Daniel S.W. Tan

Risk of hepatitis B virus (HBV) reactivation in hepatitis B surface antigen negative/hepatitis B core antibody positive patients receiving rituximab-containing combination chemotherapy without routine antiviral prophylaxis

The use of rituximab has been associated with increased risk of hepatitis B virus (HBV) reactivation in patients who are hepatitis B surface antigen (HBsAg) negative and antihepatitis B core antibody (anti-HBc) positive. We aim to determine the rate of HBV reactivation in this group of patients who received rituximab-containing combination chemotherapy without concomitant antiviral prophylaxis and to identify potential risk factors for reactivation. Sixty-two HBsAg negative/anti-HBc positive patients with B-cell lymphoma treated with rituximab-based immunochemotherapy from 2006 to 2009 were included. None of the patients received concomitant antiviral prophylaxis. In this cohort, 48 (77%) patients received rituximab with cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), eight (13%) received rituximab with cyclophosphamide, vincristine and prednisolone, and six (10%) received other chemotherapy regimens. Two patients suffered HBV reactivation; both were above 70xa0years of age, received R-CHOP chemotherapy and were negative for antihepatitis B surface antibody (anti-HBs) at baseline. One of the two patients reactivated shortly after completion of R-CHOP chemotherapy while the other reactivated during rituximab maintenance treatment. Thus, the overall reactivation rate in this cohort of patients is 3% (2/62), 4% (2/48), and 25% (1/4) in patients who received R-CHOP chemotherapy and who received rituximab maintenance, respectively. The rate of HBV reactivation is low in patients who are HBsAg negative/anti-HBc positive receiving rituximab-based combination chemotherapy without concomitant antiviral prophylaxis. However, elderly patients, particularly those without anti-HBs, seemed particularly at risk.

Microfluidic enrichment for the single cell analysis of circulating tumor cells

Resistance to drug therapy is a major concern in cancer treatment. To probe clones resistant to chemotherapy, the current approach is to conduct pooled cell analysis. However, this can yield false negative outcomes, especially when we are analyzing a rare number of circulating tumor cells (CTCs) among an abundance of other cell types. Here, we develop a microfluidic device that is able to perform high throughput, selective picking and isolation of single CTC to 100% purity from a larger population of other cells. This microfluidic device can effectively separate the very rare CTCs from blood samples from as few as 1 in 20,000 white blood cells. We first demonstrate isolation of pure tumor cells from a mixed population and track variations of acquired T790M mutations before and after drug treatment using a model PC9 cell line. With clinical CTC samples, we then show that the isolated single CTCs are representative of dominant EGFR mutations such as T790M and L858R found in the primary tumor. With this single cell recovery device, we can potentially implement personalized treatment not only through detecting genetic aberrations at the single cell level, but also through tracking such changes during an anticancer therapy.

Randomized trial comparing surgery and adjuvant radiotherapy versus concurrent chemoradiotherapy in patients with advanced, nonmetastatic squamous cell carcinoma of the head and neck: 10-year update and subset analysis.

The current study was performed to report the long‐term results of a trial comparing concurrent chemotherapy and radiotherapy (CCRT) with surgery and adjuvant radiotherapy (RT) in patients with stage III/IV nonmetastatic head and neck squamous cell carcinoma.

An eleven gene molecular signature for extra-capsular spread in oral squamous cell carcinoma serves as a prognosticator of outcome in patients without nodal metastases.

OBJECTIVESnExtracapsular spread (ECS) is an important prognostic factor for oral squamous cell carcinoma (OSCC) and is used to guide management. In this study, we aimed to identify an expression profile signature for ECS in node-positive OSCC using data derived from two different sources: a cohort of OSCC patients from our institution (National Cancer Centre Singapore) and The Cancer Genome Atlas (TCGA) head and neck squamous cell carcinoma (HNSCC) cohort. We also sought to determine if this signature could serve as a prognostic factor in node negative cancers.nnnMATERIALS AND METHODSnPatients with a histological diagnosis of OSCC were identified from an institutional database and fresh tumor samples were retrieved. RNA was extracted and gene expression profiling was performed using the Affymetrix GeneChip Human Genome U133 Plus 2.0 microarray platform. RNA sequence data and corresponding clinical data for the TCGA HNSCC cohort were downloaded from the TCGA Data Portal. All data analyses were conducted using R package and SPSS.nnnRESULTSnWe identified an 11 gene signature (GGH, MTFR1, CDKN3, PSRC1, SMIM3, CA9, IRX4, CPA3, ZSCAN16, CBX7 and ZFP3) which was robust in segregating tumors by ECS status. In node negative patients, patients harboring this ECS signature had a significantly worse overall survival (p=0.04).nnnCONCLUSIONSnAn eleven gene signature for ECS was derived. Our results also suggest that this signature is prognostic in a separate subset of patients with no nodal metastasis Further validation of this signature on other datasets and immunohistochemical studies are required to establish utility of this signature in stratifying early stage OSCC patients.

Long noncoding RNA EGFR-AS1 mediates epidermal growth factor receptor addiction and modulates treatment response in squamous cell carcinoma

Targeting EGFR is a validated approach in the treatment of squamous-cell cancers (SCCs), although there are no established biomarkers for predicting response. We have identified a synonymous mutation in EGFR, c.2361G>A (encoding p.Gln787Gln), in two patients with head and neck SCC (HNSCC) who were exceptional responders to gefitinib, and we showed in patient-derived cultures that the A/A genotype was associated with greater sensitivity to tyrosine kinase inhibitors (TKIs) as compared to the G/A and G/G genotypes. Remarkably, single-copy G>A nucleotide editing in isogenic models conferred a 70-fold increase in sensitivity due to decreased stability of the EGFR-AS1 long noncoding RNA (lncRNA). In the appropriate context, sensitivity could be recapitulated through EGFR-AS1 knockdown in vitro and in vivo, whereas overexpression was sufficient to induce resistance to TKIs. Reduced EGFR-AS1 levels shifted splicing toward EGFR isoform D, leading to ligand-mediated pathway activation. In co-clinical trials involving patients and patient-derived xenograft (PDX) models, tumor shrinkage was most pronounced in the context of the A/A genotype for EGFR-Q787Q, low expression of EGFR-AS1 and high expression of EGFR isoform D. Our study reveals how a silent mutation influences the levels of a lncRNA, resulting in noncanonical EGFR addiction, and delineates a new predictive biomarker suite for response to EGFR TKIs.

Targeting Cancer Stem Cell Plasticity Through Modulation of Epidermal Growth Factor and Insulin-Like Growth Factor Receptor Signaling in Head and Neck Squamous Cell Cancer

Emerging data suggest that cancer stem cells (CSCs) exist in equilibrium with differentiated cells and that stochastic transitions between these states can account for tumor heterogeneity and drug resistance. The aim of this study was to establish an in vitro system that recapitulates stem cell plasticity in head and neck squamous cell cancers (HNSCCs) and identify the factors that play a role in the maintenance and repopulation of CSCs. Tumor spheres were established using patient‐derived cell lines via anchorage‐independent cell culture techniques. These tumor spheres were found to have higher aldehyde dehydrogenase (ALD) cell fractions and increased expression of Kruppel‐like factor 4, SRY (sex determining region Y)‐box 2, and Nanog and were resistant to γ‐radiation, 5‐fluorouracil, cisplatin, and etoposide treatment compared with monolayer culture cells. Monolayer cultures were subject to single cell cloning to generate clones with high and low ALD fractions. ALDHigh clones showed higher expression of stem cell and epithelial‐mesenchymal transition markers compared with ALDLow clones. ALD fractions, representing stem cell fractions, fluctuated with serial passaging, equilibrating at a level specific to each cell line, and could be augmented by the addition of epidermal growth factor (EGF) and/or insulin. ALDHigh clones showed increased EGF receptor (EGFR) and insulin‐like growth factor‐1 receptor (IGF‐1R) phosphorylation, with increased activation of downstream pathways compared with ALDLow clones. Importantly, blocking these pathways using specific inhibitors against EGFR and IGF‐1R reduced stem cell fractions drastically. Taken together, these results show that HNSCC CSCs exhibit plasticity, with the maintenance of the stem cell fraction dependent on the EGFR and IGF‐1R pathways and potentially amenable to targeted therapeutics.

Phenotype-driven precision oncology as a guide for clinical decisions one patient at a time

Genomics-driven cancer therapeutics has gained prominence in personalized cancer treatment. However, its utility in indications lacking biomarker-driven treatment strategies remains limited. Here we present a “phenotype-driven precision-oncology” approach, based on the notion that biological response to perturbations, chemical or genetic, in ex vivo patient-individualized models can serve as predictive biomarkers for therapeutic response in the clinic. We generated a library of “screenable” patient-derived primary cultures (PDCs) for head and neck squamous cell carcinomas that reproducibly predicted treatment response in matched patient-derived-xenograft models. Importantly, PDCs could guide clinical practice and predict tumour progression in two nu2009=u20091 co-clinical trials. Comprehensive “-omics” interrogation of PDCs derived from one of these models revealed YAP1 as a putative biomarker for treatment response and survival in ~24% of oral squamous cell carcinoma. We envision that scaling of the proposed PDC approach could uncover biomarkers for therapeutic stratification and guide real-time therapeutic decisions in the future.Treatment response in patient-derived models may serve as a biomarker for response in the clinic. Here, the authors use paired patient-derived mouse xenografts and patient-derived primary culture models from head and neck squamous cell carcinomas, including metastasis, as models for high-throughput screening of anti-cancer drugs.

Truth About ERCC1 in Lung Cancer

sion, as evaluated by immunohistochemistry, was associated with worse outcomes in inoperable non–small-cell lung cancer (NSCLC), particularly in men. However, like most previous literature pertaining to ERCC1 in NSCLC—the majority of which are retrospective in nature—there are methodological issues that require clarification. From the data presented, stage IIA, IIIA, and IIIB constitute 44% of the cohort. It is unclear from the survival analysis if stage was evenly distributed between groups, as well as the proportion of patients that were treated with curative intent— both of which have distinct prognostic implications. In addition, the use concurrent treatments, such as radiotherapy or gemcitabine and its known interaction with RRM1 expression, 2 can further influence outcome, confounding the interpretation of ERCC1 status on survival. Technical issues also require urgent resolution, especially pertaining to the measurement of ERCC1 expression with assays that are inadequately validated and have undefined thresholds of functional significance. Indeed, it can be challenging to equate expression levels of a single protein or mRNA transcript with repair capacity, which is a dynamic process comprising of interaction between multiple factors. 3 Moreover, the 8F1 antibody used for detection of ERCC1 in Holm et al 1 and other groups 4 has been shown to be by no means discriminatory. 5 In elegant validation experiments using formalinfixed, paraffin-embedded normal (negative control) and ERCC1XPF– deficient (positive control) cell lines, it was recently confirmed that 8F1 antibody was unable to differentiate between normal and ERCC1-deficient cells by immunohistochemistry. 6 In the same study, immunofluorescence experiments conducted on UV-C– damaged fibroblasts revealed diffuse cytoplasmic staining with the 8F1 antibody, as opposed to specific localization to the nucleus where DNA damage occurs. Nuclear-specific staining using alternative antibodies such as FL297 (anti-ERCC1) and 3F2 (anti-XPF) has subsequently been suggested to be more accurate in depicting DNA repair. 6

Mutational signature analysis of Asian OSCCs reveals novel mutational signature with exceptional sequence context specificity

Mutational signatures can reveal the history of mutagenic processes that cells were exposed to prior to and during tumourigenesis. We expect that as-yet-undiscovered mutational processes will shed further light on mutagenesis leading to carcinogenesis. With this in mind, we analyzed the mutational spectra of 36 Asian oral squamous cell carcinomas. The mutational spectra of two samples from patients who presented with oral bacterial infections, showed novel mutational signatures. One of these novel signatures, SBS_AnT, is characterized by a preponderance of thymine mutations, strong transcriptional strand bias, and striking enrichment for adenines in the 4 base pairs 5’ of mutation sites. Examination of publicly available sequencing data revealed SBS_AnT in 25 tumours from several mucosal tissue types, all of which harbour human symbionts or are adjacent to tissues that harbour symbionts. Data in a preprint released while this manuscript was in revision strongly suggest that the bacterial compound colibactin causes SBS_AnT.Study of mutational signatures in cancers can reveal the history of mutagenic processes that cells were exposed to prior and during tumourigenesis. Although recent work has identified 65 distinct single nucleotide substitution signatures, we expect that as-yet-undiscovered mutational processes will be able to shed further light on mutagenesis leading to carcinogenesis. For this reason we analyzed mutational signatures in whole-exome sequencing data of 36 Asian oral squamous cell carcinomas. The mutational spectra of most of the carcinomas could be explained by known mutational signatures, but one sample showed a novel mutational signature. Whole-genome sequencing revealed that this mutational signature was characterized by a preponderance of thymine mutations, strong transcriptional strand bias, and a striking enrichment for adenines in the 4 base pairs 5′ of mutation sites. The enrichment was strongest 3 base pairs 5′ of mutated thymines, where 93.5% of bases were adenines. We also observed deletions of single thymines inside and outside of thymine repeats with similar 5′ enrichment for adenines. Examination of publicly available whole-genome and whole-exome sequencing data of 23,829 tumours revealed this signature in 23 tumours from sites including the bile-duct, bladder, pancreas and rectum. Although the aetiology of this mutational signature remains unknown, the transcriptional strand bias suggests adduct formation on adenines. We speculate that the signature may have been caused by duocarmycins, naturally occurring large DNA-crosslinking molecules, which were also used in the past in clinical trials. We call this novel mutational signature SBS_AnT.

A three gene immunohistochemical panel serves as an adjunct to clinical staging of patients with head and neck cancer

Background Current management of head and neck squamous cell carcinoma (HNSCC) depends on tumor staging. Despite refinements in clinical staging algorithms, outcomes remain unchanged for the last two decades. In this study, we set out to identify a small, clinically applicable molecular panel to aid prognostication of patients with HNSCC. Materials and Methods Data from The Cancer Genome Atlas (TCGA) was used to derive copy number aberrations and expression changes to identify putative prognostic genes. To account for cross entity relevance of the biomarkers, HNSCC (n = 276), breast (n = 808) and lung cancer (n = 282) datasets were used to identify robust and reproducible markers with prognostic potential. Validation was performed using immunohistochemistry (IHC) on tissue microarrays of an independent cohort of HNSCC (n = 333). Findings Using GISTIC algorithm together with gene expression analysis, we identified six putative prognostic genes in at least two out of three cancers analyzed, of which four were successfully optimized for automated IHC. Of these, three were successfully validated; each molecular target being significantly prognostic on univariate analysis. Patients were differentially segregated into four prognostic groups based on the number of genes dysregulated (p < 0.001). The IHC panel remained an independent predictor of survival after adjusting for known survival covariates including clinical staging criteria in a multivariate Cox regression model (p < 0.001).u2003 Interpretation We have identified and validated a clinically applicable IHC biomarker panel that is independently associated with overall survival. This panel is readily applicable, serving as a useful adjunct to current staging systems and provides novel targets for future therapeutic strategies.BACKGROUNDnCurrent management of head and neck squamous cell carcinoma (HNSCC) depends on tumor staging. Despite refinements in clinical staging algorithms, outcomes remain unchanged for the last two decades. In this study, we set out to identify a small, clinically applicable molecular panel to aid prognostication of patients with HNSCC.nnnMATERIALS AND METHODSnData from The Cancer Genome Atlas (TCGA) was used to derive copy number aberrations and expression changes to identify putative prognostic genes. To account for cross entity relevance of the biomarkers, HNSCC (n = 276), breast (n = 808) and lung cancer (n = 282) datasets were used to identify robust and reproducible markers with prognostic potential. Validation was performed using immunohistochemistry (IHC) on tissue microarrays of an independent cohort of HNSCC (n = 333).nnnFINDINGSnUsing GISTIC algorithm together with gene expression analysis, we identified six putative prognostic genes in at least two out of three cancers analyzed, of which four were successfully optimized for automated IHC. Of these, three were successfully validated; each molecular target being significantly prognostic on univariate analysis. Patients were differentially segregated into four prognostic groups based on the number of genes dysregulated (p < 0.001). The IHC panel remained an independent predictor of survival after adjusting for known survival covariates including clinical staging criteria in a multivariate Cox regression model (p < 0.001).nnnINTERPRETATIONnWe have identified and validated a clinically applicable IHC biomarker panel that is independently associated with overall survival. This panel is readily applicable, serving as a useful adjunct to current staging systems and provides novel targets for future therapeutic strategies.