Practical management of chronic lymphocytic leukemia with acalabrutinib.

Treatment of chronic lymphocytic leukemia (CLL) has been transformed in the past two decades. The introduction of targeted therapies has improved patient outcomes and the deliverability of effective therapies. Making the best use of the next wave of Bruton's tyrosine kinase (BTK) inhibitors requires an understanding of the nuances that separate the drugs in this class of agents. This paper reviews the newer BTK inhibitors and provides practical guidance on the management of CLL using acalabrutinib. Acalabrutinib is a safe and efficacious BTKi in the treatment of CLL. While some side effects appear to be an "on-target" effect of BTK inhibition, the selectivity of second-generation covalent BTK inhibitors such as acalabrutinib may result in a favorable safety profile due to less off-target kinase inhibition. Acalabrutinib represents a well-tolerated and effective alternative to ibrutinib in the management of CLL.

Recommended testing algorithms for NTRK gene fusions in pediatric and selected adult cancers: Consensus of a Singapore Task Force.

The occurrence of neurotrophic tyrosine receptor kinase (NTRK) gene fusions in a wide range of tumor types presents an attractive opportunity for using a tropomyosin receptor kinase (TRK) inhibitor as cancer therapy. Recent clinical studies have demonstrated highly efficacious outcomes associated with the use of TRK inhibitors, such as larotrectinib and entrectinib in NTRK fusion-bearing cancers, in both adult and pediatric populations. While NTRK gene fusions are commonly found in some uncommon adult and pediatric malignancies, they are also found, albeit rarely, in a wide range of more common malignancies. The potential value of testing for NTRK gene fusions in practically all advanced malignancies is underpinned by the remarkable therapeutic outcomes that TRK inhibitors offer. This requirement presents practical and financial challenges in real-world oncological practice. Furthermore, different testing platforms exist to detect NTRK gene fusions, each with its advantages and disadvantages. It is, therefore, imperative to develop strategies for NTRK gene fusion testing in an attempt to optimize the use of limited tissue specimen and financial resources, and to minimize the turnaround time. A multidisciplinary task force of Singapore medical experts in both public and private sectors was convened in late 2020 to propose testing algorithms for adult colorectal tumors, sarcomas, non-small cell lung cancer, and pediatric cancers, with particular adaptation to the Singapore oncological practice. The recommendations presented here highlight the heterogeneity of NTRK-fusion positive cancers, and emphasize the need to customize the testing methods to each tumor type to optimize the workflow.

CpG methylation in cell-free Epstein-Barr virus DNA in patients with EBV-Hodgkin lymphoma.

Epstein-Barr virus (EBV) is associated with a variety of tumors and nonmalignant conditions. Latent EBV genomes in cells, including tumor cells, are often CpG methylated, whereas virion DNA is not CpG methylated. We demonstrate that methyl CpG binding magnetic beads can be used to fractionate among sources of EBV DNA (DNA extracted from laboratory-purified virions vs DNA extracted from latently infected cell lines). We then applied the technique to plasma specimens and showed that this technique can distinguish EBV DNA from patients with EBV-associated tumors (nasopharyngeal carcinoma, Hodgkin lymphoma) and viral DNA from patients without EBV-associated tumors, including immunocompromised patients and patients with EBV(-) Hodgkin lymphoma.

Receptor Tyrosine Kinase Fusions and BRAF Kinase Fusions are Rare but Actionable Resistance Mechanisms to EGFR Tyrosine Kinase Inhibitors.

INTRODUCTION: We analyzed a large set of EGFR-mutated (EGFR+) NSCLC to identify and characterize cases with co-occurring kinase fusions as potential resistance mechanisms to EGFR tyrosine kinase inhibitors (TKIs). METHODS: EGFR+ (del 19, L858R, G719X, S768I, L851Q) NSCLC clinical samples (formalin-fixed paraffin-embedded tumor and blood) were analyzed for the presence of receptor tyrosine kinase (RTK) and BRAF fusions. Treatment history and response were obtained from provided pathology reports and treating clinicians. RESULTS: Clinical samples from 3505 unique EGFR+ NSCLCs were identified from June 2012 to October 2017. A total of 31 EGFR+ cases had concurrent kinase fusions detected: 10 (32%) BRAF, 7 (23%) ALK receptor tyrosine kinase (ALK), 6 (19%) ret proto-oncogene (RET), 6 (19%) fibroblast growth factor receptor 3 (FGFR3), 1 (3.2%) EGFR, and 1 (3.2%) neurotrophic receptor tyrosine kinase 1 (NTRK1), including two novel fusions (SALL2-BRAF and PLEKHA7-ALK). Twenty-seven of 31 patients had either a known history of EGFR+ NSCLC diagnosis or prior treatment with an EGFR TKI before the fusion+ sample was collected. Twelve of the 27 patients had paired pre-treatment samples where the fusion was not present before treatment with an EGFR TKI. Multiple patients treated with combination therapy targeting EGFR and the acquired fusion had clinical benefit, including one patient with osimertinib resistance due to an acquired PLEKHA7-ALK fusion achieving a durable partial response with combination of full-dose osimertinib and alectinib. CONCLUSIONS: RTK and BRAF fusions are rare but potentially druggable resistance mechanisms to EGFR TKIs. Detection of RTK and BRAF fusions should be part of comprehensive profiling panels to determine resistance to EGFR TKIs and direct appropriate combination therapeutic strategies.

Pan-CDK inhibition augments cisplatin lethality in nasopharyngeal carcinoma cell lines and xenograft models.

In addition to their canonical roles in regulating cell cycle transition and transcription, cyclin-dependent kinases (CDKs) have been shown to coordinate DNA damage response pathways, suggesting a rational pairing of CDK inhibitors with genotoxic chemotherapeutic agents in the treatment of human malignancies. Here, we report that roniciclib (BAY1000394), a potent pan-CDK inhibitor, displays promising anti-neoplastic activity as a single agent and potentiates cisplatin lethality in preclinical nasopharyngeal carcinoma (NPC) models. Proliferation of the NPC cell lines HONE-1, CNE-2, C666-1, and HK-1 was effectively curbed by roniciclib treatment, with IC50 values between 11 and 38 nmol/L. These anticancer effects were mediated by pleiotropic mechanisms consistent with successful blockade of cell cycle CDKs 1, 2, 3, and 4 and transcriptional CDKs 7 and 9, ultimately resulting in arrest at G1/S and G2/M, downregulation of the transcriptional apparatus, and repression of anti-apoptotic proteins. Considerably enhanced tumor cell apoptosis was achieved following combined treatment with 10 nmol/L roniciclib and 2.0 µmol/L cisplatin; this combination therapy achieved a response over 250% greater than either drug alone. Although roniciclib chemosensitized NPC cells to cisplatin, it did not sensitize untransformed (NP69) cells. The administration of 0.5 mg/kg roniciclib to BALB/c xenograft mice was well tolerated and effectively restrained tumor growth comparable to treatment with 6 mg/kg cisplatin, whereas combining these two agents produced far greater tumor suppression than either of the monotherapies. In summary, these data demonstrate that roniciclib has strong anti-NPC activity and synergizes with cisplatin chemotherapy at clinically relevant doses, thus justifying further evaluation of this combinatorial approach in clinical settings.

A phase II study of the efficacy and safety of the combination therapy of the MEK inhibitor refametinib (BAY 86-9766) plus sorafenib for Asian patients with unresectable hepatocellular carcinoma.

PURPOSE: There is an unmet need for treatment options in hepatocellular carcinoma (HCC). Sorafenib is currently the only approved systemic treatment for HCC. Refametinib, an oral, allosteric MEK inhibitor, has demonstrated antitumor activity in combination with sorafenib in vitro and in vivo. A phase II study evaluated efficacy and safety of refametinib plus sorafenib in Asian patients with HCC (NCT01204177). EXPERIMENTAL DESIGN: Eligible patients received twice-daily refametinib 50 mg plus twice-daily sorafenib 200 mg (morning)/400 mg (evening), with dose escalation to sorafenib 400 mg twice daily from cycle 2 if no grade ≥ 2 hand-foot skin reaction, fatigue, or gastrointestinal toxicity occurred. Primary efficacy endpoint: disease control rate. Secondary endpoints: time to progression, overall survival, pharmacokinetic assessment, biomarker analysis, safety, and tolerability. RESULTS: Of 95 enrolled patients, 70 received study treatment. Most patients had liver cirrhosis (82.9%) and hepatitis B viral infection (75.7%). Disease control rate was 44.8% (primary efficacy analysis; n = 58). Median time to progression was 122 days, median overall survival was 290 days (n = 70). Best clinical responders had RAS mutations; majority of poor responders had wild-type RAS. Most frequent drug-related adverse events were diarrhea, rash, aspartate aminotransferase elevation, vomiting, and nausea. Dose modifications due to adverse events were necessary in almost all patients. CONCLUSIONS: Refametinib plus sorafenib showed antitumor activity in patients with HCC and was tolerated at reduced doses by most patients. Frequent dose modifications due to grade 3 adverse events may have contributed to limited treatment effect. Patients with RAS mutations appear to benefit from refametinib/sorafenib combination.

Combination of vaccine-strain measles and mumps virus synergistically kills a wide range of human hematological cancer cells: Special focus on acute myeloid leukemia.

Through combining vaccine-derived measles and mumps viruses (MM), we efficiently targeted a wide range of hematopoietic cancer cell lines. MM synergistically killed many cell lines including acute myeloid leukemia (AML) cell lines. Further investigation suggested that enhanced oncolytic effect of MM was due to increased apoptosis induction. In an U937 xenograft AML mouse model, MM displayed greater tumor suppression and prolonged survival. Furthermore, MM efficiently killed blasts from 16 out of 20 AML patients and elicited more efficient killing effect on 11 patients when co-administered with Ara-C. Our results demonstrate that MM is a promising therapeutic candidate for hematological malignancies.

Diagnostic and prognostic utility of a DNA hypermethylated gene signature in prostate cancer.

We aimed to identify a prostate cancer DNA hypermethylation microarray signature (denoted as PHYMA) that differentiates prostate cancer from benign prostate hyperplasia (BPH), high from low-grade and lethal from non-lethal cancers. This is a non-randomized retrospective study in 111 local Asian men (87 prostate cancers and 24 BPH) treated from 1995 to 2009 in our institution. Archival prostate epithelia were laser-capture microdissected and genomic DNA extracted and bisulfite-converted. Samples were profiled using Illumina GoldenGate Methylation microarray, with raw data processed by GenomeStudio. A classification model was generated using support vector machine, consisting of a 55-probe DNA methylation signature of 46 genes. The model was independently validated on an internal testing dataset which yielded cancer detection sensitivity and specificity of 95.3% and 100% respectively, with overall accuracy of 96.4%. Second validation on another independent western cohort yielded 89.8% sensitivity and 66.7% specificity, with overall accuracy of 88.7%. A PHYMA score was developed for each sample based on the state of methylation in the PHYMA signature. Increasing PHYMA score was significantly associated with higher Gleason score and Gleason primary grade. Men with higher PHYMA scores have poorer survival on univariate (p = 0.0038, HR = 3.89) and multivariate analyses when controlled for (i) clinical stage (p = 0.055, HR = 2.57), and (ii) clinical stage and Gleason score (p = 0.043, HR = 2.61). We further performed bisulfite genomic sequencing on 2 relatively unknown genes to demonstrate robustness of the assay results. PHYMA is thus a signature with high sensitivity and specificity for discriminating tumors from BPH, and has a potential role in early detection and in predicting survival.

Malignant cells derived from 3T3 fibroblast feeder layer in cell culture for nasopharyngeal carcinoma.

In this study, we discovered a subpopulation of 3T3 feeder cells were malignantly transformed by nasopharyngeal carcinoma (NPC) tumor cells during co-culture. The transformed 3T3 cells acquired an accelerated growth rate, displayed loosely attached multilayer growth in vitro and highly tumorigenic in vivo. Most strikingly, instead of forming sarcomas, they developed into carcinoma-like tumors somewhat resembling the original NPC. We further demonstrated the transformation is not a single isolated event, rather a common reproducible, cell contact dispensable phenomena among NPC tumor cells. However, NPC tumor cells alone were not sufficient to confer the transformed characteristics onto normal human cells.

Plasma Epstein-Barr virus DNA predicts outcome in advanced Hodgkin lymphoma: correlative analysis from a large North American cooperative group trial.

Epstein-Barr virus (EBV) is associated with Hodgkin lymphoma (HL) and can be detected by in situ hybridization (ISH) of viral nucleic acid (EBER) in tumor cells. We sought to determine whether plasma EBV-DNA could serve as a surrogate for EBER-ISH and to explore its prognostic utility in HL. Specimens from the Cancer Cooperative Intergroup Trial E2496 were used to compare pretreatment plasma EBV-DNA quantification with EBV tumor status by EBER-ISH. A cutoff of >60 viral copies/100 µL plasma yielded 96% concordance with EBER-ISH. Pretreatment and month 6 plasma specimens were designated EBV(-) or EBV(+) by this cutoff. Patients with pretreatment EBV(+) plasma (n = 54) had inferior failure-free survival (FFS) compared with those with pretreatment EBV(-) plasma (n = 274), log-rank P = .009. By contrast, no difference in FFS was observed when patients were stratified by EBER-ISH. Pretreatment plasma EBV positivity was an independent predictor of treatment failure on multivariate analyses. At month 6, plasma EBV(+) patients (n = 7) had inferior FFS compared with plasma EBV(-) patients (n = 125), log-rank P = .007. These results confirm that plasma EBV-DNA is highly concordant with EBER-ISH in HL and suggest that it may have prognostic utility both at baseline and after therapy. This trial was registered at www.clinicaltrials.gov as #NCT00003389.

Pharmacodynamic effects of seliciclib, an orally administered cell cycle modulator, in undifferentiated nasopharyngeal cancer.

PURPOSE: Cell cycle dysregulation resulting in expression of antiapoptotic genes and uncontrolled proliferation is a feature of undifferentiated nasopharyngeal carcinoma. The pharmacodynamic effects of seliciclib, a cyclin-dependent kinase (CDK) inhibitor, were studied in patients with nasopharyngeal carcinoma. EXPERIMENTAL DESIGN: Patients with treatment-naïve locally advanced nasopharyngeal carcinoma received seliciclib at 800 mg or 400 mg twice daily on days 1 to 3 and 8 to 12. Paired tumor samples obtained at baseline and on day 13 were assessed by light microscopy, immunohistochemistry, and transcriptional profiling using real-time PCR low-density array consisting of a panel of 380 genes related to cell cycle inhibition, apoptosis, signal transduction, and cell proliferation. RESULTS: At 800 mg bd, one patient experienced grade 3 liver toxicity and another had grade 2 vomiting; no significant toxicities were experienced in 13 patients treated at 400 mg bd. Seven of fourteen evaluable patients had clinical evidence of tumor reduction. Some of these responses were associated with increased tumor apoptosis, necrosis, and decreases in plasma EBV DNA posttreatment. Reduced protein expression of Mcl-1, cyclin D1, phosphorylated retinoblastoma protein pRB (T821), and significant transcriptional down-regulation of genes related to cellular proliferation and survival were shown in some patients posttreatment, indicative of cell cycle modulation by seliciclib, more specifically inhibition of cdk2/cyclin E, cdk7/cyclin H, and cdk9/cyclin T. CONCLUSIONS: Brief treatment with this regimen of seliciclib in patients with nasopharyngeal carcinoma is tolerable at 400 mg bd and associated with tumor pharmacodynamic changes consistent with cdk inhibition, and warrants further efficacy studies in this tumor.

Familial nasopharyngeal carcinoma in a cohort of 200 patients.

OBJECTIVES: To describe the characteristics of familial nasopharyngeal carcinoma (NPC) in a high-risk population and to determine the role of screening first-degree relations. DESIGN: An analysis on a cohort of 200 patients newly diagnosed as having NPC. SETTING: A tertiary-level institution. PATIENTS: The patients were divided into 2 groups. Patients in group 1 had a first-degree relative with NPC, and those in group 2 did not. For patients in group 1, the relationship and the time interval between affected relatives were noted. The clinical and pathological factors of the 2 groups were obtained and statistically analyzed. RESULTS: There were 15.5% of NPC patients who had an affected first-degree relative. Of the affected relatives, 71% were siblings and 29% were parents. The mean interval between affected siblings was 5.3 years, while that between an affected parent and a child was 24.5 years. No differences were noted in the clinical factors between familial and nonfamilial NPC patients. Most patients in both groups were diagnosed as having stage III or IV NPC. CONCLUSIONS: The rate of familial NPC in our study is 15.5%. Siblings are more commonly affected, and the interval between 2 affected siblings is relatively short. No distinct clinical pattern exists in familial NPC. We recommend that siblings of NPC patients be screened as soon as possible once the index case is diagnosed.

Lack of somatic mutations in EGFR tyrosine kinase domain in hepatocellular and nasopharyngeal carcinoma.

Activating EGFR somatic mutations have been shown to predict treatment response to small molecules targeting the EGFR intracellular tyrosine kinase domain. Recent work on cell-lines and animal models had demonstrated an inhibitory effect of EGFR tyrosine kinase inhibitors in hepatocellular and nasopharyngeal carcinoma, and clinical trials in these tumour types are ongoing. There are few data on the presence or prevalence of EGFR mutations in hepatocellular and nasopharyngeal carcinomas. We studied exons 18-21 of the EGFR gene from 100 hepatocellular and 102 nasopharyngeal carcinomas, and found no exonic mutations of potential significance. Alternative mechanisms may be important for the observed activity of small molecule EGFR tyrosine kinase inhibitors in hepatocellular and nasopharyngeal carcinomas.

The stress-responsive gene GADD45G is a functional tumor suppressor, with its response to environmental stresses frequently disrupted epigenetically in multiple tumors.

The CpG island of GADD45G was identified as a target sequence during the identification of hypermethylated genes using methylation-sensitive representational difference analysis combined with 5-aza-2'-deoxycytidine demethylation. Located at the commonly deleted region 9q22, GADD45G is a member of the DNA damage-inducible gene family. In response to stress shock, GADD45G inhibits cell growth and induces apoptosis. Same as other GADD45 members, GADD45G is ubiquitously expressed in all normal adult and fetal tissues. However, its transcriptional silencing or down-regulation and promoter hypermethylation were frequently detected in tumor cell lines, including 11 of 13 (85%) non-Hodgkin's lymphoma, 3 of 6 (50%) Hodgkin's lymphoma, 8 of 11 (73%) nasopharyngeal carcinoma, 2 of 4 (50%) cervical carcinoma, 5 of 17 (29%) esophageal carcinoma, and 2 of 5 (40%) lung carcinoma and other cell lines but not in any immortalized normal epithelial cell line, normal tissue, or peripheral blood mononuclear cells. The silencing of GADD45G could be reversed by 5-aza-2'-deoxycytidine or genetic double knockout of DNMT1 and DNMT3B, indicating a direct epigenetic mechanism. Aberrant methylation was further frequently detected in primary lymphomas although less frequently in primary carcinomas. Only one single sequence change in the coding region was detected in 1 of 25 cell lines examined, indicating that genetic inactivation of GADD45G is very rare. GADD45G could be induced by heat shock or UV irradiation in unmethylated cell lines; however, this stress response was abolished when its promoter becomes hypermethylated. Ectopic expression of GADD45G strongly suppressed tumor cell growth and colony formation in silenced cell lines. These results show that GADD45G can act as a functional new-age tumor suppressor but being frequently inactivated epigenetically in multiple tumors.

Overexpression of cyclooxygenase-2 in nasopharyngeal carcinoma and association with epidermal growth factor receptor expression.

OBJECTIVES: To examine the association between cyclooxygenase-2 (COX-2) expression with epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (iNOS), and latent membrane protein 1 (LMP-1) expression and with COX-2 promoter methylation status in primary nasopharyngeal cancer (NPC) tumors and to determine COX-2 promoter methylation status in NPC cell lines. DESIGN: Retrospective study. SETTING: Patients with NPC were referred to the Department of Otolaryngology-Head and Neck Surgery for treatment. PATIENTS: Formalin-fixed, paraffin-embedded NPC specimens from 42 patients were obtained. INTERVENTIONS: Immunohistochemical expression of COX-2, EGFR, VEGF, iNOS, and LMP-1 was performed in 42 NPC samples. COX-2 promoter methylation status was studied in 20 separate specimens and in 4 NPC cell lines. MAIN OUTCOME MEASURES: (1) COX-2, EGFR, VEGF, iNOS, and LMP-1 expression; and (2) COX-2 promotor methylation status. RESULTS: COX-2 was overexpressed in 79% of NPC specimens and was associated with EGFR status (P = .03) but not with LMP-1 or iNOS. In primary NPC tissue, methylation of the COX-2 promoter was seen in 4 of 7 COX-2-negative and 1 of 13 COX-2-positive immunohistochemical cases. COX-2 promoter methylation was found in the CNE-1 cell line. CONCLUSIONS: Nasopharyngeal cancer may be a useful target for selective COX-2 inhibition. The absence of promoter methylation may be a necessary component of COX-2 overexpression, and promoter methylation may be one of the mechanisms that regulate COX-2 expression.