Palliative treatment of patients with inoperable locally advanced, recurrent or metastatic head and neck squamous cell cancer, using a low-dose and personalized chemotherapeutic regimen.

Inoperable or metastatic head and neck squamous cell cancer (HNSCC) is known to be associated with a poor patient prognosis. First line therapies include a Taxol, platinum-based antineoplastic and fluorouracil (FU) treatment regimen (TPF) or a platinum-based antineoplastic, FU and EGFR inhibitor treatment regimen (PFE). The toxicity of these regimens is one of the major limiting factors, particularly for palliative treatment. The present study is a retrospective study of 15 patients with HNSCC, where the treatment goal was palliative. Of the 15 patients, 8 received a TPF, while 7 received a PFE. A total of 129 treatment cycles were administered with a median of 9 cycles (range, 3-14). Chemotherapy began with low doses and was subsequently titrated up based on tolerance and response. Positive responses were noted with the lower doses compared with the conventional doses, and maximal doses were not required. The median dose of cisplatin, paclitaxel and 5-FU administered was 40 mg/m2, 80 mg/m2 and 360 mg/m2/day for 5 days, respectively. Cetuximab was used at a standard dose. At the initial follow-up (mean, 64 days; 3 cycles), a 100% disease control rate (DCR) and 80% overall response rate (ORR) was achieved. A positive response, 60% DCR and 60% ORR, was maintained until the late stages of the study (mean, 217 days; 9 cycles). Following termination of chemotherapy after >9 cycles, 4 patients remained disease free for ~1 year. A total of 3 patients exhibited a pathologic complete response despite radiologically exhibiting residual disease. The median progression-free survival time was 10.03 months and the overall survival time was 15.77 months. The only grade 3 hematologic toxicity noted was neutropenia in 3 (20%) patients. Grade 3 vomiting was noted in 1 (6.67%) patient and grade 3 stomatitis was noted in 1 (6.67%) patient. Due to low toxicity patients exhibited improved tolerance to this approach, particularly in terms of palliative care. Furthermore, these results are in contrast to the axiom that increased doses are more effective.

BRM Promoter Polymorphisms and Survival of Advanced Non-Small Cell Lung Cancer Patients in the Princess Margaret Cohort and CCTG BR.24 Trial.

Introduction: BRM, a key catalytic subunit of the SWI/SNF chromatin remodeling complex, is a putative tumor susceptibility gene that is silenced in 15% of non-small cell lung cancer (NSCLC). Two novel BRM promoter polymorphisms (BRM-741 and BRM-1321) are associated with reversible epigenetic silencing of BRM protein expression.Experimental Design: Advanced NSCLC patients from the Princess Margaret (PM) cohort study and from the CCTG BR.24 clinical trial were genotyped for BRM promoter polymorphisms. Associations of BRM variants with survival were assessed using log-rank tests, the method of Kaplan and Meier, and Cox proportional hazards models. Promoter swap, luciferase assays, and chromatin immunoprecipitation (ChIP) experiments evaluated polymorphism function. In silico analysis of publicly available gene expression datasets with outcome were performed.Results: Carrying the homozygous variants of both polymorphisms ("double homozygotes", DH) when compared with those carrying the double wild-type was associated with worse overall survival, with an adjusted hazard ratios (aHR) of 2.74 (95% CI, 1.9-4.0). This was confirmed in the BR.24 trial (aHR, 8.97; 95% CI, 3.3-18.5). Lower BRM gene expression (by RNA-Seq or microarray) was associated with worse outcome (P < 0.04). ChIP and promoter swap experiments confirmed binding of MEF2D and HDAC9 only to homozygotes of each polymorphism, associated with reduced promoter activity in the DH.Conclusions: Epigenetic regulatory molecules bind to two BRM promoter sequence variants but not to their wild-type sequences. These variants are associated with adverse overall and progression-free survival. Decreased BRM gene expression, seen with these variants, is also associated with worse overall survival. Clin Cancer Res; 23(10); 2460-70. ©2016 AACR.

An alternative approach with a low dose and prolonged chemotherapy for palliative treatment of locally advanced, metastatic or recurrent squamous cell head and neck cancer

Background: It is expected that about 65,000 new patients will be diagnosed with head and neck cancer in 2017 in the United States. Patients with recurrent or advanced or metastatic head and neck do not have good survival due to aggressive and recurrent nature of this cancer. Moreover, cumulative and residual toxicities from previous and ongoing treatments significantly impede quality of remaining part of their life. Currently available chemotherapeutic regimens for this group are derived from the treatments used for the potentially curable disease. These regimens and associated toxicity are obviously not the best matches for the treatment with palliative intent. We here present a retrospective study where we used dose-adjusted chemotherapy specifically for palliative treatment this sub-group of head and neck cancer patients. Methods: Study population was identified from the University of Florida, and IRB approval was obtained. We used currently available and approved chemotherapeutic agents (including Taxols, Platins, 5-Fluorouracil and Epidermal Growth Factor Receptor inhibitors) for treatment of head and neck cancer but dose-adjusted at approximate 50% dose of currently recommended doses. We then gave personalized doses for a prolonged period by titrating doses based on response and tolerability of each patient. Data was collected for treatment, response, side effects, and outcomes. KM analysis was performed for survival data. Results: Total of 32 patients were included in this study with a median age of 65.2 years and a median follow-up of 10.1 months. 62.5% (n = 20) had locally advanced disease and rest had metastatic disease. 37.5% (n = 12) had new disease while rest had recurrent cancer. Of 32 patients, 14 patients received TPF based while 18 patients received PFE based chemotherapy. Total of 270 chemotherapy cycles were delivered among these 32 patients. They received a median of 9 cycles (range 3­14) over a median of 6.2 months (range 1.8­21.1). With this treatment approach, we noted median progression-free survival of 14.0 months and median overall survival of 15.7 months. Notable grade 3 toxicities were generalized fatigue in 12.5% (n = 4), nausea/vomiting in 6.3% (n = 2), diarrhea in in 6.3% (n = 2), mouth soreness in 6.3% (n = 2), rash in 3.1% (n = 1), neutropenia in 18% (n = 6) and anemia in 15.6% (n = 5) while notable grade 4 toxicities were neutropenia and anaphylaxis in 3.1% (n = 1) patient each (AU)

Base of tongue squamous cell carcinoma with infiltrative bone marrow carcinomatosis after definitive chemoradiation: A case report.

BACKGROUND: Oropharyngeal squamous cell carcinoma (SCC) is known for its propensity for aggressive local progression and regional lymphatic spread. Distant metastases are relatively uncommon and the likelihood of hematogenous dissemination is primarily related to the extent and location of cervical lymph node metastases. Common sites of distant metastasis include the liver and lung. METHODS: We report an unusual case of base of tongue SCC with infiltrative bone marrow carcinomatosis presenting months after definitive chemoradiation despite locoregional control. RESULTS: Our patient exhibited an unusual pattern of distant dissemination after definitive chemoradiation had resulted in locoregional control. CONCLUSION: Patients who present with bone marrow failure after definitive treatment with apparent disease control should be monitored for bone marrow infiltration by the tumor and, if such infiltration is present, should be evaluated for palliative chemotherapy. Unfortunately, the prognosis for such patients is poor. © 2016 Wiley Periodicals, Inc. Head Neck 38: E2449-E2453, 2016.

Loss of the SWI/SNF ATPase subunits BRM and BRG1 drives lung cancer development.

Inactivation of Brg1 and Brm accelerated lung tumor development, shortened tumor latency, and caused a loss of differentiation. Tumors with Brg1 and/or Brm loss recapitulated the evolution of human lung cancer as observed by the development of local tumor invasion as well as distal tumor metastasis, thereby making this model useful in lung cancer studies. Brg1 loss contributed to metastasis in part by driving E-cadherin loss and Vimentin up-regulation. By changing more than 6% of the murine genome with the down-regulation of tumor suppressors, DNA repair, differentiation and cell adhesion genes, and the concomitant up-regulation of oncogenes, angiogenesis, metastasis and antiapoptosis genes, caused by the dual loss of Brg1/Brm further accelerated tumor development. Additionally, this Brg1/Brm-driven change in gene expression resulted in a nearly two-fold increase in tumorigenicity in Brg1/Brm knockout mice compared with wild type mice. Most importantly, Brg1/Brm-driven lung cancer development histologically and clinically reflects human lung cancer development thereby making this GEMM model potentially useful.

BRG1 and BRM loss selectively impacts RB and P53, respectively: BRG1 and BRM have differential functions in vivo.

The SWI/SNF complex is an important regulator of gene expression that functions by interacting with a diverse array of cellular proteins. The catalytic subunits of SWI/SNF, BRG1 and BRM, are frequently lost alone or concomitantly in a range of different cancer types. This loss abrogates SWI/SNF complex function as well as the functions of proteins that are required for SWI/SNF function, such as RB1 and TP53. Yet while both proteins are known to be dependent on SWI/SNF, we found that BRG1, but not BRM, is functionally linked to RB1, such that loss of BRG1 can directly or indirectly inactivate the RB1 pathway. This newly discovered dependence of RB1 on BRG1 is important because it explains why BRG1 loss can blunt the growth-inhibitory effect of tyrosine kinase inhibitors (TKIs). We also observed that selection for Trp53 mutations occurred in Brm-positive tumors but did not occur in Brm-negative tumors. Hence, these data indicate that, during cancer development, Trp53 is functionally dependent on Brm but not Brg1. Our findings show for the first time the key differences in Brm- and Brg1-specific SWI/SNF complexes and help explain why concomitant loss of Brg1 and Brm frequently occurs in cancer, as well as how their loss impacts cancer development.

Association of two BRM promoter polymorphisms with head and neck squamous cell carcinoma risk.

The SWI/SNF chromatin remodeling complex is an important regulator of gene expression that has been linked to cancer development. Expression of Brahma (BRM), a critical catalytic subunit of SWI/SNF, is lost in a variety of solid tumors. Two novel BRM promoter polymorphisms (BRM-741 and BRM-1321) have been correlated with BRM loss and elevated cancer risk. The aim(s) of this study were to examine BRM expression in head and neck squamous cell carcinoma (HNSCC) and to correlate BRM polymorphisms with HNSCC risk. BRM expression studies were performed on eight HNSCC cell lines and 76 surgically resected tumor samples. A case-control study was conducted on 668 HNSCC patients (oral cavity, oropharynx, larynx and hypopharynx) and 700 healthy matched controls. BRM expression was lost in 25% of cell lines and 16% of tumors. The homozygous genotype of each polymorphism was significantly associated with increased HNSCC risk [BRM-741: adjusted odds ratio (aOR) 1.75, 95% CI 1.2-2.3, P < 0.001; BRM-1321: aOR 1.65, 95% CI 1.2-2.2, P < 0.001]. Individuals that were homozygous for both BRM polymorphisms had a more than 2-fold increase in the risk of HNSCC (aOR 2.23, 95% CI 1.5-3.4, P < 0.001). A particularly elevated risk was seen within the oropharynx, human papillomavirus-positive subgroup for carriers of both homozygous variants (aOR 3.09, 95% CI 1.5-6.8, P = 0.004). BRM promoter polymorphisms appear to act as susceptibility markers of HNSCC with potential utility in screening, prevention and treatment.

Deep Sequence Analysis of Non-Small Cell Lung Cancer: Integrated Analysis of Gene Expression, Alternative Splicing, and Single Nucleotide Variations in Lung Adenocarcinomas with and without Oncogenic KRAS Mutations.

KRAS mutations are highly prevalent in non-small cell lung cancer (NSCLC), and tumors harboring these mutations tend to be aggressive and resistant to chemotherapy. We used next-generation sequencing technology to identify pathways that are specifically altered in lung tumors harboring a KRAS mutation. Paired-end RNA-sequencing of 15 primary lung adenocarcinoma tumors (8 harboring mutant KRAS and 7 with wild-type KRAS) were performed. Sequences were mapped to the human genome, and genomic features, including differentially expressed genes, alternate splicing isoforms and single nucleotide variants, were determined for tumors with and without KRAS mutation using a variety of computational methods. Network analysis was carried out on genes showing differential expression (374 genes), alternate splicing (259 genes), and SNV-related changes (65 genes) in NSCLC tumors harboring a KRAS mutation. Genes exhibiting two or more connections from the lung adenocarcinoma network were used to carry out integrated pathway analysis. The most significant signaling pathways identified through this analysis were the NFκB, ERK1/2, and AKT pathways. A 27 gene mutant KRAS-specific sub network was extracted based on gene-gene connections from the integrated network, and interrogated for druggable targets. Our results confirm previous evidence that mutant KRAS tumors exhibit activated NFκB, ERK1/2, and AKT pathways and may be preferentially sensitive to target therapeutics toward these pathways. In addition, our analysis indicates novel, previously unappreciated links between mutant KRAS and the TNFR and PPARγ signaling pathways, suggesting that targeted PPARγ antagonists and TNFR inhibitors may be useful therapeutic strategies for treatment of mutant KRAS lung tumors. Our study is the first to integrate genomic features from RNA-Seq data from NSCLC and to define a first draft genomic landscape model that is unique to tumors with oncogenic KRAS mutations.

The chromatin remodelling factor BRG1 is a novel binding partner of the tumor suppressor p16INK4a.

BACKGROUND: CDKN2A/p16INK4a is frequently altered in human cancers and it is the most important melanoma susceptibility gene identified to date. p16INK4a inhibits pRb phosphorylation and induces cell cycle arrest, which is considered its main tumour suppressor function. Nevertheless, additional activities may contribute to the tumour suppressor role of p16INK4a and could help explain its specific association with melanoma predisposition. To identify such functions we conducted a yeast-two-hybrid screen for novel p16INK4a binding partners. RESULTS: We now report that p16INK4a interacts with the chromatin remodelling factor BRG1. We investigated the cooperative roles of p16INK4a and BRG1 using a panel of cell lines and a melanoma cell model with inducible p16INK4a expression and BRG1 silencing. We found evidence that BRG1 is not required for p16INK4a-induced cell cycle inhibition and propose that the p16INK4a-BRG1 complex regulates BRG1 chromatin remodelling activity. Importantly, we found frequent loss of BRG1 expression in primary and metastatic melanomas, implicating this novel p16INK4a binding partner as an important tumour suppressor in melanoma. CONCLUSION: This data adds to the increasing evidence implicating the SWI/SNF chromatin remodelling complex in tumour development and the association of p16INK4a with chromatin remodelling highlights potentially new functions that may be important in melanoma predisposition and chemoresistance.

The expression of the SWI/SNF ATPase subunits BRG1 and BRM in normal human tissues.

SWI/SNF is a chromatin-remodeling complex important in gene regulation, cytokine responses, tumorigenesis, differentiation, and development. As a multitude of signaling pathways require SWI/SNF, loss of SWI/SNF function is expected to have an impact on cellular phenotypes. The SWI/SNF ATPase subunits, BRG1 and BRM, have been shown to be lost in a subset of human cancer cell lines and human primary cancers and may represent tumor suppressor proteins. To better understand the biology of these proteins, the authors examined the expression pattern of BRG1 and BRM in a variety of normal tissues. BRG1 expression was predominantly seen in cell types that constantly undergo proliferation or self-renewal; in contrast, BRM was preferentially expressed in brain, liver, fibromuscular stroma, and endothelial cell types, cell types not constantly engaged in proliferation or self-renewal. This differential expression suggests that these proteins serve distinct functions in human tissues.

Loss of BRG1/BRM in human lung cancer cell lines and primary lung cancers: correlation with poor prognosis.

A role for the SWI/SNF complex in tumorigenesis based on its requirement for retinoblastoma induced growth arrest and p53-mediated transcription and the appearance of tumors in SWI/SNF-deficient mice. In addition, Western blot data have shown that the SWI/SNF ATPase subunits cell, BRG1 and BRM (BRG1/BRM), are lost in approximately 30% of human non-small lung cancer cell lines. To determine whether loss of expression of these proteins occurs in primary tumors, we examined their expression in 41 primary lung adenocarcinomas and 19 primary lung squamous carcinomas by immunohistochemistry. These analyses showed that 10% of tumors show a concomitant loss of BRG1 and BRM expression. Moreover, patients with BRG1/BRM-negative carcinomas, independent of stage, have a statistically significant decrease in survival compared with patients with BRG1/BRM. This report provides supportive evidence that BRG1 and BRM act as tumor suppressor proteins and implicates a role for their loss in the development of non-small cell lung cancers.

Re-expression of hSNF5/INI1/BAF47 in pediatric tumor cells leads to G1 arrest associated with induction of p16ink4a and activation of RB.

Truncating mutations and homozygous deletions in the hSNF5/INI1/BAF47 subunit of human SWI/SNF complexes occur in most malignant rhabdoid tumors and some other malignancies. How loss of hSNF5 contributes to tumorigenesis remains unknown. Because the SWI/SNF subunit BRG1 is required for RB-mediated cell cycle arrest, we hypothesized that hSNF5 deficiency disrupts RB signaling. Here we demonstrate that unlike BRG1, hSNF5 deficient cells retain functional RB since ectopic expression of either p16ink4a or a constitutively active form of RB (PSM-RB) led to cell cycle arrest. To determine how hSNF5 loss might contribute to tumorigenesis, we used a retrovirus to introduce hSNF5 into multiple deficient cell lines. In all cases, re-expression inhibited colony formation and induced cell cycle arrest characterized by a flattened morphology. Flow cytometry revealed that these cells accumulated in G0/G1. Importantly, arrested cells exhibited strong induction of p16ink4a, hypophosphorylated RB, and down-regulation of cyclin A, suggesting that hSNF5 signals upstream of RB to induce growth arrest. Co-expression of SV40 T/t abolished hSNF5-induced G1 arrest and activation of RB. Likewise, HPV-16 E7 was sufficient to partially overcome cell cycle arrest. These results suggest that hSNF5 loss is not equivalent to BRG1/BRM loss in human tumor cell lines. Furthermore, hSNF5-induced cell cycle arrest of deficient cells is mediated in part through activation of p16ink4a expression. These findings provide insight into mechanisms of hSNF5-mediated tumor suppression.

Concomitant down-regulation of BRM and BRG1 in human tumor cell lines: differential effects on RB-mediated growth arrest vs CD44 expression.

Mammalian cells express two homologs of the SWI2 subunit of the SWI/SNF chromatin-remodeling complex called BRG1 and BRM. Whether the SWI/SNF complexes formed by these two subunits perform identical or different functions remains an important question. In this report, we show concomitant down-regulation of BRG1 and BRM in six human tumor cell lines. This down-regulation occurs at the level of mRNA abundance. We tested whether BRM could affect aberrant cellular functions attributed to BRG1 in tumor cell lines. By transient transfection, we found that BRM can restore RB-mediated cell cycle arrest, induce expression of CD44 protein and suppress Cyclin A expression. Therefore, BRM may be consistently down-regulated with BRG1 during neoplastic progression because they share some redundant functions. However, assorted tissues from BRM null/BRG1-positive mice lack CD44 expression, suggesting that BRM-containing SWI/SNF complexes regulate expression of this gene under physiological conditions. Our studies further define the mechanism by which chromatin-remodeling complexes participate in RB-mediated cell cycle arrest and provide additional novel evidence that the functions of SWI/SNF complexes containing BRG1 or BRM are not completely interchangeable.

Compensation of BRG-1 function by Brm: insight into the role of the core SWI-SNF subunits in retinoblastoma tumor suppressor signaling.

The BRG-1 subunit of the SWI-SNF complex is involved in chromatin remodeling and has been implicated in the action of the retinoblastoma tumor suppressor (RB). Given the importance of BRG-1 in RB function, germ line BRG-1 mutations in tumorigenesis may be tantamount to RB inactivation. Therefore, in this study we assessed the behavior of cells harboring discrete BRG-1 alleles for the RB-signaling pathway. Using p16ink4a, an upstream activator of endogenous RB, or a constitutively active RB construct (PSM-RB), we determined that the majority of tumor lines with germ line defects in BRG-1 were sensitive to RB-mediated cell cycle arrest. By contrast, A427 (lung carcinoma) cells were resistant to expression of p16ink4a and PSM-RB. Analysis of the SWI-SNF subunits in the different tumor lines revealed that A427 are deficient for BRG-1 and its homologue, Brm, whereas RB-sensitive cell lines retained Brm expression. Similarly, the RB-resistant SW13 and C33A cell lines were also deficient for both BRG-1/Brm. Reintroduction of either BRG-1 or Brm into A427 or C33A cells restored RB-mediated signaling to cyclin A to cause cell cycle arrest. Consistent with this compensatory role, we observed that Brm could also drive expression of CD44. We also determined that loss of these core SWI-SNF subunits renders SW13 cells resistant to activation of the RB pathway by the chemotherapeutic agent cisplatin, since reintroduction of either BRG-1 or Brm into SW13 cells restored the cisplatin DNA-damage checkpoint. Together, these data demonstrate that Brm can compensate for BRG-1 loss as pertains to RB sensitivity.