- Email: [email protected]
Cell kinetics and programmed chemotherapy for gynecologic cancer
Cell kinetics and programmed chemotherapy for gynecologic cancer I. Squamous-cell carcinoma HERVY E. AVERETTE, M.D. GERALD D. WEINSTEIN, M.D. JOHN H. FORD. JR., M.D. ROBERT E. GIRTANNER, M.D.* WILLIAM
J.
HOSKINS, M.D.**
REINALDO RAMOS, B.S.
Miami, Florida
The results of cu·rrent investigation into cellular kirutics of both normal and abno1mal human genital tissue have stimulated interest in programming chemotherapeutic drug regimens for the treatment of genital cancer. This report outlines the results of cell kinetic studies of normal and abnormal squamous epithelium of the female genital tract and presents preliminary results of a regimen utilizing methotrexate, hydroxyurea, and vincristine in patients 'with Jar-advanced squamous-cell cancer of the vulva, vagina, and cervix. A total of 92 patients have received 444 courses of programmed chemotherapy by either pelvic intra-arterial infusion or systemic administration of the drugs. Our results are prPSented and the theoretical basis of the research is discussed.
exceptional tumor regression in four patients following the continuous 24 hour administration of the antimetabolite, methotrexate, by way of arterial infusion of the pelvis. In one patient with Stage IV squamouscell carcinoma of the cervix treated only by chemotherapy, complete tumor regression was obtained and the patient was apparently free of disease $7 months after treatment." To our knowledge, this is the only reported case of a long-term total remission of Stage IV squamous-cell carcinoma of the cervix treated only by chemotherapy. Subsequently others have utilized regional infusion chemotherapy for squamous-cell carcinoma of the pelvis with varying degrees of success. 6 • 7 Our interest in programmed chemotherapy was stimulated by our studies of normal and abnormal cell proliferation kinetics in human genital tissues. 8 It was our feeling that chemotherapy utilizing various cytotoxic agents could be programmed on a scientific basis if knowledge of the cell generation cycle in normal and malignant human tissues was available. Therefore, we have used a technique of local tissue injection of minute amounts of thymidine-3 H to study
ALTHOUGH it is accepted that systemic chemotherapy is effective in metastatic trophoblastic disease, and at times is of value in the treatment of advanced carcinoma of the ovary, breast, and endometrium, it is generally agreed that chemotherapy has been ineffective in the treatment of squamous-cell carcinoma arising in the female genital tract. l-a However, a single report by Sullivan and co-workers4 in 1960 indicated
Frorn the Division of Gynecologic Oncology and Department of Dermatology, Unit'ersity of Miami School of Medicine. This investigation was supported in part by Grants CA 101192 and AM 14887 from the National lnstituJes of Health, and a research grant from the John A. Hartford Fouruiation, Inc. Presented by invitation at the Eighty-sixth Annual Meeting of the American Association of Obstetricians and Gynecolo{fists, Hot Springs, Virginia, September 4-6, 1975. Reprint requests: Dr. Henry E. Averette, Department of Obstetrics and Gynecology, .Jackson Memorial Hospital, Miami. Florida 33136. *Advanced Clinical Fellow, American CarlCeT Society. **U. S. Navy sponsored Fellow in Gynecologic Oncology.
912
Volume 124 Number 8
Cell kinetics and chemotherapy for gynecologic cancer. I 913
1000..,111-
__
,._
_....., I
$-'·- -
--~--~~--~~~ I!J,.,._MIIotlfo
\
I
~L-·L•
I
\
'
Fig. 1. Schematic representation of intra-aortic infusion of pelvis with percutaneous femoral catheterization and Bowman infusion pump.
normal and abnormal cell proliferation kinetics in human cervical, vaginal, and vulvar tissues in vivo. This report will summarize our experience with cell proliferation kinetics in human genital tissues as well as present preliminary results with programmed chemotherapy utilizing methotrexate (MTX), hydroxyurea (HU), and vincristine (VINC).
Materials and methods Studies of cell proliferation kinetics are obtained on patients admitted to the Gynecologic Oncology Service at the University of Miami School of Medicine. Conditions such as dysplasia, in situ carcinoma, and invasive carcinoma of the cervix, vagina, and vulva have been studied. Candidates for programmed chemotherapy have cell kinetic studies both prior to and during various phases of chemotherapy. Cells in human genital tissues that are actively synthesizing DNA are labeled by injecting 5 p,c (in 0.1 mi.) of tritiated thymidine into marked sites in the tissues. Punch biopsy specimens (4 mm.) are obtained from normal or neoplastic cervix, vagina, or vulva at appropriate time intervals depending upon the information desired. Our methods for analysis of autoradiographs in the study of the generation cycle in normal human cervix and vagina have been published previously. 8 In studying cell kinetics in abnormal tissues, similar techniques are used with the exception that cell counts are made from enlarged photographs of autoradiographs to ensure that cell counts are accurately made. All patients for pelvic infusion chemotherapy have a tissue diagnosis of persistent or recurrent squamous-
cell carcinoma following complete treatment in the conventional manner with surgery and/ or radiation therapy. Although it is not possible to completely exclude distant metastases, each patient treated is evaluated with intravenous pyelography, renal and liver function studies, and other radiographic techniques to exclude distant metastases prior to chemotherapy. No patient with findings consistent with tumor spread beyond the pelvis is included in the infusion group. Patients undergoing programmed intra-arterial infusion chJ>motherapy have percutaneous placement of a catheter into the femoral artery by the Seldinger9 technique. The catheter is secured to the skin with a silk suture after the tip has been placed just above the bifurcation of the aorta. The accuracy of the placement is checked by both arteriography and radioactive pelvic scan with macroaggregates of radioiodinated serum albumin. A schematic representation of the infusion setup is shown in Fig. l. A constant infusion of parenteral fluid must be maintained, after the catheter is placed in the artery, with a Model 4 Bowman infusion pump or a blood pressure pump surrounding a plastic blood transfer pack filled with 5 per cent dextrose and water. The chemotherapeutic program for patients with disease limited to the pelvis consists of three sequential steps: (I) MTX (50 mg.) in 600 mi. of 5 per cent dextrose and water is infused over a 24 hour period, thus delivering a high concentration of methotrexate to the pelvis. To prevent systemic toxicity of MTX that escapes into the circulation, folinic acid is administered intramuscularly in doses of 6 mg. every 6 hours during
April l.'i, 1976 Am . .J. Obstet. Gynecol.
914 Averette et al.
100
Tg 2 o 6.4 hrs.
§....
.... >=
:=
•
80
60
:iii
""'
....~~
40
..._
"" Q..e
Tc =Mean cell cycle time Tc
= Tg1+
T , =5.4 hrs. 9
.I
Differentiated cells that leave proliferative pool
• 24
12 16 20 4 HOURS AFTER IMIECTION Of LABELL£D THYIIIIIIE
Ts + Tg2+ Tm
Fig. 2. Schematic representation of cell generation cycle and its component parts.
~
•
20
Fig. 4. DNA synthesis curve for squamous-cell carcinoma of vulva in situ. 120
T5 =9 hrs.
Tt2 ' 4.2 hrs. 108
100
...~
V>
-J ..... w u u i= 0 1-
-;...
80
~
i ii
-
~
&8
~
~
0 w
..._
::::l
UJ
""'
a::l
< ..... u...
0
~
0 4 8 12 16 20 24 HOURS AFTER INJECTION OF LABELLED THYMIDINE
4
12
1&
20
24
HOURS AFTER INIECTIOI OF LABULED THYIUIIIIE
Fig. 3. Composite DNA synthesis curve of normal human cervical and vaginal epithelium based on mean values of per cent mitosis in 10 patients. Bars represent 95 per cent confidence levels.
Fig. 5. DNA synthesis curves for invasive squamous-cell carcinoma of cervix and normal squamous epithelium of vagina in the same patient.
the treatment period. MTX is administered for 4 consecutive days, delivering a total dose of 200 mg. (2) One hour after completing the MTX therapy, HU (4.0 Gm.) is given by mouth as a single dose. (3) Eight hours after the MTX infusion is completed, VINC (2.0 mg.) is given by way of the femoral catheter and infused over a l to 2 hour period. The triple therapy of methotrexate, hydroxyurea, and vincristine constitutes one course of therapy. The usual interval between courses of therapy is 48 hours,
during which time the femoral catheter is maintained in place and 5 per cent dextrose is infused. The total number of chemotherapeutic infusions varies, depending upon the patient's ability to tolerate the procedure and/or the appearance of toxic manifestations of the drug program. Patients selected for programmed systemic chemotherapy are patients with persistent, recurrent, or metastatic squamous-cell carcinoma following complete treatment in the conventional manner with
Volume 124 Number 8
Cell kinetics and chemotherapy for gynecologic cancer. I.
915
Fig. 6. Autoradiograph of squamous-cell carcinoma 20 minutes after local injection of thymidine- 3H. Labeling index was 27 per cent prior to infusion of MTX.
Fig. 7. Autoradiograph of squamous-cell carcinoma demonstrating marked increase in labeled cells (72 per cent) that were thymidine deficient after 96 hours of intra-aortic infusion of MTX.
surgery, radiation therapy, or both. Some of these patients have presented only with distant metastasis. Some have had local recurrence, but have been selected for systemic chemotherapy rather than infusion chemotherapy because of medical problems such as peripheral vascular disease. A third group of patients have both local recurrence and distant metastasis. All patients have had either biopsy-proved persistent or recurrent cancer or radiologic evidence of distant metastasis such as lung lesions or metastasis to bone. Methotrexate, hydroxyurea, and vincristine are given systemically by the following schedule. i. Patients receive 15 mg. of MTX daily for 4 consecutive days followed by an intramuscular injection of 6 mg. of folinic acid on the morning of the fifth day. The dosage of MTX must be reduced if there is impairment of renal or hepatic function . 2. Following the folinic acid, 4 Gm. of HU are given by mouth during a 1 hour period. 3. Approximately 6 hours after HU, the patient receives an intravenous injection of 2 mg. of VINC. This course of therapy is administered every 3 weeks if the white blood count is above 3;000 and the platelet count above 100,000. If the hemogram is depressed it is repeated weekly until the critical values are reached, when another course of therapy is instituted. Periodic evaluation of liver and renal function is necessary so that the correct dose of MTX may be determined . It is mandatory that blood urea nitrogen (BUN) and creatinine levels be normal since MTX is excreted primarily by the kidney and severe toxicity will result if
a full dose of MTX is given when renal function is poor. Results
Cell kinetics. The four phases of the proliferative cell cycle are illustrated in Fig. 2. Following mitosis (M), the daughter cells enter the postmitotic G 1 phase, where one of the cells may become a differentiated cell leaving the proliferative pool while the other cell may continue as a progenitor cell to reproduce again. In order for this cell to reproduce it duplicates its content of DNA during the DNA synthesis (S) phase. After DNA synthesis, there is a relatively short postsynthetic G 2 period before the next mitotic phase. A balance between dividing and nondividing cells has been defined in the normal human cervical and vaginal epithelium under varying degrees of estrogen stimulation.8 In Fig. 3, a composite DNA synthesis curve for normal human vagina and cervix from 10 patients is plotted. From this curve, the S phase and G 2 phase can be measured as 9 and 5.4 hours, respectively. DNA synthesis curves are also plotted for in situ and invasive squamous-cell carcinoma (Figs. 4 and 5). Very little difference has been found in the S and G 2 phases between normal and neoplastic tissues. In an effort to determine a mean cell generation cycle for squamous·cell neoplasia in the cervix and vagina, labeling indices have been tabulated for 50 patients. The mean labeling indices for patients with cervical dysplasia, carcinoma in situ, and invasive squamous-cell carcinoma are 16.6, 21.2, and 27.2
916 Averette et al. Am. J
Aprill 5, 1976 Obstet. Gynecol.
Fig. 8. Squamous-cell carcinoma in patient 24 hours after pelvic infusion of vincristine. Note numerous cells arrested in metaphase of mitosis (17 per cent). Table I. Means of the labeling indices of squamous-cell neoplasia
No. of patients
Tissue
Dysplasia Cancer in situ Invasive cancer
Labeling index of whole proliferative pool(%)
16.6 ± (4.16)* 21.2 ± (2.46) 27.2 ± (2.78)
9 12
29
*Values in parentheses represent twice the S.E. Table II. Germinative cell cycles in normal and neoplastic squamous tissues Tc
Tissue
Normal Cancer in situ Invasive cancer
(days)
14.3 21.2 27.2
9 9 9
5.4 5.8 6.3
0.17 48 0.4 27.8 0.5 15.2
63 44 33
2.6 1.8 1.4
*L.l. = labelling index.
respectively (Table I). Using the determined values for S phase (in hours) and labeling indices, cell generation cycles for the various degrees of squamous neoplasia have been calculated (Table II). It will be noted that T 5 , T 82 , and T m vary only slightly when one compares normal to neoplastic tissues. However, there is progressive shortening of the entire generation cycle, T "' when one compares normal tissue to dysplastic, and
then to neoplastic, tissues. Shortening is primarily due to a decrease in the postmitotic G 1 phase. Chemotherapy and cell cycle kinetics. Cytotoxic agents have been chosen for the treatment of invasive squamous-cell carcinoma based on their mode of action and the cellular kinetics of the tumor. The effects of methotrexate on squamous-cell carcinoma have been srudied by obtaining labeling indices prio r to infusion of MTX and after 96 hours of infusion. As see n in an autoradiograph of squamous-cell carcinoma prior to infusion of MTX, the labeling index before chemotherapy is 27 per cent (Fig. 6). However, after 96 hours of pelvic infusion chemotherapy with MTX, autoradiographs demonstrate marked uptake of tritiated thymidine (Fig. 7). The increase in labeling index to 72 per cent reflects the blockade of cells entering or already in the S phase that became thymidine deficient and are unable to complete DNA synthesis. 10 • 11 Hydroxyurea was chosen as the second cyclic agent since its specific action appears in cells synthesizing DNA by inhibition of ribonucleotide reductase. Cells in the S phase are lethally damaged by HU, whereas cells in the G1 phase are prevented from entering the S phase. 12 The third agent, vincristine, was selected because of its action during either the G2 or M period producing a metaphase arrest. 13 Since the tumor population would be substantially synchronized in S phase after a 96 hour infusion of methotrexate or, as in the case of systemic
Volume 124 Number 8
Cell kinetics and chemotherapy for gynecologic cancer. I.
917
Fig. 9. Composite of patient with squamous-cell carcinoma of vagina with extension to vulva. A, Photograph taken before infusion chemotherapy; B, photograph taken 4 months after infusion chemotherapy.
therapy, by the 4 day course of oral methotrexate, it was then planned to manipulate the majority of these cells synchronously into the G 2 and M phases where vincristine should be very effective on a relatively high percentage of tumor cells. Vincristine-produced metaphase arrest in squamous-cell carcinomas is seen ;~
J;";~
Q
.llJ.
.L
Uo
15•
Clinical applications of programmed chemotherapy Patients with regional disease. Thirty-six patients with regional disease received 100 programmed infusions with relatively few complications. The first 12 patients received MTX only. The second group of nine patients received MTX and VINC. The third group consisted of 15 patients who received the full programmed regimen of MTX, HU, and VINC, as previously outlined. The average number of infusions per patient was 2.8, with a maximum of 11 and a minimum of l. The average survival time of all patients receiving infusion chemotherapy was 12.8 months, with a maximum of 8 years and a minimum of less than I month. Patients were evaluated objectively and subjectively, with the most dramatic subjective response being pain relief. Twenty-two of 36 (61.1 per cent) patients have had significant or total relief of pain, usually after the first infusion. Patients receiving large doses of morphine every 2 to 3 hours usually required little or no analgesic 48 to 72 hours after the infusion was started. In six of 14 patients who were judged to have little or no relief of pain, a revie\v of the arteriograms and radioactive pelvic scans demonstrated that little infusate had reached the pelvic tumor masses. These treatment failures occurred in patients who had been treated previously with
radiotherapy and it is likely that radiation fibrosis or arterial obstruction secondary to the tumor masses prevented adequate infusion of cytotoxic agents to the tumor. Although initially several courses of therapy were given, it was later learned that if no response was noted during the first 5 day infusion, subsequent therapy \-vas of little value. Therefore, if no response was obtained with the first infusion, chemotherapy was discontinued. Objective response of the tumor was defined as (1) greater than 50 per cent regression, (2) 50 per cent regression, (3) 25 per cent regression, (4) no change (i.e., neither progression nor regression), and (5) progression of tumor growth. Six patients had greater than 50 per cent regression, two patients had 50 per cent regression, one patient had 25 per cent regression, and four patients remained stable. Twenty-three patients had progression of disease despite chemotherapy. Of the six paiients having greater than 50 per cent regression of tumor, two had total regression of tumor and are alive 8 and 7 years following completion of the infusion chemotherapy. Five patients died prior to discharge from the hospital subsequent to pelvic infusion. One died from congestive heart failure, two from sepsis, one from pulmonary embolization, and one died following amputation of an extremity secondary to peripheral embolization related to catheter placement. With the exception of the patient who suffered peripheral embolization, these deaths may or may not have been related to the infusion procedures. However, it is emphasized that all patients have had advanced, unresectable, squan1ouscell cancer that has been treated completely in the conventional manner with surgery, radiotherapy, or both. Only three of the 36 patients are alive at this time, but some of the patients who died have had apparent
918
Averette et al. Am.
J.
April 15, 1976 Obstet. Gynecol.
Fig. 10. Radiographs of chest demonstrating pleural effusion secondary to metastatic squamous-cell carcinoma (A) and disappearance of effusion after systemic programmed chemotherapy (B).
complete control of pelvic disease, dying due to distant metastasis. Of the two patients who have been long-term survivors, one was treated with radiotherapy for Stage III squamous-cell carcinoma of the cervix in July and August, I 965. Recurrence of carcinoma was detected \AJhen she presented \Alith hydronephrosis and weight loss of 66 pounds. Exploratory celiotomy revealed that pelvic exenteration was impossible due to tumor fixation to the pelvic walls. In January, 1967, a 5 day infusion was followed by marked tumor regression and disappearance of pain. Five separate infusions were given and in August, 1967, intravenous pyelography demonstrated a normal collecting system without hydronephrosis. At the present time she is working, without complaints, has gained 70 pounds, and is clinically free of cancer. Another survivor was treated by radiotherapy for carcinoma of the vagina in ~"1ay. 1967. Tumor recurrence with involvement of the vulva and fixation to the pubic rami was noted in October, I 967. Two infusions, each lasting 6 days, were given in October. 1967, and January, 1968. After the second infusion, the vulva appeared normal (Fig. 9). At present, the patient is without complaints except for excessive weight gain and vaginal stenosis. Patients with disseminated disease. Thirty-four patients with systemic disease received 315 courses of programmed chemotherapy. An additional 22 patients received 29 courses of therapy, but were excluded from the study as they received less than three courses and therefore represent insufficient data for detailed analysis. Of these 22 patients, two are alive but have not
completed three courses, three refused therapy prior to completing three courses, five patients were lost to follow-up. 10 patients died of their disease in less than 3 months, and two patients died secondary to toxicity of the chemotherapy. Of those patients who completed at least three courses of therapy, the average number of courses has been 9.1, with the maximum being 27 courses and the minimum being three courses. The average survival of these patients was 9.6 months, with a maximum of26 months and a minimum of3 months. Observations were again subdivided into subjective and objective response. Subjectively, 13 patients were felt to have improved with total or partial relief of presenting symptomatology. Seven patients have experienced no significant improvement hut have not deteriorated for at least 3 months and are considered to have remained stable. Fourteen patients had tumor progression and their condition \vas felt to have· worsened despite the chemotherapy. Objectively, six patients have had greater than 50 per cent regression of tumor masses, three patients have had 50 per cent regression, three patients have had 25 per cent regression, and seven patients have remained stable with neither progression nor regression over at least 3 months. Fifteen of 34 patients have experienced definite progression of disease. Five patients illustrated significant tumor response to systemic chemotherapy. Two patients with squamous-cell carcinoma were found to have lung metastasis vvith pleural effusions and were placed on the systemic regimen of MTX, H U, and VINC. Both patients had lesions that could be visualized radiographically. After two complete courses
Volume 124 Number 8
Cell kinetics and chemotherapy for gynecologic cancer. I. 919
Fig. ll. Radiographs (tomograms) of patient with metastatic squamous-cell carcinoma (A) and disappearance of metastatic nod u]e after systemic programmed chemotherapy (B) (20 courses).
of therapy, the pleural effusions in both patients disappeared and significant decrease in the lung lesions was noted (Fig. 10). Both patients subsequently died due to disseminated tumor, one surviving for 8 months and the other for 14 months. A third patient was discovered to have recurrent carcinoma of the cervix metastatic to the lung and after seven courses of systemic chemotherapy, the previously noted lung lesion could no longer be identified. She received a total of 20 courses of systemic chemotherapy before she died of myocardial infarction. At autopsy a small focus of squamous-cell carcinoma was found in the lung. This area of carcinoma corresponded to the nodule seen earlier which had decreased to the point that it was no longer visible radiographically (Fig. 11). A fourth patient with lung metastasis has had complete disappearance of lesions after 17 courses of systemic chemotherapy. Ten weeks after discontinuing therapy a radiograph of the chest is normal. The fifth patient is included through the courtesy of Dr. Myron Lutz at the Medical University of South Carolina. This patient underwent radical vulvectomy for Stage III carcinoma of the vulva. Approximately I year after radical vulvectomy, local recurrence was noted, but she refused further treatment by surgery or radiation therapy. After a single course of systemic MTX, HU, and VINC, the tumor exhibited marked regression, as seen in Fig. 12. Unfortunately, severe bone marrow depression with sepsis resulted and subsequently she died. Of the 34 patients who received programmed
systemic chemotherapy, 24 have died and 10 are alive at the present time . The longest survival to date is 26 months. The over-all results for the 34 evaluable patients who received programmed systemic chemotherapy are shown in Fig. 13. The survival time for patients responding to therapy has been compared to the survival time for patients who failed to respond. The survival curve for the untreated group was obtained from hospital records of patients who died of recurrent squamous-cell carcinoma prior to our study of programmed chemotherapy. It is apparent that nonresponders have a survival time that is nearly identical to that of the untreated group whereas responders to chemotherapy have experienced longer survival and a longer interval free of tumor progression. When delineating significant toxicity related to systemic chemotherapy, all patients have experienced hair loss, nausea, and stomatitis. Eight patients experienced vomiting or diarrhea requiring hospitalization. Three patients suffered sufficient depression of bone marrow to require admission and reverse isolation. One patient developed a peripheral neuropathy which probably was secondary to prolonged use of vincristine. One patient in the group who received at least three courses of therapy has died as a direct result of the chemotherapy due to overwhelming sepsis secondary to depression of the bone marrow. Two additional patients, mentioned previously, died as a result of the chemotherapy prior to completion of three courses.
920 Averette et al.
April 15, 1976 Am . .J. Obstet. Gynecol.
Fig. 12. Composite of patient with recurrent squamous-cell carcinoma of vulva (A) and marked regression of tumor after one course of systemic programmed chemotherapy (B). (Photographs courtesy of Dr. Myron Lutz, Medical University of South Carolina.)
100 - - - response(19) •••••• no reSponse (15) -------- untreated (30)
80 70 60
"> 50 C( ~
40
30 20
Q
12
15
24
Months
Fig. 13. Survival curve (actuarial survival) of responders to programmed chemotherapy compared to nonresponders and untreated patients.
Comment The advantage of using cell proliferation kinetics of tumor tissue to program chemotherapy is to place chemotherapy on a more scientific basis than the empiricism of the past. Cell proliferation studies have been used to determine the life cycle of genital tumor cells so that the cycle could be pharmacologically manipulated by drugs like MTX, HU, and VINC. The principle of the triple-therapy schedule was developed to maximize the cytotoxic effects of chemotherapeutic agents on the tumor cell population by using cell synchronization techniques for both regional infusion
and systemic chemotherapy. T he initial infusion or oral administration of MTX attempts to synchronize most of the tumor cells into the DNA synthesis (S) phase. While these cells are trapped in the S phase. they are exposed to HU, which is also an S-phasespecific agent, and can preferentially d estroy a larger portion of the tumor cell population. The third part of the schedule is to push the surviving cells out of the S phase synchronously with folinic acid and into another susceptible phase (G 2 and/or M phase) where VINC is cell-cycle specific. Regional infusion with programmed chemotherapy permits a maximum concentration of drugs to reach th e tumor mass without significant systemic toxicity. Where metastatic disease is present beyond the pelvis, it is reasonable to assume that the kinetic activity of the distant tumor cell behaves in a similar manner to the primary tumor and would respond to chemothe rapeutic scheduling. Systemic programmed chemotherapy has the advantage of prolonged outpatient usage : however, potential systemic toxicity becomes a greater problem and often doses must be adjusted. Several aspects of the material presented here should be emphasized. The data regarding cell kinetics of human squamous-cell genital carcinoma are preliminary. Factors considered important clinically, such as histologic cell type (i.e., large vs. small, keratinizing vs. nonkeratinizing, mature vs. immature), have not been investigated with respect to possible kinetic differences. The cell generation cycle has been defined only as an average estimate since it is possible that in an asynchronous cell population there will be considerable
Volume 124 Number 8
vanauon from one cell to another. Theoretically, if each tumor cell had the same generation cycle, one course of chemotherapy covering an entire cell cycle while utilizing the right cytotoxic agents sequentially should eliminate the tumor. Although some tumors regressed rather dramatically, it is clear that others were unresponsive. Clinically, the evaluation of patients with squamouscell carcinomas of the genital tract is difficult. Unlike patients who have ovarian carcinoma and often have readily measurable parameters, such as ascites or palpable masses, patients with recurrent or persistent squamous-cell cancer often have central disease where regression or progression is difficult to evaluate. Also, only those patients who have received the full extent of conventional therapy in the form of surgery,
Cell kinetics and chemotherapy for gynecologic cancer. I. 921
radiotherapy, or both have been selected for therapy. Another limitation of the current regimen is the exclusion of patients in whom there was significant impairment of renal or hepatic function since the frequency of toxic reactions would be unacceptable. Although these studies indicate that rational, programmed chemotherapy can, in some instances, modify the progression of squamous-cell cancer, this is a preliminary report where a limited number of drugs in a select group of patients has be~n utilized and is not recommended for standard clinical usage. It is our aim to continue these investigations of rational chemotherapy based on continuing research in the cell kinetic and pharmacokinetic characteristics of the tumor cell population.
REFERENCES I. Malkasian, G. D., Decker, D. G., Mussey, E.. and johnson, C. E.: Clin. Obstet. Gynecol. 11: 367, 1968. 2. Frick, H. C., Atchoo, N., Adamsoms, K., and Taylor, H. C.: AM. J. OBSTET. GYNECOL. 93: 1112, 1965. 3. Tindel, S.: J. A.M. A. 200:913, 1967. 4. Sullivan, R. D., Wood, A. M., Clifford, P., Duff, J. K., Trussel, R., Nary, D. K., and Burchendal, ]. H.: Ca. Chemother. Rep. 8: 1, 1960. 5. Watloms. E., and Sullivan, R. D.: Surg. Gynec. Obstet. ll8: 3, 1964. 6. Bateman, J. R., Hazen, J. G., Stolinsky, D. C., and Steinfield,J. L.: AM.j. 0BSTET. GYNECOL. 96: 181, 1966.
7. Rogers, L. S., eta!.: Cancer 17: 1365, 1964. 8. Averette, H. E., Weinstein, G. D., and :Frost, P.: AM. J. 0BSTET. GYNECOL. 108: 8, 1970. 9. Se1dinger, S. 1.: Acta Radio!. (Stockholm) 39: 368, 1953. 10. Bertino, J. R.: Cancer Res 23: 1286, 1963. 11. Borsa, J., and Whitmore, G. F.: Cancer Res. 29: 737, 1969. 12. Sinclair, W. F.: Science 150: 1729, 1965. 13. Pfeiffer, S. E., and To1mach, L. F.: Nature (London) 213: 139, 1967.
Discussion DR. GEoRGE D. WILBANKS, Chicago, Illinois. It is exciting to read the second installment of this interesting work being done by Averette and his group in Miami. When one analyzes the amount of man hours involved in this study one understands the 5 year gap since the study of normal tissue kinetics of 1970. This is a key work in three important areas; first, the basic cell kinetics of normal epithelium, dysplasia, carcinoma in situ, and invasive squamous-cell carcinoma of the cervix and vulva; second, the effectiveness of local infusion vs. systemic chemotherapy in recurrent squamous-cell cancers; finally, and perhaps the most important practicality, the use of these cell kinetic observations in the programming of chernotheraputic drug regimens for the treatment of gynecologic cancers. In spite of a wealth of data on cell kinetics from in vitro systems and from animal in vivo systems, little information has been accumulated in human in vivo systems because of the problem of the amount of radioactive material needed to label the cells. In a recent report using somewhat different techniques,
Nitze, Ganzer, and Vosteen 1 in Frankfurt studied the cell kinetics of patients with ENT squamous lesions and used these data in programming radiation therapy. Their study corroborates most of the observations of Dr. Averette. They found that both normal and malignant tissues could be synchronized equally well and that after synchronization no differences occur between normal and malignant tissues in the timing of the cycle phases. They also had a remarkable 50 per cent long-term survival rate of these advanced lesions. Averette found rather similar times in S, G2 , and M phases of the cycle of normal and malignant cells, with the difference in the total cycle time found between normal and malignant cells being a decrease in the G 1 phase in the malignant cells. Two interesting ancillary observations made in this study have been supported by in vitro observations on human cervical epithelium in both Richart's and our laboratories. These are that CIS seems to behave in a manner intermediate between normal and invasive tissues and that the time in mitosis is longer in CIS than in normal epithelial cells. Averette's calculated time in mitosis is shorter than that observed by others in similar studies and in our actual
922
Averette et al. Am.
observations by time lapse cinematography in vitro. This is a minor point. The more important times of S and G2 are consistent with other studies in the literature. Averette's studies were performed in the surfaces of the tumors and from specific areas. Have samples been taken from any metastatic lesions to see if these cells behave similarly and do samples from several areas of the same tumor behave in a similar fashion? The question of the effectiveness oflocal infusion vs. systemic chemotherapy apparently is still open from the data presented. Is Dr. Averette's group still using arterial infusion? His results of the two long-term cures with this technique, and the relatively low complication rate presented, seem somewhat at odds with other observations in the literature. Do the authors have any data on infusion therapy not given using the kinetic techniques for comparison? Was the three-drug regimen used on the last 15 patients more effective than the initial one- or two-drug regimens? The use of the programming of systemic chemotherapy with the cell cycle kinetics seemed to be effective in the tissue studies. The survival curves of those patients that responded seem different from those that did not respond or had no treatment. Would Dr. Averette comment on any statistical analysis of this difference? Finally, I think Dr. Averette and his group should be commended for their persistence in this important work. We, as gynecologists, should capitalize on this work and institute programmed studies in the use of both chemotherapy and radiation therapy on less advanced gynecologic malignancies in a large cooperative study. REFERENCES I. Nitze, H. R., Ganzer, U., and Vosteen, K. H.: Synchronization of human tissue and its consequences for cancer therapy in ENT. Cell kinetic and clinical studies, Adv. Oto Rhino Laryngol. 21:82, 1974.
DR. jAMES H. NELSON, JR., Brooklyn, New York. The cell generation time method which Dr. Averette has described concerns me from a technical standpoint. First of all, a 0. I mm. fluid volume is quite small and a 4 mm. punch biopsy is relatively large. Could Dr. Averette be certain of the diffusion of the injected fluid and did he take more than one biopsy from the same cervix or the same tumor after a single injection? I realize that with the long half-life of tritium one could not afford to inject the material intravenously or intra-arteriorly in any significant amount. A fine point in the description of technique: I wonder why Dr. Averette did not infuse saline between courses to maintain patency of the catheter rather than a glucose solution. Glucose carries greater risk of infection, but also saline would prevent obstruction of the catheter by a clot.
J.
April 15, 1976 Obsret. GvnecoL
What is the dose of methotrexate actually infused? The 15 mg. given in the paper I received does not seem to be a very high dose, since doses higher than that are routinely used in the treatment of malignant trophoblastic lesions. There was also a contradictory statement in the paper concerning how often courses were given. On one page it was stated that a course was repeated every 48 hours, while on the next page it was indicated that the course of therapy was repeated even :~ weeks. The difference between groups of responses seems too finely drawn to be accurate. For example, the difference between a 50 per cent reduction in size of the tumor and 25 per cent reduction in size seems as though. at the very best, it would have to be an estimate. Of course, it may be that Dr. Averette is measuring this by some more objective means, such as sonography. but no mention was made in the paper of such methods. As one reads the results, the success rate is not overwhelming but it must be remembered that the patients selected for this study had all received conventional therapy and were classified as terminal patients or patients for whom no other therapy was available. When placed in that context, even two long-term survivors of 7 and 8 years must be viewed with some interest and, indeed, they are impressive. A complication mentioned in one of these long-term survivors was that of vaginal stenosis and I wonder if Dr. Averette thought that it was due to the chemotherapv? It occurred to me as I read this paper that most of the patients had had radiation therapy, which we all know is an extremely destructive form of treatment. Dr. Averette himself mentioned the fact that due to the fibrosis secondary to radiotherapy it may be that the chemotherapeutic agents were not actually delivered to the tumor bed in concentrated levels. In the late 1950's and early 1960's, when we were doing a lot of intra-arterial chemotherapy, fluorescein was injected through the catheter and the area supplied by the artery injected was viewed with an ultraviolet light to see if the area fluoresced. In this wav one could determine whether or not the catheter was properly placed and also whether, in fact, the arterial system was patent. If the area fluoresced one could be certain the agents to be infused would reach the tumor under treatment. Dr. Averette does not mention this anywhere in the paper and I wonder if he has used this method to determine whether or not his agents are getting to the tumor. If he has not done that, I would recommend it to him in the future because it might very well correlate with the ultimate response. In other words, it might explain in part why some patients are complete failures with progression of the tumor under treatment while others give good remissions. The approach is well thought out and the work carefully done. While I might question the definitions
Volume 124 Number 8
of response, an attempt has been made to document the results in an objective manner. The attempt to use chemotherapy in a more rational way is also a most desirable step in the right direction. DR. BRuCE H. DRUKKER, Detroit, Michigan. Dr. Averette and his colleagues' detailed study moves the modality of chemotherapy to a practical area. Two question arise, one speculative and the other inquiring. Is it feasible to consider dose reduction with triple therapy based on the high cell labeling index seen following methotrexate treatment? Potentially this would produce less alteration in the patient's remaining immunity as well as reduction in the toxic side effects. It would appear that the dosage of the chemotherapeutic agents utilized in the study is equal to that which would be utilized if each was used alone. Second, although this was not mentioned in the paper, has Dr. Averette had an opportunity to study any patients who had been given adriamycin by means of autoradiography? On the basis of Dr. Pattillo's work, one might find a high degree of labeling with adriamycin. DR. AVERETTE (Closing). Drs. Nelson and Wilbanks raised the question regarding possible cell kinetic differences between primary tumors and metastatic lesions. Thus far, we have found no difference between primary cervical squamous cancer that has extended to the vulva or vulvar carcinoma that has extended to the groin. I think it is important to recognize that what we are measuring really is a rough estimate of the average cell generation cycle in an asynchronous cell population. Also, it was questioned whether or not we found differences in kinetic activity in different parts of the same tumor. With our local injection technique we infiltrated the tumor in multiple areas and we found no uptake of labeled thymidine in areas of necrosis, as might be expected. Also, in areas where there apparently were viable tumor cells, we could detect no uptake in some areas and active incorporation in other areas. Possibly the i~jected thymidine did not reach all of the tumor cells, or the viable nonlabeled cells were in the Go or nonactive clonogenic population. However, our studies have been confined to areas within the
Cell kinetics and chemotherapy for gynecologic cancer. I. 923
tumor showing active uptake of thymidine. This seems reasonable, since our aim is to study the actively dividing tumor cells so that we can program therapy based upon the proliferative compartments within the tumor. With respect to systemic vs. infusion chemotherapy, I do not think we have treated enough patients to determine which technique is best. Certainly infusion is more complicated, requiring hospitalization for prolonged periods. In general, large tumors confined to the pelvis were treated by pelvic infusion and patients with disseminated disease were treated systemically. Dr. Nelson raised the question of vaginal stenosis in the patient treated first for vaginal carcinoma by radiotherapy and later by infusion chemotherapy. I think there is no doubt that stenosis was due to radiotherapy since she was treated rather radically with radiation prior to recurrence and chemotherapy. We have not used fluoroescence to determine infusate distribution. Instead, we utilize pelvic arteriography as well as a scan of the pelvis following a slow drip infusion of macroaggregates of radioiodinated human serum albumin. Unfortunately, at times we do find poor infusion in areas where there is active tumor associated with extensive radiation fibrosis. Drs. Wilbanks and Nelson commented on our methods for evaluating clinical response to chemotherapy. Certainly it is difficult to accurately judge objectively and subjectively response to treatment, especially with squamous carcinoma of the pelvis. Usually we did not have large tumor masses or ascites that could be measured as one might do with ovarian cancer. However, we made the best observations that we could with the disease that we were treating. Our method for analysis of the survival curve was based on the life-table method of Cutler and Erlderer. Dr. Drukker mentioned the possibility of reducing the total dose of drugs after initial synchronization. This might be possible. However, severe toxicity occurred usually in the early phase of the study when we treated patients with compromised renal or hepatic function. Adriamycin has been used on several occasions recently but we have no conclusions regarding the drug at this time.
J.
HOSKINS, M.D.**
REINALDO RAMOS, B.S.
Miami, Florida
The results of cu·rrent investigation into cellular kirutics of both normal and abno1mal human genital tissue have stimulated interest in programming chemotherapeutic drug regimens for the treatment of genital cancer. This report outlines the results of cell kinetic studies of normal and abnormal squamous epithelium of the female genital tract and presents preliminary results of a regimen utilizing methotrexate, hydroxyurea, and vincristine in patients 'with Jar-advanced squamous-cell cancer of the vulva, vagina, and cervix. A total of 92 patients have received 444 courses of programmed chemotherapy by either pelvic intra-arterial infusion or systemic administration of the drugs. Our results are prPSented and the theoretical basis of the research is discussed.
exceptional tumor regression in four patients following the continuous 24 hour administration of the antimetabolite, methotrexate, by way of arterial infusion of the pelvis. In one patient with Stage IV squamouscell carcinoma of the cervix treated only by chemotherapy, complete tumor regression was obtained and the patient was apparently free of disease $7 months after treatment." To our knowledge, this is the only reported case of a long-term total remission of Stage IV squamous-cell carcinoma of the cervix treated only by chemotherapy. Subsequently others have utilized regional infusion chemotherapy for squamous-cell carcinoma of the pelvis with varying degrees of success. 6 • 7 Our interest in programmed chemotherapy was stimulated by our studies of normal and abnormal cell proliferation kinetics in human genital tissues. 8 It was our feeling that chemotherapy utilizing various cytotoxic agents could be programmed on a scientific basis if knowledge of the cell generation cycle in normal and malignant human tissues was available. Therefore, we have used a technique of local tissue injection of minute amounts of thymidine-3 H to study
ALTHOUGH it is accepted that systemic chemotherapy is effective in metastatic trophoblastic disease, and at times is of value in the treatment of advanced carcinoma of the ovary, breast, and endometrium, it is generally agreed that chemotherapy has been ineffective in the treatment of squamous-cell carcinoma arising in the female genital tract. l-a However, a single report by Sullivan and co-workers4 in 1960 indicated
Frorn the Division of Gynecologic Oncology and Department of Dermatology, Unit'ersity of Miami School of Medicine. This investigation was supported in part by Grants CA 101192 and AM 14887 from the National lnstituJes of Health, and a research grant from the John A. Hartford Fouruiation, Inc. Presented by invitation at the Eighty-sixth Annual Meeting of the American Association of Obstetricians and Gynecolo{fists, Hot Springs, Virginia, September 4-6, 1975. Reprint requests: Dr. Henry E. Averette, Department of Obstetrics and Gynecology, .Jackson Memorial Hospital, Miami. Florida 33136. *Advanced Clinical Fellow, American CarlCeT Society. **U. S. Navy sponsored Fellow in Gynecologic Oncology.
912
Volume 124 Number 8
Cell kinetics and chemotherapy for gynecologic cancer. I 913
1000..,111-
__
,._
_....., I
$-'·- -
--~--~~--~~~ I!J,.,._MIIotlfo
\
I
~L-·L•
I
\
'
Fig. 1. Schematic representation of intra-aortic infusion of pelvis with percutaneous femoral catheterization and Bowman infusion pump.
normal and abnormal cell proliferation kinetics in human cervical, vaginal, and vulvar tissues in vivo. This report will summarize our experience with cell proliferation kinetics in human genital tissues as well as present preliminary results with programmed chemotherapy utilizing methotrexate (MTX), hydroxyurea (HU), and vincristine (VINC).
Materials and methods Studies of cell proliferation kinetics are obtained on patients admitted to the Gynecologic Oncology Service at the University of Miami School of Medicine. Conditions such as dysplasia, in situ carcinoma, and invasive carcinoma of the cervix, vagina, and vulva have been studied. Candidates for programmed chemotherapy have cell kinetic studies both prior to and during various phases of chemotherapy. Cells in human genital tissues that are actively synthesizing DNA are labeled by injecting 5 p,c (in 0.1 mi.) of tritiated thymidine into marked sites in the tissues. Punch biopsy specimens (4 mm.) are obtained from normal or neoplastic cervix, vagina, or vulva at appropriate time intervals depending upon the information desired. Our methods for analysis of autoradiographs in the study of the generation cycle in normal human cervix and vagina have been published previously. 8 In studying cell kinetics in abnormal tissues, similar techniques are used with the exception that cell counts are made from enlarged photographs of autoradiographs to ensure that cell counts are accurately made. All patients for pelvic infusion chemotherapy have a tissue diagnosis of persistent or recurrent squamous-
cell carcinoma following complete treatment in the conventional manner with surgery and/ or radiation therapy. Although it is not possible to completely exclude distant metastases, each patient treated is evaluated with intravenous pyelography, renal and liver function studies, and other radiographic techniques to exclude distant metastases prior to chemotherapy. No patient with findings consistent with tumor spread beyond the pelvis is included in the infusion group. Patients undergoing programmed intra-arterial infusion chJ>motherapy have percutaneous placement of a catheter into the femoral artery by the Seldinger9 technique. The catheter is secured to the skin with a silk suture after the tip has been placed just above the bifurcation of the aorta. The accuracy of the placement is checked by both arteriography and radioactive pelvic scan with macroaggregates of radioiodinated serum albumin. A schematic representation of the infusion setup is shown in Fig. l. A constant infusion of parenteral fluid must be maintained, after the catheter is placed in the artery, with a Model 4 Bowman infusion pump or a blood pressure pump surrounding a plastic blood transfer pack filled with 5 per cent dextrose and water. The chemotherapeutic program for patients with disease limited to the pelvis consists of three sequential steps: (I) MTX (50 mg.) in 600 mi. of 5 per cent dextrose and water is infused over a 24 hour period, thus delivering a high concentration of methotrexate to the pelvis. To prevent systemic toxicity of MTX that escapes into the circulation, folinic acid is administered intramuscularly in doses of 6 mg. every 6 hours during
April l.'i, 1976 Am . .J. Obstet. Gynecol.
914 Averette et al.
100
Tg 2 o 6.4 hrs.
§....
.... >=
:=
•
80
60
:iii
""'
....~~
40
..._
"" Q..e
Tc =Mean cell cycle time Tc
= Tg1+
T , =5.4 hrs. 9
.I
Differentiated cells that leave proliferative pool
• 24
12 16 20 4 HOURS AFTER IMIECTION Of LABELL£D THYIIIIIIE
Ts + Tg2+ Tm
Fig. 2. Schematic representation of cell generation cycle and its component parts.
~
•
20
Fig. 4. DNA synthesis curve for squamous-cell carcinoma of vulva in situ. 120
T5 =9 hrs.
Tt2 ' 4.2 hrs. 108
100
...~
V>
-J ..... w u u i= 0 1-
-;...
80
~
i ii
-
~
&8
~
~
0 w
..._
::::l
UJ
""'
a::l
< ..... u...
0
~
0 4 8 12 16 20 24 HOURS AFTER INJECTION OF LABELLED THYMIDINE
4
12
1&
20
24
HOURS AFTER INIECTIOI OF LABULED THYIUIIIIE
Fig. 3. Composite DNA synthesis curve of normal human cervical and vaginal epithelium based on mean values of per cent mitosis in 10 patients. Bars represent 95 per cent confidence levels.
Fig. 5. DNA synthesis curves for invasive squamous-cell carcinoma of cervix and normal squamous epithelium of vagina in the same patient.
the treatment period. MTX is administered for 4 consecutive days, delivering a total dose of 200 mg. (2) One hour after completing the MTX therapy, HU (4.0 Gm.) is given by mouth as a single dose. (3) Eight hours after the MTX infusion is completed, VINC (2.0 mg.) is given by way of the femoral catheter and infused over a l to 2 hour period. The triple therapy of methotrexate, hydroxyurea, and vincristine constitutes one course of therapy. The usual interval between courses of therapy is 48 hours,
during which time the femoral catheter is maintained in place and 5 per cent dextrose is infused. The total number of chemotherapeutic infusions varies, depending upon the patient's ability to tolerate the procedure and/or the appearance of toxic manifestations of the drug program. Patients selected for programmed systemic chemotherapy are patients with persistent, recurrent, or metastatic squamous-cell carcinoma following complete treatment in the conventional manner with
Volume 124 Number 8
Cell kinetics and chemotherapy for gynecologic cancer. I.
915
Fig. 6. Autoradiograph of squamous-cell carcinoma 20 minutes after local injection of thymidine- 3H. Labeling index was 27 per cent prior to infusion of MTX.
Fig. 7. Autoradiograph of squamous-cell carcinoma demonstrating marked increase in labeled cells (72 per cent) that were thymidine deficient after 96 hours of intra-aortic infusion of MTX.
surgery, radiation therapy, or both. Some of these patients have presented only with distant metastasis. Some have had local recurrence, but have been selected for systemic chemotherapy rather than infusion chemotherapy because of medical problems such as peripheral vascular disease. A third group of patients have both local recurrence and distant metastasis. All patients have had either biopsy-proved persistent or recurrent cancer or radiologic evidence of distant metastasis such as lung lesions or metastasis to bone. Methotrexate, hydroxyurea, and vincristine are given systemically by the following schedule. i. Patients receive 15 mg. of MTX daily for 4 consecutive days followed by an intramuscular injection of 6 mg. of folinic acid on the morning of the fifth day. The dosage of MTX must be reduced if there is impairment of renal or hepatic function . 2. Following the folinic acid, 4 Gm. of HU are given by mouth during a 1 hour period. 3. Approximately 6 hours after HU, the patient receives an intravenous injection of 2 mg. of VINC. This course of therapy is administered every 3 weeks if the white blood count is above 3;000 and the platelet count above 100,000. If the hemogram is depressed it is repeated weekly until the critical values are reached, when another course of therapy is instituted. Periodic evaluation of liver and renal function is necessary so that the correct dose of MTX may be determined . It is mandatory that blood urea nitrogen (BUN) and creatinine levels be normal since MTX is excreted primarily by the kidney and severe toxicity will result if
a full dose of MTX is given when renal function is poor. Results
Cell kinetics. The four phases of the proliferative cell cycle are illustrated in Fig. 2. Following mitosis (M), the daughter cells enter the postmitotic G 1 phase, where one of the cells may become a differentiated cell leaving the proliferative pool while the other cell may continue as a progenitor cell to reproduce again. In order for this cell to reproduce it duplicates its content of DNA during the DNA synthesis (S) phase. After DNA synthesis, there is a relatively short postsynthetic G 2 period before the next mitotic phase. A balance between dividing and nondividing cells has been defined in the normal human cervical and vaginal epithelium under varying degrees of estrogen stimulation.8 In Fig. 3, a composite DNA synthesis curve for normal human vagina and cervix from 10 patients is plotted. From this curve, the S phase and G 2 phase can be measured as 9 and 5.4 hours, respectively. DNA synthesis curves are also plotted for in situ and invasive squamous-cell carcinoma (Figs. 4 and 5). Very little difference has been found in the S and G 2 phases between normal and neoplastic tissues. In an effort to determine a mean cell generation cycle for squamous·cell neoplasia in the cervix and vagina, labeling indices have been tabulated for 50 patients. The mean labeling indices for patients with cervical dysplasia, carcinoma in situ, and invasive squamous-cell carcinoma are 16.6, 21.2, and 27.2
916 Averette et al. Am. J
Aprill 5, 1976 Obstet. Gynecol.
Fig. 8. Squamous-cell carcinoma in patient 24 hours after pelvic infusion of vincristine. Note numerous cells arrested in metaphase of mitosis (17 per cent). Table I. Means of the labeling indices of squamous-cell neoplasia
No. of patients
Tissue
Dysplasia Cancer in situ Invasive cancer
Labeling index of whole proliferative pool(%)
16.6 ± (4.16)* 21.2 ± (2.46) 27.2 ± (2.78)
9 12
29
*Values in parentheses represent twice the S.E. Table II. Germinative cell cycles in normal and neoplastic squamous tissues Tc
Tissue
Normal Cancer in situ Invasive cancer
(days)
14.3 21.2 27.2
9 9 9
5.4 5.8 6.3
0.17 48 0.4 27.8 0.5 15.2
63 44 33
2.6 1.8 1.4
*L.l. = labelling index.
respectively (Table I). Using the determined values for S phase (in hours) and labeling indices, cell generation cycles for the various degrees of squamous neoplasia have been calculated (Table II). It will be noted that T 5 , T 82 , and T m vary only slightly when one compares normal to neoplastic tissues. However, there is progressive shortening of the entire generation cycle, T "' when one compares normal tissue to dysplastic, and
then to neoplastic, tissues. Shortening is primarily due to a decrease in the postmitotic G 1 phase. Chemotherapy and cell cycle kinetics. Cytotoxic agents have been chosen for the treatment of invasive squamous-cell carcinoma based on their mode of action and the cellular kinetics of the tumor. The effects of methotrexate on squamous-cell carcinoma have been srudied by obtaining labeling indices prio r to infusion of MTX and after 96 hours of infusion. As see n in an autoradiograph of squamous-cell carcinoma prior to infusion of MTX, the labeling index before chemotherapy is 27 per cent (Fig. 6). However, after 96 hours of pelvic infusion chemotherapy with MTX, autoradiographs demonstrate marked uptake of tritiated thymidine (Fig. 7). The increase in labeling index to 72 per cent reflects the blockade of cells entering or already in the S phase that became thymidine deficient and are unable to complete DNA synthesis. 10 • 11 Hydroxyurea was chosen as the second cyclic agent since its specific action appears in cells synthesizing DNA by inhibition of ribonucleotide reductase. Cells in the S phase are lethally damaged by HU, whereas cells in the G1 phase are prevented from entering the S phase. 12 The third agent, vincristine, was selected because of its action during either the G2 or M period producing a metaphase arrest. 13 Since the tumor population would be substantially synchronized in S phase after a 96 hour infusion of methotrexate or, as in the case of systemic
Volume 124 Number 8
Cell kinetics and chemotherapy for gynecologic cancer. I.
917
Fig. 9. Composite of patient with squamous-cell carcinoma of vagina with extension to vulva. A, Photograph taken before infusion chemotherapy; B, photograph taken 4 months after infusion chemotherapy.
therapy, by the 4 day course of oral methotrexate, it was then planned to manipulate the majority of these cells synchronously into the G 2 and M phases where vincristine should be very effective on a relatively high percentage of tumor cells. Vincristine-produced metaphase arrest in squamous-cell carcinomas is seen ;~
J;";~
Q
.llJ.
.L
Uo
15•
Clinical applications of programmed chemotherapy Patients with regional disease. Thirty-six patients with regional disease received 100 programmed infusions with relatively few complications. The first 12 patients received MTX only. The second group of nine patients received MTX and VINC. The third group consisted of 15 patients who received the full programmed regimen of MTX, HU, and VINC, as previously outlined. The average number of infusions per patient was 2.8, with a maximum of 11 and a minimum of l. The average survival time of all patients receiving infusion chemotherapy was 12.8 months, with a maximum of 8 years and a minimum of less than I month. Patients were evaluated objectively and subjectively, with the most dramatic subjective response being pain relief. Twenty-two of 36 (61.1 per cent) patients have had significant or total relief of pain, usually after the first infusion. Patients receiving large doses of morphine every 2 to 3 hours usually required little or no analgesic 48 to 72 hours after the infusion was started. In six of 14 patients who were judged to have little or no relief of pain, a revie\v of the arteriograms and radioactive pelvic scans demonstrated that little infusate had reached the pelvic tumor masses. These treatment failures occurred in patients who had been treated previously with
radiotherapy and it is likely that radiation fibrosis or arterial obstruction secondary to the tumor masses prevented adequate infusion of cytotoxic agents to the tumor. Although initially several courses of therapy were given, it was later learned that if no response was noted during the first 5 day infusion, subsequent therapy \-vas of little value. Therefore, if no response was obtained with the first infusion, chemotherapy was discontinued. Objective response of the tumor was defined as (1) greater than 50 per cent regression, (2) 50 per cent regression, (3) 25 per cent regression, (4) no change (i.e., neither progression nor regression), and (5) progression of tumor growth. Six patients had greater than 50 per cent regression, two patients had 50 per cent regression, one patient had 25 per cent regression, and four patients remained stable. Twenty-three patients had progression of disease despite chemotherapy. Of the six paiients having greater than 50 per cent regression of tumor, two had total regression of tumor and are alive 8 and 7 years following completion of the infusion chemotherapy. Five patients died prior to discharge from the hospital subsequent to pelvic infusion. One died from congestive heart failure, two from sepsis, one from pulmonary embolization, and one died following amputation of an extremity secondary to peripheral embolization related to catheter placement. With the exception of the patient who suffered peripheral embolization, these deaths may or may not have been related to the infusion procedures. However, it is emphasized that all patients have had advanced, unresectable, squan1ouscell cancer that has been treated completely in the conventional manner with surgery, radiotherapy, or both. Only three of the 36 patients are alive at this time, but some of the patients who died have had apparent
918
Averette et al. Am.
J.
April 15, 1976 Obstet. Gynecol.
Fig. 10. Radiographs of chest demonstrating pleural effusion secondary to metastatic squamous-cell carcinoma (A) and disappearance of effusion after systemic programmed chemotherapy (B).
complete control of pelvic disease, dying due to distant metastasis. Of the two patients who have been long-term survivors, one was treated with radiotherapy for Stage III squamous-cell carcinoma of the cervix in July and August, I 965. Recurrence of carcinoma was detected \AJhen she presented \Alith hydronephrosis and weight loss of 66 pounds. Exploratory celiotomy revealed that pelvic exenteration was impossible due to tumor fixation to the pelvic walls. In January, 1967, a 5 day infusion was followed by marked tumor regression and disappearance of pain. Five separate infusions were given and in August, 1967, intravenous pyelography demonstrated a normal collecting system without hydronephrosis. At the present time she is working, without complaints, has gained 70 pounds, and is clinically free of cancer. Another survivor was treated by radiotherapy for carcinoma of the vagina in ~"1ay. 1967. Tumor recurrence with involvement of the vulva and fixation to the pubic rami was noted in October, I 967. Two infusions, each lasting 6 days, were given in October. 1967, and January, 1968. After the second infusion, the vulva appeared normal (Fig. 9). At present, the patient is without complaints except for excessive weight gain and vaginal stenosis. Patients with disseminated disease. Thirty-four patients with systemic disease received 315 courses of programmed chemotherapy. An additional 22 patients received 29 courses of therapy, but were excluded from the study as they received less than three courses and therefore represent insufficient data for detailed analysis. Of these 22 patients, two are alive but have not
completed three courses, three refused therapy prior to completing three courses, five patients were lost to follow-up. 10 patients died of their disease in less than 3 months, and two patients died secondary to toxicity of the chemotherapy. Of those patients who completed at least three courses of therapy, the average number of courses has been 9.1, with the maximum being 27 courses and the minimum being three courses. The average survival of these patients was 9.6 months, with a maximum of26 months and a minimum of3 months. Observations were again subdivided into subjective and objective response. Subjectively, 13 patients were felt to have improved with total or partial relief of presenting symptomatology. Seven patients have experienced no significant improvement hut have not deteriorated for at least 3 months and are considered to have remained stable. Fourteen patients had tumor progression and their condition \vas felt to have· worsened despite the chemotherapy. Objectively, six patients have had greater than 50 per cent regression of tumor masses, three patients have had 50 per cent regression, three patients have had 25 per cent regression, and seven patients have remained stable with neither progression nor regression over at least 3 months. Fifteen of 34 patients have experienced definite progression of disease. Five patients illustrated significant tumor response to systemic chemotherapy. Two patients with squamous-cell carcinoma were found to have lung metastasis vvith pleural effusions and were placed on the systemic regimen of MTX, H U, and VINC. Both patients had lesions that could be visualized radiographically. After two complete courses
Volume 124 Number 8
Cell kinetics and chemotherapy for gynecologic cancer. I. 919
Fig. ll. Radiographs (tomograms) of patient with metastatic squamous-cell carcinoma (A) and disappearance of metastatic nod u]e after systemic programmed chemotherapy (B) (20 courses).
of therapy, the pleural effusions in both patients disappeared and significant decrease in the lung lesions was noted (Fig. 10). Both patients subsequently died due to disseminated tumor, one surviving for 8 months and the other for 14 months. A third patient was discovered to have recurrent carcinoma of the cervix metastatic to the lung and after seven courses of systemic chemotherapy, the previously noted lung lesion could no longer be identified. She received a total of 20 courses of systemic chemotherapy before she died of myocardial infarction. At autopsy a small focus of squamous-cell carcinoma was found in the lung. This area of carcinoma corresponded to the nodule seen earlier which had decreased to the point that it was no longer visible radiographically (Fig. 11). A fourth patient with lung metastasis has had complete disappearance of lesions after 17 courses of systemic chemotherapy. Ten weeks after discontinuing therapy a radiograph of the chest is normal. The fifth patient is included through the courtesy of Dr. Myron Lutz at the Medical University of South Carolina. This patient underwent radical vulvectomy for Stage III carcinoma of the vulva. Approximately I year after radical vulvectomy, local recurrence was noted, but she refused further treatment by surgery or radiation therapy. After a single course of systemic MTX, HU, and VINC, the tumor exhibited marked regression, as seen in Fig. 12. Unfortunately, severe bone marrow depression with sepsis resulted and subsequently she died. Of the 34 patients who received programmed
systemic chemotherapy, 24 have died and 10 are alive at the present time . The longest survival to date is 26 months. The over-all results for the 34 evaluable patients who received programmed systemic chemotherapy are shown in Fig. 13. The survival time for patients responding to therapy has been compared to the survival time for patients who failed to respond. The survival curve for the untreated group was obtained from hospital records of patients who died of recurrent squamous-cell carcinoma prior to our study of programmed chemotherapy. It is apparent that nonresponders have a survival time that is nearly identical to that of the untreated group whereas responders to chemotherapy have experienced longer survival and a longer interval free of tumor progression. When delineating significant toxicity related to systemic chemotherapy, all patients have experienced hair loss, nausea, and stomatitis. Eight patients experienced vomiting or diarrhea requiring hospitalization. Three patients suffered sufficient depression of bone marrow to require admission and reverse isolation. One patient developed a peripheral neuropathy which probably was secondary to prolonged use of vincristine. One patient in the group who received at least three courses of therapy has died as a direct result of the chemotherapy due to overwhelming sepsis secondary to depression of the bone marrow. Two additional patients, mentioned previously, died as a result of the chemotherapy prior to completion of three courses.
920 Averette et al.
April 15, 1976 Am . .J. Obstet. Gynecol.
Fig. 12. Composite of patient with recurrent squamous-cell carcinoma of vulva (A) and marked regression of tumor after one course of systemic programmed chemotherapy (B). (Photographs courtesy of Dr. Myron Lutz, Medical University of South Carolina.)
100 - - - response(19) •••••• no reSponse (15) -------- untreated (30)
80 70 60
"> 50 C( ~
40
30 20
Q
12
15
24
Months
Fig. 13. Survival curve (actuarial survival) of responders to programmed chemotherapy compared to nonresponders and untreated patients.
Comment The advantage of using cell proliferation kinetics of tumor tissue to program chemotherapy is to place chemotherapy on a more scientific basis than the empiricism of the past. Cell proliferation studies have been used to determine the life cycle of genital tumor cells so that the cycle could be pharmacologically manipulated by drugs like MTX, HU, and VINC. The principle of the triple-therapy schedule was developed to maximize the cytotoxic effects of chemotherapeutic agents on the tumor cell population by using cell synchronization techniques for both regional infusion
and systemic chemotherapy. T he initial infusion or oral administration of MTX attempts to synchronize most of the tumor cells into the DNA synthesis (S) phase. While these cells are trapped in the S phase. they are exposed to HU, which is also an S-phasespecific agent, and can preferentially d estroy a larger portion of the tumor cell population. The third part of the schedule is to push the surviving cells out of the S phase synchronously with folinic acid and into another susceptible phase (G 2 and/or M phase) where VINC is cell-cycle specific. Regional infusion with programmed chemotherapy permits a maximum concentration of drugs to reach th e tumor mass without significant systemic toxicity. Where metastatic disease is present beyond the pelvis, it is reasonable to assume that the kinetic activity of the distant tumor cell behaves in a similar manner to the primary tumor and would respond to chemothe rapeutic scheduling. Systemic programmed chemotherapy has the advantage of prolonged outpatient usage : however, potential systemic toxicity becomes a greater problem and often doses must be adjusted. Several aspects of the material presented here should be emphasized. The data regarding cell kinetics of human squamous-cell genital carcinoma are preliminary. Factors considered important clinically, such as histologic cell type (i.e., large vs. small, keratinizing vs. nonkeratinizing, mature vs. immature), have not been investigated with respect to possible kinetic differences. The cell generation cycle has been defined only as an average estimate since it is possible that in an asynchronous cell population there will be considerable
Volume 124 Number 8
vanauon from one cell to another. Theoretically, if each tumor cell had the same generation cycle, one course of chemotherapy covering an entire cell cycle while utilizing the right cytotoxic agents sequentially should eliminate the tumor. Although some tumors regressed rather dramatically, it is clear that others were unresponsive. Clinically, the evaluation of patients with squamouscell carcinomas of the genital tract is difficult. Unlike patients who have ovarian carcinoma and often have readily measurable parameters, such as ascites or palpable masses, patients with recurrent or persistent squamous-cell cancer often have central disease where regression or progression is difficult to evaluate. Also, only those patients who have received the full extent of conventional therapy in the form of surgery,
Cell kinetics and chemotherapy for gynecologic cancer. I. 921
radiotherapy, or both have been selected for therapy. Another limitation of the current regimen is the exclusion of patients in whom there was significant impairment of renal or hepatic function since the frequency of toxic reactions would be unacceptable. Although these studies indicate that rational, programmed chemotherapy can, in some instances, modify the progression of squamous-cell cancer, this is a preliminary report where a limited number of drugs in a select group of patients has be~n utilized and is not recommended for standard clinical usage. It is our aim to continue these investigations of rational chemotherapy based on continuing research in the cell kinetic and pharmacokinetic characteristics of the tumor cell population.
REFERENCES I. Malkasian, G. D., Decker, D. G., Mussey, E.. and johnson, C. E.: Clin. Obstet. Gynecol. 11: 367, 1968. 2. Frick, H. C., Atchoo, N., Adamsoms, K., and Taylor, H. C.: AM. J. OBSTET. GYNECOL. 93: 1112, 1965. 3. Tindel, S.: J. A.M. A. 200:913, 1967. 4. Sullivan, R. D., Wood, A. M., Clifford, P., Duff, J. K., Trussel, R., Nary, D. K., and Burchendal, ]. H.: Ca. Chemother. Rep. 8: 1, 1960. 5. Watloms. E., and Sullivan, R. D.: Surg. Gynec. Obstet. ll8: 3, 1964. 6. Bateman, J. R., Hazen, J. G., Stolinsky, D. C., and Steinfield,J. L.: AM.j. 0BSTET. GYNECOL. 96: 181, 1966.
7. Rogers, L. S., eta!.: Cancer 17: 1365, 1964. 8. Averette, H. E., Weinstein, G. D., and :Frost, P.: AM. J. 0BSTET. GYNECOL. 108: 8, 1970. 9. Se1dinger, S. 1.: Acta Radio!. (Stockholm) 39: 368, 1953. 10. Bertino, J. R.: Cancer Res 23: 1286, 1963. 11. Borsa, J., and Whitmore, G. F.: Cancer Res. 29: 737, 1969. 12. Sinclair, W. F.: Science 150: 1729, 1965. 13. Pfeiffer, S. E., and To1mach, L. F.: Nature (London) 213: 139, 1967.
Discussion DR. GEoRGE D. WILBANKS, Chicago, Illinois. It is exciting to read the second installment of this interesting work being done by Averette and his group in Miami. When one analyzes the amount of man hours involved in this study one understands the 5 year gap since the study of normal tissue kinetics of 1970. This is a key work in three important areas; first, the basic cell kinetics of normal epithelium, dysplasia, carcinoma in situ, and invasive squamous-cell carcinoma of the cervix and vulva; second, the effectiveness of local infusion vs. systemic chemotherapy in recurrent squamous-cell cancers; finally, and perhaps the most important practicality, the use of these cell kinetic observations in the programming of chernotheraputic drug regimens for the treatment of gynecologic cancers. In spite of a wealth of data on cell kinetics from in vitro systems and from animal in vivo systems, little information has been accumulated in human in vivo systems because of the problem of the amount of radioactive material needed to label the cells. In a recent report using somewhat different techniques,
Nitze, Ganzer, and Vosteen 1 in Frankfurt studied the cell kinetics of patients with ENT squamous lesions and used these data in programming radiation therapy. Their study corroborates most of the observations of Dr. Averette. They found that both normal and malignant tissues could be synchronized equally well and that after synchronization no differences occur between normal and malignant tissues in the timing of the cycle phases. They also had a remarkable 50 per cent long-term survival rate of these advanced lesions. Averette found rather similar times in S, G2 , and M phases of the cycle of normal and malignant cells, with the difference in the total cycle time found between normal and malignant cells being a decrease in the G 1 phase in the malignant cells. Two interesting ancillary observations made in this study have been supported by in vitro observations on human cervical epithelium in both Richart's and our laboratories. These are that CIS seems to behave in a manner intermediate between normal and invasive tissues and that the time in mitosis is longer in CIS than in normal epithelial cells. Averette's calculated time in mitosis is shorter than that observed by others in similar studies and in our actual
922
Averette et al. Am.
observations by time lapse cinematography in vitro. This is a minor point. The more important times of S and G2 are consistent with other studies in the literature. Averette's studies were performed in the surfaces of the tumors and from specific areas. Have samples been taken from any metastatic lesions to see if these cells behave similarly and do samples from several areas of the same tumor behave in a similar fashion? The question of the effectiveness oflocal infusion vs. systemic chemotherapy apparently is still open from the data presented. Is Dr. Averette's group still using arterial infusion? His results of the two long-term cures with this technique, and the relatively low complication rate presented, seem somewhat at odds with other observations in the literature. Do the authors have any data on infusion therapy not given using the kinetic techniques for comparison? Was the three-drug regimen used on the last 15 patients more effective than the initial one- or two-drug regimens? The use of the programming of systemic chemotherapy with the cell cycle kinetics seemed to be effective in the tissue studies. The survival curves of those patients that responded seem different from those that did not respond or had no treatment. Would Dr. Averette comment on any statistical analysis of this difference? Finally, I think Dr. Averette and his group should be commended for their persistence in this important work. We, as gynecologists, should capitalize on this work and institute programmed studies in the use of both chemotherapy and radiation therapy on less advanced gynecologic malignancies in a large cooperative study. REFERENCES I. Nitze, H. R., Ganzer, U., and Vosteen, K. H.: Synchronization of human tissue and its consequences for cancer therapy in ENT. Cell kinetic and clinical studies, Adv. Oto Rhino Laryngol. 21:82, 1974.
DR. jAMES H. NELSON, JR., Brooklyn, New York. The cell generation time method which Dr. Averette has described concerns me from a technical standpoint. First of all, a 0. I mm. fluid volume is quite small and a 4 mm. punch biopsy is relatively large. Could Dr. Averette be certain of the diffusion of the injected fluid and did he take more than one biopsy from the same cervix or the same tumor after a single injection? I realize that with the long half-life of tritium one could not afford to inject the material intravenously or intra-arteriorly in any significant amount. A fine point in the description of technique: I wonder why Dr. Averette did not infuse saline between courses to maintain patency of the catheter rather than a glucose solution. Glucose carries greater risk of infection, but also saline would prevent obstruction of the catheter by a clot.
J.
April 15, 1976 Obsret. GvnecoL
What is the dose of methotrexate actually infused? The 15 mg. given in the paper I received does not seem to be a very high dose, since doses higher than that are routinely used in the treatment of malignant trophoblastic lesions. There was also a contradictory statement in the paper concerning how often courses were given. On one page it was stated that a course was repeated every 48 hours, while on the next page it was indicated that the course of therapy was repeated even :~ weeks. The difference between groups of responses seems too finely drawn to be accurate. For example, the difference between a 50 per cent reduction in size of the tumor and 25 per cent reduction in size seems as though. at the very best, it would have to be an estimate. Of course, it may be that Dr. Averette is measuring this by some more objective means, such as sonography. but no mention was made in the paper of such methods. As one reads the results, the success rate is not overwhelming but it must be remembered that the patients selected for this study had all received conventional therapy and were classified as terminal patients or patients for whom no other therapy was available. When placed in that context, even two long-term survivors of 7 and 8 years must be viewed with some interest and, indeed, they are impressive. A complication mentioned in one of these long-term survivors was that of vaginal stenosis and I wonder if Dr. Averette thought that it was due to the chemotherapv? It occurred to me as I read this paper that most of the patients had had radiation therapy, which we all know is an extremely destructive form of treatment. Dr. Averette himself mentioned the fact that due to the fibrosis secondary to radiotherapy it may be that the chemotherapeutic agents were not actually delivered to the tumor bed in concentrated levels. In the late 1950's and early 1960's, when we were doing a lot of intra-arterial chemotherapy, fluorescein was injected through the catheter and the area supplied by the artery injected was viewed with an ultraviolet light to see if the area fluoresced. In this wav one could determine whether or not the catheter was properly placed and also whether, in fact, the arterial system was patent. If the area fluoresced one could be certain the agents to be infused would reach the tumor under treatment. Dr. Averette does not mention this anywhere in the paper and I wonder if he has used this method to determine whether or not his agents are getting to the tumor. If he has not done that, I would recommend it to him in the future because it might very well correlate with the ultimate response. In other words, it might explain in part why some patients are complete failures with progression of the tumor under treatment while others give good remissions. The approach is well thought out and the work carefully done. While I might question the definitions
Volume 124 Number 8
of response, an attempt has been made to document the results in an objective manner. The attempt to use chemotherapy in a more rational way is also a most desirable step in the right direction. DR. BRuCE H. DRUKKER, Detroit, Michigan. Dr. Averette and his colleagues' detailed study moves the modality of chemotherapy to a practical area. Two question arise, one speculative and the other inquiring. Is it feasible to consider dose reduction with triple therapy based on the high cell labeling index seen following methotrexate treatment? Potentially this would produce less alteration in the patient's remaining immunity as well as reduction in the toxic side effects. It would appear that the dosage of the chemotherapeutic agents utilized in the study is equal to that which would be utilized if each was used alone. Second, although this was not mentioned in the paper, has Dr. Averette had an opportunity to study any patients who had been given adriamycin by means of autoradiography? On the basis of Dr. Pattillo's work, one might find a high degree of labeling with adriamycin. DR. AVERETTE (Closing). Drs. Nelson and Wilbanks raised the question regarding possible cell kinetic differences between primary tumors and metastatic lesions. Thus far, we have found no difference between primary cervical squamous cancer that has extended to the vulva or vulvar carcinoma that has extended to the groin. I think it is important to recognize that what we are measuring really is a rough estimate of the average cell generation cycle in an asynchronous cell population. Also, it was questioned whether or not we found differences in kinetic activity in different parts of the same tumor. With our local injection technique we infiltrated the tumor in multiple areas and we found no uptake of labeled thymidine in areas of necrosis, as might be expected. Also, in areas where there apparently were viable tumor cells, we could detect no uptake in some areas and active incorporation in other areas. Possibly the i~jected thymidine did not reach all of the tumor cells, or the viable nonlabeled cells were in the Go or nonactive clonogenic population. However, our studies have been confined to areas within the
Cell kinetics and chemotherapy for gynecologic cancer. I. 923
tumor showing active uptake of thymidine. This seems reasonable, since our aim is to study the actively dividing tumor cells so that we can program therapy based upon the proliferative compartments within the tumor. With respect to systemic vs. infusion chemotherapy, I do not think we have treated enough patients to determine which technique is best. Certainly infusion is more complicated, requiring hospitalization for prolonged periods. In general, large tumors confined to the pelvis were treated by pelvic infusion and patients with disseminated disease were treated systemically. Dr. Nelson raised the question of vaginal stenosis in the patient treated first for vaginal carcinoma by radiotherapy and later by infusion chemotherapy. I think there is no doubt that stenosis was due to radiotherapy since she was treated rather radically with radiation prior to recurrence and chemotherapy. We have not used fluoroescence to determine infusate distribution. Instead, we utilize pelvic arteriography as well as a scan of the pelvis following a slow drip infusion of macroaggregates of radioiodinated human serum albumin. Unfortunately, at times we do find poor infusion in areas where there is active tumor associated with extensive radiation fibrosis. Drs. Wilbanks and Nelson commented on our methods for evaluating clinical response to chemotherapy. Certainly it is difficult to accurately judge objectively and subjectively response to treatment, especially with squamous carcinoma of the pelvis. Usually we did not have large tumor masses or ascites that could be measured as one might do with ovarian cancer. However, we made the best observations that we could with the disease that we were treating. Our method for analysis of the survival curve was based on the life-table method of Cutler and Erlderer. Dr. Drukker mentioned the possibility of reducing the total dose of drugs after initial synchronization. This might be possible. However, severe toxicity occurred usually in the early phase of the study when we treated patients with compromised renal or hepatic function. Adriamycin has been used on several occasions recently but we have no conclusions regarding the drug at this time.