Am I Otolaryngol 11:393-397.1990

DNA Content in Nasopharyngeal Carcinoma DAVID S. CHENG, MD, BRUCEH. CAMPBELL,MD, LAWRENCEJ. CLOWRY,MD, LARRYE. HOPWOOD,PhD, KEVIN J. MURRAY,MD, ROBERTJ. TOOHILL, MD, AND RAYMONDG. HOFFMANN,PhD DNA analysis by flow cytometry was performed on tissue blocks from 41 patients with nasopharyngeal carcinoma. The histologic slides were reviewed by a pathologist and blindly classified according to the World Health Organization classification. The paraffinembedded blocks were processed to obtain individual nuclei, which were then stained with propidium iodide. The nuclei were analyzed on a flow cytometer. Excluding IO uninterpretable histograms, the remainder were interpreted blindly and classified as diploid or aneuploid. The Cox proportional hazards survival model was used to analyze stage, histology, radiation dose, and ploidy. We observed more diploids (23 of 31; 74%) than aneuploids (eight of 31; 26%). The 2-year survival rate of diploids was 55%, compared with 25% of aneuploids (P < -05). We conclude that ploidy status is an independent prognostic factor in nasopharyngeal carcinoma. AM J OTOLARYNGOL 11:393-397. 0 1990 by W.B. Saunders Company. Key words: nasopharyngeal carcinoma, DNA content, flow cytometry.

Nasopharyngeal carcinoma (NPC) prognosis depends on the stage at presentation. For patients with stage I or II disease, the s-year survival rate is 93%. However, for stage III or IV disease, the 5year survival rate drops to 43%.l Most patients present in stage III or IV. Investigators have examined factors besides tumor stage that might provide insight into prognosis. For example, Applebaum et al2 confirmed that squamous cell carcinoma carries a worse prognosis than lymphoepithelioma. Recently, Neel et al3 examined the antibody-dependent cellular cytotoxicity (ADCC) titer against the Epstein-Barr virus-induced membrane antigen, and concluded that in World Health Organization types 2 and 3 tumors, a low ADCC titer correlated with a poorer prognosis. On the other hand, Looi found that tumor-associated tissue eosinophilia had no prognostic significance. Recently, there has been interest in flow

Received September 27, 1988,from the Departments of Otolaryngology and Human Communication, Radiation Oncology and Pathology and the Division of Biostatistics and Clinical Epidemiology, Medical College of Wisconsin, Milwaukee, WI. Accepted for publication June 20, 1990. Presented at the Second International Conference on Head and Neck Cancer, Boston, MA, August 3, 1988. Address correspondence and reprint requests to Bruce H. Campbell, MD, Medical College of Wisconsin, Department of Otolaryngology and Human Communication, 8700 W Wisconsin Ave, Milwaukee, WI 53226. 0 1990 by W.B. Saunders Company. 0196-0709/90/1106-0006$5.00/O

cytometry as a method of DNA analysis and as a potential predictive assay. The purpose of this study was to analyze the DNA content of NPC and to determine if ploidy status in NPC might be helpful in determining prognosis. METHODS We reviewed 53 patients with NPC who were treated between the years 1959 and 1986 at three of the Medical College of Wisconsin-affiliated hospitals. The patients were treated with varying doses of radiation ranging from 1,900 to 7,660 cGy to the nasopharynx and 4,500 to 5,000 cGy to the neck. If adjuvant chemotherapy was given, it consisted of two to three courses of cisplatin and 5-fluorouracil. The stage of the disease, according to the American Joint Committee on Cancer classification,’ was retrospectively determined by assessing the patient’s history, physical examination, operative report, and radiographs (either computerized tomography scan or lateral skull films). Distant metastases were assessed by examining the history, physical examination, and radiographs (either computerized tomography of the liver or liver-spleen scan]. HOW Cytometry. Paraffin-embedded blocks of tissue from the nasopharynx were obtained for 41 of the 53 patients. A hematoxylin-eosin-stained section was cut to ensure that more than 25% tumor cells were present. The technique of Bauer et al” was used to process the blocks. Multiple 50-km sections of the paraffin blocks were cut and dewaxed in two lo-mL washes of xylene for 10 minutes. The tissue was rehydrated in lo-mL of ethanol at concentrations of 100% (twice), 95%, 70%, and 50% for 10 minutes each, and left in distilled water. The next day, the tissue was minced

DNA CONTENT IN NASOPHARYNGEAL CARCINOMA

394

into tiny pieces with a dissecting scissors and suspended in 1 mL of 0.5% pepsin at pH 1.5 for 30 minutes at 37°C and vortexed every 5 minutes. The reaction was stopped with 100 ~1 of pepstatin A (0.51 mg/mL). A pipet was used to break up large pieces of tissue. The suspension was then filtered through a 62-pm filter. The bare nuclei were neutralized with 15 mL of HEPESHanks medium and centrifuged at 1,000 rpm for 10 minutes. The pellet was resuspended with 2 mL of HEPES-Hanks medium and counted to ensure adequate recovery. The nuclei were then centrifuged at 1,000 rpm for 10 minutes. After removal of the supernatant, they were stained with propidium iodide (2.5 kg/l to 2 million nuclei) solution also containing RNAase (180 U/mL), PEG 6000 (3%), Triton X (O.l%], and citrate buffer (pH 7.8) at 37°C for 20 minutes. An equal amount of salt solution consisting of sodium chloride (0.4 mall L, PEG 6000 (3%), and Triton X (0.1%) was then added, and the nuclei were left overnight at 4%. An EPICS model flow cytometer, using the 488 nm Argon laser at 400 mW, was used to analyze DNA content. When recoverable, at least 10,000 to 15,000 nuclei were counted for each patient. Two blocks of tissue from cervical lymph nodes obtained between the years 1975 and 1986 were used as external controls, Both of these histograms were diploid. When there were several blocks of tissue from the same patient, all blocks were submitted. Of the 41 patients with paraffin blocks, 10 did not have any blocks of tissue with interpretable histograms. Flow cytometry histograms from the remaining 31 patients were analyzed blindly as either diploid or aneuploid according to Murray et al.’ The definition of aneuploid applies when any of following three criteria are met: the presence of two distinct Gl/GO peaks, the presence of a single broad GlGO peak whose ratio of % width to height exceeded 0.2, and the presence of tetraploidy as defined by a GYM peak exceeding 20% of the total population of nuclei. Figure 1 shows a histogram that was classified as diploid because the first peak was identified as Cl/GO, and no other distinct peaks were noted except for a GYM peak that was not more than 20% of the total population. Figure 2 shows a histogram that was classified as aneuploid because there was a definite peak distinct from the first Gl/GO peak, fulfilling the first criterion. The hematoxylin-eosin-stained sections were blindly reviewed by a pathologist. Tumors were classified according to the WHO classification for NPC8: class 1 is keratinizing squamous cell carcinoma showing sheets of stratified squamous epithelial cells that resemble normal keratinizing squamous epithelium; class 2 is nonkeratinizing squamous cell carcinoma with no ker-

Figure 1. An example of diploidy (patient no.

10).

Figure 2. An example of aneuploidy

(patient no.

27).

atin, growing in a random fashion resembling transitional cell carcinoma of the urinary bladder; and class 8 is lymphoepithelioma resembling anaplastic carcinoma or clear cell carcinoma lacking differentiation, with a large lymphoid background. Actuarial and determinate survival statistics were calculated. The Cox proportional hazards survival model9 was used to assess the combined effect of stage, histologic type, radiation dose, and ploidy status for actuarial and disease-free survival. Patients with inadequate follow-up were excluded from the survival statistics calculations. RESULTS

We reviewed 53 patients with NPC. Patients ranged in age from 7 to 87 years, with a mean age of 54 years. There were eight black patients and 45 white patients; 36 were men and 17 were women. Forty-two of the 53 patients were stage IV (Table 1). Only two patients (nos. 5 and 44) presented with distant metastases, and both died of their disease. Of the 53 patients, 15 (28%) were WHO class 1, 17 (32%) were class 2, and 21 (40%) were class 3. The primary treatment was irradiation to the nasopharynx and neck. Eleven patients received adjuvant chemotherapy and nine received elective neck dissections (Table 1). Only one patient (no. 10) received preradiation neck dissection; the remaining dissections were performed after radiation therapy on patients who had clinically positive neck disease prior to treatment. The neck contents for the postradiation neck dissections were all pathologically negative for viable tumor cells. Of the 53 patients with NPC, blocks were located for 41 patients. Of these, 10 patients did not have interpretable histograms either because of initial fixation in Bouin’s solution or inadequate numbers of nuclei after tissue processing. Many blocks contained only tiny amounts of tissue. Thirty-one patients had interpretable histograms; eight were aneuploid and 23 were diploid. Patient no. 10 had both the neck mass and the primary tumor analyzed by flow cytometry, and both were diploid. Patient no. 27 had both the

CHENG ET AL

395

TABLE 1.

Patient

Statistics

RADIATION

PATIENT No.

AJCC STAGE

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53

4 4 4 4 4 4 4 2 3 4 4 4 3 2 4 4 4 4 1 3 4 4 4 4 4 3 4 4 4 4 3 4 4 4 4 4 4 3 4 4 4 2 4 4 4 4 4 4 4 3 4 4 4

-

CHEMOTHERAPY

Abbreviations: D, diploid; evidence of disease.

Yes

Yes

Yes Yes Yes

Yes Yes

Yes Yes Yes Yes A, aneuploid;

DOSE TO THE NASOPHARYNX

NECK DISSICTION

1,900 3,000 3,800 4,800 5,000 5,940 5,950 6,000 6,000 6,000 6,400 6,480 6,560 6,600 6,600 6,760 6,900 6,960 7,000 7,020 7,020 7,400 7,660 0 3,000 6,000 6,480 6,600 6,840 7,020 7,560 1,500 2,150 5,100 5,200 5,400 5,800 6,000 6,000 6,120 6,460 6,460 6,500 6,600 6,620 6,880 7,000 7,000 7,000 7,000 7,000 7,150 7,500

WHO HISTOLOGIC CLASSIFICATION FORNPC

Yes

Yes Yes Yes Yes

Yes

Yes

2 3 3 1 1 2 3 1 1 2 1 3 3 2 1 2 3 3 2

SURVIVAL PLOIDY

(YRJ

OUTCOME

D D D D D D D D D D D D D D D D D D D D D D D A A A A A A A A

1.25 0.5 0.5 2.75 2.25

DOD DOD DOD DOD DOD DOC NED DOD DOD DOD DOC DOD NED DOD DOD NED NED NED NED NED NED DOC AWD DOD DOD NED DOD DOD DOD DOD NED DOD DOD DOD DOD NED NED DOD DOD NED DOD NED DOD DOD DOD DOD DOC DOD DOD NED DOC DOD DOD

10

8 0.5 1.5 8 0.5 3.5 8 8.5 8 1.25 1.25 0.75 1.25 0

15 0.75 1.25 0.75 3 0 0 0.5 0.5 11 10 2 1.5 1.5 9

Yes

Yes

2.5 0.5 0.5 14 1 1.75 4 2.5 2 0.75

DOD, dead of disease; DOC, dead of other causes; AWD, alive with disease; NED, no

neck mass and the primary tumor analyzed by flow cytometry, and both were aneuploid. Three patients had diploid and aneuploid histograms when different sites of the same nasopharyngeal carcinoma were sampled (nos. 26, 29, and 31); these three patients were therefore classified as aneuploid. Two patients (nos. 15 and 23) had histograms from different sites of the same nasopharyngeal carcinoma and both showed diploid.

Actuarial survivals are shown in Table 2. Of the 53 patients, three were excluded from the Z-year and six from the 5-year actuarial survival calculations because of inadequate follow-up. The X-year actuarial survival rates for patients with diploid and aneuploid tumors were 55% and 25%, respectively. Both surviving patients with aneuploid histograms had WHO class 3 tumors (Table 3). The 5-year actuarial survival rates were 32% for

DNA CONTENT IN NASOPHARYNGEAL CARCINOMA

396

TABLE 2.

Actuarial z-YR

WHO Class 1 Class 2 Class 3 Diploid Aneuploid No flow Stage l&2 3 4

TABLE 4. Probability Values in Cox Analysis

Survival

(%I

3114 (21) 5/16 (31) 12/20(65) 11/20 (55) 2/8 (25) i3/22(36)

o/12 (0) 2/16 (13) lo/19 (53) 6119 (32) l/7 (14) 5121 (24)

3/4 (75) 4/6 (67) 14/40(35)

2/3 (67) 2/4 (50) 8140 (20)

diploid and 14% for aneuploid. For patients who died of their disease, the mean survival was 15 months. The Zyear actuarial survival rate was better for WHO class 3 (65%) than for WHO class 1 (21%) patients (Table 3). There were no survivors at 2 years in the group with aneuploid WHO class 1 or 2 tumors, compared with four of nine survivors of diploid WHO class 1 or 2 tumors. The Cox proportional hazards model was used to examine the simultaneous effect of stage, histology, radiation dose, and ploidy status. The significance of each factor is presented in Table 4, showing that ploidy status has a significant effect on survival even after adjustment for stage, histologic type, and radiation dose. DISCUSSION Prior to 1983, flow cytometry was only applicable to fresh tissue. Hedley et al” described a method of applying DNA flow cytometry to paraffin-embedded archival blocks, and investigators have reported results on a par with fresh tissue Flow cytometry DNA analysis flow cytometry.ll data have been reported for several tumors. Generally, aneuploidy tends to correlate with a poorer prognosis for patients with tumors of the breast,7 prostate, I2 and colon.13 Data is emerging for head and neck tumors. Abnormal (nondiploid) DNA content in head and neck cancers has ranged from 46%14 to 75 0/o. I5 Goldsmith et all6 examined squamous cell carcinoma of the tongue, larynx, and hypopharynx and found that aneuploidy correlated with a poorer prognosis in tongue cancers TABLE 3.

Actuarial

DIPLOID (%I

Survival

ANEUPLOID (%I

at 2

215 (40) 2/4 (50) 7/11(64) 11/20(55)

o/4 (0) o/2 (0) 2/2 (100) 218 (25)

DISEASE-FREE SURVIVAL

Stage Histology Radiation dose Ploidy

NS .02 .006 .03

Abbreviation:

NS, not significant.

ACTUARIAL SURVIVAL

NS .03

.0005 .02

but a better prognosis in larynx and hypopharynx cancers. However, Gussack et all’ correlated aneuploidy with a poorer prognosis in larynx cancers. Farrar et al” and Tytor et all9 agreed that aneuploid oral cavity cancers had a worse prognosis. Advanced NPC generally has a poor prognosis, but some predictors of survival have emerged. The addition of DNA analysis to the clinical and laboratory evaluation may help to assess the status of the patient and the tumor, and may be a useful prognostic tool. The incidence of aneuploid tumors in this study was 26%; this rate is lower than the 46% to 75% reported for other head and neck sites.14g15 Statistically, we found that the group with diploid tumors had a significantly better survival than the group with aneuploid tumors. The survival curve for ploidy status is shown in Fig 3. Our study also examined the prognostic significance of staging, histology, and radiation dosages. Stage IV disease connotes a worse prognosis; however, this correlation was not statistically significant. As with other studies, almost 90% of patients presented with stage III or IV disease. There was a significantly better prognosis for lymphoepithelioma (WHO 3) than for the other histologies (Fig 4). This is consistent with p,revious studies.’ As shown in the past, radiation dose has a significant influence on prognosis. In Cox analysis, radiation dosage was the most significant factor (P = .005) and reflects the evolution of the treatment of NPC. Ploidy status and histology

o dlplold 0

aneuploid

Years

NOFLOW (%I

TOTAL (%I

WHO

Class 1 Class 2 Class 3 Total

FACTOR

5-YR (%)

l/5 (20) 3/10 (30) 4/7 (57) 8/22 (36)

3/14 (21) 5116 (31) 13120 (651

OJ

,

,

,

0

5

10

15

,

,

,

20 25 30 35

,

,

/

40

45

(

Survival (months) Figure 3.

Kaplan-Meier

,

50 55

plot for survival by ploidy.

,

60

397

CHENG ET AL

who3

:;I, , ,~iK 0

5

IO 15 20

25 30

35 40 45

50 55 60

Survival (months)

Figure 4.

Kaplan-Meier

plot for survival by histology.

were also statistically significant independent factors. Our results indicate that aneuploidy correlates with diminished survival. In our study, ploidy status emerged as a statistically significant independent variable after correcting for stage, histology, and radiation dose. Ploidy status has become an independent factor” in the prognosis of head and neck carcinoma and is a cofactor, in conjunction with histology, ADCC titer, and staging, in the prognosis of patients with NPC. Acknowledgment. The authors thank Pat McFadden and Kris Daenhert for their technical assistance. References 1. Neel HB III: Nasopharyngeal carcinoma. Otolaryngol Clin North Am 1985; 18:479-490 2. Applebaum EL, Mantravadi P, Haas R: Lymphoepithelioma of the nasopharynx. Laryngoscope 1982; 92510-514 3. Neel HB III, Pearson BR, Taylor WF: Antibody-dependent cellular cytotoxicity: Relation to stage and disease course in North American patients with nasopharyngeal carcinoma. Arch Otolaryngol Head Neck Surg 1984; llO:742-747 4. Looi L: Tumor-associated tissue eosinophilia in nasopharyngeal carcinoma. Cancer 1987; 59:466-470 5. Staging of Cancer of Head and Neck Sites and of Melanoma. American Joint Committee on Cancer, Chicago, IL, 1980

6. Bauer KD, Clevenger CV, Engelhard HH: DNA and nuclear antigen analysis of tissue specimens. Department of Pathology, Northwestern University McGaw Medical Center, Chicago, IL 7. Murray K, Hopwood L, Volk D, et al: Cytofluorometric analvsis of the DNA content in ovarian Ca and its relationshiu to patient survival. Presented at the Conference on Predictioh of Tumor Response in Banff, Alberta, Canada, April 21-24, 1987 8. Weiland LH: Nasopharyngeal carcinoma, in Barnes L (ed): Pathology of the Head and Neck. Dekker, New York, NY, 1985, pp 453-466 9. Kalbfleisch JD, Prentice RL: The Statistical Analysis of Failure Time Data, Wiley, New York, NY, 1980 10. Hedley DW, Friedlander ML, Taylor IW, et al: Method for analysis of cellular DNA content of paraffin-embedded pathological material using flow Cytometry. J Histochem Cytochem 1983; 31:1333-1335 11. Berlinger NT, Malone BN, Kay NE: A comparison of flow cytometric DNA analyses of fresh and fixed squamous cell carcinomas. Arch Otolaryngol Head Neck Surg 1987; 113:1301-1305 12. Tribukait B, Gustafson

H, Esposti P: Ploidy and proliferation in human bladder tumors as measured by flow cytofluorometric DNA-analysis and its reactions to histopathology and cytology. Cancer 1979; 43:1742-1751 13. Woiley RC, Schreiber K, Koss LG, et al: DNA distribution in human colon carcinoma and its relationship to clinical behavior. JNCI 1982; 69:15-22 14. Kaplan AS, Caldarelli DD, Chacho MS, et al: Retrospective DNA analysis of head and neck squamous cell carcinoma. Arch Otolaryngol Head Neck Surg 1986; 112;1159-1162 15. Holm LE: Cellular DNA amounts of squamous cell carcinomas of the head and neck region in relation to prognosis. Laryngoscope 1982; 92:1064-1069 16. Goldsmith MM, Cresson DH, Arnold LA, et al: Part I. DNA flow cytometry as a prognostic indicator in head and neck cancer. Otolaryngol Head Neck Surg 1987; 96:307-318 17. Gussack GS, Donelly K, Hester R, et al: Flow cytometric DNA analysis of laryngeal carcinomas. Presented at the 1987 Meeting of the American Academy of Otolaryngology-Head and Neck Surgery, Chicago, IL 18. Farrar WB, Artman S, Sickle-Santenello B, et al: Flow cytometric analysis of DNA content as a prognostic indicator in squamous cell carcinoma of the oral cavity. Presented at the 1987 Meeting of the American Academy of OtolaryngologyHead and Neck Surgery, Chicago, IL 19. Tytor M, Franzen G, Olofsson J, et al: DNA content, malignancy grading and prognosis in Tl and T2 oral cavity carcinomas. Br J Cancer 1987; 56:647-752 20. Kokal WA, Gardine RL, Sheibani K, et al: Tumor DNA content as a prognostic indicator in squamous cell carcinoma of the head and neck region. Am J Surg 1989; 156:276-280

DNA content in nasopharyngeal carcinoma.

DNA analysis by flow cytometry was performed on tissue blocks from 41 patients with nasopharyngeal carcinoma. The histologic slides were reviewed by a...
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