SIGNIFICANCE OF GROWTH RATES, CELL KINETICS, AND HISTOLOGY IN T H E IRRADIATION AND CHEMOTHERAPY OF

SQUAMOUS CELL CARCINOMA OF T H E MOUTH MILTON FRIEDMAN, MD, CARLO NERVI, MD, CARLO CASALE, MD,

GIUSEPPE STARACE, PHD, GIORGIO ARCANCELI, MD, GIORGIO PAGE, MD, AND ELIO ZIPARO

In eight cases of extended tumors of oral cavity, we have studied the growth rate, the cell kinetics parameters, and the histology before and during treat- ment. We have found that growth rate and cell kinetics are not useful for clinical purposes because the first is not related to the response to treatment, while the second is unable to give relevant parameters in solid human tumors. New information was obtained from examination of serial biopsies before and throughout the treatment. These data suggest that multiple subpopulation groups of cancer cells with different intrinsic sensitivity to chemotherapy and/ or radiotherapy may exist in the same tumor. Resistance of the tumor is related to some groups of cells scarcely sensitive to treatment.

HIS STUDY WAS UNDERTAKEN TO INVESTIGATE T some biological and clinical aspects of radioresistance and chemoresistance of human solid tumors in order to obtain more infor- mation for an optimal therapeutic approach to cancer treatment. It was based on a pros- pective study of a number of biological and clinical parameters before, during, and after combined treatment with chemotherapy (methotrexate) and irradiation of some ad-

vanced tumors of the oral cavity. The aim of this study was to relate tumor

growth rate and cell kinetics to response to treatment. Additional observations were made about the dynamic histologic changes result- ing from chemotherapy and irradiation.

MATERIALS AND METHODS

Up to now, all the data in eight unselected

Presented at the First Joint Meeting of the James Ewing Society, the Society of Head and Neck Surgeons, and the American Radium Society, Boca Raton, Fla., May 14-19, 1972.

From the Departments of Radiation Therapy and Pathology, Istituto Regina Elena per lo Studio e la Cura dei Tumori, Rome, Italy.

.4ddress for reprints: Prof. Carlo Nervi, Department of Radiotherapy, Istituto Regina Elena per lo Studio e la Cura dei Tumori, Viale Regina Elena, 291, 00161 Rome, Italy.

Received for publication August 31, 1972.

cases of extensive squamous cell carcinoma of the mouth have been harvested. In each pa- tient, the following parameters were studied:

1. Growth rate obtained from clinical his- tory and periodic measurement of the tumor prior to treatment. These observations were made available during the period needed for tooth removal, oral hygiene, and insertion of intra-arterial catheters into the external caro- tid artery (through the superficial temporal ar- tery). The catheter was used for injection of tritiated thymidine and, in 5 cases, also for methotrexate (MTX) infusion chemotherapy. ’

2. Cell kinetics studies before treatment by autoradiography of serial biopsy specimens, averaging 3 per day for 6 days, beginning one- half hour after a pulse intra-arterial injection of 2 mCi of tritiated thymidine. Autoradio- graphs were prepared using the “squash” and “dipping” techniques.2 In one case, a second cell kinetics study was carried out during M T X treatment, while, in another case, i t was performed on a post-treatment recurrence.

3. Serial biopsies for light microscopy study before, during, and after treatment. After a traditional histologic examination, cell counts were performed on each biopsy specimen. An average of eight high-power fields ( ~ 2 5 0 ) was counted in each sample. 4. Response following intra-arterial infusion

of MTX administered at a daily dose of 3 mg,

10

No. 1 GROWTH RATES, CELL KINETICS, AND HISTOLOGY - Friedman et al. 11

-3

w m

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24 hours a day, for a period ranging between 14 and 22 days.

5. Response following CO-60 teletherapy (in 7 cases) and conventional 250 kv orthovoltage (in one case). Tumor dose ranged between 6500 and 7500 rads in 7-8 weeks.

6. Rate of regrowth in recurrent tumors.

RESULTS

The data are summarized in Tables 1 and 2. Growth rates were derived from clinical his- tory and from periodic measurement of tumor volume. Because of difficulties in determining an extremely accurate estimation of irregu- larly shaped volumes, it was decided to give a rough evaluation and consider only “rapid,” “moderate,” or “slow” rates. The growth rate was rapid in three patients, moderate in one, and slow in four others.

Concerning cell kinetics, the per cent la- belled mitoses (PLM) curves and the labelling index (LI) were drawn plotting the data col- lected on specimens taken before treatment.

The PLM curves were designed as a family of curves based on counting the number of grains per mitosis. This procedure was done to aid in minimizing the influence of back- ground. The curve based on more than 2 grains per mitosis (heavy line) was chosen as the most representative. The feature of the PLM curves was so complex that it prevented calculation of the intermitotic time and other cell cycle parameters. T h e LI demonstrates some sudden and remarkable variation during the whole experiment (Fig. 1.).

When a second cell kinetics study was per- formed following treatment, the PLM curves were more similar to the classical form war- ranting the calculation of some parameters. In fact, it was possible to derive the DNA syn- thesis time (Ts) and the cell cycle time (Tc) according to the methods suggested by Men- delsohn and Takahashi‘ (Fig. 2).

TABLE 2. Case 1 (C.A.) Case 2 ( M . R . ) Ca lower gum

TJVz T3N3 Ca lower lip

Rate of regrowth rapid rapid Cell cycle time 63 hrs. 71 hrs. DNA synthesis time 26 hrs. 26 hrs. Response to chemother-

Response to radio- aPY - moderate

- moderate therapy

12 CANCER January 1973 Vol. 31

Concerning response after chemotherapy, a rapid regression of the tumor, ranging be-

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tween 65% and 90% of the initial volume, was observed in four patients, while in one case no results were obtained.

After radiotherapy, a complete disappear- ance of the primary lesion was reached in four cases; it was incomplete in the others.

Concerning the relationship between growth rate and clinical response, it can be noticed that two rapidly growing tumors and two slowly growing tumors, which were submitted to chemotherapy, showed a rapid regression while another one did not respond at all.

Among the tumors which disappeared after radiotherapy, two grew rapidly and two slowly,

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FIG. la-c. Cell kinetics studies of cases 1 and 2 (radioresistant) and case 7 (radiosensitive) before treat- ment. The family of curves is based on the number of grains per mitotic figure. In the upper half, the heavy line represents the cells with more than two grains per mitosis and was selected to symbolize the PLM. The lower group of curves is the LI.

a 0 a 0 * o 110 0

WOUi8 AFTER ' w e d IWJICTIOY

FIG. 2a (top) and U (bottom). Second cell kinetics study of recurring tumor. T h e PLM curves are more similar to a classical form.

No. 1 GROWTH RATES, CELL KINETICS, AND HISTOLOGY Friedman et al. 13

while among those incompletely reduced, two grew rapidly and two slowly (Figs. 3,4).

Information was made available by means of serial biopsies during the treatment. As it is difficult to establish, from the microscopic ap- pearance, whether or not a particular cell is viable, the same criteria were used in all speci- mens for defining the “probably viable” and the “probably nonviable” or killed cells. As generally accepted, a good estimation of via- bility is the regularity of size, shape, and stain- ing characteristics of the cellular components.

In four tumors later recurring, viable cells sometimes grouped in islands were observed even at the end of the treatment (Fig. 5a, b).

A total of five tumors recurred within a maximum of 8 months after completion of treatment. In two cases, there was no evidence of disease after 18 and 20 months, respectively, and, in case 3, only recurrent nodes were observed after 21 months. Two of these (cases 4, 7) had slowly growing tumors and one (case 3) had rapidly growing tumor.

The rate of regrowth of the recurrent dis- ease was rapid in four cases and slow in one. The latter case grew rapidly initially, but it was typically necrotizing. The others were one rapidly, one moderate, and two slowly grow- ing tumors, respectively.

As shown in Table 2, a second cell kinetics study was performed in one case when the tumor was regrowing after chemotherapy and in another 3 months after irradiation. In both cases, the rate of regrowth was rapid.

In these cases, the value of T c can be de- rived and is the same when calculated with two different methods. In one case, no re- sponse was obtained after either chemother- apy or radiotherapy, while in the second case only a moderate regression was observed.

DISCUSSION

The growth rate of a neoplasm has been considered an important parameter for plan- ning the treatment and predicting the prognos i~ .~

It is evident in this study that the clinical response of the primary tumor is not corre- lated with its growth rate. The cases showing slow growth were either radiosensitive (cases 3, 4) or radioresistant (cases 1-8), while in the same way other rapidly growing tumors were equally either radiosensitive (case 7) or radio- resistant (cases 2-6).

No relationship was attempted between re-

Ifebruard march I aprll I may 1 June Iseptemb.l

FIG. 3. Case 1. Diagram of the treatment and re- sponse of the tumor. Note that there was no detectable increase in tumor volume during the 4-week pretreat- ment period. Following intra-arterial MTX, there was rapid and almost total shrinkage of the tumor volume only to be followed by rapid regrowth immediately afterward. Subsequent irradiation with Co-60, giving a total tumor dose of 7000 rads in 47 days, produced slow shrinkage. Subsequently, there was almost com- plete recurrence in 3 months at a faster rate than originally. C. A., carcinoma of gum (TINZ).

sponse to chemotherapy and growth rate be- cause of the incompleteness and temporariness of regressions.

On the other hand, the absence of this rela- tionship could be expected because the growth rate is solely the result of all dynamic progressive and regressive phenomena occur- ring in a tumor (i.e., cell cycle time, growth fraction, cell loss).

Cell kinetics studies were undertaken for a better evaluation of these processes. Neverthe- less, they are unable to offer us any suggestion in predicting the response to treatment. In fact, the PLM curves in the squamous cell car-

march i FIG. 4. Case 7. Diagram of the treatment and re-

sponse of the tumor. The curve depicts the pretreat- ment growth of the tumor volume and its marked shrinkage as a result of sequential MTX and irradia- tion. L. F., Carcinoma of lip (T,N,).

14 CANCER January 197 3 Vol. 31

cinomas under study are so complex that it is impossible to extract relevant parameters with the current methods of analysis.4 According to the classical interpretation of PLM curves, the second smaller wave, following the larger first one, should signify a second generation wave while the peaks of all the subsequent mitotic waves should be progressively lower until the curve reaches a plateau, indicative of de- synchronization.' This is difficult to per- ceive in our cases because we find a complex curve with many irregular waves. Further-

FIG. 5a. Case 2. One day after administra- tion of 4500 rads in 22 days (250 kv x-rays). T h e r e are tumor- viable cells in an area of severely injured cells (x250).

FIG. 5b. Case 6. Three days after ad- ministration of 7000 rads in 40 days (Go-60 teletherapy). Note the presence of residual viable cells (x250).

more, the second one is always followed by one or more waves which are higher.

All these findings do not correspond to the proliferative behavior of a relatively homoge- neous population composed of a single type of cells.9

A stimulating hypothesis is that the irregu- larity in shape of PLM curve is due to the presence of groups of cells with widely scattered proliferating characteristics. This hy- pothesis could also be supported by the behav- ior of the mean grain count which is not pro- gressively lowered during the experiment as it

No. 1 GROWTH RATES, CELL KINETICS, AND HISTOLOGY - Friedman et al. 15

should be at the time when most or all the cells divide.4 Another finding is that following tre,itmcnt either with MTX (case 2) or irra- diation (case I), the second PLM curve of the same tumors is different, more resembling a classical curve, so that some parameters can be calculated (Table 2).

In two other cases, not included in this study because they were post-irradiation recur- rences, the PLM curves showed similar “cleaner” characteristics, so that the cell kinet- ics parameters could be calculated.

A reasonable explanation is that the hetero geneous population becomes more homogene- ous almost as if successive subpopulation groups of cells are extracted from the tumor.

The peculiar pattern of the LI curve-that sometimes changes so rapidly and so much within a few hours-suggests that the prolifer- ative activity has wide variations in different areas of the same tumor and even in the same 5pecimen.

The histologic aspect of the tumor is an- other classical parameter on which the thera- peutic and prognostic criteria are commonly based. Nevei theless, only a limited predicta- bility can be obtained from histologic type, gi ading of differentiation, and the number ot mitotic figures.0 It may be that more informa- tion can be derived by studying other aspects commonly overlooked. For instance, accord- ing to the experience of one of us (M.F.), the presence of a particular architecture or nu- clear abnormalities is an expression of a lesser radiosensitivity of the tumor.5

By employing serial biopsies throughout the whole treatment, it was possible to find viable cells in four recurring cases out of five. It is likely that these residual survival cells become supplying centers of malignant cells later gen- erating the recurrence.

These cells are representative of a selected subpopulation which has different characteris- tics in being less chemosensi tive or radiosensi- tive.

In an attempt to quantitize this finding, the variation in the per cent of pretreatment den-

sity of viable cells (i.e., their number per unit microscopic field) was measured. N o relation with clinical result was found because of poor reliability of this type of analysis due to great nonhomogeneity of distribution of viable cells.

The rate of regrowth of recurrent disease has been faster in all cases except in case 6 which had necrotizing characteristics.

The combination of chemotherapy with ra- diotherapy, in this limited experience, does not seem to have improved the final results.

CONCILJSIONS

1 . The growth rate of a primary tumor has no practical usefulness in the clinical ap- proach to treatment planning because it is not related to the future response to therapy.

2 . For clinical purposes, the significance of cell kinetics studies is seriously questionable. Furthermore, it is impossible to obtain any information before treatment because of the prolonged time required with the present techniques of autoradiography.

3. Histologic study of serial biopsy speci- mens provides corroborative information tlur- ing the treatment and may disclose the persist- ence of less sensitive groups of cells.

Frbm all new information collected during this study, a hypothesis arises which might es- plain the observed differences in response to therapy.

It could be that such a wide spectrum of sensitivity exists in the cancer cells that the cure can be achieved only when the treatment overcomes those cells with the lowest sensitiv- ity. In fact, cell kinetics suggests the presence of multiple subpopulation groups of cells in the same tumor, and dynamic histology dem- onstrates viable cells surviving to treatmeqt.

Radioresistance of a tumor appears to be es- sentially related to some intrinsic characteris- tics of specific cells. Thus, on this particular aspect, the quality is perhaps more important than the quantity of the tumor cells.

REFERENCES

1 . Baserga, R.: T h e relationship of the cell cycle to tunior growth and control of cell division. Cnnrer Res. 25: 581 -595, 1965.

2. Bresciani, F.: A comparison of the cell generative cycle in normal, hyperplastic, and neoplastic mammary

gland of the C3H mouse. In Cellular Radiation Biol- ogy. Baltimore, Williams and Wilkins, 1965; p. 547.

3. Breur, K.: Growth rate and radiosensitivity of human tumors. 11. Radiosensitivity of human tumors. Ef t rop . J. Cancer 4:343-347, 1968.

16 CANCER January 1973 Vol. 31

4. Casale, C., Starace, G., Nervi, C., and Ziparo, E.: .4n evaluation of cell kinetics in squamous cell carci- noma of oral cavity. To be published.

5. Freidman, M.. and Daly, J. F.: The treatment of ~ i i u a m o u ~ cell carcinoma of the head and neck with m'ethotrexate and irradiation. Anz. J . Roentgenol. 99: 289-301, 1967.

6 . Glucksmann, A,: LighL micioscope information: Pre-treatment prediction and radiation effects. In Bio- logical and Clinical Basis of Tumor Radiosensitivity, M. Friedman and C. Nervi, Eds. Springfield, Charles C Thomas ( In press).

7 . Mendelsohn, M . L., and Takahashi, M.: A critical evaluation of the fraction of labeled mitoses method as applied to the analysis of tumor and other cell cycle. In T h e Cell C cle and Cancer, R. Baserga, Ed. Dekker, New York, 1971; pp. 58-95.

8. Nervi, C., Arcangeli, G . , Casale, C., Cortese, M., Guadagni, A, , and Le Pera, V.: A reappraisal of intra- arterial chemotherapy. Cancer 26:577-582, 1970.

9. Steel, G. G.: The cell cycle in tumors: A n erami- nation of data gained by the rechnique of labelled mi- toses. C e l l 7i. isue K z n e t . 5:87-100, 1972.