Jas C. Lang

Significance of circulating tumor cells in patients with squamous cell carcinoma of the head and neck: initial results.

OBJECTIVES to present and discuss a high-performance negative depletion method for the isolation of circulating tumor cells (CTCs) in the blood of patients with head and neck cancer and to determine the correlation between the presence of CTCs and early clinical outcome in these patients. DESIGN prospective clinical follow-up study of patients with squamous cell carcinoma of the head and neck (SCCHN) undergoing surgical intervention, who had peripheral blood examined for the presence of CTCs. PATIENTS the study population comprised 48 patients diagnosed as having SCCHN and undergoing surgical intervention. INTERVENTION a negative depletion process to isolate and quantify CTCs from the blood of patients with SCCHN using immunomagnetic separation was developed and validated. Immunostaining for cytokeratin was performed on the enriched samples to determine the number of CTCs extracted from each patients blood sample. Correlation of the presence of CTCs, tumor stage, nodal status, clinical characteristics, and outcome was made. MAIN OUTCOME MEASURE disease-free survival. RESULTS our initial data, that have a mean follow-up of 19.0 months, suggest that patients with no detectable CTCs per milliliter of blood had a significantly higher probability of disease-free survival (P = .01). There was no correlation between the presence of CTCs with regard to age, sex, tumor site, stage, or nodal involvement. CONCLUSIONS our enrichment technology, based on the removal of normal cells, has been used on the peripheral blood of patients with head and neck cancer for which follow-up data were collected. If no CTCs were present, a statistically significant improved disease-free survival was observed in SCCHN. A blood test with such a prognostic capability could have important implications in the treatment of patients with head and neck cancer.

Somatic INK4a‐ARF locus mutations: A significant mechanism of gene inactivation in squamous cell carcinomas of the head and neck

The INK4a‐ARF locus is located on human chromosome 9p21 and is known to encode two functionally distinct tumor‐suppressor genes. The p16INK4a (p16) tumor‐suppressor gene product is a negative regulator of cyclin‐dependent kinases 4 and 6, which in turn positively regulate progression of mammalian cells through the cell cycle. The p14ARF tumor‐suppressor gene product specifically interacts with human double minute 2, leading to the subsequent stabilization of p53 and G1 arrest. Previous investigations analyzing the p16 gene in squamous cell carcinomas of the head and neck (SCCHNs) have suggested the predominate inactivating events to be homozygous gene deletions and hypermethylation of the p16 promoter. Somatic mutational inactivation of p16 has been reported to be low (0–10%, with a combined incidence of 25 of 279, or 9%) and to play only a minor role in the development of SCCHN. The present study examined whether this particular mechanism of INK4a/ARF inactivation, specifically somatic mutation, has been underestimated in SCCHN by determining the mutational status of the p16 and p14ARF genes in 100 primary SCCHNs with the use of polymerase chain reaction technology and a highly sensitive, nonradioactive modification of single‐stranded conformational polymorphism (SSCP) analysis termed “cold” SSCP. Exons 1α, 1β, and 2 of INK4a/ARF were amplified using intron‐based primers or a combination of intron‐ and exon‐based primers. A total of 27 SCCHNs (27%) exhibited sequence alterations in this locus, 22 (22%) of which were somatic sequence alterations and five (5%) of which were a single polymorphism in codon 148. Of the 22 somatic alterations, 20 (91%) directly or indirectly involved exon 2, and two (9%) were located within exon 1α. No mutations were found in exon 1β. All 22 somatic mutations would be expected to yield altered p16 proteins, but only 15 of them should affect p14ARF proteins. Specific somatic alterations included microdeletions or insertions (nine of 22, 41%), a microrearrangement (one of 22, 5%), and single nucleotide substitutions (12 of 22, 56%). In addition, we analyzed the functional characteristics of seven unique mutant p16 proteins identified in this study by assessing their ability to inhibit cyclin‐dependent kinase 4 activity. Six of the seven mutant proteins tested exhibited reduced function compared with wild‐type p16, ranging from minor decreases of function (twofold to eightfold) in four samples to total loss of function (29‐ to 38‐fold decrease) in two other samples. Overall, somatic mutation of the INK4a/ARF tumor suppressor locus, resulting in functionally deficient p16 and possibly p14ARF proteins, seems to be a prevalent event in the development of SCCHN. Mol. Carcinog. 30:26–36, 2001.

p16 Mutation Frequency and Clinical Correlation in Head and Neck Cancer

The tumor suppressor gene p16, when altered, has been shown to play a role in oncogenesis in many different tumor types including head and neck cancer. The goal of this study was to analyse alterations to p16 in squamous cell carcinoma (SCC) of the head and neck and to correlate these with clinical outcome. RNA was isolated from 26 SCC head and neck tumors and from 24 matched controls. A reverse transcription polymerase chain reaction was utilized to generate p16 cDNA, which was sequenced and analysed for alterations. In the 26 patient group 58% of the tumors had a p16 alteration, which were characterized by: 8 deletions, 1 insertion/deletion, 4 point mutations and 2 with no p16 expression. In 24 matched normal tissue samples there were no p16 alterations. Those patients with p16 alterations appear to have survival rates comparable to those without p16 alterations, although patients with p16 alterations appear to have more recurrences.

GRS, a novel member of the Bcl-2 gene family, is highly expressed in multiple cancer cell lines and in normal leukocytes.

Our laboratory previously described the independent isolation of the fibroblast growth factor 4 (FGF-4) gene by NIH3T3 transformation assay using DNA from a patient with CML leukemia (Lucas et al., 1994). The FGF-4 gene was truncated by DNA rearrangement with a novel gene named GRS. In this manuscript we describe isolation of GRS cDNA and show by sequence comparison that GRS is a novel member of the Bcl-2 gene family. Northern analysis shows expression of the gene in normal human tissue to be largely restricted to the hematopoietic compartment. Analysis of the pattern of gene expression in cancer cell lines demonstrates GRS is expressed in hematopoietic malignancies and in melanoma. The chromosomal location of GRS has also been determined. The gene is positioned on chromosome 15 within bands q24-25.

Frequent Mutation of p16 in Squamous Cell Carcinoma of the Head and Neck

RNA was isolated from 22 squamous cell carcinomas (SCCs) obtained from diverse sites within the head and neck and from matched normal tissue where available. Tissue samples were then screened for expression of RNA from tumor suppressor gene p16 by utilizing semiquantitative reverse transcriptase polymerase chain reaction (RT‐PCR) analysis. p16‐Specific PCR amplification products generated from tumor samples were subject to further analysis by direct DNA sequencing to determine if any tumor sample harbored a p16 mutation. The results show the presence of mutations in 10 of 22 (45%) of the tumor samples. Mutations comprise two identical point mutations, two small deletions (1 bp and 2 bp), one single‐nucleotide insertion, four larger deletions, and an insertion/deletion. No mutations in p16 have been identified by analysis of PCR products generated from normal matched tissue, suggesting that p16 alterations are generated by somatic mutation and are not germline in origin. All 22 samples were analyzed additionally by immunohistochemistry for nuclear expression of the retinoblastoma (RB) tumor suppressor gene product. Results show lack of RB nuclear expression in only one sample, suggesting that mutation of RB is an infrequent event in the development of SCC of the head and neck (SCCHN).

Identification of circulating tumor cells: a prognostic marker in squamous cell carcinoma of the head and neck?

In 2010, according to the NCI, 49,260 new cases of oral cavity, pharyngeal, and laryngeal cancer were estimated to occur in the United States; approximately 11,480 deaths were attributed to these cases. More than 95% of these cases are squamous cell carcinomas (1, 2). For all stages combined, the 5-year survival rate is approximately 50% (3), and this rate has not changed significantly in the last several decades. Treatment failure in patients with squamous cell carcinoma of the head and neck (SCCHN) can include local recurrence, regional recurrence (cervical lymph nodes), distant metastasis, or development of a second primary cancer. There is certainly a need for a reliable blood test to determine prognosis in SCCHN patients, specifically those patients who may be at increased risk of locoregional recurrence or distant metastasis.

Activation of fibroblast growth factor 8 gene expression in human embryonal carcinoma cells.

To study the role of fibroblast growth factor 8 (FGF-8) in human development and malignancies, we have isolated and characterized its gene. The gene spans 6.0 kbp and is comprised of five exons. Using reverse transcription-polymerase chain reaction, we were able to show that FGF-8 is expressed in two of the seven human mammary carcinoma cell lines tested and in only one of nine breast tumors. In contrast, both of the two normal breast tissues tested express FGF-8. FGF-8 was previously shown to be present in adult testis and ovary. Surprisingly, only one of the seven testis carcinomas and one of 12 ovary carcinomas express FGF-8, whereas all three kidney carcinomas tested express FGF-8. We further showed that fetal brain and lung express FGF-8, whereas fetal intestine and liver do not. Finally, we showed that a teratocarcinoma cell line, Tera-2, can be induced to express FGF-8 mRNA by fetal bovine serum.

Effect of surgical intervention on circulating tumor cells in patients with squamous cell carcinoma of the head and neck using a negative enrichment technology

The purpose of this study was to investigate the impact of surgical intervention on detection of circulating tumor cells (CTCs) in patients with squamous cell carcinoma of the head and neck (SCCHN.)

Circulating tumor cells in head and neck cancer: A review

Carcinoma of the head and neck represents 3.5% of all cancers, and the vast majority of these tumors are squamous cell carcinoma (HNSCC). With a stable overall survival rate of 50% among all stages, there is continued interested in developing measures for early detection and disease aggressiveness. Circulating tumor cells (CTCs) have been identified as a potential marker for early metastatic disease, response to treatment, and surveillance in head and neck squamous cell carcinoma. In this article, techniques of CTC detection, applications of CTC technology, and outcomes of HNSCC patients will be discussed.

Abstract 3284: Isolation of circulating tumor cells (CTCs) with mesenchymal and stem cell markers in localized and metastatic breast cancer using a novel negative selection enrichment

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: Breast circulating tumor cells (CTCs) are commonly isolated by positive selection enrichment technology, which targets the epithelial cell adhesion activating molecule, EpCAM. However, CTCs with low or no EpCAM expression, such as those from basal and normal-like subtypes, are more likely to be missed by this method. We developed a novel negative enrichment technology to detect CTCs with mesenchymal and stem cell markers in localized and metastatic breast cancer (BC). Patients and Methods: Twenty nine patients with localized and metastatic BC initiating chemotherapy were enrolled. CTCs were isolated in 10 mL of peripheral blood using negative selection with immunomagnetic tagging and removal of CD45 positive cells at 3 time points: pretreatment, after one cycle of treatment (TP1) and at end of treatment or at disease progression in metastatic patients (TP2). Immunocytochemical staining for nucleus, cytokeratin (8,18,19), vimentin, and CD44 was completed on available samples. Double staining for nucleus and cytokeratin with high nucleus to cytoplasm ratio defined a CTC. Enrollment is ongoing. Results: Median age was 57 yrs (range 28-78 yrs); stage distributions were I-2 (7%), II-10 (35%), III-3 (10%), and IV-14 (48%); 19 (66%) were estrogen receptor positive (ER+), 4 (14%) estrogen and progesterone receptor negative (ER-PR-HER2 non-overexpressing) and 6 (21%) HER2 overexpressing. Negative enrichment yielded an average log10 depletion of nucleated cells of 2.74 and an overall, average log10 depletion of 5.2 (>100,000 enrichment). No CTCs were identified in 5 healthy volunteers or in buffy coats purchased from the Red Cross. CTCs were identified in all stages at pretreatment but decreased to 0 in three patients by TP1. Baseline median CTC level was 373/mL in localized BC (range 2.2-1975/mL) and 761/mL in metastatic BC (range 9.9-47513/mL). In localized BC, the median percent (%) change in CTCs was +2% at TP1 (n= 13; range −100% to +14945%) and −95% at TP2 (n=5; range −100% to −13%). In metastatic BC, median CTC numbers decreased by 41% at TP1 (n=11; range −100% to +4222%) and increased at TP2 in 2 patients by 231% and 730%. Baseline CTC levels and changes at TP1 were not significantly different between localized and metastatic groups by the Wilcoxen Rank Sum test. All tested CTCs expressed vimentin and CD44. In addition to CTCs, a population of cells without detectable cytokeratin expression but positive for the other markers was identified in some samples. Further characterization of these cells for epithelial mesenchymal transition markers is underway. Conclusions: CTCs identified with this novel negative selection method have both mesenchymal and stem cell markers in localized and metastatic BC. Higher CTC numbers with epithelial characteristics are detected with this method relative to what is reported with positive selection. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3284.