- Email: [email protected]
Heterogeneity, histological features and DNA ploidy in oral carcinoma by image-based analysis
Oral Oncology (2005) 41, 416–422
http://intl.elsevierhealth.com/journals/oron/
Heterogeneity, histological features and DNA ploidy in oral carcinoma by image-based analysis N. Diwakar a, M. Sperandio a, M. Sherriff b, A. Brown a, E.W. Odell
a,*
a
Department of Oral Pathology, Guy’s Hospital, King’s College, Floor 28 Guy’s Tower, London SE1 9RT, UK Department of Biomaterials Science, Guy’s Hospital, King’s College, Floor 17 Guy’s Tower, London SE1 9RT, UK b
Received 13 August 2004; accepted 22 October 2004
KEYWORDS
Summary Oral squamous carcinomas appear heterogeneous on DNA ploidy analysis. However, this may be partly a result of sample dilution or the detection limit of techniques. The aim of this study was to determine whether oral squamous carcinomas are heterogeneous for ploidy status using image-based ploidy analysis and to determine whether ploidy status correlates with histological parameters. Multiple samples from 42 oral squamous carcinomas were analysed for DNA ploidy using an image-based system and scored for histological parameters. 22 were uniformly aneuploid, 1 uniformly tetraploid and 3 uniformly diploid. 16 appeared heterogeneous but only 8 appeared to be genuinely heterogeneous when minor ploidy histogram peaks were taken into account. Ploidy was closely related to nuclear pleomorphism but not differentiation. Sample variation, detection limits and diagnostic criteria account for much of the ploidy heterogeneity observed. Confident diagnosis of diploid status in an oral squamous cell carcinoma requires a minimum of 5 samples. c 2004 Elsevier Ltd. All rights reserved.
Oral carcinoma; Ploidy analysis; Large scale genomic status
Introduction
Cells from the majority of human malignant neoplasms are aneuploid as a result of chromosomal * Corresponding author. Tel.: +44 020 7188 4367; fax: +44 020 7188 4375. E-mail address: [email protected] (E.W. Odell).
instability (imbalance). Chromosomal instability arises from non-disjunction and other mitotic errors and may result in loss or, more frequently gain, of whole or part chromosomes. This acquired genetic instability originates in mutations in genes controlling a number of processes including cell cycle checkpoints, mismatch repair, apoptosis, telomere maintenance, centrosome and spindle function.1
1368-8375/$ - see front matter c 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.oraloncology.2004.10.009
Heterogeneity, histological features and DNA ploidy in oral carcinoma Chromosomal instability is now considered fundamental to the malignant phenotype and is proposed as a cause rather than the result of malignant transformation.2,3 Aneuploidy can be detected in oral carcinomas4,5 and specific chromosomes are more frequently duplicated than others.6 Aneuploidy has been extensively studied in oral and other head and neck carcinomas in relation to prognosis and metastasis, though no convincing relationship has emerged.7,8 In contrast, the study of aneuploidy in oral premalignant lesions shows great promise as a predictor of malignant transformation.9 As part of a larger study of image-based ploidy analysis in oral premalignancy, we have identified a possible population of diploid lesions that may develop into diploid carcinomas. However, this possibility cannot be accepted without an understanding of ploidy heterogeneity within oral carcinomas, because sample variation and other factors could account for spurious diploid results. It is well recognised that, although most carcinomas are clonal in origin, they are heterogeneous with respect to the karyotype and phenotype of clonal populations within them. Oral and other head and neck carcinomas show heterogeneity on the basis of ploidy when assessed by flow cytometry,10–13 comparative genomic hybridisation14 and fluorescent in situ hybridisation.15 Thus, sampling may be critical when assessing the true ploidy status of a carcinoma.13 It is unclear whether heterogeneity might influence the results of image-based ploidy analysis. To address these concerns, we have undertaken an analysis of DNA ploidy heterogeneity in oral carcinoma using an image-based ploidy analysis system. The study was approved by the Guy’s Hospital Research Ethics Committee and the UK Patient Information Advisory Group.
Material and methods Oral squamous carcinomas Multiple blocks of paraffin-embedded, formalinfixed, tissue from 45 consecutive cases of untreated primary oral squamous cell carcinoma were selected from the Oral Pathology Department archive at King’s College, Guy’s Hospital, London. All were accessioned between 2000 and 2003 and exclusion criteria were previous treatment by radiotherapy or chemotherapy, insufficient material remaining for analysis or carcinomas too small to analyse multiple sites within them. The primary
417
sites were the floor of the mouth (n = 15), tongue (n = 15), retromolar region (n = 4), buccal mucosa (n = 2), soft palate, tonsil and fauces (n = 3) and gingiva/tuberosity (n = 3). Carcinoma size was determined from both macroscopic and microscopic pathological staging data. Routine haematoxylin and eosin sections were examined and between 2 and 8 sample areas approximately 5 · 2 mm were selected from each carcinoma (mean 5.02, n = 226). Each sample contained as high a proportion of carcinoma as possible and was of a uniform histological appearance. Samples were selected to include as diverse a range of histological appearances as possible. Each sample was graded histologically for the following parameters: invasive front according to Bryne16,17 differentiation (grades: prominent keratinisation with minority basaloid component, focal keratinisation only, no keratinisation, basaloid, basaloid squamous carcinoma), invasive outline (scored at ·20 magnification, grades: well-defined or irregular) and nuclear pleomorphism (scored in basal and suprabasal cells only: mild, moderate, marked). All grading was performed blind to the ploidy result.
Ploidy analysis Ploidy analysis was performed as previously reported by Sudbø et al.9 Briefly, 6 · 50 lm sections were cut from each selected area, dewaxed in xylene, rehydrated and nuclei extracted using protease type XXIV (Sigma Chemical Co., Poole, UK) according to Hedley.18 Nuclear monolayers were prepared in a cytospin 4 (Thermo Shandon, UK) and stained by the Feulgen–Schiff method. Ploidy analysis was performed on a Fairfield image-based ploidy analyser comprising a high resolution camera (C4742-95, Hamamatsu Photonics K.K., Hamamatsu, Japan) attached to a Zeiss Axioplan II microscope (Zeiss, Oberkochen, Germany) with a 4 slide scanning stage. A total of 226 monolayers were analysed, each with a minimum of 300 nuclei sampled using the automated scanning option. Diagnostic criteria were as previously published for this system and oral lesions.9 A sample was classified as diploid if only one G0/G1 (2c) peak was present, or if the number of nuclei in the G2 (4c) peak did not exceed 10% of the total number of epithelial cells. A sample was defined as DNA tetraploid when the fraction of nuclei in the 4c region exceeded 10% of the total, without corresponding S phase. The tetraploid group thus includes cases intermediate between diploid and aneuploid (oral carcinomas being rarely chromosomally
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tetraploid). A lesion was defined as aneuploid if non-euploid peaks were present, of if the number of nuclei with a DNA content greater than 5c exceeded 1%. All analysis was performed blind to the histological results.
Statistical analysis The interrelationships between ploidy and histological parameters were assessed using JK biplot analysis.19 Specific correlations between histological parameters and ploidy status were determined using Somers’ D in the whole dataset and within the sub groups of ploidy-homogeneous and ploidy-heterogeneous carcinomas. All analyses were performed using Stata 8.2.
Results No ploidy result could be obtained from 13 monolayers, eliminating 3 carcinomas because insufficient samples remained to assess heterogeneity. Twenty two of 42 carcinomas appeared uniformly
Table 1
aneuploid, 1 uniformly tetraploid and 3 uniformly diploid. The site, size, number of samples analysed and ploidy status are shown in Table 1. Sixteen carcinomas appeared heterogeneous and their characteristics and the number of samples diagnosed as aneuploid, tetraploid or diploid is shown in Table 2. Normal and hyperplastic mucosal lesions were uniformly diploid. Histograms revealed subtle ploidy anomalies that were insufficient to meet diagnostic criteria for non-euploidy in all diploid samples from 8 carcinomas. The small peaks often matched the pattern seen in aneuploid samples from the same lesion, suggesting that sample variation accounted for the apparent heterogeneity and that these samples were not truly diploid. The remaining 8 cases that appeared genuinely heterogeneous on the basis of different histogram patterns are in bold in Table 2. This group includes both carcinomas with low and high numbers of samples analysed. An example of a carcinoma classified as ploidy heterogeneous is illustrated in Fig. 1, together with the histological appearances of the two sample sites shown. The JK biplot analysis for the whole series is shown in Fig. 2 indicating that ploidy status is clo-
Origin, size, sampling and ploidy status of apparently homogeneous carcinomas
Case no.
Site
Diameter mm.
No. of samples
Ploidy status
28 33 34 38 35 42 31 43 40 29 04 05 06 08 09 13 16 17 21 23 24 22 18 19 25 02
Tongue Tongue Tongue Tongue Tongue Tongue Tongue Tongue Tongue Tongue Floor of mouth Floor of mouth Floor of mouth Floor of mouth Floor of mouth Floor of mouth Floor of mouth Floor of mouth Retromolar Retromolar Retromolar Retromolar Gingiva Gingiva Soft palate Buccal mucosa
8·8 30 · 18 10 · 8 10 · 18 12 · 13 40 · 35 17 · 12 20 · 13 60 · 40 16 · 3 70 · 30 12 · 10 50 · 15 30 · 60 20 · 7 30 · 20 38 · 40 9 · 17 150 · 25 35 · 20 30 · 25 8·5 25 · 12 14 · 11 20 · 20 40 · 15
5 5 5 2 4 6 2 7 5 6 5 3 9 4 4 6 5 4 5 8 5 3 4 5 2 6
A A A A A A A A D D A A A A A A A A A A A D A T A A
Ploidy status: A, aneuploid; T, tetraploid; D, diploid.
Heterogeneity, histological features and DNA ploidy in oral carcinoma
419
Table 2 Origin, size, sampling and ploidy status of apparently heterogeneous carcinomas showing the number of samples analysed as aneuploid, tetraploid and diploid Case no.
Site
Diameter mm.
Total no. of samples
No. of diploid
No. of tetraploid
No. of aneuploid
36 37 39 41 27 03 10 11 15 14 07 12 01 44 20 45
Tongue Tongue Tongue Tongue Tongue Floor of mouth Floor of mouth Floor of mouth Floor of mouth Floor of mouth Floor of mouth Floor of mouth Buccal mucosa Tonsil Pillar of fauces Tuberosity
20 · 25 50 · 40 40 · 10 12 · 10 73 · 28 15 · 5 50 · 60 33 · 17 43 · 30 17 · 12 60 · 60 13 · 7 95 · 55 24 · 10 45 · 40 40 · 35
8 4 7 2 5 4 8 4 4 2 7 4 5 8 3 9
1 3 1 1 0 0 4 1 1 0 0 3 4 3 1 2
2 0 0 0 1 2 4 0 2 1 1 0 0 3 2 0
5 1 6 1 4 2 0 3 1 1 6 1 1 2 0 7
Apparent heterogeneity in cases in bold appeared to be accounted for by sample variation.
sely related to nuclear morphology but poorly to differentiation. The same relationships were shown in GH biplots and separately in the homogeneous and heterogeneous subgroups (data not shown). The correlations with low power invasive outline and nuclear pleomorphism were highly significant with P = 0.003 and P = 0.001 respectively.
Discussion There have been many investigations of the ploidy status of oral and other head and neck carcinomas, mostly attempts to correlate ploidy with recurrence, metastasis or survival. The majority have been performed using flow cytometry and have analysed a single sample or whole paraffin block. These studies have established that a proportion of carcinomas appear diploid, though the proportion varied widely. Published studies have reported 24%,20 27%,21 32%,24 45%,22 50%,23 58%,24 63%,25 and 70%26 to be diploid. One group suggested that diploid carcinomas were more frequent on the tongue using both flow cytometry and image cytometry4 but no site correlation has been confirmed by others. The very wide variation in the proportion that have been found to be diploid is unlikely to be due to differences between flow cytometry and cytophotometry,4,27 sample processing or varying diagnostic criteria for aneuploidy. Heterogeneity within lesions or sample variation appear more
likely causes. This suggestion is supported by the finding that more sensitive techniques such as comparative genomic hybridisation show that all oral carcinomas are aneuploid but that many are not detected as such by flow cytometry or image cytometry because of their detection limit.28 Few papers have addressed the question of ploidy heterogeneity within oral carcinomas and the proportion found to be heterogeneous varies. Slootweg et al.5 found 8 of 21 primary oral carcinomas to be heterogeneous on the basis of DNA index, Mahmood et al.,10 9 of 15 aneuploid oral carcinomas lesions and Oya et al.29 14 of 31. Studies using more extensive sampling tend to detect more heterogeneity.10,13,29 However, some have ascribed this to dilution of the sample by normal tissue or inflammatory cells or sample variation rather than true ploidy heterogeneity within the carcinoma.13 Image-based ploidy analysis is better than flow cytometry for investigation of heterogeneity. Small samples can be taken under histological control, ensuring minimal irrelevant tissue is included and small non-euploid populations are more readily identified in the histograms. In the present study, we have analysed multiple small samples of approximately 3.5 cubic millimetres selected to contain solid carcinoma with a uniform histological appearance. The results show that although 16 of 42 carcinomas appeared heterogeneous, only 8 of these were likely to be truly heterogeneous. Sampling and lesions lying near the divisions of diagnostic
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Figure 1 Ploidy histograms and tissue samples from an apparently truly ploidy-heterogeneous carcinoma. Lower panel: histograms, black bar epithelial nuclei, white bar normal diploid control lymphocytes. The left sample is classified as tetraploid with a 4c peak at DI 1.78 of 24.6% of the total epithelial nuclei and 0.4% of nuclei exceeding 5c (coefficient of variation 5.6). The right sample is classified as aneuploid, with peaks at 3c and a large proportion of cells with aneuploid DNA content. The histological appearances of both samples were identical, shown above, with large and medium sized islands of relatively poorly differentiated carcinoma with central necrosis. Approximately 10% of the sampled area is shown.
categories appear to have generated false negative diploid results and apparent heterogeneity. On the basis of this study, it would be necessary to analyse 5 samples to ensure that any carcinoma appearing diploid in one sample was correctly diagnosed as aneuploid (Table 2). The remaining 8 cases may be aneuploid but at a level below the detection limit of image based ploidy analysis,14,15 approximately a 1% change of nuclear DNA content per cell. Many previous studies attempted to correlate histological parameters with ploidy status. Many have used only a single sample, often a whole
paraffin block from which only either an average or a worst histological grade could be taken. Such studies have shown that aneuploidy is more likely to be detected in higher grade and larger carcinomas11,21,25 though no consistent relationship is evident.4,24,30 Observations have often been limited to mitotic activity and an average grade. In the present study, the use of small samples of uniform histological appearance has made a more detailed analysis possible. Biplot analysis is a derivative of principal component analysis that allows visual inspection of the data to identify patterns and outliers. The relationship between two
Heterogeneity, histological features and DNA ploidy in oral carcinoma
421
noma as diploid, a minimum of 5 samples must be analysed and histological parameters do not indicate specific areas to sample. DNA ploidy status is closely related to nuclear pleomorphism but not to differentiation in oral squamous carcinoma.
Acknowledgements We are grateful to the Charitable Foundation of Guy’s and St Thomas’ Hospitals for funding and for a Commonwealth Scholarship awarded to N. Diwakar, to the Commonwealth Scholarship Commission in the UK and the Department of Human Resources Development, India.
Figure 2 JK biplot for the whole series. 1. Apparently homogeneous and 2. apparently heterogeneous samples. There are no outliers. Correlations between variables are given by the cosine of the angle between the lines. Ploidy status is closely related to nuclear pleomorphism but not to differentiation.
variables is shown by the direction of the lines and the cosine of the angle between the lines shows the degree of correlation between them. Using this method, ploidy has been shown to be closely related to nuclear pleomorphism and invasive outline but less so to the invasive front morphology or degree of differentiation. Correlations with low power invasive outline and nuclear pleomorphism were highly significant. Nuclear pleomorphism cannot be scored in other than a subjective manner as it is a complex combination of size, staining, shape and frequency of abnormal nuclei. Nevertheless the correlation with ploidy is informative, plausible and consistent with the fact that DNA ploidy is independent of traditional grading based on cellular differentiation. After analysis, each sample was reassessed to determine whether the identified sampling errors could be related to histological heterogeneity. There was no detectable association with the degree of keratinisation, proportion of basaloid cells or any other parameter. Samples with apparently identical histological appearances generated diploid, tetraploid and aneuploid results unpredictably. In conclusion, using image-based ploidy analysis, 16 of 42 oral carcinomas appeared heterogeneous for DNA ploidy status. In the majority of cases this could be ascribed to a failure to detect non-euploid populations as a result of sample variation. In order to confidently diagnose an oral squamous cell carci-
References 1. Pihan GA, Doxsey SJ. The mitotic machinery as a source of genetic instability in cancer. Sem Cancer Biol 1999;9(4): 289–302. 2. Nigg EA. Centrosome aberrations: cause or consequence of cancer progression. Nat Rev Cancer 2002;2(11):815–25. 3. Rajagopalan H, Nowak MA, Vogelstein B, Lengauer C. The significance of unstable chromosomes in colorectal cancer. Nat Rev Cancer 2003;3(9):695–701. 4. Baretton G, Li X, Stoll C, Fischer-Brandies E, Schmidt M, Lohrs U. Prognostic significance of DNA ploidy in oral squamous cell carcinomas. A retrospective flow and image cytometric study with comparison of DNA ploidy in excisional biopsy specimens and resection specimens, primary tumours, and lymph node metastases. Oral Surg Oral Med Oral Pathol Oral Radiol Endodontics 1995;79:68–76. 5. Slootweg PJ, Giessen CA, Rutgers DH, Wils IS. DNA heterogeneity in metastasising squamous cell head and neck cancer. J Craniomaxillofac Surg 1993;21(8):348–50. 6. Soder AI, Hopman AH, Ramaekers FC, Conradt C, Bosch FX. Distinct nonrandom patterns of chromosomal aberrations in the progression of squamous cell carcinomas of the head and neck. Cancer Res 1995;55(21):5030–7. 7. Stell PM. Ploidy in head and neck cancer: a review and meta-analysis. Clin Otolaryngol 1991;16(5):510–6. 8. Baak JP, Janssen E. DNA ploidy analysis in histopathology. Morphometry and DNA cytometry reproducibility conditions and clinical applications. Histopathology 2004;44(6): 603–14. 9. Sudbø J, Kildal W, Risberg B, Koppang HS, Danielsen HE. DNA content as a prognostic marker in patients with oral leukoplakia. N Eng J Med 2001;344(17):1270–8. 10. Mahmood JU, Nomura T, Suzuki K, Shingaki S, Nkajima T. Heterogeneity of squamous cell carcinomas of head and neck in relation to clinicopathological variables. Br J Oral Maxillofac Surg 1998;36:446–52. 11. Tytor M, Wingren S, Olofsoon J. Heterogeneity of squamous cell carcinoma of oral cavity studied by flow cytometry. Pathol Res Pract 1991;187(1):30–5. 12. Fukushima T, Yasuda N, Nakai S, Murakami Y, Matsumono H. DNA heterogeneity in oral cavity and hypopharyngeal carcinoma. Gan To Kagaku Ryoho 1994;21(1):6–13. 13. Westerbeek HA, Mooi WJ, Begg C, Dessing M, Balm AJM. DNA-flow cytometry of head and neck carcinoma; the
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N. Diwakar et al. importance of uniform tissue sampling and tumour sites. Eur Arch Otorhinolaryngol 1992;249:140–3. Tremmel SC, Gotte K, Popp S, Weber S, Hormann K, Bartman CR, et al. Intratumoral genomic heterogeneity in advanced head and neck cancer detected by comparative genomic hybridisation. Cancer Genet Cytogenet 2003; 144(2):165–74. Hardisson D, Alvarez-Marcos C, Salas-Bustamante A, AlonsoGuervos M, Sastre N, Sampedro A. Numerical aberrations of chromosomes 8, 9, 11, and 17 in squamous cell carcinoma of the pharynx and larynx: a fluorescence in situ hybridization and DNA flow cytometric analysis of 50 cases. Oral Oncol 2004;40(4):409–17. Bryne M, Koppang HS, Lilleng R, Kjaerheim A. Malignancy grading of the deep invasive margins of oral squamous cell carcinomas has high prognostic value. J Pathol 1992; 166(4):375–81. Odell EW, Jani P, Sherriff M, Ahluwalia SM, Hibbert J, Levison DA, et al. The prognostic value of individual histologic grading parameters in small lingual squamous cell carcinomas. The importance of the pattern of invasion. Cancer 1994;74(3):789–94. Hedley DW. DNA analysis from paraffin-embedded blocks. Methods Cell Biol 1994;41:231–40. Gabriel KR. The biplot graphical display of matrices with application to principal component analysis. Biometrika 1971;58:453–67. Feichter GE, Maier H, Adler D, Born IA, Abel U, Haag D, et al. S-phase fractions and DNA-ploidy of oropharyngeal squamous epithelium carcinomas compared with histologic grade, stage, response to chemotherapy and survival. Acta Otolaryngol 1987;104(3–4):377–84. Hemmer J, Schon E, Kreidler J, Haase S. Prognostic implications of DNA ploidy in squamous cell carcinoma of the tongue assessed by flow cytometry. J Clin Oncol 1990; 16(1):83–6.
22. Bueno PR, Delgado RG, Gonzalez FJD. Tumour DNA content as a prognostic indicator in squamous cell carcinoma of the oral cavity and tongue base. Head Neck 1998;5:232–9. 23. Mahmood JU, Nomura T, Suzuki K, Shingaki S, Nkajima T. Heterogeneity of squamous cell carcinomas of head and neck in relation to clinicopathological variables. Br J Oralmaxillofac Surg 1998;36:446–52. 24. Seoane J, Asenjo JA, Bascones A, Varela-Centelles PI, Romero MA. Flow cytometric DNA ploidy analysis of oral cancer comparison with histologic grading. Oral Oncol 1999;35:266–72. 25. Gomez R, el-Naggar AK, Byers RM, Garnsey L, Luna MA, Batsakis JG. Squamous carcinoma of oral tongue: prognostic significance of flow-cytometric DNA content. Mod Pathol 1992;5(2):141–5. 26. Farrar WB, Sickle-Santanello BJ, Keyhani-Rofagha S, DeCenzo JF, O’Toole RV. Follow-up on flow cytometric DNA analysis of squamous cell carcinoma of the tongue. Am J Surg 1989;157(4):377–80. 27. Gandour-Edwards RF, Donald PJ, Yu TLC, Howard RR, Teplitz RL. DNA Content of head and neck squamous carcinoma by flow and image cytometry. Arch Otolaryngol Head Neck Surg 1994;120:294–8. 28. Okafuji M, Ita M, Oga A, Hayatsu Y, Matsu A, et al. The relationship of genetic aberrations detected by comparative genomic hybridisation to DNA ploidy and tumour size in human oral squamous cell carcinomas. J Oral Pathol Med 2000;29:226–31. 29. Oya R, Ikemura K. Can flow cytometrically determined DNA ploidy and S-phase fraction predict regional metastasis in squamous cell carcinoma of the oral cavity? Head Neck 2002;24(2):136–42. 30. Sarker S, Ghugoor K, Patel KS, Tolley NS, Coleman DV. Nuclear DNA content using computerized image cytometry of squamous cell carcinoma of the head and neck. J Laryngol Otol 1997;111:43–7.
http://intl.elsevierhealth.com/journals/oron/
Heterogeneity, histological features and DNA ploidy in oral carcinoma by image-based analysis N. Diwakar a, M. Sperandio a, M. Sherriff b, A. Brown a, E.W. Odell
a,*
a
Department of Oral Pathology, Guy’s Hospital, King’s College, Floor 28 Guy’s Tower, London SE1 9RT, UK Department of Biomaterials Science, Guy’s Hospital, King’s College, Floor 17 Guy’s Tower, London SE1 9RT, UK b
Received 13 August 2004; accepted 22 October 2004
KEYWORDS
Summary Oral squamous carcinomas appear heterogeneous on DNA ploidy analysis. However, this may be partly a result of sample dilution or the detection limit of techniques. The aim of this study was to determine whether oral squamous carcinomas are heterogeneous for ploidy status using image-based ploidy analysis and to determine whether ploidy status correlates with histological parameters. Multiple samples from 42 oral squamous carcinomas were analysed for DNA ploidy using an image-based system and scored for histological parameters. 22 were uniformly aneuploid, 1 uniformly tetraploid and 3 uniformly diploid. 16 appeared heterogeneous but only 8 appeared to be genuinely heterogeneous when minor ploidy histogram peaks were taken into account. Ploidy was closely related to nuclear pleomorphism but not differentiation. Sample variation, detection limits and diagnostic criteria account for much of the ploidy heterogeneity observed. Confident diagnosis of diploid status in an oral squamous cell carcinoma requires a minimum of 5 samples. c 2004 Elsevier Ltd. All rights reserved.
Oral carcinoma; Ploidy analysis; Large scale genomic status
Introduction
Cells from the majority of human malignant neoplasms are aneuploid as a result of chromosomal * Corresponding author. Tel.: +44 020 7188 4367; fax: +44 020 7188 4375. E-mail address: [email protected] (E.W. Odell).
instability (imbalance). Chromosomal instability arises from non-disjunction and other mitotic errors and may result in loss or, more frequently gain, of whole or part chromosomes. This acquired genetic instability originates in mutations in genes controlling a number of processes including cell cycle checkpoints, mismatch repair, apoptosis, telomere maintenance, centrosome and spindle function.1
1368-8375/$ - see front matter c 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.oraloncology.2004.10.009
Heterogeneity, histological features and DNA ploidy in oral carcinoma Chromosomal instability is now considered fundamental to the malignant phenotype and is proposed as a cause rather than the result of malignant transformation.2,3 Aneuploidy can be detected in oral carcinomas4,5 and specific chromosomes are more frequently duplicated than others.6 Aneuploidy has been extensively studied in oral and other head and neck carcinomas in relation to prognosis and metastasis, though no convincing relationship has emerged.7,8 In contrast, the study of aneuploidy in oral premalignant lesions shows great promise as a predictor of malignant transformation.9 As part of a larger study of image-based ploidy analysis in oral premalignancy, we have identified a possible population of diploid lesions that may develop into diploid carcinomas. However, this possibility cannot be accepted without an understanding of ploidy heterogeneity within oral carcinomas, because sample variation and other factors could account for spurious diploid results. It is well recognised that, although most carcinomas are clonal in origin, they are heterogeneous with respect to the karyotype and phenotype of clonal populations within them. Oral and other head and neck carcinomas show heterogeneity on the basis of ploidy when assessed by flow cytometry,10–13 comparative genomic hybridisation14 and fluorescent in situ hybridisation.15 Thus, sampling may be critical when assessing the true ploidy status of a carcinoma.13 It is unclear whether heterogeneity might influence the results of image-based ploidy analysis. To address these concerns, we have undertaken an analysis of DNA ploidy heterogeneity in oral carcinoma using an image-based ploidy analysis system. The study was approved by the Guy’s Hospital Research Ethics Committee and the UK Patient Information Advisory Group.
Material and methods Oral squamous carcinomas Multiple blocks of paraffin-embedded, formalinfixed, tissue from 45 consecutive cases of untreated primary oral squamous cell carcinoma were selected from the Oral Pathology Department archive at King’s College, Guy’s Hospital, London. All were accessioned between 2000 and 2003 and exclusion criteria were previous treatment by radiotherapy or chemotherapy, insufficient material remaining for analysis or carcinomas too small to analyse multiple sites within them. The primary
417
sites were the floor of the mouth (n = 15), tongue (n = 15), retromolar region (n = 4), buccal mucosa (n = 2), soft palate, tonsil and fauces (n = 3) and gingiva/tuberosity (n = 3). Carcinoma size was determined from both macroscopic and microscopic pathological staging data. Routine haematoxylin and eosin sections were examined and between 2 and 8 sample areas approximately 5 · 2 mm were selected from each carcinoma (mean 5.02, n = 226). Each sample contained as high a proportion of carcinoma as possible and was of a uniform histological appearance. Samples were selected to include as diverse a range of histological appearances as possible. Each sample was graded histologically for the following parameters: invasive front according to Bryne16,17 differentiation (grades: prominent keratinisation with minority basaloid component, focal keratinisation only, no keratinisation, basaloid, basaloid squamous carcinoma), invasive outline (scored at ·20 magnification, grades: well-defined or irregular) and nuclear pleomorphism (scored in basal and suprabasal cells only: mild, moderate, marked). All grading was performed blind to the ploidy result.
Ploidy analysis Ploidy analysis was performed as previously reported by Sudbø et al.9 Briefly, 6 · 50 lm sections were cut from each selected area, dewaxed in xylene, rehydrated and nuclei extracted using protease type XXIV (Sigma Chemical Co., Poole, UK) according to Hedley.18 Nuclear monolayers were prepared in a cytospin 4 (Thermo Shandon, UK) and stained by the Feulgen–Schiff method. Ploidy analysis was performed on a Fairfield image-based ploidy analyser comprising a high resolution camera (C4742-95, Hamamatsu Photonics K.K., Hamamatsu, Japan) attached to a Zeiss Axioplan II microscope (Zeiss, Oberkochen, Germany) with a 4 slide scanning stage. A total of 226 monolayers were analysed, each with a minimum of 300 nuclei sampled using the automated scanning option. Diagnostic criteria were as previously published for this system and oral lesions.9 A sample was classified as diploid if only one G0/G1 (2c) peak was present, or if the number of nuclei in the G2 (4c) peak did not exceed 10% of the total number of epithelial cells. A sample was defined as DNA tetraploid when the fraction of nuclei in the 4c region exceeded 10% of the total, without corresponding S phase. The tetraploid group thus includes cases intermediate between diploid and aneuploid (oral carcinomas being rarely chromosomally
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tetraploid). A lesion was defined as aneuploid if non-euploid peaks were present, of if the number of nuclei with a DNA content greater than 5c exceeded 1%. All analysis was performed blind to the histological results.
Statistical analysis The interrelationships between ploidy and histological parameters were assessed using JK biplot analysis.19 Specific correlations between histological parameters and ploidy status were determined using Somers’ D in the whole dataset and within the sub groups of ploidy-homogeneous and ploidy-heterogeneous carcinomas. All analyses were performed using Stata 8.2.
Results No ploidy result could be obtained from 13 monolayers, eliminating 3 carcinomas because insufficient samples remained to assess heterogeneity. Twenty two of 42 carcinomas appeared uniformly
Table 1
aneuploid, 1 uniformly tetraploid and 3 uniformly diploid. The site, size, number of samples analysed and ploidy status are shown in Table 1. Sixteen carcinomas appeared heterogeneous and their characteristics and the number of samples diagnosed as aneuploid, tetraploid or diploid is shown in Table 2. Normal and hyperplastic mucosal lesions were uniformly diploid. Histograms revealed subtle ploidy anomalies that were insufficient to meet diagnostic criteria for non-euploidy in all diploid samples from 8 carcinomas. The small peaks often matched the pattern seen in aneuploid samples from the same lesion, suggesting that sample variation accounted for the apparent heterogeneity and that these samples were not truly diploid. The remaining 8 cases that appeared genuinely heterogeneous on the basis of different histogram patterns are in bold in Table 2. This group includes both carcinomas with low and high numbers of samples analysed. An example of a carcinoma classified as ploidy heterogeneous is illustrated in Fig. 1, together with the histological appearances of the two sample sites shown. The JK biplot analysis for the whole series is shown in Fig. 2 indicating that ploidy status is clo-
Origin, size, sampling and ploidy status of apparently homogeneous carcinomas
Case no.
Site
Diameter mm.
No. of samples
Ploidy status
28 33 34 38 35 42 31 43 40 29 04 05 06 08 09 13 16 17 21 23 24 22 18 19 25 02
Tongue Tongue Tongue Tongue Tongue Tongue Tongue Tongue Tongue Tongue Floor of mouth Floor of mouth Floor of mouth Floor of mouth Floor of mouth Floor of mouth Floor of mouth Floor of mouth Retromolar Retromolar Retromolar Retromolar Gingiva Gingiva Soft palate Buccal mucosa
8·8 30 · 18 10 · 8 10 · 18 12 · 13 40 · 35 17 · 12 20 · 13 60 · 40 16 · 3 70 · 30 12 · 10 50 · 15 30 · 60 20 · 7 30 · 20 38 · 40 9 · 17 150 · 25 35 · 20 30 · 25 8·5 25 · 12 14 · 11 20 · 20 40 · 15
5 5 5 2 4 6 2 7 5 6 5 3 9 4 4 6 5 4 5 8 5 3 4 5 2 6
A A A A A A A A D D A A A A A A A A A A A D A T A A
Ploidy status: A, aneuploid; T, tetraploid; D, diploid.
Heterogeneity, histological features and DNA ploidy in oral carcinoma
419
Table 2 Origin, size, sampling and ploidy status of apparently heterogeneous carcinomas showing the number of samples analysed as aneuploid, tetraploid and diploid Case no.
Site
Diameter mm.
Total no. of samples
No. of diploid
No. of tetraploid
No. of aneuploid
36 37 39 41 27 03 10 11 15 14 07 12 01 44 20 45
Tongue Tongue Tongue Tongue Tongue Floor of mouth Floor of mouth Floor of mouth Floor of mouth Floor of mouth Floor of mouth Floor of mouth Buccal mucosa Tonsil Pillar of fauces Tuberosity
20 · 25 50 · 40 40 · 10 12 · 10 73 · 28 15 · 5 50 · 60 33 · 17 43 · 30 17 · 12 60 · 60 13 · 7 95 · 55 24 · 10 45 · 40 40 · 35
8 4 7 2 5 4 8 4 4 2 7 4 5 8 3 9
1 3 1 1 0 0 4 1 1 0 0 3 4 3 1 2
2 0 0 0 1 2 4 0 2 1 1 0 0 3 2 0
5 1 6 1 4 2 0 3 1 1 6 1 1 2 0 7
Apparent heterogeneity in cases in bold appeared to be accounted for by sample variation.
sely related to nuclear morphology but poorly to differentiation. The same relationships were shown in GH biplots and separately in the homogeneous and heterogeneous subgroups (data not shown). The correlations with low power invasive outline and nuclear pleomorphism were highly significant with P = 0.003 and P = 0.001 respectively.
Discussion There have been many investigations of the ploidy status of oral and other head and neck carcinomas, mostly attempts to correlate ploidy with recurrence, metastasis or survival. The majority have been performed using flow cytometry and have analysed a single sample or whole paraffin block. These studies have established that a proportion of carcinomas appear diploid, though the proportion varied widely. Published studies have reported 24%,20 27%,21 32%,24 45%,22 50%,23 58%,24 63%,25 and 70%26 to be diploid. One group suggested that diploid carcinomas were more frequent on the tongue using both flow cytometry and image cytometry4 but no site correlation has been confirmed by others. The very wide variation in the proportion that have been found to be diploid is unlikely to be due to differences between flow cytometry and cytophotometry,4,27 sample processing or varying diagnostic criteria for aneuploidy. Heterogeneity within lesions or sample variation appear more
likely causes. This suggestion is supported by the finding that more sensitive techniques such as comparative genomic hybridisation show that all oral carcinomas are aneuploid but that many are not detected as such by flow cytometry or image cytometry because of their detection limit.28 Few papers have addressed the question of ploidy heterogeneity within oral carcinomas and the proportion found to be heterogeneous varies. Slootweg et al.5 found 8 of 21 primary oral carcinomas to be heterogeneous on the basis of DNA index, Mahmood et al.,10 9 of 15 aneuploid oral carcinomas lesions and Oya et al.29 14 of 31. Studies using more extensive sampling tend to detect more heterogeneity.10,13,29 However, some have ascribed this to dilution of the sample by normal tissue or inflammatory cells or sample variation rather than true ploidy heterogeneity within the carcinoma.13 Image-based ploidy analysis is better than flow cytometry for investigation of heterogeneity. Small samples can be taken under histological control, ensuring minimal irrelevant tissue is included and small non-euploid populations are more readily identified in the histograms. In the present study, we have analysed multiple small samples of approximately 3.5 cubic millimetres selected to contain solid carcinoma with a uniform histological appearance. The results show that although 16 of 42 carcinomas appeared heterogeneous, only 8 of these were likely to be truly heterogeneous. Sampling and lesions lying near the divisions of diagnostic
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Figure 1 Ploidy histograms and tissue samples from an apparently truly ploidy-heterogeneous carcinoma. Lower panel: histograms, black bar epithelial nuclei, white bar normal diploid control lymphocytes. The left sample is classified as tetraploid with a 4c peak at DI 1.78 of 24.6% of the total epithelial nuclei and 0.4% of nuclei exceeding 5c (coefficient of variation 5.6). The right sample is classified as aneuploid, with peaks at 3c and a large proportion of cells with aneuploid DNA content. The histological appearances of both samples were identical, shown above, with large and medium sized islands of relatively poorly differentiated carcinoma with central necrosis. Approximately 10% of the sampled area is shown.
categories appear to have generated false negative diploid results and apparent heterogeneity. On the basis of this study, it would be necessary to analyse 5 samples to ensure that any carcinoma appearing diploid in one sample was correctly diagnosed as aneuploid (Table 2). The remaining 8 cases may be aneuploid but at a level below the detection limit of image based ploidy analysis,14,15 approximately a 1% change of nuclear DNA content per cell. Many previous studies attempted to correlate histological parameters with ploidy status. Many have used only a single sample, often a whole
paraffin block from which only either an average or a worst histological grade could be taken. Such studies have shown that aneuploidy is more likely to be detected in higher grade and larger carcinomas11,21,25 though no consistent relationship is evident.4,24,30 Observations have often been limited to mitotic activity and an average grade. In the present study, the use of small samples of uniform histological appearance has made a more detailed analysis possible. Biplot analysis is a derivative of principal component analysis that allows visual inspection of the data to identify patterns and outliers. The relationship between two
Heterogeneity, histological features and DNA ploidy in oral carcinoma
421
noma as diploid, a minimum of 5 samples must be analysed and histological parameters do not indicate specific areas to sample. DNA ploidy status is closely related to nuclear pleomorphism but not to differentiation in oral squamous carcinoma.
Acknowledgements We are grateful to the Charitable Foundation of Guy’s and St Thomas’ Hospitals for funding and for a Commonwealth Scholarship awarded to N. Diwakar, to the Commonwealth Scholarship Commission in the UK and the Department of Human Resources Development, India.
Figure 2 JK biplot for the whole series. 1. Apparently homogeneous and 2. apparently heterogeneous samples. There are no outliers. Correlations between variables are given by the cosine of the angle between the lines. Ploidy status is closely related to nuclear pleomorphism but not to differentiation.
variables is shown by the direction of the lines and the cosine of the angle between the lines shows the degree of correlation between them. Using this method, ploidy has been shown to be closely related to nuclear pleomorphism and invasive outline but less so to the invasive front morphology or degree of differentiation. Correlations with low power invasive outline and nuclear pleomorphism were highly significant. Nuclear pleomorphism cannot be scored in other than a subjective manner as it is a complex combination of size, staining, shape and frequency of abnormal nuclei. Nevertheless the correlation with ploidy is informative, plausible and consistent with the fact that DNA ploidy is independent of traditional grading based on cellular differentiation. After analysis, each sample was reassessed to determine whether the identified sampling errors could be related to histological heterogeneity. There was no detectable association with the degree of keratinisation, proportion of basaloid cells or any other parameter. Samples with apparently identical histological appearances generated diploid, tetraploid and aneuploid results unpredictably. In conclusion, using image-based ploidy analysis, 16 of 42 oral carcinomas appeared heterogeneous for DNA ploidy status. In the majority of cases this could be ascribed to a failure to detect non-euploid populations as a result of sample variation. In order to confidently diagnose an oral squamous cell carci-
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