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Keywords:

  • cancer;
  • DNA;
  • ploidy;
  • parotid gland tumors;
  • FNAB;
  • LSC;
  • prediction;
  • diagnostic value

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSIONS
  7. Acknowledgements
  8. LITERATURE CITED

Background

To minimize hospitalization and morbidity for a patient with a solid tumor of a salivary gland, malignancy must be confirmed or excluded as soon as possible. This information cannot be obtained preoperatively by existing standard procedures. Minimal-invasive approaches with adequate diagnostic analysis represent a promising precondition for optimized therapy.

Methods

For fine needle aspirate biopsies (FNABs), laser scanning cytometry (LSC) offers a semi-automated slide-based technology for objective and quantitative analysis. We have established an assay for FNABs from salivary gland tumors. FNAB cells were stained for cytokeratin and DNA followed by LSC analysis. The cells were subsequently HE-stained and were relocalized on the slide. The LSC analysis quantitatively determines the DNA index (DI) of the tumor cells taking leukocytes as internal DNA diploid standard. Histograms with 0.95 < DI < 1.05 and 1.9 <DI <2.1 were defined as DNA euploid, whereas any other DI was defined as DNA aneuploid. The percentage of cytokeratin positive cells with DI > 2.5 (i.e., 5c exceeding rate, 5cER) was calculated. Samples with DNA aneuploid peaks or with 5cER > 5% were classified as malignant. Routine histopathology was performed as a control.

Results

FNABs from 51 solid salivary gland tumors (41 parotid gland, six submandibular, four parapharyngeal) were analyzed with this assay. Eleven of 14 malignant tumors were DNA aneuploid by LSC analysis. All benign tumors showed diploid DNA content. The positive predictive value for malignancy was 1.0, the negative predictive value was 0.93, the correlation with routine histopathology was highly significant (p = 7.6 × 10-9, Fisher's exact test). The calculated specificity of LSC analysis was 1.0 and the sensitivity was 0.79.

Conclusions

This pilot study demonstrates the validity of slide-based cytometry for the preoperative prediction of malignancy in solid tumors being inaccessible for incision biopsy but suitable for FNABs such as those of the parotid gland. Cytometry Part B (Clin. Cytometry) 53B:20–25, 2003. © 2003 Wiley-Liss, Inc.

The establishment of individualized disease course predictions for patients, rather than prognosis for a population of patients with this disorder (1), will increasingly influence clinical decision making. For example, in the treatment of tumors of the parotid and other salivary glands it is important to exclude or confirm malignancy as soon as possible. The reasons are obvious, since surgery is more extensive for malignant tumors than for benign tumors. Using the parotid gland as an example, lateral parotidectomy is performed for benign tumors, whereas malignant tumors require subtotal or radical parotidectomy including primary resection plus reconstruction of the facial nerve. In addition, patients with malignant tumors may benefit from neck dissection for preventing delayed clinical manifestation of occult metastasis even if preoperative diagnostic procedures, such as palpation, sonography, or CT do not provide evidence of lymph node involvement (2). As extended surgery bears a high risk of complications, such as temporary or permanent facial nerve palsy, patients with parotid tumors of uncertain dignity currently undergo inaugural lateral parotidectomy in order to obtain a definitive histopathological diagnosis. In case of malignancy, radical resection of the medial parotid lobe with or without facial nerve resection and neck dissection is performed in a subsequent session. Submandibular gland tumors and those of the small glands within the mucosa are treated analogously. This strategy avoids overtreatment for benign tumors, while patients with definite malignancy are exposed to higher risk of increased morbidity and loss of facial nerve function. This approach also contributes to overhead costs for the health care system.

Clinical indicators of malignancy include facial nerve palsy as a sign of infiltrative growth, clinical loco-regional lymph node metastases, and infiltrative growth to neighboring tissues like the mandible. Other symptoms, such as rapid tumor growth and pain, are weak indicators and not recognized as proofs of malignancy. Many tumors are accessible for incision biopsies; but facial nerve injury is a crucial point, particularly in the large salivary glands. In addition, in the case of the most common benign tumor, the pleomorphic adenoma, it is well known that rupture of the capsule and tumor cell spread leads to multiple recurrences with the potential for malignant transformation (3). In search for novel approaches to pre-therapeutically assess the dignity of salivary gland tumors, preference should be given to minimal invasive procedures such as FNABs and swabs (4).

An increasing number of diagnoses are currently analyzed by cytopathology. This is an inexpensive and quick procedure, but due to the overall rare occurrence of salivary gland tumors, final diagnosis is often committed to a few specialized pathologists (5). Specimens can, furthermore, be analyzed by semi-automated cytometric measurements, yielding quantitative data such as cellular DNA content. DNA ploidy has been shown as a prognostic marker in several tumor entities including the parotid gland (6–8). Up to now, the DNA ploidy of solid carcinomas is determined by image analysis or flow cytometry. Both methods are well described and numerous standardized protocols have been published (9–11). Both methods have, however, certain weaknesses. Rather few cells (150–300) are analyzed by image analysis, and only one parameter (i.e., DNA content) is determined in most protocols; analysis is time-consuming and may give rise to inter-observer variation, since the target cells are selected individually. Flow cytometry, in contrast, allows multiparameter analysis of bulk cell numbers, but physiologic characteristics cannot be attributed to cellular morphology.

In the present study we make use of the laser scanning cytometer (LSC) to determine the DNA ploidy. This microscope-based instrument combines high throughput multiparametric cytometry and morphological analysis. LSC was commercially introduced in the early 1990s; its instrumentation and software are explained elsewhere (12–14). Numerous assays have been developed for different experimental applications, but only few assays are applicable in a routine clinical context (15).

The most important feature of the LSC concerns the recording of the exact position of every object together with the fluorescence data. Among other capabilities this allows 1) verification whether objects are single cells, doublets, debris or artifacts; and 2) documentation of the cell morphology. To this end, the slide can be removed from the stage, stained by conventional cytological methods (Giemsa, Hematoxylin, and Eosin), and followed by re-analysis on the stage (16).

In conclusion, this slide-based design makes it very suitable for analyzing hypocellular specimens such as FNABs. The data can be evaluated in a standardized and objective way, which is extremely important for clinical decision making. We and others have compared DNA ploidy analysis by LSC with image analysis after Feulgen staining or with flow cytometry, and showed a good correlation of both methods (14, 17, 18). It was therefore investigated in this pilot study whether the LSC has the potential to provide preoperative prediction of tumor dignity in solid salivary gland tumors. Cellular DNA was stained by propidium iodide (PI) and cytokeratin by immunofluorescence (19). Similar assays have already been described for specimens from voided urine and other hypocellular samples (20).

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSIONS
  7. Acknowledgements
  8. LITERATURE CITED

Specimen Preparation

Specimens.

The design of this study was approved by the local ethics committee. After written informed consent FNABs from 51 patients were taken with a 27G needle and a 20-mL syringe, either in the out-patient department or in the operation theatre immediately after resection. Tumors were included that were classified as solid by clinical examination or sonography, whereas cystic lesions were excluded. Obtained tissue fragments were suspended in a 1.5-mL Eppendorf tube (Eppendorf, Hamburg, Germany) pre-filled with 100 μl phosphate buffered saline (PBS; Gibco BRL, Paisley, Scotland), pH 7.4, supplemented with 1% bovine serum albumin (BSA; Sigma, St. Louis, MO) and stored at 4°C for up to 24 h until further preparation.

Slide preparation.

One mL FACSLysis (BD Biosciences, San Jose, CA) was added to each tube for double-staining of cytokeratin and DNA. After 15 min incubation at room temperature, the suspension was spun down (250 × g, five min) and the pellet was resuspended in 50 μl PBS supplemented with 0.5% BSA. To maintain identical conditions for both diagnostic specimen and negative control, two aliquots of 10–20 μl of cell suspensions were placed onto a single microscopic glass slide with each sample covering a 1.2-cm square. Slides were air-dried for one hour and stored in 70% ethanol at room temperature.

Staining.

Slides were washed with PBS for 10 min and then 200 μl of PBS with 1% BSA were pipetted onto each area of the slide. After incubation for 15 min, supernatant was carefully discarded. Correspondent sample areas were loaded with either 9 μl of anti-cytokeratin antibody solution (3 μl Clone MNF116, Code M0821; 3 μl Clone 34ßE12, Code M0630; 3 μl Clone AE1/AE3, Code M3515) diluted in 91 μl PBS containing 0.5% BSA or with 9 μl unspecific control antibody solution (Code X0931; all: Dako, Carpinteria, CA) diluted in 91 μl PBS containing 0.5% BSA. After 20 min incubation, slides were washed twice with 0.5-mL PBS prior to 20 min incubation with 91 μl PBS with 0.5% BSA containing 9 μl biotinylated anti-mouse antibody (Caltag, Hamburg, Germany, Code M32015). Slides were washed twice with 0.5-mL PBS and were then incubated for 20 min in a solution containing 91 μl PBS/0.5% BSA and 9 μl streptavidin conjugated to allophycocyanin (APC; Caltag, Hamburg, Germany, Code SA1005) supplemented with 50 μg propidium iodide (PI)/ml (Sigma, St. Louis, MO) and 100 μg RNase/ml (Sigma, St. Louis, MO). Slides were washed twice with 0.5-mL PBS and covered with 40 μl of glycerol in PBS 75%/25% with 25 μg PI/mL. Following microscopic analysis, the coverslip was carefully removed and a conventional hematoxilin eosin (HE) staining was performed. Slides were then covered using Eukitt (Kindler GmbH and Co, Freiburg, Germany) and were stored as normal cytological samples.

Analysis by LSC

Slides were fixed on the microscope stage of the LSC and were analyzed with the 20× objective as detailed earlier (16). In general, 5,000–10,000 cells were measured per area with the PI signal as trigger. The minimal area size was set to 10 μm2. Signals were acquired for the forward scatter and the green and the red channel excited by the argon-laser, and for the far-red channel excited by the helium-neon-laser. Dynamic background calculation was activated for all parameters. The power of the argon-laser was set to 5 mW. The PMTs were adjusted in such a way that signals did not exceed the range of sensitivity. Data were then interpreted with the proprietary WinCyte software.

Data Interpretation

Data interpretation is exemplified in Figure 1. A cutoff level of 5% positive cells in the APC-channel of the control sample was set for the detection of cytokeratin positive cells in the specifically immunostained sample. The 2c-channel for DNA diploid cells was set in the gated DNA-histogram for cytokeratin negative cells such as infiltrating/contaminating leukocytes or bare nuclei. The DNA-index (DI) of the different peaks for cytokeratin positive and cytokeratin negative cells was calculated. Cells or nuclei with 0.95<DI<1.05 were defined as DNA diploid; 1.9<DI<2.1 was defined as DNA tetraploid. Any other DI was defined as DNA aneuploid. A gate was set for cytokeratin positive cells with DI>2.5 and their relative number was calculated (i.e., 5c exceeding rate, 5cER). Relocalization of single cells after HE-staining with the 20× objective was used to confirm the proper assignment of different cell subsets. Twenty consecutive cells were relocalized for each subset to estimate the loss of cells during HE-staining. A LSC sample was classified as malignant if DNA aneuploid peaks were detected or if the 5cER exceeded 5%. Otherwise it was classified as benign. Fisher's exact probability test was used to compare routine histopathology and LSC classification. In addition to the sensitivity and the specificity, the positive predictive value (PPV) and the negative predictive value (NPV) of the LSC analysis was calculated based on a confusion matrix.

thumbnail image

Figure 1. An FNAB from a solid tumor of the parotid gland (#38) was analyzed in the LSC and H&E restained for single cell relocalization. The dot plots (a) of the control (left) and of the specific immunostaining (center) display DNA-content (x-axis, linear) versus APC-fluorescence per area (y-axis, logarithmic) of cytokeratin positive epithelial cells. The 5% cutoff in the control sample (left) permits to gate on cytokeratin positive cell populations (2–5) in the DNA histogram (right). The DI of the subpopulations is determined from the DNA histogram. Single cells (b) from the sub-populations are relocalized under the 20× objective: 1 and 2: reference cells; 3–5: tumor cells. The histologic diagnosis of this sample was squamous cell carcinoma.

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RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSIONS
  7. Acknowledgements
  8. LITERATURE CITED

FNABs yielded typically between 5,000 and 50,000 cells. Erythrocytes were effectively destroyed by treatment with lysis buffer. We did not experience problems related to insufficient lysis of erythrocytes. The morphology of the cells was well preserved.

Cover slip removal and HE restaining did not lead to a significant cell loss, since none of 20 regularly relocalized cells within the different cell subsets (typically between 100 and 200 cells per sample) was missing. Even sporadically analyzed sets of up to 50 consecutive cells were relocalized without missing cells.

All tumors were examined by an experienced pathologist (A. Tannapfel) by routine histopathologic diagnosis. A data sheet of all 51 tumors was prepared (Table 1) comparing LSC and routine histopathology. Three of 14 histologically malignant tumors turned out to be DNA diploid by LSC. None of the histologically benign tumors was misdiagnosed as malignant by LSC. Overall, LSC data showed an excellent correlation with histopathology as determined by Fisher's exact test with a positive predictive value of 1.0 and a negative predictive value of 0.93. Sensitivity was 0.79 and specificity was 1.0 (Table 2).

Table 1. Patient Data on LSC and Histopathology
Patient numberLocalizationDI-peaks5cERLSC classificationPathology classificationHistology
  1. Fifty one solid salivary gland tumors from different localization were analyzed by LSC. The DI-peaks in each specimen were determined, and the percentage of cells with a DI exceeding 2.5 (i.e., the 5c exceeding rate = 5cER) was calculated for the cytokeratin-positive sub-population. In patient 29, the 5cER of the tumor is given for the cytokeratin-positive (5.8%) and the cytokeratin-negative (13.1%) sub-population.

1Parotid gland1.000.4BenignBenignPleomorphic adenoma
2Parotid gland1.00/2.003.6BenignBenignPleomorphic adenoma
3Parotid gland1.04/2.081.8BenignBenignPleomorphic adenoma
4Parotid gland1.00/2.082.1BenignBenignPleomorphic adenoma
5Parotid gland1.00/2.004.7BenignBenignPleomorphic adenoma
6Parotid gland1.00/2.001.2BenignBenignBenign lymphoepithelial lesion
7Parotid gland1.00/2.001.3BenignBenignPleomorphic adenoma
8Parotid gland1.00/2.063.3BenignMalignantSquamous cell carcinoma
9Parotid gland1.00/2.002.3BenignMalignantMALT-lymphoma
10Parotid gland1.00/1.12/2.163.3MalignantMalignantAzinic cell carcinoma
11Parotid gland1.00/2.000.9BenignBenignPleomorphic adenoma
12Parotid gland1.00/2.000.9BenignBenignBenign lymphoepithelial lesion
13Parotid gland1.00/2.000.8BenignBenignPleomorphic adenoma
14Parotid gland1.00/2.001.6BenignBenignPleomorphic adenoma
15Parotid gland1.00/2.002.1BenignBenignPleomorphic adenoma
16Parotid gland1.00/1.24/1.681.7MalignantMalignantAdenocarcinoma
17Parotid gland1.00/2.000.7BenignBenignPleomorphic adenoma
18Parotid gland1.00/2.001.4BenignBenignChronic inflammation
19Parotid gland1.00/1.62/1.86/3.5213.5MalignantMalignantMetastasis of melanoma
20Parotid gland1.00/2.000.7BenignBenignPleomorphic adenoma
21Parotid gland1.002.0BenignBenignLipoma
22Parotid gland0.70/1.00/2.001.8MalignantMalignantMalignant hidradenoma
23Parotid gland1.00/2.000.9BenignBenignPleomorphic adenoma
24Parotid gland1.00/2.003.3BenignBenignPleomorphic adenoma
25Parotid gland1.00/2.001.0BenignBenignPleomorphic adenoma
26Parotid gland1.00/2.000.8BenignBenignPleomorphic adenoma
27Parotid gland1.00/2.002.9BenignBenignBenign granular tumor
28Parotid gland1.00/2.002.4BenignBenignIntraglandular lymph node
29Parotid gland0.96/1.66/2.22/2.465.8/13.1MalignantMalignantCarcinosarkoma
30Parotid gland0.56/1.24/1.84/2.461.3MalignantMalignantMetastasis of melanoma
31Parotid gland1.002.8BenignBenignPleomorphic adenoma
32Parotid gland1.00/1.08/1.48/2.281.3MalignantMalignantMucoepidermoid carcinoma
33Parotid gland1.000.3BenignBenignIntraglandular lymph node
34Parotid gland1.000.1BenignBenignIntraglandular lymph node
35Parotid gland1.00/1.20/1.66/2.245.5MalignantMalignantSquamous cell carcinoma
36Parotid gland0.36/1.002.2MalignantMalignantSquamous cell carcinoma
37Parotid gland1.00/2.083.0BenignBenignPleomorphic adenoma
38Parotid gland0.52/1.00/1.40/2.100.5MalignantMalignantSquamous cell carcinoma
39Parotid gland1.00/2.000.0BenignMalignantMALT-lymphoma
40Parotid gland1.00/2.001.2BenignBenignPleomorphic adenoma
41Parotid gland1.00/2.003.6BenignBenignPleomorphic adenoma
42Submandibular1.00/2.002.4BenignBenignPleomorphic adenoma
43Submandibular1.08/1.54/2.16/3.2410.8MalignantMalignantMetastasis of Merkelcell-carcinoma
44Submandibular1.000.7BenignBenignMyoepithelioma
45Submandibular1.001.3BenignBenignChronic sclerosing sialadenitis
46Submandibular1.001.7BenignBenignChronic sclerosing sialadenitis
47Submandibular1.00/2.082.8BenignBenignEctopic thyroid
48Pharyngeal1.00/1.30/2.00/3.103.3MalignantMalignantMyxoid liposarkoma
49Pharyngeal1.00/2.001.1BenignBenignGanglioneuromy
50Pharyngeal1.00/2.003.3BenignBenignPleomorphic adenoma
51Pharyngeal1.00/1.26/1.64/2.046.7MalignantMalignantRhabdomyosarkoma
Table 2. Classification of LSC Analyzed FNAB Samples From Solid Salivary Gland Tumors and Their Histopathology
 Histology malignantHistology benignSum
  1. The confusion matrix LSC versus histopathology of 51 FNABs from human solid salivary gland tumors provides a positive (PPV) and negative (NPV) predictive values of 1.00 and 0.93. Samples were analyzed by LSC and classified as benign or malignant as described in Material and Methods. The resected specimens were sent to routine histopathology as golden standard. Fishers exact test served for the determination of P. Specificity = 1.0, PPV = 1.0; Sensitivity = 0.79, NPV = 0.93. P = 7.6 × 10−9 (Fishers exact test).

LSC malignant11011
LSC benign33740
Sum143751

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSIONS
  7. Acknowledgements
  8. LITERATURE CITED

The setup of our assay includes only one washing and spinning step prior to immobilization of the cells on the slide. This is important because FNABs are hypocellular specimens, and repeated washing steps may reduce cell numbers. All staining steps are performed on the slide after fixation of the cells. Immobilization was stable enough to allow a second staining procedure, i.e., HE, without considerable cell loss.

Some samples were analyzed as multiple aliquots at different times after FNAB preparation. No differences in cytokeratin expression or DNA ploidy after prolonged storage even for up to 18 months were observed. This indicates that cell constituents are very stable once cells are immobilized and fixed on the slide. Specimen stability may be of special value in medicolegal issues since specimens can be used in aliquots for a second analysis and stored as conventional cytological specimens.

Although a relative small number of tumors (N = 51) was analyzed is this study, a broad spectrum of histopathological diagnoses was covered by the benign as well as by the malignant tumors. This broad spectrum is typical for salivary gland tumors and explains why analysis by conventional cytology is difficult. Nevertheless, all tumors diagnosed as benign by routine histopathology showed DNA diploid tumor cells by LSC analysis. Some malignant tumors were DNA diploid as well, but most importantly DNA aneuploidy was restricted to malignancy. In addition to DNA ploidy, the 5cER was evaluated as another sign of malignancy. The 5cER is a long established prognosis-related feature in image cytometry of squamous cell carcinoma of the head and neck (21). It was shown on Feulgen-stained cytological specimens that the 5cER increases with higher grade of malignancy.

Keeping the limitations of an initial study - relative small patient number, heterogeneity of tumors - in mind, the assay described here promises to generate objective preoperative data (DNA ploidy and 5cER) on solid salivary gland tumors. The excellent correlation of LSC classification with routine histopathology and the PPV and NPV, as well as the specificity and sensitivity of the LSC classification, could make this analysis a valuable tool for the clinician and surgical oncologist. If these data were confirmed by a large-scale multi-center study, they could be included in the therapeutic strategy decision-making process. Three out of 14 malignant tumors were missed by LSC (Table 1); these three tumors comprised one squamous cell carcinoma (#8) and two MALT-lymphomas (#9 and #39). This indicates the present limitation of FNAB analysis solely by LSC in case of DNA diploid malignant tumors. DNA diploid tumors could additionally be stained for surface markers, or other innovative techniques such as the detection of DNA strand breaks (22) and of certain other features (23) could be employed. Not only the cytokeratin positive population, but also the cytokeratin negative population can be used for the detection of DNA aneuploid cells or nuclei as exemplified by patient 29, where a mixed malignant tumor was present. Additionally, specimens could be analyzed by conventional cytology after LSC analysis. In conventional cytology, tissue fragments rather than single cells are analyzed. On the single cell level, nuclear to cytoplasmic ratios in HE-stained cells may be used as an additional indicator for the detection of malignant cytokeratin positive DNA diploid cells. In tissue fragments, DNA diploid malignant tumors may exhibit further distinctive features of malignancy, such as growth patterns being not apparent at the single-cell level.

CONCLUSIONS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSIONS
  7. Acknowledgements
  8. LITERATURE CITED

We conclude that LSC analysis of FNABs represents a promising method for the determination of DNA ploidy and 5cER in solid salivary gland tumors. It could help to detect malignancy preoperatively in a clinical setting and therefore may pave the way for an individualized management of patients, such as single-step operation including radical surgery, nerve reconstruction, and neck dissection. A large-scale, out-patient based, multi-center study is required to spread this procedure. If the data of this study were confirmed, positive consequences could yield shorter hospitalization and faster recovery for the patient at cost reduction for the health care system.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSIONS
  7. Acknowledgements
  8. LITERATURE CITED

We acknowledge the help of the team in the surgical ward headed by Mrs. C. Scheppukat in acquiring the samples and the assistance of Prof. Dr. rer. nat. O. Gerstner, Dept. of Mathematics, University of Erlangen, Germany, in performing statistical analyses.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSIONS
  7. Acknowledgements
  8. LITERATURE CITED
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