Molecular Cytogenetic Characterization of Head and Neck Squamous Cell Carcinoma and Refinement of 3q Amplification

Cell Lines.

Eleven human cell lines derived from HNSCC (584, MDA686, MDA886, MSK921, MSK922, MDA1186, MDA1386, 1483, MDA1586, MDA1986, and MSKQLL2) and one cell line derived from premalignant oral epithelium (MSK Leuk1) were used in the present study. Of these cell lines, MSK921, MSK922, MSKQLL2, and MSK Leuk1 were established at Memorial Sloan-Kettering Cancer Center; MDA686, MDA886, MDA1186, MDA1386, MDA1586, and MDA1986 were established at The University of Texas M. D. Anderson Cancer Center (Houston, TX); and 584 and 1483 were established at Wadsworth Laboratories, New York State Department of Health (Albany, NY). The cells were grown to 80% confluence at 37°C in a 5% CO₂ atmosphere in RPMI 1640 supplemented with 10% FCS and penicillin-streptomycin. MSK Leuk1 was grown in serum-free KGM medium (BioWittaker, Rockland, ME). Metaphase chromosome preparations were obtained from each cell line by standard methods. The metaphase preparations were aged for a week before SKY analysis.

CGH.

The DNA from each cell line was labeled using nick translation (Life Technologies, Inc., Rockville, MD) with fluorescein-12-dUTP. As a control, we used DNA extracted from normal placenta and labeled it with Texas red-5-uUTP (New England Nuclear-DuPont, Boston, MA). CGH was performed using previously published methods (6 , 7) . Then, 7–10 separate metaphases were captured and processed using the Quantitative Image Processing System (Quips Pathvysion System; Applied Imaging, Santa Clara, CA). Red, green, and blue fluorescence intensities were analyzed for all metaphase spreads, normalized to a standard length, and statistically combined to show the red:green signal ratio and 95% confidence intervals for the entire chromosome. Copy number changes were detected based on the variance of the red:green ratio profile from the standard of 1. Ratio values of 1.2 and 2.0 were defined as thresholds for gains and amplifications, respectively, and losses were defined as a ratio value of 0.8 or less.

SKY.

The mixture of human chromosome paints was obtained from ASI (Carlsbad, CA). Hybridization and detection were carried out according to the manufacturer’s protocol with slight modifications (6) . Chromosomes were counterstained with DAPI. For each case, a minimum of five metaphases was analyzed by SKY. Images were acquired with a SD200 Spectra cube (ASI) mounted on a Nikon Eclipse 800 microscope using a custom-designed optical filter (SKY-1; Chroma Technology, Brattleboro, VT) and analyzed using SKY View 1.2 software (ASI).

Breakpoints on the SKY-painted chromosomes were determined by comparison of corresponding DAPI banding of the same chromosome and by comparison to the G-banded karyotype from the same tumor (4) . A breakpoint was considered recurring if it was identified in two or more cases. A breakpoint cluster was defined as the occurrence of four or more breakpoints on the same chromosomal band.

FISH.

We generated previously a physical map of the region of 3q amplification using 73 YAC clones. The boundaries of 3q amplification were mapped by sequential FISH analysis, identifying an amplification peak contained within three YAC clones. 4 FISH was performed as described previously (13) . In brief, DNA extracted from YAC clones (Research Genetics, Huntsville, AL) was labeled individually by nick translocation using Spectrum green dUTP (Vysis, Downers Grove, IL) and hybridized to the interphase and metaphase spreads from the cell lines. The slides were counterstained with DAPI, and the images were captured using the Quips Pathvysion System (Applied Imaging). To determine the amplification status, 200 individual interphase nuclei and 5 metaphase spreads were analyzed for each cell line. The presence of amplification was accepted if more than 10% of tumor nuclei displayed an increased copy number relative to the chromosome 3 centromeric probe signals and/or tumor ploidy. In cases in which there is a discrepancy between the number of centromeric signals and ploidy, the ploidy number was used to estimate the relative copy number at 3q26.3.

Statistical Analysis.

Statistical comparisons were performed using the JPM software (SAS Institute, Inc., Cary, NC). Pearson correlation coefficient was used to determine the association between the number of structural aberrations identified by SKY and CGH for each cell line.

DNA Copy Number Changes Identified by CGH.

DNA extracted from 12 cell lines was subjected to CGH analysis to identify chromosomal gains and losses. Fig. 1a ⇓ summarizes the copy number changes identified in the present study. Partial CGH karyotypes from MDA1386 and 1483 showing high-level amplification at 3q are shown in Fig. 2a, A and C ⇓ . Underrepresentation of chromosomal regions was noted at 1p, 3p, 4, 8p, 10p, and 18q. The most frequently gained chromosomal regions were 3q, 5p, 8q, 9q, and 14q. High-level amplification was detected at nine chromosomal sites including 3q13, 3q25-q26, 5q22-q23, 7q21, 8q24, 11q13-q14, 12p13, 14q24, and 20q13.1. Recurrent sites of amplification were noted at four sites including 3q25-q26 (42%) in MDA686, MDA886, MDA1186, MDA1386, and 1483; 11q13 (33%) in 584, MDA886, MSK921, and MSK922; 8q24 (17%) in MSK922 and MDA1586; and 9q22-q34 (17%) in MSK922 and MDA1386. Nonrecurrent amplifications were noted at 3q13.3, 7q21, and 12p13 (MDA886); 14q24 (MSK922); and 20q13.1 (MDA1986).

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Fig. 1.

a, ideogram showing DNA copy number changes identified by CGH in the cell lines. Thin vertical lines on either side of the ideogram indicate losses (left) and gains (right) of the chromosomal region. The chromosomal regions of the high-level amplification are shown by thick lines (right). b, ideogram showing all of the breakpoints noted in the cell lines identified by SKY (inverted DAPI). The number of breakpoints in each chromosome that were identified by SKY but could not be precisely assigned to a chromosomal band are noted in the box on top of the chromosome.

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Fig. 2.

a. A and C, partial CGH karyotypes for chromosome 3 (left) and the corresponding ratio profiles (right) illustrating high-level amplification in cell lines MDA1386 (A) and 1483 (C), respectively. The blue line in the ratio profiles represents the mean of 8–10 chromosomes, and the yellow lines indicate the SD. The threshold values were 0.80 and 1.20 for loss and gain, respectively. B and D, detection of 3q amplification in interphase nuclei from cell lines MDA1386 (B) and 1483 (D) by FISH. An increased copy number from the 3q26.3 region (green) was seen in interphase cells (nine copies for MDA1386 and eight copies for 1483) compared with chromosome 3 centromeric signals (five copies for MDA1386 and three copies for 1483). b, A and B, a representative metaphase spread from cell line MDA1386 after hybridization with 24 fluorescence-labeled painting probes showing spectral classification colors (A) and the corresponding inverted DAPI image (B). C and D, a metaphase spread from cell line MDA1386 hybridized with 3q mixture probe (green) and chromosome 3 centromeric probe (red) showing the duplicated region of chromosome 3q26.3. A blowup image of duplicated chromosome 3 is shown in the inset. c, A and B, a representative metaphase spread from the cell line 1483 after hybridization with 24 fluorescence-labeled painting probes showing spectral classification colors (A) and the corresponding inverted DAPI image (B). C and D, a metaphase spread from cell line 1483 hybridized with 3q mixture probe (green) and chromosome 3 centromeric probe (red) showing the duplicated region of chromosome 3q26.3. A blowup image of duplicated chromosome 3 is shown in the inset (for a complete description of chromosome 3 abnormalities, refer to Table 1 ⇓ ). d, A, partial CGH karyotype for chromosome 8 (left) and corresponding ratio profiles (right) showing loss of 8p and gain of 8q in MDA1386. Vertical red and green bars on the right of the ideogram represent the threshold values of 0.80 and 1.20 for loss and gain, respectively. B, partial spectral karyotype (classified images) for chromosomes del(8)(p11), der(8), der(11), and der(14) showing translocated chromosome 8 material.

SKY Analysis.

Spectral analysis identified a range of simple and complex chromosomal rearrangements, which included reciprocal and nonreciprocal translocations, Robertsonian translocations, deletions, and insertions. Representative metaphase spreads from cell lines from MDA1386 and 1483 showing classification colors after hybridization with 24 fluorescence-labeled painting probes are depicted in Fig. 2, b and c ⇓ , respectively.

Analysis of Structural Rearrangements.

A total of 185 translocations, 33 deletions, 4 insertions, and 3 duplications were described in the present study. Recurrent deletions were noted at 3p11, 4q21, 5q13, 9p11, and 12q11. Two reciprocal translocations were noted at t(8;9)(q24;q34) in MSK922 and t(5;11)(q13;q13) in 584. Robertsonian translocations were identified by SKY, involving t(13;13)(q10;q10) in four cell lines (MSK921, MDA1386, 1483, and MDA1986), t(14;14)(q10;q10) in two cell lines (MDA686 and MDA1386), t(14;21)(q10;q10) in 584, t(15;15)(q10;q10) in MDA1186, and t(15;22)(q10;q10) in 1483. Translocations involving the whole chromosomal arm were identified in two cell lines [namely, t(1;13)(q10;q10) in 584 and MSKQLL2, t(3;13)(p10;q10) in MSKQLL2, and t(7;15)(p10;q10) t(10;15) (p10;q10) and t(1;15)(q10;q10) in 584]. Nine isochromosomes were noted, including i(1)(p10) in MSKQLL2; i(2)(p10) in MDA1186; i(3)(q10) in MSK922; i(5)(p10) in MDA686, MDA886, and 1483; i(8)(q10) in 584 and MDA1586; i(9)(q10) in MSK922 and MDA1386; i(10)(p10) in MSK Leuk1; i(10)(q10) in MSK Leuk1; and i(18)(q10) in MDA1986, of which i(5)(p10), i(8)(q10), and i(9)(q10) were recurrent.

Analysis of Breakpoints.

By SKY, 242 breakpoints were identified in rearranged chromosomes, with another 166 breakpoints assigned to chromosomes based on identification of chromosomal segments attached to or inserted into add or der chromosomes but whose specific breakpoints could not be determined. Fig. 1b ⇓ summarizes the breakpoints identified by SKY in these cell lines. Overall, recurring breaks (two or more) were noted in 62 sites. Breakpoint clusters (four or more) were seen at 1p11, 1q21, 3p11, 5q11, 5q13, 6q23, 8p11, 8q11, 9p13, 9q13, 10q11, 11q13, 13q10, 14q10, and 15q10 (Fig. 1b) ⇓ .

Comparison of CGH and SKY Findings.

A summary of CGH and SKY analyses is presented in Fig. 1 ⇓ . The median number of copy number changes identified by CGH was 16 (range, 5–29). The median number of aberrations identified by SKY was 19 (range, 7–26). There was a good correlation between the number of structural aberrations identified by SKY and CGH (r = 0.69; P = 0.05).

The combination of these analyses was confirmatory and complementary in the assessment of chromosomal aberrations. SKY helped to define the chromosomal imbalances identified by CGH as detailed in Table 1 ⇓ ⇓ . As an example, the genomic imbalances affecting chromosome 9 were identified in six cell lines (MSK921, MSK922, MDA1186, MDA1386, MDA1586, and MDA1986). Whole chromosomal gains identified by CGH coincided with the presence of four copies of der(9) in MDA1986 [der(9) t(1;9) t(p?;p24), der(9) t(3;9)(?;p24), der(9)t(9;10)(p?;q10), and der(9)t(9;11)(q13;q13)], and gains at 9q could be explained by the presence of translocation of 9q to chromosome 8 in MDA1186 and the presence of two normal chromosomes and two additional copies of isochromosome for 9q in MSK922. The CGH ratio profiles from the remaining cell lines showed a loss of 9p and a gain of 9q. These imbalances could be attributed to the presence of i(9)(q10) in MDA1386, two copies of normal chromosome 9 and two copies of del(9)(p11) in MSK921, and two copies of normal chromosome 9 and three copies of translocated 9q [der(4)t(4;9)(q11;q12),der(11) t(9;11) (q13;q10), der(14)t(9;14)(q13;q10), and der(20) t(9;20)] and one copy of der(9)t(9;11)(p11;?)t(X;11)(?;?) in MDA1586.

Similarly, the loss of 8p and gain of 8q in cell lines 584 and MDA1386 were correlated with the presence of i(8)(q10). Conversely, the imbalances of chromosome 8 identified by CGH (−8p and +8q) in cell lines MSK921 and MDA1986 were explained by the presence of derivative chromosome 8 - der(8)t(8;8)(p11;q?). An example of comparative CGH and SKY data is depicted in Fig. 2d ⇓ , which displays a loss and gain of the short and long arms of chromosome 8, respectively, in MDA1386.

Comparable symbiosis was seen in the assessment of amplification events, including refinement of amplification at 11q13-q14 identified by CGH in four cases (584, MDA886, MSK921, and MSK922). In the majority of cases, the amplification resulted from translocation of chromosomal material from this region to the other chromosomes in the genome, with the exception of MSK922, where a duplication of the 11q13-q14 region was identified.

The true strength of the combination of SKY and CGH analyses was observed in the refinement of complex rearrangements. As an example, the gain of 7q11-q32, including amplification at 7q21 in MDA886, resulted from the presence of a complex marker chromosome consisting of chromosomes 3, 5, 7, and 10 and two additional copies of translocated 7q chromosome material to chromosome 5.

Moreover, several chromosomal abnormalities were uniquely identified by either CGH or SKY. As an example, amplification at 11q13 and 12p13 in MDA886 was only visible by CGH. Conversely, isochromosomes at i(1)(p10) and i(5)(p10) identified by SKY in MSKQLL2 and 1483, respectively, were not detected in the corresponding CGH profiles. These discrepancies may be due to a low prevalence of these clones in the tumor genome leading to a lack of detection by CGH or sampling limitations by SKY. In addition, in MSK Leuk1, SKY identified i(10)(p10) and i(10)(q10), but CGH could not detect any imbalance on chromosome 10. This assessment highlights the limitation of CGH analysis in detecting balanced abnormalities because there was no apparent loss or gain of DNA sequences from chromosome 10 in this cell line.

Refinement of 3q Amplification by FISH.

The combination of CGH and SKY was insufficient to precisely define several subtle chromosomal aberrations. To illustrate the utility of FISH as a supplementary approach, an analysis of 3q aberrations was performed. Amplification at 3q was identified in 42% of the cell lines in this study, and gains were identified in 25% of the cell lines in this study. The chromosomal derivation of 3q overrepresentation in these cell lines identified by CGH included duplications, insertions, and complex translocations, as defined by SKY (Table 2) ⇓ . High-level amplification of chromosome 3 at the 3q26-q29 region coincided with translocated material from chromosome 3q in MDA686, MDA886, MDA1186, MDA1386, and 1483 (Table 2) ⇓ . In MDA886, 3q26.1-q26.3 amplification results from translocation of chromosome 3q material to chromosomes 2 and 5 and an additional copy of 3q. In the MDA1386 cell line, the amplicon was derived from the translocation of 3q material to chromosomes 2 and 17 and duplication of the 3q25-q26 region (Fig. 2b) ⇓ . However, the quantification of the extent of amplification was not discernible, and precise 3q breakpoints could not be defined by SKY analysis.'

We have characterized previously the 3q26-q27 locus by generating a physical map of the minimal common region of amplification defined by CGH, using 73 YAC clones covering a 38-cM region between sequence-tagged site markers D3S3650 and WI7174.⁴ We identified three highly overlapping, nonchimeric YAC clones containing the apex of amplification. Using a mixture of these clones as a probe for FISH analysis, amplification at 3q26.3 was confirmed and quantified, and the specific breakpoints were defined in each case (Table 2) ⇓ . In cases with high-level amplification, 6–12 copies were identified by FISH using the YAC probe (Fig. 2a, B and D) ⇓ , compared with 4–5 copies in cell lines with gain of 3q and 1–4 copies in cell lines with no gain or amplification detected by CGH. A discrepancy was seen in the number of FISH signals between interphase nuclei and metaphase spreads from the cell lines MDA886 and 1483, which can be attributed to the presence of multiple clones and the number of copies of der(3) chromosomes. Similarly, discrepancies between the number of 3q centromeric signals and tumor ploidy in two cases could be attributed to the presence of duplicated chromosome der(3).

Unassigned chromosome 3 breakpoints identified by SKY were refined as shown in Table 2 ⇓ . As an example, a duplication of 3q in cell line MDA1386 and an insertion of chromosome 3 material into chromosome 2 in MSK922 were noted by SKY. FISH analysis identified the precise breakpoint in both cell lines, identifying the origin of the duplication and insertion as 3q26.3 in MDA1386 (Fig. 2b, C) ⇓ . In addition, in cell line 1483, we identified a der(3)t(3;14)(q26∼q27;?) by SKY, but FISH analysis identified this as a cryptic duplication at 3q26.3 on the der(3) (Fig. 2c, C) ⇓ .