal swab and peripheral blood (PB) sample. In the younger brother DNA was extracted from normal and tumor tissue from archived formalin fixed paraffin embedded (FFPE) samples. DNA from the unaffected mother, older brother and paternal aunt were obtained from PB and buccal swab samples. We performed whole exome sequencing (WES) on the younger affected brother's tumor, the older affected…

Background We have shown that the JAK/STAT pathway is dysregulated in Natural-killer/T-cell lymphomas (NKTL) and that the JAK3A572 and JAK3A573 activating mutations were found in 35% of NKTL. JAK3 mutant (MT) NKTL cell lines demonstrated IL-2 independent growth and constitutive activation of downstream JAK/STAT signaling molecules. When these cell lines were treated with JAK3 siRNA, downstream JAK/STAT signaling is inhibited. Reciprocally, transient expression of in JAK3A572 in wild-type (WT) NKTL resulted in IL-2 independent growth and constitutive activation of JAK/STAT signaling. The main aim of this study was to determine the prognostic significance of JAK3 mutation in NKTL. Methods Patients with NKTL treated at the National Cancer Centre Singapore and Singapore General Hospital were identified through clinical and pathology databases. All cases of NKTL were centrally reviewed by hematopathologists to confirm the diagnosis of NKTL based on the 2008 WHO classification. Whenever possible, FFPE tumor samples were examined for JAK3A572 and JAK3A573 mutations using Sanger sequencing and results confirmed with high-resolution melt. Patient demographics, treatment details and survival outcomes were obtained from medical records. Data for hemophagocytic lymphohistiocytosis (HLH) and site of disease involvement were specifically captured. Treatment was stratified according to curative or palliative intents and whether radiotherapy (RT) or stem cell transplant (SCT) was administered. The primary endpoint was to compare survival; OS and PFS, between JAK3 MT and WT patients. Secondary endpoints were to compare clinical characteristics between JAK3 MT and WT patients and identify poor prognostic features amongst NKTL patients who were treated with curative intent. Results Between 1997 and 2011, 98 patients were diagnosed with NKTL. Of these, JAK3 mutation testing was successful in 58 tumor samples: 37.9% (22/58) were JAK3 MT and 62.1% were JAK3 WT. There were 10% with HLH. The 2-year PFS for JAK3 MT and WT patients were 17% and 30% respectively (p=0.91) and the 2-year OS for JAK3 MT and WT patients were 31% and 40% respectively (p=0.45). There was no difference in the baseline clinical characteristics between JAK3 MT and WT patients. (Table 1) In our cohort of NKTL patients, the median OS for patients for stage I/II was 74.2 months (95% CI, 21.7-NR) and the median OS for stage III/IV was 5.2 months (95% CI, 2.93 - 7.86). Amongst the 73 patients treated with curative intent, on univariate analysis, age >60 years, stage III/IV disease, ≥2 extra-nodal sites, non-nasal extranodal involvement, B symptoms, higher IPI scores, HLH and the absence of RT were associated with a poorer OS. On multivariate analysis, age >60years, stage III/IV disease and presence of HLH were independently associated with a poorer survival. Discussion This study showed that JAK3 mutation was not prognostic for poorer OS or PFS and JAK3 MT and WT patients are clinically indistinguishable. Since in vitro, treatment of both JAK3 MT and WT cell lines with a pan-JAK inhibitor resulted in a reduction in cell viability and current treatment strategies against NKTL are poor, future clinical trials should focus on targeting the JAK/STAT pathway in NKTL patients. We also report that HLH was found in 10% of NKTL patients and it was independently prognostic of a poorer OS. The association of HLH and NKT, in addition to them being associated with EBV may point towards a shared etiology. Disclosures: No relevant conflicts of interest to declare.

Cancers often overexpress multiple clinically relevant oncogenes, but it is not known if combinations of oncogenes in cellular subpopulations within a cancer influence clinical outcomes. Using quantitative multispectral imaging of the prognostically relevant oncogenes MYC, BCL2, and BCL6 in diffuse large B-cell lymphoma (DLBCL), we show that the percentage of cells with a unique combination MYC+BCL2+BCL6− (M+2+6−) consistently predicts survival across four independent cohorts (n = 449), an effect not observed with other combinations including M+2+6+. We show that the M+2+6− percentage can be mathematically derived from quantitative measurements of the individual oncogenes and correlates with survival in IHC (n = 316) and gene expression (n = 2,521) datasets. Comparative bulk/single-cell transcriptomic analyses of DLBCL samples and MYC/BCL2/BCL6-transformed primary B cells identify molecular features, including cyclin D2 and PI3K/AKT as candidate regulators of M+2+6− unfavorable biol...

Although combination therapy is the standard of care for relapsed/refractory non-Hodgkin’s lymphoma (RR-NHL), combination treatment chosen for an individual patient is empirical, and response rates remain poor in individuals with chemotherapy-resistant disease. Here, we evaluate an experimental-analytic method, quadratic phenotypic optimization platform (QPOP), for prediction of patient-specific drug combination efficacy from a limited quantity of biopsied tumor samples. In this prospective study, we enrolled 71 patients with RR-NHL (39 B cell NHL and 32 NK/T cell NHL) with a median of two prior lines of treatment, at two academic hospitals in Singapore from November 2017 to August 2021. Fresh biopsies underwent ex vivo testing using a panel of 12 drugs with known efficacy against NHL to identify effective single and combination treatments. Individualized QPOP reports were generated for 67 of 75 patient samples, with a median turnaround time of 6 days from sample collection to repor...

With lowering costs of sequencing and genetic profiling techniques, genetic drivers can now be detected readily in tumors but current prognostic models for Natural‐killer/T cell lymphoma (NKTCL) have yet to fully leverage on them for prognosticating patients. Here, we used next‐generation sequencing to sequence 260 NKTCL tumors, and trained a genomic prognostic model (GPM) with the genomic mutations and survival data from this retrospective cohort of patients using LASSO Cox regression. The GPM is defined by the mutational status of 13 prognostic genes and is weakly correlated with the risk‐features in International Prognostic Index (IPI), Prognostic Index for Natural‐Killer cell lymphoma (PINK), and PINK‐Epstein–Barr virus (PINK‐E). Cox‐proportional hazard multivariate regression also showed that the new GPM is independent and significant for both progression‐free survival (PFS, HR: 3.73, 95% CI 2.07–6.73; p < .001) and overall survival (OS, HR: 5.23, 95% CI 2.57–10.65; p = .001...

MYC, BCL2, and BCL6 are commonly used markers for immunohistochemistry of Diffuse large B-cell lymphomas (DLBCL). Coexpression of MYC and BCL2 in particular constitutes a subgroup of “double expressor lymphomas” (DEL) with a distinct poor clinical outcome. However, it is not known if MYC and BCL2/BCL6 coexpression occurs in the same cell or in different cells within the tumor, as traditional immunohistochemistry (IHC) is limited by the number of markers that can be simultaneously assessed within formalin-fixed, paraffin-embedded (FFPE) samples. We set out to discover the clinical significance of MYC, BCL2, and BCL6 colocalization at single-cell resolution using multiplexed quantitative immunofluorescence (qIF) based on sequential OPAL-TSA staining and spectral microscopy on the Vectra platform. The initial discovery cohort comprised 90 cases of DLBCL from NUH Singapore with adequate clinical follow-up after R-CHOP therapy. We stratified each DLBCL tumor into 8 “clonal fractions” bas...

Background and aims: Angioimmunoblastic T cell Lymphoma (AITL) is a subtype of peripheral T cell lymphoma that is generally felt to be aggressive and of poor prognosis. It is characterized as a lymphoma associated with inflammatory and immune conditions, typically seen in the older population and presenting at more advanced stages. The International T-Cell Lymphoma project recently reported a novel AITL score comprising of age, ECOG performance status, serum CRP level and serum B2-microglobulin level; the latter 2 variables suggesting a pro-inflammatory state. They also found that progression of disease within 24 months (POD24) to be strongly prognostic. In our Asian multicenter study, we aim to investigate the clinical prognostic factors affecting the outcomes of our AITL patients and attempt to identify a prognostic index that would be relevant to our Asian population. Methods: Patients who were consecutively diagnosed with AITL and seen at National Cancer Centre Singapore and Sin...

SummaryPeripheral T‐cell lymphomas (PTCL) and natural killer (NK)/T‐cell lymphomas (NKTCL) are a heterogeneous group of aggressive malignancies with dismal outcomes and limited treatment options. While the phosphatidylinositol 3‐kinase (PIK3) pathway has been shown to be highly activated in many B‐cell lymphomas, its therapeutic relevance in PTCL and NKTCL remains unclear. The aim of this study is to investigate the expression of PIK3 and phosphatase and tensin homolog (PTEN) in these subtypes of lymphoma and to identify potential therapeutic targets for clinical testing. Therefore, the expression of PIK3α, PIK3β, PIK3γ, PIK3δ and PTEN was analyzed in 88 cases of PTCL and NKTCL samples by immunohistochemistry. All PTCL and NKTCL samples demonstrated high expression of PIK3 isoforms. In particular, high PIK3α expression was significantly associated with poor survival, even after adjustment for age, International Prognostic Index (IPI) score and anthracycline‐based chemotherapy in fir...

Background Hodgkin lymphoma (HL) accounts for 15% of all cancer diagnosis in young adults. Standard of care is ABVD (Adriamycin, Bleomycin, Vinblastine, Dacarbazine), which has resulted in overall cures of 70-90%. In the setting of HL, bleomycin is still one of the cornerstones in its treatment with the omission of bleomycin in the randomized trial of GHSG HD13 showing poorer outcomes. A major complication of HL treatment is Bleomycin induced pneumonitis (BIP) occurring in 0.4-28% of patients, with up to 10% mortality. Currently, we are unable to predict which patients are more prone. Given that human genomic variations underlie both disease susceptibility and drug response, uncovering genetic biomarkers predisposing to BIP is imperative to predicting and preventing BIP. Methods In this first pharmacogenomics study of BIP in South-East Asian patients, we perform both candidate gene and GWAS analyses. 96 HL patients were recruited from the National University Cancer Institute, Singap...

Monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL) is a type of rare aggressive lymphoma accounting for 5.4% of primary intestinal peripheral T-cell lymphoma and 10-25% of all primary intestinal lymphoma. MEITL also has an extremely poor prognosis with the median overall survival of only seven months. No effective treatment or targeted therapies are currently available to manage this disease. Therefore, there is an urgent need to devise and establish an appropriate preclinical model for a better understanding of this disease and for new therapeutic strategies to be developed on it. Here, we present the first MEITL patient derived xenograft (PDX) as both orthotropic intestinal and as a subcutaneous model. The histological analysis demonstrated high similarity in the overall immunomorphologic features between the primary tumour and PDX tumours. Importantly, all the PDX tumors carried the distinctive MEITL immunophenotype; CD3+CD4-CD8+CD56+ and extensive nuclear expression ...

Ameloblastoma is a rare tumor of odontogenic epithelium, the low incidence rate of which precludes statistical determination of its molecular characterizations. Despite recent genomic and transcriptomic profiling, the etiology of ameloblastomas remains poorly understood. Risk factors of ameloblastoma development are also largely unknown. Whole exome sequencing was performed on 11 mandibular ameloblastoma samples. We identified 2 convergent mutational signatures in ameloblastoma: 1) a signature found in multiple types of lung cancers with probable etiology of tobacco carcinogens (COSMIC signature 4) and 2) a signature present in gingivobuccal oral squamous cell carcinoma and correlated with tobacco-chewing habits (COSMIC signature 29). These mutational signatures highlight tobacco usage or related mutagens as one possible risk factor of ameloblastoma, since the association of BRAF mutations and smoking was demonstrated in multiple studies. In addition to BRAF hotspot mutations (V600E...

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is a rare autosomal dominant disorder characterized by fumarate hydratase (FH) gene mutation. It is associated with the development of very aggressive kidney tumors, characterized by early onset and high metastatic potential, and has no effective therapy. The aim of the study was to establish a new preclinical platform for investigating morphogenetic and metabolic features, and alternative therapy of metastatic hereditary papillary renal cell carcinoma type 2 (PRCC2). Fresh cells were collected from pleural fluid of a patient with metastatic hereditary PRCC2. Morphogenetic and functional characteristics were evaluated via microscopy, FH gene sequencing analysis, real-time polymerase chaine reaction and enzymatic activity measurement. We performed bioenergetic analysis, gene-expression profiling, and cell viability assay with 19 anti-neoplastic drugs. We established a new in vitro model of hereditary PRCC2 - the NCCFH1 cell l...

Context: Cell division cycle 73 (CDC73), encoding the protein parafibromin, is the most prevalent mutated gene in familial and sporadic parathyroid carcinoma (PC). Objective: Identify additional genetic abnormalities in PCs Design: Whole-exome sequencing was performed using DNA from seven pairs of matched PCs and one triplet containing double primary tumour and normal leukocyte. Somatic variants were confirmed using Sanger sequencing and recurrently mutated genes were assessed in 13 additional PCs as well as 40 parathyroid adenomas (PA). Results: PC had an average of 51 somatic variants/tumour (range 3 -176) with approximately 58% of variants occurring as non-synonymous single nucleotide variants. The importance of CDC73 in PC is reinforced with a remarkable preferential amplification of mutant CDC73 allele. Furthermore recurrent germ line and somatic mutations in prune homolog 2 [Drosophila] (PRUNE2) were found in PC and computationally predicted to be deleterious; in addition, recurrent mutations in kinase genes related to cell migration and invasion were found. PRUNE2 showed recurrent mutations in 18% (4/22) of PCs with additional screening in 40 PAs revealing only one rare missense polymorphism (Asp1677Asn). For the first time, the mutational signature associated with apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC)-catalyzed cytosine-to-uracil deamination is found in a subset of PC. Conclusion: This study outlines the genetic landscape of PC and attempts to characterize the mutational processes shaping the PC genome.

Breast fibroepithelial tumors comprise a heterogeneous spectrum of pathological entities, from benign fibroadenomas to malignant phyllodes tumors. Although MED12 mutations have been frequently found in fibroadenomas and phyllodes tumors, the landscapes of genetic alterations across the fibroepithelial tumor spectrum remain unclear. Here, by performing exome sequencing of 22 phyllodes tumors followed by targeted sequencing of 100 breast fibroepithelial tumors, we observed three distinct somatic mutation patterns. First, we frequently observed MED12 and RARA mutations in both fibroadenomas and phyllodes tumors, emphasizing the importance of these mutations in fibroepithelial tumorigenesis. Second, phyllodes tumors exhibited mutations in FLNA, SETD2 and KMT2D, suggesting a role in driving phyllodes tumor development. Third, borderline and malignant phyllodes tumors harbored additional mutations in cancer-associated genes. RARA mutations exhibited clustering in the portion of the gene encoding the ligand-binding domain, functionally suppressed RARA-mediated transcriptional activation and enhanced RARA interactions with transcriptional co-repressors. This study provides insights into the molecular pathogenesis of breast fibroepithelial tumors, with potential clinical implications.

Growth factors and growth inhibitors play crucial roles in the growth regulation and differentiation of mammary epithelial cells. Studies have shown that during pregnancy, with the onset of terminal differentiation, there is a dramatic decrease in the proliferation of the mammary epithelial cells. Here we report the cloning and characterization of a novel pregnancy-induced cDNA, OKL38, from a human ovarian cDNA library. This cDNA encodes for a protein of approximately 34.5 kDa. Tissue distribution studies through Northern analyses revealed the ubiquitous nature of OKL38 transcripts in most tissues, with the highest levels observed in the ovary, kidney, and liver. The onset and advancement of pregnancy also gave rise to a concomitant increase in OKL38 gene expression. In situ hybridization revealed that OKL38 mRNA was further detected in mammary secretory epithelial cells. However, low levels of OKL38 transcripts were observed in the various human breast cancer cell lines studied and...

The blockade of VEGF pathway has been clinically validated as an initial treatment for renal cell carcinoma (RCC). Angiopoietin-2 (Ang-2) has been indicated as a key regulator for angiogenesis escape. The effect of a novel bispecific antibody (A2V CrossMab) against both Ang-2 and VEGF was investigated in comparison with either factor. A2V CrossMab significantly reduced tumor volume, vessel density, and interstitial fluid pressure compared to either monotherapy of anti-VEGF or anti-Ang-2. Host-derived angiogenesis-related genes have been significantly down-regulated in A2V CrossMab group. These data demonstrate that A2V CrossMab has additive anti-tumor effect for the treatment of RCC.

ABSTRACT Cholangiocarcinoma, the second most common liver cancer, is known to be caused by different etiologies such as liver fluke infection, choledochal cyst and primary sclerosing cholangitis. Thus, this cancer provides a good model to study the impact of different carcinogenic exposures on the specific patterns of somatic mutations in human tumors. To address this issue, we profiled 209 cholangiocarcinomas (CCAs) from Asia and Europe, including 108 cases caused by liver fluke Opisthorchis viverrini (O. viverrini)-infection and 101 cases due to non-O. viverrini etiologies. Whole-exome (N = 15) and prevalence screening (N = 194) revealed recurrent somatic mutations in BAP1 and ARID1A, neither of which has been previously reported to be mutated in CCA. Comparisons between intrahepatic O. viverrini and non-O. viverrini CCAs demonstrated statistically significant different mutation patterns: BAP1 and IDH1/2 were more frequently mutated in non-O. viverrini CCAs, while TP53 displayed the reciprocal pattern. Functional studies demonstrated tumor suppressive roles of BAP1 and ARID1A, establishing the role of chromatin modulators in CCA pathogenesis. These findings indicate that different causative etiologies may induce distinct somatic alterations even within the same tumor type.

Neurofibromatosis type 1 (NF1) is a relatively common tumour predisposition syndrome related to germline aberrations of NF1, a tumour suppressor gene. The gene product neurofibromin is a negative regulator of the Ras cellular proliferation pathway, and also exerts tumour suppression via other mechanisms. Recent next-generation sequencing projects have revealed somatic NF1 aberrations in various sporadic tumours. NF1 plays a critical role in a wide range of tumours. NF1 alterations appear to be associated with resistance to therapy and adverse outcomes in several tumour types. Identification of a patient's germline or somatic NF1 aberrations can be challenging, as NF1 is one of the largest human genes, with a myriad of possible mutations. Epigenetic factors may also also contribute to inadequate levels of neurofibromin in cancer cells. Clinical trials of NF1-based therapeutic approaches are currently limited. Preclinical studies on neurofibromin-deficient malignancies have mainly...

Cholangiocarcinoma (CCA) is a malignant tumour of bile duct epithelial cells with dismal prognosis and rising incidence. Chronic inflammation resulting from liver fluke infection, hepatitis and other inflammatory bowel diseases is a major contributing factor to cholangiocarcinogenesis, likely through accumulation of serial genetic and epigenetic alterations resulting in aberration of oncogenes and tumour suppressors. Recent studies making use of advances in high-throughput genomics have revealed the genetic landscape of CCA, greatly increasing our understanding of its underlying biology. A series of highly recurrent mutations in genes such as TP53, KRAS, SMAD4, BRAF, MLL3, ARID1A, PBRM1 and BAP1, which are known to be involved in cell cycle control, cell signalling pathways and chromatin dynamics, have led to investigations of their roles, through molecular to mouse modelling studies, in cholangiocarcinogenesis. This review focuses on the landscape genetic alterations in CCA and its functional relevance to the formation and progression of CCA.

Testicular germ cell tumors are the most common cancer diagnosed in young men, and seminomas are the most common type of these cancers. There have been no exome-wide examinations of genes mutated in seminomas or of overall rates of nonsilent somatic mutations in these tumors. The objective was to analyze somatic mutations in seminomas to determine which genes are affected and to determine rates of nonsilent mutations. Eight seminomas and matched normal samples were surgically obtained from eight patients. DNA was extracted from tissue samples and exome sequenced on massively parallel Illumina DNA sequencers. Single-nucleotide polymorphism chip-based copy number analysis was also performed to assess copy number alterations. The DNA sequencing read data were analyzed to detect somatic mutations including single-nucleotide substitutions and short insertions and deletions. The detected mutations were validated by independent sequencing and further checked for subclonality. The rate of n...

Context: Cell division cycle 73 (CDC73), encoding the protein parafibromin, is the most prevalent mutated gene in familial and sporadic parathyroid carcinoma (PC). Objective: Identify additional genetic abnormalities in PCs Design: Whole-exome sequencing was performed using DNA from seven pairs of matched PCs and one triplet containing double primary tumour and normal leukocyte. Somatic variants were confirmed using Sanger sequencing and recurrently mutated genes were assessed in 13 additional PCs as well as 40 parathyroid adenomas (PA). Results: PC had an average of 51 somatic variants/tumour (range 3 -176) with approximately 58% of variants occurring as non-synonymous single nucleotide variants. The importance of CDC73 in PC is reinforced with a remarkable preferential amplification of mutant CDC73 allele. Furthermore recurrent germ line and somatic mutations in prune homolog 2 [Drosophila] (PRUNE2) were found in PC and computationally predicted to be deleterious; in addition, recurrent mutations in kinase genes related to cell migration and invasion were found. PRUNE2 showed recurrent mutations in 18% (4/22) of PCs with additional screening in 40 PAs revealing only one rare missense polymorphism (Asp1677Asn). For the first time, the mutational signature associated with apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC)-catalyzed cytosine-to-uracil deamination is found in a subset of PC. Conclusion: This study outlines the genetic landscape of PC and attempts to characterize the mutational processes shaping the PC genome.