Genetic analysis of single disseminated tumor cells in the lymph nodes and bone marrow of patients with head and neck squamous cell carcinoma

Abstract

Considering the limited information on the biology and molecular characteristics of disseminated tumor cells (DTCs) in head and neck squamous cell carcinoma (HNSCC), we examined the genomic alterations in DTCs from HNSCCs and their potential clinical relevance. To analyze both the lymphatic and hematogenous routes of tumor cell dissemination, we investigated samples from lymph nodes (LNs) and bone marrow (BM) of 49 patients using immunofluorescence double staining for epithelial cells expressing cytokeratin 18 (KRT18) and/or epithelial cell adhesion molecules (EpCAM, CD326). The identified marker-positive cells were isolated by micromanipulation followed by single-cell whole-genome amplification and metaphase-based comparative genomic hybridization (mCGH) to determine genome-wide copy number alterations. The findings were correlated with clinical parameters and follow-up data. We detected chromosomal aberrations in KRT18- and EpCAM-positive cells from both compartments; BM-derived cells showed a significantly higher percentage of aberrant genome (PAG) per cell than cells detected in LNs. No significant association was found between DTC data and clinical follow-up. Genomic profiling of BM-DTCs revealed genomic alterations typical for HNSCC, suggesting hematogenous dissemination of subclones around the time of surgery. In contrast, DTC data in LNs revealed that several marker-positive cells were not of malignant origin, indicating the presence of epithelial glandular inclusions in parts of the processed neck LN samples. Therefore, DTC detection of LNs in the neck based only on epithelial markers is not advisable and requires detection of chromosomal instability (CIN), gene mutations, or additional markers, which have yet to be identified. Nevertheless, our investigation paves the way for larger studies to focus on HNSCC BM-DTCs with high-resolution methods to gain deeper insights into the biology of hematogenous metastasis in this cancer.

Keywords: bone marrow; disseminated tumor cells; genetic alterations; head and neck squamous cell carcinoma; lymph nodes; minimal residual disease.

Fig. 1

Example of a cytokeratin 18/epithelial cell adhesion molecule (KRT18/EpCAM) double‐positive cell, Pat. #30, LN 4, cell No. T3: (A) brightfield, (B) KRT18 (Cy3, red), and (C) EpCAM (Alexa 488, green). Disseminated tumor cell, detached from the adhesive slide, isolated, and captured in a microhematocrit capillary with the help of a micromanipulator (Eppendorf) at 40× magnification. (D) Bright‐field (from Pat. #49, LN 3, cell No. T3), (E) KRT18 (Cy3, red) and (F) EpCAM (Alexa 488, green). KRT18/EpCAM double immunofluorescence staining of the cell line LN1590 at 40× magnification served as a positive control. (G) Bright‐field, H. KRT18 (Cy3, red), and I. EpCAM (Alexa 488, green). Both epithelial antigens were detected in the control cell line LN1590. Scale bars correspond to 50 µm.

Fig. 2

Percentage of aberrant genome per cell (PAG) showing significantly higher values in marker‐positive cells derived from bone marrow (BM) than in those derived from lymph nodes (LN; Wilcoxon–Mann–Whitney U‐test: P = 0.0003). Box plot with median and interquartile box.

Fig. 3

(A) Metaphase‐based comparative genomic hybridization (mCGH) analysis of marker‐positive cells showing genomic gains and losses allocated to chromosomes. Cumulative mCGH plot of (a) all analyzed marker‐positive cells from bone marrow (BM) and lymph node (LN) samples, (b) 23 LN‐derived marker‐positive cells, and (c) 15 BM‐derived marker‐positive cells. Horizontal axis = chromosome number, vertical axis = percentage of genomic aberrations, green = amplifications and red = deletions. (B) Dendrogram of similarity analyses of all disseminated tumor cells (DTCs; percentage of aberrant genome (PAG) > 1%) from bone marrow (BM) and lymph nodes (LNs) using r software. In the dendrogram, the chromosomes are in the ascending order on the y‐axis from top to bottom (no visual numbering). The respective DTC is shown on the x‐axis. The dendrogram is on the top of the x‐axis. Green boxes indicate amplifications and red box indicates deletions. The first sample number (#) corresponds to the patient number; LN = LNs with the corresponding numbering; BM = BM with the corresponding numbering T = tumor cell with the corresponding numbering.

Fig. 4

Number of disseminated tumor cells with copy number alterations in chromosomal regions where known oncogenes and tumor suppressor genes for head and neck squamous cell carcinoma (HNSCC) are located. (A) Number of bone marrow‐derived (BM) and lymph node‐derived (LN) cells (y‐axis) with amplifications in HNSCC oncogenes (A) or losses in tumor suppressors (B) (Tables 3 and 4). Each point corresponds to a gene locus and the most frequently amplified, and deleted gene loci are labeled. P‐values are calculated using a paired Wilcoxon rank‐sum test.