HISTOLOGIC DIFFERENTIATION AND CHEMOSENSITIVITY OF HUMAN HEAD AND NECK SQUAMOUS CELL CARCINOMAS Tadashi Nakashima, MD, Yoshihiko Maehara, MD, Shunji Kohnoe, MD, ltsuroh Hayashi, MD, and Yasaburoh Katsuta, MD
The chernosensitivities of 42 human head and neck squamous cell carcinomas were examined using the in vitro succinate dehydrogenase inhibition (SDI) test. The tumor tissues obtained at surgery or biopsy were exposed to five different antitumor drugs: adriamycin (ADM), cisplatin (CDDP), carboquone (CQ), 5-fluorouracil (5-FU), and 1-hexylcarbamoyl-5-fluorouracil (HCFU). The results were analyzed according to the histopathologic degree of differentiation of well, moderately, and poorly differentiated squamous cell carcinoma. The average decrease in succinate dehydrogenase activity was 43.2 t 24.9 for ADM, 29.0 t 14.2 for CDDP, 32.9 f 17.6 for CQ, 64.2 ? 20.6 for 5FU, and 26.8 ? 16.9 for HCFU. There was a statistically significant difference in the decrease of succinate dehydrogenase activity between well and poorly differentiated squarnous cell carcinomas. These data suggest that, for patients with a poorly differentiated squamous cell carcinoma, the response to anticancer drugs may be more satisfactory than in those with a welldifferentiated squamous cell carcinoma. HEAD & NECK 1990; 12:406-410
From the Departments of Otolaryngology (Dr Nakashima) and Pathology (Drs. Hayashi and Katsuta). National Kyushu Cancer Center, Fukuoka. Japan; and Cancer Center of Kyushu University Hospital (Drs Maehara and Kohnoe). Fukuoka, Japan Acknowledgments. Supported by a Grant-in-Aid for scientific research from the Fukuoka Cancer Society. Japan The authors thank K. Miyarnoto and K Fukuchi tor expert technical assistance and M Ohara for helptul comments. Address reprint requests to Dr. Nakashima at the Department of Otolaryngology, National Kyushu Cancer Center, Notame 3- 1 1, Minamiku. Fukuoka 815, Japan. ~
Accepted for publication March 3. 1990 CCC 0148-64031901050406- 05 $04.00 0 1990 John Wilev 8, Sons, Inc
406
Chemosensitivity of Head and Neck Cancer
squamous cell carcinomas of the head and neck regions are among the most morbid of human cancers. Despite optimal treatment with surgery and radiotherapy, local tumor control remains a significant problem. Distant metastases occur frequently and second primaries account for the remainder of deaths. These unfavorable results, combined with functional and cosmetic disabilities associated with local or distant recurrence, have led to investigations of additional chemotherapy for head and neck cancer patients.'-3 We focused our attention on the development of a test, which can determine the sensitivity or resistance of malignant tumors to cytostatic agents, prior t o ~ h e m o t h e r a p y . In ~ , ~the present study, we examined differences in chemosensitivity of human head and neck squamous cell carcinomas to various antitumor drugs, as assessed from the point of histologic differentiation. MATERIALS AND METHODS
Materials. Cancer specimens were obtained at biopsy or surgery on 42 Japanese patients with head and neck cancer. All specimens were immediately placed in McCoy's 5A solution. The tumors consisted of primary and metastatic carcinomas (13 pharyngeal cancers, 10 laryngeal cancers, 8 oral cavity cancers, 6 maxillary sinus
HEAD & NECK
SeptemberiOctober 1990
Table 1. Number of head and neck cancer patients with well (W), moderately (M), and poorly (P) differentiated squamous cell carcinomas. Head and neck cancer
w
M
P
Pharyngeal cancer Laryngeal cancer Oral cavity cancer Maxillary sinus cancer Esophageal cancer Other
3 5 2 4 1 0
4 5 5 2 2 0
6 0 1 0 1 1
Total
15
18
9
cancers, 4 esophageal cancers, and 1 metastatic cancer of unknown origin) found in the head and neck region. Pathological examination of the same tissue specimens revealed that 15 were well differentiated, 18 moderately differentiated, and 9 poorly differentiated squamous cell carcinomas (Table 1). The antitumor drugs tested were adriamycin (ADM), cisplatin (CDDP), carboquone (CQ), 5-fluorouracil(5-FU),and l-hexylcarbamoyl-5-fluorouracil (HCFU). Each drug was tested at 10 times the peak plasma concentration6: ADM, 4 pg/ml; CDDP, 20 pg/ml; CQ, 0.5 kg/ml; 5-FU, 100 pglml; and HCFU, 198 pg/ml. HCFU was tested in an equimolar concentration with 5-FU.7 The sources of these drugs were as follows: ADM and 5-FU were from Kyowa Hakko Co., Ltd., Tokyo, Japan; CDDP from Nihon Kayaku Co., Ltd., Tokyo, Japan; C Q from Sankyo Co., Ltd., Tokyo, Japan; and HCFU from Mitsui Co., Ltd., Tokyo, Japan.
Antitumor Drugs.
The succinate dehydrogenase inhibition (SDI) test was performed as previously d e ~ c r i b e d Briefly, .~ tumor tissues were cut with scissors, passed through no. 32 stainless steel mesh into McCoy’s 5A solution containing antibiotics, and washed three times with the same solution. The fragments were suspended in minimum essential medium with Lglutamine (292 mg/ml), 10% fetal calf serum, and antibiotics, placed in 35-mm plastic dishes (3 dishes per test group and 4 to 6 dishes per control), and then incubated for 3 days while exposed to the antitumor drug in medium containing 1% dimethyl sulfoxide at 37°C in a humidified 5% CO, atmosphere. The tissue fragments were washed with phosphate-buffered saline (PBS, pH 7.2 at 4°C) and
assayed for succinate dehydrogenase activity. 3(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT18 was used as the hydrogen acceptor for the succinate dehydrogenase activity. The formazan formed from MTT was extracted with acetone containing 0.5% trichloroacetic acid, and the absorbance of formazan was measured at 565 nm. The succinate dehydrogenase activity was presented as the optical density (OD) per milligram protein. The tumor fragments with an OD of 0.5 on day 0 were plated in separated dishes. The chemosensitivity was estimated by the percentage of succinate dehydrogenase activity of the drug-treated cells, compared with that of control cells, and the sensitivity was determined as positive when the succinate dehydrogenase activity was decreased to less than 50%. Significant differences between means were determined by Student’s t test. A p value of c0.05 was considered to be statistically significant. Statistical Analysis.
RESULTS
The chemosensitivity was indicated by the percentage of succinate dehydrogenase activity of drug-treated cells as compared to that of control cells. The decrease in succinate dehydrogenase activity with each antitumor drug varied (Figure 1).The averages of succinate dehydrogenase activity of 42 head and neck squamous cells carcinomas were 43.2 -t 24.9 for ADM, 29.0 14.2 for +_
ADM
CDDP
CQ
In vitro Chemosensitivity Test.
Chernosensitivity of Head and Neck Cancer
5-FU
‘r
HCFU
4: 4
8
f
ri t 8
I
FIGURE 1. Decrease in succinate dehydrogenase activity in head and neck squamous cell carcinomas. The chemosensitivity is indicated by the percentage of succinate dehydrogenase activity of drug-treated cells compared to that of control cells. Values are means ? standard deviation (SD).
HEAD & NECK
SeptemberIOctober 1990
407
Table 2. Decrease in succinate dehydrogenase activity in well-differentiated squamous cell carcinomas. Case no./origin
ADM
CDDP
CQ
5-FU
HCFU
1. Larynx 2. Oral cavity 3. Maxillary sinus 4. Maxillary sinus 5. Pharynx 6. Esophagus 7. Larynx 8. Oral cavity 9. Pharynx 10. Larynx 11. Maxillary sinus 12. Larynx 13. Larynx 14. Pharynx 15. Maxillary sinus
62.8 58.3 95.8 39.5 32.3 60.5 23.0 30.2 39.4 38.1 17.8 91 .o 96.4 35.0 48.2
32.5 38.0 42.0 44.1 25.0 41.3 17.2 22.3 31.7 29.2 55.3 53.7 39.0 33.8 52.6
47.1
90.3 75.2 94.6 55.2 56.5 61.5 94.9 96.4 81.4 74.1 61.2 100.0 85.7 61.7 88.8
32.5 33.0 75.2 34.7 21.5 57.9 19.0 17 3 14.6 17.2 16.3 61 2 39 0 13.5 41.6
Average
-t
SD
51.2
-t
25.8
37.2 i 11.4
*
CDDP, 32.9 * 17.6 for CQ, 64.2 20.6 for 5-FU, and 26.8 * 16.9 for HCFU. Most squamous cell carcinomas showed a high sensitivity to CDDP, CQ, and HCFU and a low sensitivity to 5-FU. In certain cases, however, the decrease in succinate dehydrogenase activity by 5-FU was prominent, the level being much the same as seen with the other 4 drugs. The results of succinate dehydrogenase activity and chemosensitivity were analyzed according to the histologic differentiation (Tables 2-4).
42.4 28.4 39.0 19.0 31.7 35.1 29.6 39.1 76.1 39.8 32.7 48.2 39.1
* 13.7
78.5
15.9
33.0
* 19.1
In 15 cancer tissues diagnosed as well-differentiated squamous cell carcinoma, the averages of succinate dehydrogenase activity were 51.2 25.8 for ADM, 37.2 11.4 for CDDP, 39.1 2 13.7 for CQ, 78.5 2 15.9 for 5-FU, and 33.0 2 19.1 for HCFU. In 18 cancer tissues diagnosed as moderately differentiated squamous cell carcinomas, the averages of succinate dehydrogenase activity were 45.3 ? 24.2 for ADM, 28.7 2 12.7 for CDDP, 36.3 2 19.9 for CQ, 60.1 2 20.6 for 5-FU, and 26.5 k 14.7 for HCFU. The averages from 9
*
_+
~~
Table 3. Decrease in succinate dehydrogenase activity in moderately differentiated squamous cell carcinomas. Case no./origin
I . Maxillary sinus 2. Oral cavity 3. Pharynx 4. Pharynx 5. Larynx 6. Oral cavity 7. Larynx 8. Pharynx 9. Esophagus 10. Oral cavity 11. Pharynx 12. Oral cavity 13. Larynx 14. Oral cavity 15. Maxillary sinus 16. Larynx 17. Esophagus 18. Larynx
Average 2 SD
408
ADM
CDDP
CQ
5-FU
HCFU
24.0 19.5 88.3 26.5 71.1 70.9 33.8 17.5 36.2 14.1 66.7 54.5 88.4 20.1 42.6 49.2 34.1 58.2
18.0 15.8 44.1 26.2 49.6 17.1 30.0 13.4 9.2 28.3 23.5 30.6 51.9 14.5 38.8 37.7 34.1 34.3
54.3 25.5 42.5 35.3
54.3 38.1 74.0 97.7 73.0 62.8 55.2 23.4 31.8 47.3 63.0 73.9 94.2 49.2 33.5 65.3 65.5 78.9
8.4 13.0 37 0 25.0 43.3 34.9 12.2 16.5 13.4 25.9 32.6 21.6 58.1 5.4 39.2
28.7 2 12.7
36.3 2 19.9
45.3
C
24.2
Chemosensitivity of Head and Neck Cancer
18.8 28.1 15.1 14.3 24.1 32.5 70.4 21.3 32.1 48.5 85.6 32.5
60.1
?
20.6
HEAD & NECK
37.9 26.5
-t
14.7
Septernber/October 1990
Table 4. Decrease in succinate dehydrogenase activity in poorly differentiated squamous cell carcinomas. Case no./oriain 1. 2. 3. 4. 5. 6. 7. 8. 9.
Pharynx Oral cavity Pharynx Esophagus Pharynx Pharynx Unknown Pharynx Pharynx
Average f SD
ADM
CDDP
CQ
5-FU
HCFU
27.2 7.8 23.2 16.5 17.3 16.3 15.0 25.3 18.3
11.4 6.4 18.7 16.9 15.9
16.1 8.3 23.6 18.0 11.6
13.5 25.9 13.6
14.6 28.8 15.7
66.2 49.9 64.7 44.8 25.1 49.5 45.5 44.4 47.9
5.5 17.1 14.1 16.1 16.5 10.9 10.5 30.1 17.0
17.1 t 6.5
48.7 2 12.1
15.3 t 6.8
18.5
* 5.9
15.3
* 5.7
poorly differentiated squamous cell carcinomas were 18.5 2 5.9 for ADM, 15.3 5.7 for CDDP, 17.1 k 6.5 for CQ, 48.7 2 12.1 for 5-FU, and 15.3 I+_ 6.8 for HCFU. There was no difference in succinate dehydrogenase activity in cases of exposure to CDDP and HCFU between well and moderately differentiated squamous cells carcinomas. On exposure to ADM ( p < 0.001), CQ ( p < 0.001) and 5-FU ( p < 0.05), there was a greater decrease in succinate dehydrogenase activity in the moderately differentiated as compared to the well-differentiated squamous cell carcinomas. The decrease in succinate dehydrogenase activity was remarkable in the poorly differentiated tissues, compared with the well-differentiated tissues, exposed to ADM (p < 0.0051, CDDP ( p < 0.001), CQ ( p < 0.001),5-FU ( p < 0.001), and HCFU ( p < 0.02). Even in comparison with moderately differentiated tissues, there was a statistical difference of decrease in succinate dehydrogenase activity in cases of ADM ( p < 0.005), CDDP ( p < 0.01),CQ ( p < 0.021, and HCFU ( p < 0.05). The chemosensitivity rate to each drug was evaluated (Fig. 2). In the well-differentiated squamous cell carcinomas, the highest sensitivity was seen with exposure to CDDP (80%), CQ (92%),and HCFU (80%), but none of these examined tissues was sensitive to 5-FU. In the moderately differentiated squamous cell carcinomas, CDDP (94%) and HCFU (94%) were the most sensitive. Six of eighteen (33%) tissues were sensitive to 5-FU. In contrast, all poorly differentiated squamous cell carcinomas were sensitive to ADM, CDDP, CQ, and HCFU. Even for 5-FU, 7 of 9 (78%) tissues showed sensitivity. The number of tissues resistant to more than three drugs was 3 (20%) in the well-differentiated squamous
*
Chemosensitivity of Head and Neck Cancer
cell carcinomas and 1 (6%) in the moderately differentiated squamous cell carcinomas. DISCUSSION
Many in vivo and in vitro chemosensitivity tests using different parameters have emerged during
Succinate dehydrogenase activity (%)
1
00qoaep;w
0
' i
FIGURE 2. Comparison of decrease in succinate dehydrogenase activity in the well (W), moderately (M), and poorly (P) differentiated squamous cell carcinomas exposed to ADM (A), CDDP (B), CQ (C), 5-FU (D), and HCFU (E). Values are means f standard deviation (SD). The chemosensitivity was considered to be positive when the succinate dehydrogenase activity of the drug-treated cells decreased to less than 50% of that of control cells (shaded area).
HEAD & NECK
September/October 1990
409
attempts to acquire drugs effective for patients with cancer. Succinate dehydrogenase is an adenosine triphosphate (ATPI-producing enzyme of the citric cycle, and the activity of this enzyme correlates well with cell ~ i a b i l i t y SDI . ~ test is a simple, inexpensive, and rapid in vivo method for screening antitumor drugs. The chemosensitivity of this test correlates with that of the in vitro ATP assay" and the in vivo subrenal capsule (SRC) assay." Using the SDI test, one of the present authors reported differences in the chemosensitivity of drugs between gastric and colorectal cancers. They found that the origin of a lesion was a critical factor related to the chemosensitivity of various tumors.6 We have also examined the succinate dehydrogenase activity in various cancer tissues originating in the head and neck region and found that a malignant lymphoma was the most sensitive to antitumor drugs.5 Head and neck squamous cell carcinomas also responded well to antitumor drugs. The present analysis shows that the histopathologic degree of tissue differentiation could be an important factor for determining chem-
osensitivity of head and neck squamous cell carcinomas. As the decrease of succinate dehydrogenase activity is prominent in poorly differentiated tissues, patients with a head and neck cancer of the poorly differentiated type may be more responsive to chemotherapy, compared to those with moderately or well differentiated type. For patients with the moderately or well differentiated type of head and neck squamous cell carcinoma, sensitive drugs have to be selected. By routinely repeating the SDI test, the clinical effect of an anticancer drug, for the individual patient, can be predicted. It is interesting that most of the poorly differentiated squamous cell carcinomas were from pharyngeal cancers, whereas most of the well or moderately differentiated squamous cell carcinomas were from laryngeal, oral cavity, or maxillary sinus cancers. This tendency suggests the predominant response to chemotherapy by patients with a pharyngeal cancer. We are now collecting data to statisticaly analyze differences in sensitivity to anticancer drugs, depending on site of the lesion in the head and neck regions.
REFERENCES
1. Clark JR, Fallon BG, Dreyfuss AI, e t al. Chemotherapeutic strategies in the multidisciplinary treatment of head and neck cancer. Semin Oncol 1988;15(suppl3):35-44. 2. Adjuvant chemotherapy for advanced head and neck squamous cell carcinoma: Final report of the Head and Neck Contracts Program. Cancer 1987;60:301-311. 3. Jacobs JR, Pajak TF, Kinzie J , et al. Induction chemotherapy in advanced head and neck cancer: A radiation therapy oncology group study. Arch Otolaryngol Head Neck Surg 1987;113:193- 197. 4. Maehara Y, Kusumoto T, Kusumoto H, et al. Sodium succinate enhances the colorimetric reaction of the in vitro chemosensitivity test: MTT assay. Oncology 1988;45:434-436. 5. Nakashima T, Uemura T, Maehara Y, Sugimachi K. Succinate dehydrogenase inhibition test for evaluating head and neck tumors. Oncology 1989;46:162- 168. 6. Maehara Y, Anai H, Kusumoto H, Sugimachi K. Poorly differentiated human gastric carcinoma is more sensitive to antitumor drugs than is well differentiated carcinoma. Eur J Surg Oncol 1987;13:203-206.
410
Chemosensitivity of Head and Neck Cancer
7. Maehara Y, Kusumoto H, Anai H, et al. One-hexylcarbamoyl-5-fluorouracil is more cytostatic than S-flUOrOuracil against human tumors in vitro. Eur J Cancer Clin Oncol 1987;23:1511- 1515. 8 . Mosmann T. Rapid colorimetric assay for cellular growth and survival. Application to proliferation and cytotoxic assays. J Immunol Methods 1983;65:55 -63. 9. Kondo T, Imamura T,Ichihashi H. In vitro test sensitivity of tumor carcinostatic agents. Gann 1966;57:113121. 10. Maehara Y, Anai H, Tamada R, e t al. The ATP assay is more sensitive than the succinate dehydrogenase inhibition test for predicting cell viability. Eur J Cancer Clin Oncol 1987;25:273-276. 11. Anai H, Maehara Y, Kusumoto H, Sugimachi K. Comparison between succinate dehydrogenase inhibition test and subrenal capsule assay for chemosensitivity testing. Oncology 1987;44:115- 117.
HEAD & NECK
September/October 1990
The chernosensitivities of 42 human head and neck squamous cell carcinomas were examined using the in vitro succinate dehydrogenase inhibition (SDI) test. The tumor tissues obtained at surgery or biopsy were exposed to five different antitumor drugs: adriamycin (ADM), cisplatin (CDDP), carboquone (CQ), 5-fluorouracil (5-FU), and 1-hexylcarbamoyl-5-fluorouracil (HCFU). The results were analyzed according to the histopathologic degree of differentiation of well, moderately, and poorly differentiated squamous cell carcinoma. The average decrease in succinate dehydrogenase activity was 43.2 t 24.9 for ADM, 29.0 t 14.2 for CDDP, 32.9 f 17.6 for CQ, 64.2 ? 20.6 for 5FU, and 26.8 ? 16.9 for HCFU. There was a statistically significant difference in the decrease of succinate dehydrogenase activity between well and poorly differentiated squarnous cell carcinomas. These data suggest that, for patients with a poorly differentiated squamous cell carcinoma, the response to anticancer drugs may be more satisfactory than in those with a welldifferentiated squamous cell carcinoma. HEAD & NECK 1990; 12:406-410
From the Departments of Otolaryngology (Dr Nakashima) and Pathology (Drs. Hayashi and Katsuta). National Kyushu Cancer Center, Fukuoka. Japan; and Cancer Center of Kyushu University Hospital (Drs Maehara and Kohnoe). Fukuoka, Japan Acknowledgments. Supported by a Grant-in-Aid for scientific research from the Fukuoka Cancer Society. Japan The authors thank K. Miyarnoto and K Fukuchi tor expert technical assistance and M Ohara for helptul comments. Address reprint requests to Dr. Nakashima at the Department of Otolaryngology, National Kyushu Cancer Center, Notame 3- 1 1, Minamiku. Fukuoka 815, Japan. ~
Accepted for publication March 3. 1990 CCC 0148-64031901050406- 05 $04.00 0 1990 John Wilev 8, Sons, Inc
406
Chemosensitivity of Head and Neck Cancer
squamous cell carcinomas of the head and neck regions are among the most morbid of human cancers. Despite optimal treatment with surgery and radiotherapy, local tumor control remains a significant problem. Distant metastases occur frequently and second primaries account for the remainder of deaths. These unfavorable results, combined with functional and cosmetic disabilities associated with local or distant recurrence, have led to investigations of additional chemotherapy for head and neck cancer patients.'-3 We focused our attention on the development of a test, which can determine the sensitivity or resistance of malignant tumors to cytostatic agents, prior t o ~ h e m o t h e r a p y . In ~ , ~the present study, we examined differences in chemosensitivity of human head and neck squamous cell carcinomas to various antitumor drugs, as assessed from the point of histologic differentiation. MATERIALS AND METHODS
Materials. Cancer specimens were obtained at biopsy or surgery on 42 Japanese patients with head and neck cancer. All specimens were immediately placed in McCoy's 5A solution. The tumors consisted of primary and metastatic carcinomas (13 pharyngeal cancers, 10 laryngeal cancers, 8 oral cavity cancers, 6 maxillary sinus
HEAD & NECK
SeptemberiOctober 1990
Table 1. Number of head and neck cancer patients with well (W), moderately (M), and poorly (P) differentiated squamous cell carcinomas. Head and neck cancer
w
M
P
Pharyngeal cancer Laryngeal cancer Oral cavity cancer Maxillary sinus cancer Esophageal cancer Other
3 5 2 4 1 0
4 5 5 2 2 0
6 0 1 0 1 1
Total
15
18
9
cancers, 4 esophageal cancers, and 1 metastatic cancer of unknown origin) found in the head and neck region. Pathological examination of the same tissue specimens revealed that 15 were well differentiated, 18 moderately differentiated, and 9 poorly differentiated squamous cell carcinomas (Table 1). The antitumor drugs tested were adriamycin (ADM), cisplatin (CDDP), carboquone (CQ), 5-fluorouracil(5-FU),and l-hexylcarbamoyl-5-fluorouracil (HCFU). Each drug was tested at 10 times the peak plasma concentration6: ADM, 4 pg/ml; CDDP, 20 pg/ml; CQ, 0.5 kg/ml; 5-FU, 100 pglml; and HCFU, 198 pg/ml. HCFU was tested in an equimolar concentration with 5-FU.7 The sources of these drugs were as follows: ADM and 5-FU were from Kyowa Hakko Co., Ltd., Tokyo, Japan; CDDP from Nihon Kayaku Co., Ltd., Tokyo, Japan; C Q from Sankyo Co., Ltd., Tokyo, Japan; and HCFU from Mitsui Co., Ltd., Tokyo, Japan.
Antitumor Drugs.
The succinate dehydrogenase inhibition (SDI) test was performed as previously d e ~ c r i b e d Briefly, .~ tumor tissues were cut with scissors, passed through no. 32 stainless steel mesh into McCoy’s 5A solution containing antibiotics, and washed three times with the same solution. The fragments were suspended in minimum essential medium with Lglutamine (292 mg/ml), 10% fetal calf serum, and antibiotics, placed in 35-mm plastic dishes (3 dishes per test group and 4 to 6 dishes per control), and then incubated for 3 days while exposed to the antitumor drug in medium containing 1% dimethyl sulfoxide at 37°C in a humidified 5% CO, atmosphere. The tissue fragments were washed with phosphate-buffered saline (PBS, pH 7.2 at 4°C) and
assayed for succinate dehydrogenase activity. 3(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT18 was used as the hydrogen acceptor for the succinate dehydrogenase activity. The formazan formed from MTT was extracted with acetone containing 0.5% trichloroacetic acid, and the absorbance of formazan was measured at 565 nm. The succinate dehydrogenase activity was presented as the optical density (OD) per milligram protein. The tumor fragments with an OD of 0.5 on day 0 were plated in separated dishes. The chemosensitivity was estimated by the percentage of succinate dehydrogenase activity of the drug-treated cells, compared with that of control cells, and the sensitivity was determined as positive when the succinate dehydrogenase activity was decreased to less than 50%. Significant differences between means were determined by Student’s t test. A p value of c0.05 was considered to be statistically significant. Statistical Analysis.
RESULTS
The chemosensitivity was indicated by the percentage of succinate dehydrogenase activity of drug-treated cells as compared to that of control cells. The decrease in succinate dehydrogenase activity with each antitumor drug varied (Figure 1).The averages of succinate dehydrogenase activity of 42 head and neck squamous cells carcinomas were 43.2 -t 24.9 for ADM, 29.0 14.2 for +_
ADM
CDDP
CQ
In vitro Chemosensitivity Test.
Chernosensitivity of Head and Neck Cancer
5-FU
‘r
HCFU
4: 4
8
f
ri t 8
I
FIGURE 1. Decrease in succinate dehydrogenase activity in head and neck squamous cell carcinomas. The chemosensitivity is indicated by the percentage of succinate dehydrogenase activity of drug-treated cells compared to that of control cells. Values are means ? standard deviation (SD).
HEAD & NECK
SeptemberIOctober 1990
407
Table 2. Decrease in succinate dehydrogenase activity in well-differentiated squamous cell carcinomas. Case no./origin
ADM
CDDP
CQ
5-FU
HCFU
1. Larynx 2. Oral cavity 3. Maxillary sinus 4. Maxillary sinus 5. Pharynx 6. Esophagus 7. Larynx 8. Oral cavity 9. Pharynx 10. Larynx 11. Maxillary sinus 12. Larynx 13. Larynx 14. Pharynx 15. Maxillary sinus
62.8 58.3 95.8 39.5 32.3 60.5 23.0 30.2 39.4 38.1 17.8 91 .o 96.4 35.0 48.2
32.5 38.0 42.0 44.1 25.0 41.3 17.2 22.3 31.7 29.2 55.3 53.7 39.0 33.8 52.6
47.1
90.3 75.2 94.6 55.2 56.5 61.5 94.9 96.4 81.4 74.1 61.2 100.0 85.7 61.7 88.8
32.5 33.0 75.2 34.7 21.5 57.9 19.0 17 3 14.6 17.2 16.3 61 2 39 0 13.5 41.6
Average
-t
SD
51.2
-t
25.8
37.2 i 11.4
*
CDDP, 32.9 * 17.6 for CQ, 64.2 20.6 for 5-FU, and 26.8 * 16.9 for HCFU. Most squamous cell carcinomas showed a high sensitivity to CDDP, CQ, and HCFU and a low sensitivity to 5-FU. In certain cases, however, the decrease in succinate dehydrogenase activity by 5-FU was prominent, the level being much the same as seen with the other 4 drugs. The results of succinate dehydrogenase activity and chemosensitivity were analyzed according to the histologic differentiation (Tables 2-4).
42.4 28.4 39.0 19.0 31.7 35.1 29.6 39.1 76.1 39.8 32.7 48.2 39.1
* 13.7
78.5
15.9
33.0
* 19.1
In 15 cancer tissues diagnosed as well-differentiated squamous cell carcinoma, the averages of succinate dehydrogenase activity were 51.2 25.8 for ADM, 37.2 11.4 for CDDP, 39.1 2 13.7 for CQ, 78.5 2 15.9 for 5-FU, and 33.0 2 19.1 for HCFU. In 18 cancer tissues diagnosed as moderately differentiated squamous cell carcinomas, the averages of succinate dehydrogenase activity were 45.3 ? 24.2 for ADM, 28.7 2 12.7 for CDDP, 36.3 2 19.9 for CQ, 60.1 2 20.6 for 5-FU, and 26.5 k 14.7 for HCFU. The averages from 9
*
_+
~~
Table 3. Decrease in succinate dehydrogenase activity in moderately differentiated squamous cell carcinomas. Case no./origin
I . Maxillary sinus 2. Oral cavity 3. Pharynx 4. Pharynx 5. Larynx 6. Oral cavity 7. Larynx 8. Pharynx 9. Esophagus 10. Oral cavity 11. Pharynx 12. Oral cavity 13. Larynx 14. Oral cavity 15. Maxillary sinus 16. Larynx 17. Esophagus 18. Larynx
Average 2 SD
408
ADM
CDDP
CQ
5-FU
HCFU
24.0 19.5 88.3 26.5 71.1 70.9 33.8 17.5 36.2 14.1 66.7 54.5 88.4 20.1 42.6 49.2 34.1 58.2
18.0 15.8 44.1 26.2 49.6 17.1 30.0 13.4 9.2 28.3 23.5 30.6 51.9 14.5 38.8 37.7 34.1 34.3
54.3 25.5 42.5 35.3
54.3 38.1 74.0 97.7 73.0 62.8 55.2 23.4 31.8 47.3 63.0 73.9 94.2 49.2 33.5 65.3 65.5 78.9
8.4 13.0 37 0 25.0 43.3 34.9 12.2 16.5 13.4 25.9 32.6 21.6 58.1 5.4 39.2
28.7 2 12.7
36.3 2 19.9
45.3
C
24.2
Chemosensitivity of Head and Neck Cancer
18.8 28.1 15.1 14.3 24.1 32.5 70.4 21.3 32.1 48.5 85.6 32.5
60.1
?
20.6
HEAD & NECK
37.9 26.5
-t
14.7
Septernber/October 1990
Table 4. Decrease in succinate dehydrogenase activity in poorly differentiated squamous cell carcinomas. Case no./oriain 1. 2. 3. 4. 5. 6. 7. 8. 9.
Pharynx Oral cavity Pharynx Esophagus Pharynx Pharynx Unknown Pharynx Pharynx
Average f SD
ADM
CDDP
CQ
5-FU
HCFU
27.2 7.8 23.2 16.5 17.3 16.3 15.0 25.3 18.3
11.4 6.4 18.7 16.9 15.9
16.1 8.3 23.6 18.0 11.6
13.5 25.9 13.6
14.6 28.8 15.7
66.2 49.9 64.7 44.8 25.1 49.5 45.5 44.4 47.9
5.5 17.1 14.1 16.1 16.5 10.9 10.5 30.1 17.0
17.1 t 6.5
48.7 2 12.1
15.3 t 6.8
18.5
* 5.9
15.3
* 5.7
poorly differentiated squamous cell carcinomas were 18.5 2 5.9 for ADM, 15.3 5.7 for CDDP, 17.1 k 6.5 for CQ, 48.7 2 12.1 for 5-FU, and 15.3 I+_ 6.8 for HCFU. There was no difference in succinate dehydrogenase activity in cases of exposure to CDDP and HCFU between well and moderately differentiated squamous cells carcinomas. On exposure to ADM ( p < 0.001), CQ ( p < 0.001) and 5-FU ( p < 0.05), there was a greater decrease in succinate dehydrogenase activity in the moderately differentiated as compared to the well-differentiated squamous cell carcinomas. The decrease in succinate dehydrogenase activity was remarkable in the poorly differentiated tissues, compared with the well-differentiated tissues, exposed to ADM (p < 0.0051, CDDP ( p < 0.001), CQ ( p < 0.001),5-FU ( p < 0.001), and HCFU ( p < 0.02). Even in comparison with moderately differentiated tissues, there was a statistical difference of decrease in succinate dehydrogenase activity in cases of ADM ( p < 0.005), CDDP ( p < 0.01),CQ ( p < 0.021, and HCFU ( p < 0.05). The chemosensitivity rate to each drug was evaluated (Fig. 2). In the well-differentiated squamous cell carcinomas, the highest sensitivity was seen with exposure to CDDP (80%), CQ (92%),and HCFU (80%), but none of these examined tissues was sensitive to 5-FU. In the moderately differentiated squamous cell carcinomas, CDDP (94%) and HCFU (94%) were the most sensitive. Six of eighteen (33%) tissues were sensitive to 5-FU. In contrast, all poorly differentiated squamous cell carcinomas were sensitive to ADM, CDDP, CQ, and HCFU. Even for 5-FU, 7 of 9 (78%) tissues showed sensitivity. The number of tissues resistant to more than three drugs was 3 (20%) in the well-differentiated squamous
*
Chemosensitivity of Head and Neck Cancer
cell carcinomas and 1 (6%) in the moderately differentiated squamous cell carcinomas. DISCUSSION
Many in vivo and in vitro chemosensitivity tests using different parameters have emerged during
Succinate dehydrogenase activity (%)
1
00qoaep;w
0
' i
FIGURE 2. Comparison of decrease in succinate dehydrogenase activity in the well (W), moderately (M), and poorly (P) differentiated squamous cell carcinomas exposed to ADM (A), CDDP (B), CQ (C), 5-FU (D), and HCFU (E). Values are means f standard deviation (SD). The chemosensitivity was considered to be positive when the succinate dehydrogenase activity of the drug-treated cells decreased to less than 50% of that of control cells (shaded area).
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attempts to acquire drugs effective for patients with cancer. Succinate dehydrogenase is an adenosine triphosphate (ATPI-producing enzyme of the citric cycle, and the activity of this enzyme correlates well with cell ~ i a b i l i t y SDI . ~ test is a simple, inexpensive, and rapid in vivo method for screening antitumor drugs. The chemosensitivity of this test correlates with that of the in vitro ATP assay" and the in vivo subrenal capsule (SRC) assay." Using the SDI test, one of the present authors reported differences in the chemosensitivity of drugs between gastric and colorectal cancers. They found that the origin of a lesion was a critical factor related to the chemosensitivity of various tumors.6 We have also examined the succinate dehydrogenase activity in various cancer tissues originating in the head and neck region and found that a malignant lymphoma was the most sensitive to antitumor drugs.5 Head and neck squamous cell carcinomas also responded well to antitumor drugs. The present analysis shows that the histopathologic degree of tissue differentiation could be an important factor for determining chem-
osensitivity of head and neck squamous cell carcinomas. As the decrease of succinate dehydrogenase activity is prominent in poorly differentiated tissues, patients with a head and neck cancer of the poorly differentiated type may be more responsive to chemotherapy, compared to those with moderately or well differentiated type. For patients with the moderately or well differentiated type of head and neck squamous cell carcinoma, sensitive drugs have to be selected. By routinely repeating the SDI test, the clinical effect of an anticancer drug, for the individual patient, can be predicted. It is interesting that most of the poorly differentiated squamous cell carcinomas were from pharyngeal cancers, whereas most of the well or moderately differentiated squamous cell carcinomas were from laryngeal, oral cavity, or maxillary sinus cancers. This tendency suggests the predominant response to chemotherapy by patients with a pharyngeal cancer. We are now collecting data to statisticaly analyze differences in sensitivity to anticancer drugs, depending on site of the lesion in the head and neck regions.
REFERENCES
1. Clark JR, Fallon BG, Dreyfuss AI, e t al. Chemotherapeutic strategies in the multidisciplinary treatment of head and neck cancer. Semin Oncol 1988;15(suppl3):35-44. 2. Adjuvant chemotherapy for advanced head and neck squamous cell carcinoma: Final report of the Head and Neck Contracts Program. Cancer 1987;60:301-311. 3. Jacobs JR, Pajak TF, Kinzie J , et al. Induction chemotherapy in advanced head and neck cancer: A radiation therapy oncology group study. Arch Otolaryngol Head Neck Surg 1987;113:193- 197. 4. Maehara Y, Kusumoto T, Kusumoto H, et al. Sodium succinate enhances the colorimetric reaction of the in vitro chemosensitivity test: MTT assay. Oncology 1988;45:434-436. 5. Nakashima T, Uemura T, Maehara Y, Sugimachi K. Succinate dehydrogenase inhibition test for evaluating head and neck tumors. Oncology 1989;46:162- 168. 6. Maehara Y, Anai H, Kusumoto H, Sugimachi K. Poorly differentiated human gastric carcinoma is more sensitive to antitumor drugs than is well differentiated carcinoma. Eur J Surg Oncol 1987;13:203-206.
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7. Maehara Y, Kusumoto H, Anai H, et al. One-hexylcarbamoyl-5-fluorouracil is more cytostatic than S-flUOrOuracil against human tumors in vitro. Eur J Cancer Clin Oncol 1987;23:1511- 1515. 8 . Mosmann T. Rapid colorimetric assay for cellular growth and survival. Application to proliferation and cytotoxic assays. J Immunol Methods 1983;65:55 -63. 9. Kondo T, Imamura T,Ichihashi H. In vitro test sensitivity of tumor carcinostatic agents. Gann 1966;57:113121. 10. Maehara Y, Anai H, Tamada R, e t al. The ATP assay is more sensitive than the succinate dehydrogenase inhibition test for predicting cell viability. Eur J Cancer Clin Oncol 1987;25:273-276. 11. Anai H, Maehara Y, Kusumoto H, Sugimachi K. Comparison between succinate dehydrogenase inhibition test and subrenal capsule assay for chemosensitivity testing. Oncology 1987;44:115- 117.
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