intravenous corynebacterium parvum. an adjunct to chemotherapy for resistant advanced ovarian cancer

INTRAVENOUS CORYNEBACTERIUM PARVUM An Adjunct to Chemotherapy

f o r Resistant Advanced Ovarian Cancer BHASKAR RAO, MD, HAROLD J. WANEBO, MD, MANUEL OCHOA, J R . , MD, JOHN L.

LEWIS, JR., MD, AND HERBERT F. OETTGEN, MD

This is a preliminary report of an effort to treat women with advanced (Stage 111 and IV) ovarian cancer who had progressive disease in spite of previous surgery, chemotherapy and/or radiation by a program of reductive surgery, intensive immune stimulation and combination chemotherapy. An initial laparotomy was done where possible to reduce tumor burden, and then all patients were given intravenous corynebacterium parvum (C.P.) in escalating doses over a 10- to 14-day period. Cyclic chemotherapy with Cytoxan, adriamycin and 5-fluorouracil (CAF) was started and repeated monthly. Mainte- nance subcutaneous C.P. was given weekly. All patients had frequent follow-up clinical and laboratory examination. Immune function was measured by skin tests and in vitro tests prior to treatment and periodically during therapy. Two- thirds of the patients had depressed DNCB and PHA stimulation responses prior to treatment, and almost all had severely depressed lymphocyte counts. Thirty-nine patients entered the program. Exploratory laparotomy was done in 16 patients and in eight, successful tumor reduction was accomplished. Eleven patients received intravenous C. Parvum and all expired before receiving chemotherapy. Four patients received C. Parvum and <three cycles of CAF; all expired within 2 months. Twenty-four patients received C. Parvum and 2 three cycles of CAF. Four patients had complete regression of measurable disease and were living free of disease 9-12 months after starting CAF. Eight patients had > 50% regression for a minimum of 3 months, and five were living with disease (LWD) 5-11 months. Five patients had 25% to 50% regression and three were LWD 4-8 months. Seven patients had no regression and all expired within 4 months. Of eight patients who had successful reductive surgery prior to treatment, three were free of disease, median of 10 months, and five had partial responses and were living with disease, a median of 9 months. Although pre-treatment immune function was better in the patients who had a good response to CP and CAF (10 of 12 were DNCB+) vs that in patients with a poor response (4 of 12 were DNCBC) immune function was no.t significantly improved during therapy. The initial treatment results in this program are encouraging and suggest that this approach may be useful in patients with earlier disease.

Cancer 39:514-526, 1977.

HIS PRELIMINARY R E P O R T IN P A R T DE-

1 scribes a pilot study to treat women with advanced (Stage I11 and IV) ovarian cancer who

Presented at the 29th Annual Meeting of The Society of Surgical Oncology, New York, NY, April 14-17.

From the Departments of Surgery and Medicine, the Gynecology Service, Memorial Sloan-Kettering Cancer Cen- ter, New York, NY 10021.

Supported in part by Cancer Research Institute, Inc., New York, NY.

Address for reprints: Harold J. Wanebo, Memorial Sloan- Kettering Cancer Center, 1275 York Avenue, New York, NY 10021.

Received for publication October 22, 1976.

have progressive disease in spite of previous surgery, chemotherapy and/or radiation therapy by a program of reductive surgery, intensive immune stimulation and combination chemotherapy.

Women with Stage I11 and IV ovarian cancer have a notably poor survival rate. About 20% live 2 years" and less than 6% live 5 years.' Chemotherapy, usually in the form of alkylating agents, has been the mainstay of treatment. The response rate has ranged from 45% to 65% with 5% to 15% of the patients continuing to respond 2 years after initiation of therapy.* Although combination chemotherapy with two or more

514

No. 2 INTRAVENOUS CORYNEBACTERIUM PARVUM Ruo et al . 515

drugs appears to give higher response the duration of response is similar to

that obtained with alkylating agent^^^^'^^^ and no combination regimen has been shown to be su- perior to treatment with single alkylating agents.' The response to chemotherapy with alkylating agents has been markedly lower in patients previously treated with radiation' and/or chemotherapy, although these patients may still respond to combination chemotherapy. "J'

An additional problem in these patients is the extensive tumor burden. Although there are theoretical advantages to reducing the amount of tumor prior to initiating chemotherapy, there has been no convincing data to support this."

Another major area of concern is the severe immunosuppression in these patients. Current therapeutic approaches have attempted to coun- teract this immune suppression and potentiate the response to chemotherapy by the use of immune adjuvants." Mathe et uI. were the first to point out the benefit of immunopotentiation with BCG after cytoreductive therapy with chemotherapy in patients with leukemia. " Later, Israel and Halpern showed the advantages of non-specific immunopotentiation and combination chemotherapy. la They demonstrated that patients with metastatic solid tumors who were treated with C. Parvum plus combination chemotherapy had prolonged survival compared to patients with similar tumors who were treated with chemotherapy alone. '' It was Israel's suggestion that prompted us to de- sign our own program of intensive immune stimulation with intravenous C. Parvum followed by combination chemotherapy in an effort to re- trieve immunodepressed patients who had failed previous chemotherapy and radiation therapy.

The basic treatment plan is outlined in Fig. 1.

In patients in whom it was feasible, an initial laparotomy was performed in an attempt to remove as much tumor as possible. All patients were then treated with a course of intravenous C. Parvum given in escalating doses over a 10- to 14-day period, and continued as subcutaneous weekly injections thereafter. Cyclic chemotherapy with Cytoxan, Adriamycin and 5-fluorouracil was started and repeated at monthly intervals. Immune function in these patients was also evaluated by a battery of skin tests and in vitro tests which were performed prior to their entry into this program and were then continued on a periodic basis during therapy.

MATERIALS AND METHODS

Patient Selection : Thirty-nine patients were ac- cepted into the protocol who had demonstrated progression of disease after previous chemotherapy or chemotherapy and radiation therapy. The median age was 56 with a range of 28 to 74 years. Previous treatment included chemotherapy in 21 patients and radiation plus chemotherapy in 18 patients (Table 1). Single agent chemotherapy (Leukeran) was given in 19 patients and combination chemotherapy (sequential or concomitant) was given in 20 patients. Twenty-one patients were classified as FIG0 Stage 111 (disease involves one or both ovaries with widespread intraperitoneal metastases). Eighteen patients were classified as Stage IV on the basis of disease outside of the per- itoneal cavity or documentation of intrahepatic metastases by unequivocal liver scan or laparotomy. All patients had histologic evidence of serous, mucinous or undifferentiated carcinoma of the ovary. Patients were excluded who had germinal tumors or in whom there was a question of the primary origin of metastastic adeno-

FIG. 1. The planned treatment program for patients with advanced ovarian cancer whose disease has progressed after previous cherno- therapy and/or radiation. Ideally, all patients are explored and have reductive surgery where possible and then receive intensive immune stimulation with C. Parvurn, followed by cyclic therapy with CAF and maintenance subcutaneous C. Parvum.

J 0

R Y

Eligible - Stage I I I or I V Pts Who Fail Adequate Therapy. Program - 1. Reductive Surgery

2. I .V . C. Parvum 10-14 Doses. 3. Cyclical Chemotherapy (Cytoxan Adriamyci n 5-FI uorou raci I) 4. Maintenance Subcutaneous C,Parvum

Cytoxan 60mg/m2 - Adriamycin 30mglm2 4 5 - F l u o r o u r a c i l d

400mglm2 ~~

1 14 15 23 28 35 42 I- C. Parvum I .V . 4 A A A A

C. Parvum S.C.

516 CANCER February 1977 Vol. 39

TABLE 1. Prior Therapy in 39 Patients with Advanced Ovarian Cancer

Patients

Chemotherapy alone 21

Chemotherapy and radiation 18

Single alkylating agent* 14 Combination or sequential therapy’ 7

Single Alkylating agent* 5 Combination or Sequential Therapy’ 13

* Leukeran. Combinations used included Cytoxan, Leukeran, 5-FU,

Provera, Velhan, MTX.

carcinoma. All patients had a complete evaluation prior to entering the study, including a hemogram, coagulogram, bone marrow examination, S M A 12, chest roentgenogram, intravenous pyelogram, barium enema and GI series. Liver and bone scans and skeletal survey were performed in most cases. Exploratory laparotomy was done in 16 of 39 patients who required surgery either for alleviation of ob- struction or in an effort to remove tumor. Re- ductive surgery, with removal of > 50% of tumor, was accomplished in eight patients. Immune testing, including skin tests and in uitro immune parameters, was performed prior to the course of intravenous C. Parvum. In uitro immune tests were performed prior to each cycle of chemotherapy. (CBC, differential, platelet count and S M A 12 were repeated twice weekly during the course of the intravenous C. Parvum and a hemogram was obtained frequently during the course of the chemotherapy. A complete physical examination was done prior to initiation of chemotherapy and was repeated at regular intervals.)

Patient Evaluation

Performance Status: The Karnofsky Perform- ance Status (PS) categorizes patients into three broad classes according to their clinical state: (1) The patient is able to carry on normal activity and needs no special care (PS 80-100%); (2) The patient is unable to work but can live at home and care for his personal needs (PS 70%), or may need some assistance (PS 50-60%); (3) The patient is unable to care for self and requires institutional or hospital care (PS 30-40%).

Other categories include a PS of 20% if the patient requires active support, and a PS of 10% and 0% for a moribund patient, or death. Definition of Therapeutic Response : Determina-

tion of a response was made on the basis of clinical examination or on evidence of radiologic

or radioisotope scan studies. None of the patients had a laparoscopy or a “second look” laparotomy to gauge response to treatment.

Complete Response: indicates complete dis- appearance of all measurable disease for a period of at least three months.

Partial Response: (50% response) includes those patients in whom there was an objective regression of gross tumor of at least 50% which was maintained for three months.

Incomplete Response: (25-50%) includes patients in whom there was some decrease in tumor size (at least 25% reduction) and who had symptom- atic improvement lasting for a period of three months or more.

No Response: Those patients in whom there was no detectable response or decrease in volume of tumor mass or those in whom there was minimal subjective improvement or those who remained stable but have been followed less than three months.

Methods of Immune Testing Skin Tes ts : Patients were treated for pre-exist-

ing delayed hypersensitivity to Dermatophy- ton-0 (Hollister Stier Laboratories, Yeadon, Pa.), mumps skin test antigen (Eli Lilly and Co., Indianapolis, Ind. ), intermediate strength tu- berculin (Parke-Davis Co., Detroit, Mich.) and streptokinase/streptodornase (Lederle Labora- tories, Pearl River, N.Y.). The antigens were injected intradermally in volumes of 0.1 ml. These were read at 48 hours and considered positive only when the diameter of induration was 5 mm or more.

Sensitization and challenge with 2,4-dini- trochlorbenzene (DNCB) was carried out as described by Pinsky et al.’’ DNCB 2000 mcg, dis- solved in 0.1 ml of acetone was applied to the inner aspect of the upper arm, within the con- fines of a plastic ring 2 cm in diameter, and allowed to evaporate. It is rare for patients or controls to show de nouo sensitivity to this dose at 48 hours. Prior hypersensitivity to DNCB was excluded by control testing with 100 ug and 25 ug of DNCB in 0.1 ml acetone applied simulta- neously to the ipsilateral forearm and read at 48 hours. These sites were covered for 48 hours and kept dry. They were examined for erythema and induration after 2 weeks. If there was no reaction at the 100 and 25 ug sites, the patients were then re-challenged 2 weeks later on the ipsilateral extremity with 100, 50 and 25 ug of DNCB. A reaction was considered positive if there was induration of 5 mm or greater at 48 hours at the challenge sites.

No. 2 INTRAVENOUS CORYNEBACTERIUM PARVUM Rao et al. 517

In Vitro Tests

Stimulation of Lymphocytes by Mitogens and Anti- gens: Lymphocyte transformation in vitro was performed using a micro method. Lymphocytes were obtained from heparinized blood in Ficoll- Hypaque (“lymphoprep”) density gradients, and re-suspended in RPMT 1640 with Hepes buffer, glutamine (0.25 mg/cc), penicillin 10 units/cc, streptomycin 100 units/cc, heparin 10 i.u./cc and 15% pooled normal human serum. Lymphocyte culture was performed in flat bot- tom microtiter plates (Falcon #3040) by adding 100,000 lymphocytes in 200X of media. Trans- formation of lymphocytes was measured in terms of incorporation of C “ thymidine which was added after 72 hours (for mitogens) or 120 hours (for antigens) and then incubated 24 hours. Cultures were performed in triplicate. Dilutions of mitogens or antigens which had been prepared in advance and stored at -20°C were freshly thawed and were added to the cultures in 25X volumes. The concentrations of phytohemagglutinin (PHA-P) used ranged from 500, 250, 50, 10 and 5 mcg/100 ml, conconavlin A (Con A, Difco) at 125, 42, 15 and 5 mcg/100 ml, pokeweed mitogen (PWM, Gibco) at 2000, 400 and 80 mcg/100 ml. The following antigen preparations were used in tenfold dilutions (four dilutions each) : candida albicans (Hollister- Stier Laboratories) 1 : 10 dilution, streptokinase/streptodornase (Lederle) 25,000 units in 2 ml, staph aureus 1 X lo8 organisms/ml heat inactivated, E. coli 1 X lo9 organisms/ml, heat inactivated, mixed bacterial vaccine (MBV) 1 : 10 dilution, mumps antigen (Lilly mumps skin test antigen) 1 :10 dilution.

T Lymphocyte Rosettes ( E - R ) : T lymphocyte rosettes in the peripheral blood were determined by a modification of the method of Jondal.’’ Equal volumes (0.1 ml) of a lymphocyte suspension (3 X los cells/ml) and a suspension of washed sheep red blood cells (SRBC 0.5%) were mixed with 0.1 ml of human AB serum previously absorbed with RBC of the same sheep. The mixture was rotated at 20 rpm in a waterbath at 37°C for 5 minutes, then centrifuged at 50 rpm for 5 minutes, then finally left at 4OC for 18 hours. A drop of trypan blue was added, the cells resuspended with a Pasteur pipette, and the rosettes were counted in a hemocytometer. Two hundred viable cells were counted and big (three or more RBC) and small (one or two SRBC) rosettes were counted. Each test was done in triplicate.

B Lymphocyte Rosettes (EAC-R) : B lymphocytes

were determined as C3 receptor lymphocytes, using a modification of the technique described by Bianco.‘ Human A-1 erythrocytes (E) were sensitized with inactivated rabbit anti-A-1 serum at 37°C for 30 minutes with frequent shak- ing. Sensitized cells (EA) were washed with culture medium in the cold and the EA suspension was diluted 1 :10 in C6-deficient AKR mouse serum, and the mixture (EAC) was incubated for 30 minutes and then washed in the cold. Equal volumes (0.1 ml) of lymphocytes (3 X 10’ ml) and EAC (0.5%) were mixed and rotated at 50 rpm in a waterbath at 37OC for 30 minutes. The cells were resuspended, shaken on a vortex shaker and placed in an ice bath, a drop of trypan blue was added, and rosettes were counted within 30 minutes. Ingestion of latex particles was used as a marker for excluding phagocytic monocytes. Two hundred living cells were counted and large and small rosettes were recorded separately.

Serum Immunoglobulins: The concentration of IgG, IgM and IgA was determined by radial immune diffusion using anti-immunoglobulin plates purchased from the Behring Company (Tri-Partigen plates.) Patient sera and pre- diluted standards were placed in the wells and the diameter of the antigen-antibody precipitate was measured at 24 hours.

R Lymphocytes Bearing Surface Immunoglobulins : Approximately lo6 lymphocytes in 0.025 ml of phosphate-buffered saline with 3% bovine serum albumin (BSA-PBS) were mixed with 0.025 ml of fluorescein-conjugated polyvalent Ig-Antise- rum (purchased from Behring Diagnostics) in the presence of 0.02% sodium azide and kept in an ice bath for 30 minutes. The cells were washed in the cold four times with BSA-PBS containing 0.02% sodium azide. A drop of oil suspension on a slide, covered with a cover-glass and sealed with nail polish, was examined under a Leitz fluorescence microscope. Phase contrast microscopy for identification of cells and examination under ultraviolet light was carried out on the same preparations. A minimum of 200 lymphocytes was examined on every slide. Cells showing intracellular fluorescence in the form of a dense fluorescein deposit were considered non- viable and were not counted.

Treatment Course with Intravenous C. Parvum and Subsequent Chemotherapy

Intravenous C. Parvum was given in escalating doses as tolerated over the 10- to 14-day course. It was mixed with 50 cc normal saline and was given as an infusion over a 30 minute

518 CANCER February 1977 Vol. 39

period. O n completion of this course, and de- pending on the white blood cell count and platelet count, chemotherapy was instituted on day 15. Cyclic chemotherapy consisted of daily oral Cytoxan 60 mg/m2 on days 1-14; Adriamycin 30 mg/mz on days 1 and 8; 5-fluorouracil 400 mg/m2 on days 1 and 8. There was generally a two-week rest period and the cycle was re- started. After the initial course of intravenous C. Parvum, 4 mg of C . Parvum was given subcutaneously at weekly intervals as maintenance therapy.

RESULTS Pre-Treatment Clinical Studies

Patients were classified into three groups based on the treatment received. This was on the basis of whether they lived long enough to receive the C. Parvum and expired before receiving any chemotherapy (11 patients); received less than three courses of combination chemotherapy (four patients) or received C. Parvum plus three or more courses of combination chemotherapy (24 patients). There was an equal distribution of patients with Stage 111 and Stage IV disease in the three categories (Table 2 ) . The overall performance status of the 11 patients who received C. Parvum alone and four patients who completed the course of intravenous C. Par- vum but less than three cycles of CAF was 20% to 30% in all but three patients. The latter three had a PS of 50% to 60%. Iri contrast, the patients who received C. Parvum and three or more cycles of CAF had a much better pre-treatment performance status. Only two patients had a PS in the 20% to 30% range, whereas the rest had a PS above 40%.

TABLE 2. Advanced Ovarian Cancer Failures of Previous Treatment

C. Parvum C. Parvurn

alone < 3 cycles 2 3 cycles C. Parvum + C A F + CAF

Patients 11 4 24 Stage I11 6 2 13

IV 5 I 11

5 70% - -

3 11 S0-60% -

6 40% - -

20-3090 11 I 2

(> 50%) O/ 5 o/ 1 8/10

* 16 of 39 patients had laparotomy of which 8 had

3

Performance status

Reductive Surgery*

reductive surgery with removal of >SO% of tumor

TABLE 3. Immune Status in Patients with Advanced Ovarian Cancer Prior to Therapy with

C. Parvum and CAF

C. Parvum C. Parvum C.Parvum + C A F + C A F

alone 3 cycles 3 cycles

IINCB + 2/11 0/4 14/24 IDS + * 0/11 1/4 12/24 Absolute lymphocytic

count Normal 2 0 0 1,OW 9 4 24

Normal 0 0 11 Low 10 3 9

PHA response

* IDS - Intradermal skin tests: + indicated a reaction to any of a battery of 4 antigens: mumps, Dermatophyton, PPD, SK/SD.

Pre-Treatment Immune Status

A summary of the immune status is shown in Table 3. Of the patients receiving C. Parvum alone, only two of 11 were DNCB positive. None of the four patients who received intravenous C. Parvum and less than three cycles of CAF were DNCB positive. In contrast, of the patients who received C. Parvum and more than three cycles of CAF, 14 of 24 were DNCB positive. Skin testing with intradermal recall antigens showed a similar response to that of DNCB testing. The absolute lymphocyte count was depressed below the control tenth percentile cutoff point of 1200 cells/mm3 in all but two. The PHA response was low (less than the tenth percentile of the control group) in all the patients who had been treated with intravenous C. Parvum alone, or who had received treatment with C. Parvum and less than three cycles of CAF. In the patients who received C. Parvum and three or more cycles of CAF, the PHA response was normal in 11 and low in nine.

Response to Therapy

Of the 11 patients who received only intravenous C. Parvum, none showed a measurable response and none survived for more than one month following initiation of treatment. Of the four patients who received intravenous C. Parvum and less than three cycles of CAF, two survived 6 weeks and two survived 8 weeks after initiation of treatment. None showed a response to therapy. Only the patients who received C. Parvum and three or more cycles of CAF showed a clinical response to therapy (Table 4). None of these latter patients showed an objective


TABLE 4. Response and Survival

Response Patients NED LWD DOD Complete 4 4 (9-12mo) - -

t 50% 8 - 5 (5-1 1 mo) 3 (3-7 mo) 25550% 5 - 3 (4-8 mo) 2 (6-10 mo) No response 7 - 4 (2-4mo) 3 (4-5 mo)

TOTAL 24

NED = No evidence of disease 1.WD = Living with disease D O D = Dead of disease

response to the course of the intravenous C. Par- vum itself. Four patients had a complete regression of measurable disease and were alive and clinically free of disease 9-12 months after starting treatment. Eight patients had a greater than 50% regression of tumor volume for a minimum of 3 months and five of these were living with disease 5-11 months after initiation of treatment. Five patients had a 25%-50% response and three of these were living from 4 to 8 months. Seven patients had no tumor response and four of these were alive 2-4 months following treatment. Of the 24 patients who completed the course of intravenous C. Parvum and three or more cycles of CAF, eight patients had successful reductive surgery prior to entering the chemoimmunotherapy program. Three of these were free of disease for a median of 10 months, and five had a partial response and were living with disease for a median of 9 months.

Relation of Pre-Treatment Immune Tests to the Response in Patients Receiving C. Parvum and Greater than Three Cycles of CAF (Table 5)

There was a difference in the DNCB responses in patients who had greater than 50% tumor regression compared to those who had less of a response after therapy. Ten of the 12 patients who had greater than 50% tumor regression with therapy were DNCB positive prior to treatment. In contrast, of the patients showing minimal or no response, only four of 12 were DNCB positive (P = < .05). The pre-treatment absolute lymphocyte count was depressed below the control 10th percentile level in all patients receiving > 3 cycles of CAF and showed no corre- lation to the clinical response. There were no differences in the lymphocyte responses to PHA between the good responders and the poor responders to CAF therapy. Four of nine of the good responders (complete response or > 50% response) had normal PHA reactions vs seven of

11 poor responders (< 50% response or no response group).

Changes in Immune Function During Therapy

In the patient group that received intravenous C. Parvum alone or completed the course of intravenous C. Parvum but received < 3 cycles of CAF there was a general depression of absolute lymphocyte counts and mitogen responses concomitant with clinical deterioration.

Among the 24 patients who had completed 2 3 cycles of CAF, the changes in immune measurements during the period of immunostimula- tion with intravenous C. Parvum were variable. There was generally a fall in the absolute lymphocyte count during the treatment with intravenous C. Parvum. (All pre-treatment values were below the 10th percentile of the control group). The PHA responses were variable and showed no constant appreciable changes from pre-treatment baseline levels during the course of the intravenous C. Parvum.

The changes in immune function after intravenous C. Parvum and three cycles of CAF are listed in Table 6. There were no consistent

TABLE 5. Relation of Response to Immune Tests Response Groups - C. Parvum + C.A.F.

No Total CK >50% 25-50% response pts.

DNCB t 3 7 2 2 14

1 1 3 5 10 -

Absol Lymph Cells/mma

>1200 0 0 0 0 0 < 1200 4 8 5 7 24

Normal 2 2 4 3 11 Low 2 3 1 3 9

PHA*

* Normal > 10th Control Percentile Low < 10th Control Percentile

520 CANCER February 1977 Val. 39

TABLE 6 . Advanced Ovarian Carcinoma Changes in Immune Function After Therapy with C Parvum and 3 Cycles of CAF

~ ~~

Total Clinical patients DNCB Absolute lymphocyte count PHA response ingroup 1 NC T 1 NC T I NC T

Complete 4 1 3 - - 1 2 0 4 0 > 50% 8 2 2 2 1 1 1 0 3 3 25-50% 5 1 4 0 1 1 1 2 0 2 No Response 7 - 4 1 1 n 2 3 2 0

TOTAL 24 4 13 3 3 3 6 5 9 5

NC = No change.

changes in DNCB responses. Thirteen of 20 patients showed no change in DNCB response. Three patients who were previously DNCB negative became DNCB positive and four patients who were initially DNCB positive became DNCB negative. The absolute lymphocyte

Count in 100'sl 42 30

28

26

24

22

20

I8

16

I4

12

10

8

6

4

2

0

_ _ _ _ -

ABSOLUTE LYMPHOCYTE COUNT iCel lr lmmI

Contrrl I . Median b= 1Mh Percentile

b b b

mtrol Total C . Parvuk NO 25%-50% > 50* Pis. Pts. Alone Response

b

Complete

Group -Response Groups-J

FIG. 2. The pre-treatment absolute lymphocyte counts for all patient groups is shown abovr. All but two patients had depressed counts and were below the 10th percentile of the control group.

count was increased above pre-treatment base- lines in six of 12 patients but was increased into the normal range in only one patient after three cycles of CAF. The PHA responses remained the same in nine patients, were increased in five patients and were depressed in five patients after three cycles of CAF (Figs. 2-5). In 10 patients with evaluable PHA measurements who showed complete or > 50% tumor regression the PHA response remained stable in seven and increased in three. In the patients who had < 50% tumor regression or no regression the PHA response was depressed in five, unchanged in two and increased in two.

Toxicity from C. Parvum Administration

C. Parvum in escalating doses was generally well-tolerated. Individual doses ranged from 0.5 mg to 23 mg and were given in saline over a 30 minute period. The total dosage during the 10- to 14-day course ranged from 10 mg to 177 mg. Fever, headache and chills were the most com- mon symptoms. Transient rises in blood pressure and respiratory rate were also noted in a majority of the patients. Four patients had marked hypertension with elevations of > 60 mm Hg systolic and 40 mm diastolic. In most of the patients who developed hypertension, elevations of < 30 mm Hg systolic and < 20 mm Hg diastolic were noted. Only two patients developed hypotension which responded to fluid therapy. Nausea and vomiting were very rare. About 50% of the patients showed a drop in the white cell or platelet counts which occasionally necessitated a delay in the white cell or platelet counts which occasionally necessitated a delay in the initiation of chemotherapy. I n only two patients was it necessary to discontinue the C. Parvum for a few days because of the leukopenia and thrombocytopenia. However, coagulograms, measurement of fibrin split products, and evaluation of the bone marrow aspiration in those patients did not document a specific coagulation

INTRAVENOUS CORYNEBACTERIUM PARVUM 9 Rao et al .

20

No. 2

Control Median __--__-_-_________-_--------------- -

40 38

36

34 32

30

28

26

24

FIG. 3. Pre-treatment lym- 22

phocyte stimulation studies with 20 PHA as performed in two different '*P.M. laboratories are shown for all treat- 'Oo0 rnent groups. 16

14

12

10

8

6

4

2

0

clophosphamide, adriamycin and " 5-fluorouracil.

MAXIMUM PHA STIMULATION fPRE TREATMENT)

Control 10th Percentile 8_-------------_----_------------_--

52 1

Control Lab 1 Median

D 1Mh Percentile cut Off

Control Cab 2 H Median

1Mh Percentile cut Off

D D D

Control All Pts. c. P. No 25-50 ~ 5 0 Complete Lab I - Lab 2 Alone Response

abnormality. In most patients there was a tran- travenous C. Parvum. The majority of the sient elevation of the SGOT, LDH and alkaline patients tolerated the weekly subcutaneous dos- phosphatase, but these values reverted to nor- age of 4 mg but occasional patients had fever. ma1 a few days after the completion of the in- and some had discomfort from these painful

24 r

i.v. C Parvum Subq C. Parvum + C.A.F. I t

I I I I I I 1 I I ; / ' I 'P++ 2 4 6 8 10 12 14 16 1 2 3'6 9

Days Months

522

4-

CANCER February 1977

A+ S.C.

C. Parvum i.v. C Parvum ---A + C.A.F. I I I

Vol. 39

0 LWD stable A Progression of disease

24 + Dead of disease

/-/ 4 6 0 10 1 2 1 4 16 1 ’ 4 4 5 6’5 Days

nodules at injection sites. Occasionally it was necessary to reduce the dose because of severe local reactions or generalized symptoms of an incapacitating nature.

DISCUSSION

This preliminary study attempted to max- imize the treatment of patients with advanced ovarian cancer who had already failed conven- tional therapy with radiation therapy and chemotherapy or chemotherapy alone. The preliminary clinical results in the 24 patients who had an adequate trial of the combined C. Par- vum and CAF program are encouraging. Twelve patients showed greater than 50% tumor regression lasting over three months and four patients were living free of disease 9-12 months, and five were alive with disease 5-11 months after start of therapy.

In general, the ovarian cancer patients could be categorized into two groups according to the overall response to this treatment program. The more favorable or retrievable group consisted of patients who initially had good performance status, intact immunity and were able to have reductive surgery. The unfavorable or non-re-

Months

trievable group consisted of patients who generally had a poor performance status, impaired immunity and did not have reductive surgery. Of 15 patients in this unfavorable group, 11 completed the period of initial immunostimula- tion with intravenous C. Parvum but succumbed before receiving chemotherapy. Four others survived long enough to receive only one or two courses of chemotherapy.

In an effort to remove all or to markedly decrease tumor burden, surgery was carried out in 16 patients prior to initiating chemoimmunotherapy. Reductive surgery, removal of more than 50% of the tumor, was possible in eight of 16. Of importance, three of these patients showed a complete response and were without evidence of disease for a median of 10 months. Five have had > 50% tumor regression and were living with disease a median of 9 months. Although these numbers are small and the length of follow-up short, this patient group will merit close scrutiny to see if improved survival was obtained by use of reductive surgery followed by chemoimmunotherapy. Although there are theoretical advantages to reductive surgery in patients with advanced ovarian cancer, there has been no conclusive data. Indeed,

No. 2 INTRAVENOUS CORYNEBACTERIUM PARVUM Rao et al.

0 LWD stable A Progression of disease

28 + Dead of disease

523

FIG. 6. Shown are the sequential lymphocyte responses to PHA in patients who showed a minimum response to therapy with C. Par- vum and CAF.

Q 7

X

E a u

/ \ Control V . 10th Percentile /

I i.v "

'. C Parvum S.C. C. Parvum +

_ _ _ _

C.A.F.

w i 4 6 8 10 12 14 16/ 1 ' 3 4 5 6 5 Days Months

Piver et al. have reported a highly select group of eight patients with advanced ovarian cancer who initially showed a favorable response to chemotherapy and were then subjected to a second look laparotomy, with resection of all possible residual cancer followed by total abdominal radiation with a linear accelerator. Seven developed recurrence within three to 20 months and five of these died of disease (3-19 months) and only one patient was free of disease at 17 month^.^" Underwood et al. were more encour- aged about their results using reductive surgery. 37 They treated 70 consecutive previously untreated ovarian cancer patients by surgical removal of all gross tumor, and subsequent total abdominal irradiation with a pelvic boost. Their 2-year disease-free survival in 20 patients with Stage I1 disease was 50%.37 No information is available on the use of this approach in patients who have failed previous chemotherapy or radiation after primary surgery.

The treatment of patients who have failed therapy with single alkylating agents and/or irradiation is a formidable problem which cur-

rently would appear best approached by the use of combination chemotherapy. Theoretical interest in the use of combination chemotherapy was initially stimulated by the reports of Goldin" and Skipper;36 it was clinically first demonstrated by Li et a/." in the treatment of metastatic cancer of the testis and later by Greenspan l2 in the treatment of advanced ovarian cancer. More recently, Smith et al.33 were able to obtain a 38% response in patients with ovarian cancer refractory to previous therapy by using Cytoxan, actinomycin D and 5-fluorouracil. Using Provera, 5-fluorouracil and vinblas- tine, OchoaZS has been able to obtain a 30% response rate in patients who have relapsed on alkylating agents.28 It would appear that combination chemotherapy may not only result in substantially better results when used as a first line a p p r a ~ c h ~ - ' ~ ~ ' ' * ~ ~ but may also permit sal- vage of patient failures of previous radiation or single agent chemotherapy.

In designing a therapy program for patients who have failed previous chemotherapy and radiation it is essential to consider the immuno-

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Flc. 7. Shown are the sequential lymphocyte responses to PHA in patients who showed no response to C. Parvum and CAF. There is a general depression of the PHA response which parallels clinical deterioration.

i.v. C Parvum S.C. C Parvum + C.A.F. t I I

ty 2 4 6 8 10 12 14 16' 1 5 i 5 6 5

Days Months

suppression that exists in these patients second- ary to extensive tumor burden and previous therapy. 7*13,16 Intermittent cyclic chemotherapy has some theoretical advantages in that the rest period can permit rebound of the immune function and recovery of the bone marrow." Many authors have pointed out the need to combine immunopotentiating agents with chemotherapy to amplify this immune rebound as well as to stimulate marrow recovery. 16*18,18 Initial interest in an immunochemotherapy program was stir- red by Mathe's report which showed that BCG scarification alone or in combination with irra- diated allogeneic leukemia cells prolonged chemotherapy-induced remissions in children with lymphoblastic le~kemia . '~ Numerous chemoimmunotherapy programs have now been initiated (recently summarized by Gutterman P t a / . ) . l3 C. Parvum has been shown to be an active immunopotentiating agent in various animal tumor models.",3s In humans, C. Parvum given subcutaneously with chemotherapy has been ef- fective in prolonging the survival of patients with

metastatic solid tumor as compared to patients who received chemotherapy alone. Based on Israel's unpublished observation that there was increased clinical benefit from the use of intravenous C. Parvum in ovarian cancer patients, we were prompted to embark on this pilot study combining a period of intensive stimulation by intravenous C. Parvum followed by a cyclic chemotherapy with CAF and weekly subcutaneous C. Parvum.

A major issue in this preliminary trial was also to assess the immune effects of intravenous C. Parvum and subsequent weekly subcutaneous C. Parvum in conjunction with CAF. Pre-treatment immune testing showed marked immune depression in most of the patients. Two-thirds of the patients were DNCB negative and two-thirds had depressed PHA responses. All but two patients had markedly depressed lymphocyte counts. Pre-treatment immune function also appeared to correlate with the clinical response to therapy. Patients who showed a favorable response (> 50% tumor regression)

had better pre-treatment immune responses than patients with poor responses (minimal or no tumor regression). Ten of 12 patients in the good response group were DNCB positive compared to four of 12 patients in the poor response group (P < .05). There were no significant differences in the absolute lymphocyte counts nor in the lymphocyte responses to PHA between good responders and the poor responders. At this point there were no consistent patterns of change in the immune tests performed either during the period of intravenous C. Parvum nor in the overall treatment with CAF and maintenance subcutaneous C. Parvum. Whether C. Parvum has a beneficial effect on immune function when it is combined with CAF will hope- fully be determined by the randomized trial that is under way.

Although it has been stated by Israel et al . that stimulation with C. Parvum will increase toler- ance for chemotherapy, we have not seen this in our limited study. In less than half of the patients who were maintained on subcutaneous C. Parvum and combination chemotherapy

(CAF) was it possible to maintain a full dosage schedule of chemotherapy. Again the question whether C. Parvum can effectively stimulate bone marrow and facilitate more aggressive chemotherapy can only be answered by a randomized trial.

Our initial concern about toxicity from intravenous C. Parvum in these very ill patients was not substantiated. Intravenous C. Parvum in escalating doses was generally well-tolerated. Individual doses ranged from 0.5 to 23 mg and the total dosage during the 10- to 14-day course ranged from 10 to 177 mg. Patients commonly had fever, headache, chills and transient rise in blood pressure and respiratory rate. Nausea and vomiting were very rare. About 50% of the patients showed a drop in the white cell count or platelet count which occasionally necessitated a delay in the institution of chemotherapy. Sub- sequent maintenance therapy with subcutaneous C. Parvum in 4 mg weekly doses was tolerated by the majority of patients, although in occasional patients the dose was reduced because of severe discomfort at injection sites.


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therapy in treatment of primary ovarian malignancy. ObJlet. Gynecol. Suro. 24.:1307-1342. 1969.

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intravenous corynebacterium parvum. an adjunct to chemotherapy for resistant advanced ovarian cancer

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