Radiotherapy and Oncology, 3 (1985) 1-9 Elsevier 1

RTO 00080

What is known about tumour proliferation rates to choose between accelerated fractionation or hyperfractionation?

Klaus -Rf id ige r T r o t t an d J o h a n n K u m m e r m e h r

Strahlenbiologisches Institut der Universitiit, Schillerstrasse 42, D-8000 Miinchen 2, Abteilung fiir Strahlenbiologie der GSF, D-8042 Neuherberg, F.R.G.

(Received 12 April 1984, revision received 9 July 1984, accepted 10 July 1984)

Key words: Repopulation: Accelerated fractionation; Cell proliferation

Summary

Repopulation of surviving clonogenic tumour cells during fractionated radiotherapy is one of the crucial factors determining cure probability in radiotherapy. Clinical and experimental data suggest that repopula- tion rates vary considerably between different tumours but may be similar to the cell production rates in the untreated tumour. For those tumours which repopulate fast, such as squamous cell carcinomas,, bladder cancer and colorectal carcinomas, accelerated fractionation may be indicated.

Introduction

Cure of a tumour by radiotherapy is achieved if the number of clonogenic tumour cells, that is those tumour cells capable of infinite proliferation and thus capable of giving rise to a local recurrence, is decreased to a certain level, in the ideal case to zero. With increasing dose, the number of surviving clon- ogenic tumour cells decreases exponentially until their concentration in the total tumour mass is so far diluted that with further increasing dose the probability of achieving zero clonogenic cells rises

This article is based on a lecture held at the second Annual Meet- ing of the European Society for Therapeutic Radiology and On- cology, Bordeaux, 1983.

according to a Poisson function. The dose to achieve a certain cure probability therefore depends on only two factors: the initial number of clono- genic tumour cells (which may be proportional to tumour size) and the slope of the survival curve for fractionated irradiation (which is influenced by sev- eral factors, two of which are of the greatest prac- tical importance: split-dose recovery and repopu- lation) (Fig. 1).

The more repopulation of clonogenic cells occurs during fractionation intervals the more additional dose is needed to compensate for the increase in clonogenic cell number. As a reasonable estimate 2 Gy per cell doubling can be assumed as the slope of the survival curve of Hela cells to multiple frac- tions of 2 Gy conforms to this value (Trott, unpub- lished). On the other hand, this increase in dose

0167-8140/85/$03.30 �9 1985 Elsevier Science Publishers B.V.

2

cell number

I0 S

10 4

10 3

10 2

10 ~

10 0

dose [rod }

~ A I ~ fractionctted / / / / / , / / / / / ~ / . / / / / / / / / cure

Fig. 1. Schematic representation of the decrease in the number of clonogenic tumour cells with increasing total dose given as single dose, as fractionated irradiation with intervals short enough to prevent repopulation and with repopulation taking place during the intervals. The dose difference at equal cure rate between the concentrated and the extended course of radio- therapy with equal numbers of fractions is proportional to the number of cell doublings which took place during radiotherapy.

necessary to achieve the same effect on a tumour when radiation is given with the same number of fractions but in longer overall t reatment times is the only reliable method of measuring repopulation in vivo. All other methods, especially cell kinetic tech- niques, give only non-functional information de- scribing the behaviour of mostly non-clonogenic tumour cells, yet there are indications that such da- ta might be of some help in deciding which tumours should receive accelerated fractionation: the greater the repopulation rate the shorter should be the in- tervals between fractions, in some tumours which repopulate rapidly it might even be necessary to give several fractions of normal size every day. If, however, repopulation rates are slow, radiotherapy might safely be extended over 6 weeks or even longer, making full use of the differences in split- dose recovery between tumours and normal tissues in a course of hyperfractionated radiotherapy.

It is therefore of considerable importance to de- velop methods to predict or measure the repopu- lation potential of the individual human tumour in order to select the adequate interval between dose fractions or the optimal overall treatment time.

Direct measurements of repopulation

Published experimental data on the kinetics of re- populat ion in tumours are scarce. Suit et al. [37] measured repopulation in a mouse tumour by ex- tending interfraction intervals between 10 or 20 fractions f rom one to several days. However, the data were difficult to interpret since effective doses of about 5 Gy per daily fraction (12 Gy clamped) were compared with even higher doses per fraction given at intervals of more than one day. Yet one important conclusion of these experiments was that the dose per fraction is crucial for the amount of repopulation f rom day to day.

The results of our multifractionated irradiation experiments to measure repopulat ion between daily equal doses in three different mouse tumours [14] allowed the following conclusions:

(1)Repopulat ion rates in different tumours were different.

(2) Average repopulation rates over 2 weeks of daily fractionated radiotherapy were faster than the clinical growth rates before t reatment but equal to potential doubling rates in two out of three tumours.

(3) Repopulat ion rates were not constant through- out the course of daily fractionated irradiation; in a sarcoma it was faster in the first week than in the second week, in both carcinomas it was faster in the second week than in the first week.

Clinical data on larynx cancer [17] also suggested that in some human carcinomas repopulation rates are similar to the potential doubling rate: The dose to control 50% of T3/4 larynx cancer increased by about 0.5 Gy per day which is consistent with a repopulation doubling time of 4 days which, more- over, is the potential doubling time of the untreated tumours calculated from the average labelling index of 15% observed in squamous cell carcinomas of the head and neck (Table I).

Da ta of Friedman et al. [9] on local control of Hodgkin ' s disease show a significant increase of 50% control dose (TCDso) for overall treatment times of over 3 weeks and doses per fraction of less

3

aBLE I

otential doubling times of human tumours calculated from in vitro labelling of S-phase cells with tritiated thymidine, assuming 18 h S-phase duration and 2 of 0.8 (from Steel, 1977).

Tumour LI Tvot Author (%) (days)

Lymphomas Hodgkin 20 3 non-Hodgkin high grade

cutaneous 20 3 Burkitt 28 2.5 Others 30 2 non-Hodgkin low grade

cutaneous 1.5 40 Myeloma 2.8 22 Others 3 20

Carcinomas Bladder grade I 4 15

II 7 9 III 22 3

Small cell lung 16 4 Squamous cell cervix 15 4

ENT 15 4 Adenocarcinomas

colorectal 16 4 parotis 6 10 breast: nuclear atypicality

minimal 1.2 50 moderate 3.5 17 severe 8 8

all 5 12 Kidney 2 30 Prostate 1 60

Malignant melanomas lymph node metastasis 7 8 nodular melanomas 4 15 superficically spreading 3 20

Astrocytomas grade II 1 60 grade III 5.3 17 glioblastomas 10 6

Peckham and Cooper [24]

Kummermehr and Burg [13] Cooper et al. [4] Peckham and Cooper [23]

Kummermehr and Burg [13] Sasaki et al. [30] Silvestrini et al. [31]

Hainau and Dombernowski [10]

Muggia et al. [21] Wehweck* [40] Brenner* [2]

Stahl* [32] Brenner* [2]

Meyer et al. [19]

Rabes et al. [26] Helpap et al. [11]

Enthammer* [6]

Hoshino et al. [12]

* M.D. students who worked in our group using the described technique. Their data are presented in full detail in their thesis, obtainable from the authors.

t h a n 1.5 G y (Fig. 2). T h e l ikely r an g e o f doses pe r

f rac t ion in the first 3 weeks is b e t ween 4 G y a n d 1.3

G y b u t n o c h a n g e in T C D s o is obse rved d u r i n g this

per iod. Ye t wi th l onge r overa l l t r e a t m e n t t imes a n d

s u b s e q u e n t decrease in the dose per f r ac t ion to less

t h a n 1 Gy , T C D s 0 rises. Th i s is s t r o n g ev idence

aga ins t sp l i t -dose recovery a n d in f a v o u r o f r epo-

p u l a t i o n as the u n d e r l y i n g m e c h a n i s m . The s lope in

4

50~ 4 0 Icaca[ control " dose 30

(TCD-50)

2O

10-

I 03 5y/d

10 20 30 40 days 50 overcall treotmenf time

Fig. 2. The dependence of the TCDso (with 95% confidence lim- its) on the overall treatment time, calculated from data of Fried- mann et al. (1967).

Fig. 2 fitted to the longer overall treatment times defines a dose equivalent of 0.30 Gy repopulated per day. Considering the higher cellular radiosen- sitivity of Hodgkin's cells compared with squamous cell carcinoma cells, repopulation in Hodgkin's dis- ease appears comparable to squamous cell carci- nomas and consistent with the potential doubling time calculated from in vitro labelling data.

Even shorter potential doubling times have been measured in Burkitt's lymphoma where clinical da- ta [22] indeed showed a marked increase in com- plete response rate if the interval between fractions and thus the time available for repopulation was decreased. There are not many more clinical data on the effective repopulation of human tumours. Clinical experience with concentrated fractionation will certainly produce more information soon. Yet the available data suggest that the cell production rate of the tumour before treatment may be an in- dicator of the repopulation potential and might therefore serve as a guide to selecting tumours for accelerated fractionation schedules.

Cell production rates in human tumours

The cell production rate in a tumour can be esti- mated from the percentage of tumour cells which incorporate tritiated thymidine into newly synthe- tized DNA. In experimental animals, tritiated thymidine is usually injected intra-peritoneally and all S-phase cells in the body become labelled. In

man, the systemic application of tritiated thymidine has only rarely been done owing to problems of radiation protection and high cost [1]. Yet the in vitro labelling technique developed by Rajewski [27] and Steel and Bensted [34] allowed, on a large scale, the determination of average labelling indices and thus of the cell production rates in a variety of human tumours. The collected data have repeatedly been reviewed, e.g. by Malaise et al. [18] and by Sasaki et al. [30].

The commonly used in vitro labelling technique counts the labelling index in the outer cell layers of tumour fragments of about 1 mm 3 and this poses problems of how representative these tiny samples are for the whole tumour [5]~ Moreover, the relationship between tumour cell proliferation and tissue structure cannot be investigated with this method. Rabes et al. [26] therefore used extracor- poreal perfusion of the tumour to deliver the tri- tiated thymidine to the proliferating cells through the vasculature. This elegant method, however, is restricted to the few tumour types which have one afferent and one efferent vessel, e.g. tumours in the kidney, ovary or testis. In these cases, however, the method gives superb autoradiographs of the topical proliferation pattern. We modified the usual in vi- tro labelling technique in order to study the relationship between cell proliferation and tissue structure in human tumours [13]. The incorpora- tion of tritiated thymidine into the DNA of S-phase cell depends on sufficient oxygenation. In the ab- sence of functioning microvasculature, oxygen pen- etration into the tissue is limited by diffusion which can only slightly be raised by increasing the oxygen pressure in the incubation medium, the maximum range being about 200 #m [27]. With a simple cut- ting device (an array of 3 parallel razor blades) slices 370/~m thick and up to 3 cm 2 in area were cut from the fresh tumour tissue. These are thin enough to allow homogenous labelling of all S- phase cells when incubated at 2.5 atm oxygen in Eagle's medium containing tritiated thymidine. After incubation, the tumour slices were washed, fixed between two stainless steel meshes to keep them fiat and embedded in methacrylate. 5/~m sec- tions were cut and subjected to standard auto-ra-

diographic procedures. Table I lists the median la- belling indices and derived potential doubling times of different types of human tumours determined by various authors and in our laboratory.

High grade lymphomas have the fastest cell pro- duction rate of all human tumours. Cooper et al. [4] reported a median labelling index of 30% in 26 patients with Burkitt lymphoma, Peckham and Cooper found a median labelling index of 28% in 25 patients with high grade non-Hodgkin lympho- mas [23] and of 20% in 10 patients with Hodgkin's disease [24]. In high grade lymphomas of the skin, the labelling index was about 20% [13,25]. These data as well as other recent publications [16,31] sug- gest that more than 80% of high grade lymphomas, irrespective of histological type and location, have very high cell production rates with potential doubling times of 3 days or less. In low grade lym- phomas, however, the labelling index rarely exceeds 3-5% as has been shown for cutaneous lymphomas I13], for myelomas [30], and for other non-Hodgkin lymphomas [31]. In centrocytic-centroblastic non- Hodgkin lymphomas the average labelling index in- creased with increasing proportion of centroblastic cells from less than 1% to more than 10%. For low grade malignant lymphomas in general the calcu- lated potential doubling times are longer than 2 weeks.

Squamous cell carcinomas of different sites are remarkably uniform with regard to their cell pro- duction rates. Irrespective of the degree of differ- entiation we measured the same labelling index in 14 squamous cell carcinomas of the head and neck [2] and 16 squamous cell carcinomas of the cervix and vulva [40]. Moreover, the heterogeneity be- tween individual tumours with regard to cell pro- liferation rates is smaller in squamous cell carci- nomas than in most other malignant tumours (Fig. 3). Sasaki et al. [30] collected data from the litera- ture on 198 squamous cell carcinomas, 160 of which had a labelling index between 6% and 30%. It can therefore be concluded that in 50% of squamous cell carcinomas potential doubling times are 4 days or less and in 90% they are less than one week. High labelling indices of 20% and more have also been published for undifferentiated bladder cancer

9s 1 90

80 c

~- 50 4 - -

30-

20

10

o

o ~

o

o

o

o

o

I , i !

5 10 15 20 % LI

Fig. 3. The cumulative frequency of the labelling indices counted in 30 untreated squamous cell carcinomas of the head and neck (O) and the cervix and vulva (O). Data from Brenner (1984) and Wehweck (1982).

by Hainau and Dombernowsky [10]. In contrast to the homogenously high cell pro-

duction rates in squamous cell carcinomas, cell pro- duction varies considerably between different ad- enocarcinomas. In our material of 18 cases of co- lorectal carcinoma [32] as well as in 71 cases from the literature [30] and in 90 cases of Meyer and Pri- oleau [20], the average labelling index was higher than in squamous cell carcinomas. In breast cancer, the median value of the labelling index in the 131 NOS cases studied by Meyer et al. [19] was about 5%, only medullary carcinomas showing cell pro- duction rates similar to those in colorectal carci- nomas.

Even lower cell production rates have been mea- sured in 38 adenocarcinomas of the prostate by Helpap et al. [11] with an average labelling index of less than 1%. In 16 adenocarcinomas of the kidney, Rabes et al. [26] counted higher labelling indices in granular cells than in clear cells. Despite the pro- nounced heterogeneity of cell proliferation within a single tumour the average cell production rate appeared to be low with a potential doubling time of about one month. The labelling indices of ad-

enocarcinomas of the salivary glands were similar to those of breast cancer [2] while in some cases of well differentiated thyroid cancer the labelling in- dices were as low as in prostatic cancer.

In malignant melanomas as well as in gliomas cell production rates varied according to the histologi- cal type and grade. Metastatic melanomas had an average labelling index of 7%, primary nodular malignant melanomas of 4% and superficially spreading melanomas of 3% [6]. These values are surprisingly low considering the fast clinical growth of many lesions. The glioblastomas studied by Hoshino et al. [12] had an average labelling index of 10% and thus lower cell production rates than expected from clinical observations. Whereas in squamous cell carcinomas and colorectal carcino- mas no influence of differentiation and anaplasia on cell production rates could be determined, such dependence was very obvious in gliomas, malignant melanomas, bladder cancer and breast cancer. In clinical treatment of these tumours where infor- mation on the individual labelling index is absent, the degree of anaplasia should be considered as cri- terium for selecting the adequate fractionation schedule.

It is difficult to decide whether the reported vari- ation of labelling indices between individual tu- mours of the same type reflects the biological var- iability of cell production and thus potential repo-

pulation rates between tumours or is a sampling error arising from spatial heterogeneity within tu- mours. This uncertainty arises especially in those studies where few tiny samples taken randomly were counted. Even in our material where relatively large pieces of a tumour were available, consider- able variability was observed between different tu- mours. In our material of 30 squamous cell carci- nomas (Fig. 3) the fastest 20% had a median po- tential doubling time of 3 days and the slowest 20% of 9 days. If the cell production rate of the untreat- ed tumour is a useful indicator of the potential for repopulation, individual determination of the per- centage of S-phase tumour cells might be useful in identifying those individuals who might really gain from accelerated fractionation.

Repopulation rates in tumours after radiotherapy

An independent estimate of the repopulation po- tential of human tumours can be gained from the latency time to local recurrence after potentially curative radiotherapy [38]. The method is based on the assumption that post-irradiation subclinical growth is not much different from effective prolif- eration during radiotherapy and that after poten- tially curative radiation doses, recurrent growth of the uncontrolled tumours starts from very few sur-

TABLE II

Median latency time to local recurrence after curative radiotherapy and derived effective post-treatment doubling times.

Tumour Latency Effective Author (mths) doubling time

(weeks)

Bladder 9 < 2 Squamous cell cervix 12 2

ENT less than 12 mths 2

Adenocarcinomas colorectal 14 2 3 parotis 13 2-3 breast 18 3 prostate 3 5 thyroid 6 10

Maciejewski, unpublished Ries and Breitner [29] Fletcher [8]

Kutzner et al. [15] Stauch et al. [33] Biinemann [3] Rangala et al. [28] Tubiana et al. [39]

viving clonogenic cells. Table II shows the median latency times to local recurrence after curative radiotherapy and the effective post-treatment doubling times assuming exponential growth for 30 doublings. These values, however, are only the low- er limits of repopulation as the clinical growth rates of the overt recurrences are much slower and this has to be compensated for by faster initial growth rates at subclinical sizes.

The latency times of recurrent carcinomas vary according to histology and location. Maciejewski (unpublished) studied 14 local recurrences of T2/3 bladder carcinomas given definitive radiotherapy with 65-70 Gy in 30-35 fractions, and the distri- bution of latency times he obtained is plotted in Fig. 4. There was very little variation and a median latency of 9 months.

Several studies have been published on the laten- cy to local recurrence in squamous cell carcinomas. After post-operative radiotherapy for squamous cell carcinomas of oral cavity or oropharynx [7,38], half of the 38 local recurrences in 102 patients ap- peared within 6 months and three quarters within 12 months. Two-thirds of the 15 local recurrences after radiotherapy of T1/2 larynx cancer appeared within 12 months [8]. From these data, Thames et al. [38] calculated effective doubling times of about 3 weeks or less during regrowth, even taking into account the fact that geographical misses (with an unknown but large number of surviving clonogenic

cumutohve incidence of Local recurrence

100

50 k after 68 Qy / 3~ f

0" 6 12 1fl months after RI

(otency times

Fig. 4. The cumulative frequency of latency times to local re- currence of 14 local recurrences of Tz/3 bladder carcinomas given definite radiotherapy. Data from Maciejwski (unpublished).

cells) may be responsible for most of the early ap- pearing recurrences. Squamous cell carcinomas of the cervix are somewhat slower to recur. Ries and Breitner [29] reported that more than 40% of local recurrences were clinically manifest within 12 months, 90% within 3 years. Stegmann and Hanger [36] observed even 54% of local recurrences within one year. The longer latencies of central recurrences of cervix cancers compared with head and neck can- cers may be partly due to more geographic misses in head and neck lesions than in cervix cancer [7] and partly due to the smaller size at which a re- currence is clinically detectable in the neck com- pared with the pelvis. Therefore, a median latency of 12 months and a calculated effective average doubling time of 12 days appear to be a reasonable value for squamous carcinomas.

The latency times to local recurrence in adeno- carcinomas are much less well documented. After pre-operative radiotherapy of 60 patients with ad- enocarcinoma of the rectum and rectosigmoid with 34.5 Gy, Kutzner et al. [15] observed eight local recurrences after a median latency of 14 months. Stauch et al. [33] found 11 local recurrences after definitive radiotherapy of 18 salivary gland carci- nomas. The latency times varied between 6 months and 4 years with a median of 13 months. Biinemann [3] studied the latency time distribution for 43 chest wall recurrences after post-mastectomy radio- therapy in T1 breast cancer and found a median value of 18 months. In 32 T2 cases the median la- tency was only 13 months. On the other hand, ad- enocarcinomas of the prostate have a much longer latency to local recurrence. The median latency in 18 local recurrences of 128 patients studied by Ran- gala et al. [28] was 3 years.

The longest latency times to local recurrence have been published for thyroid cancer. Tubiana et al. [39] saw local recurrences as late as 12 years after radiotherapy; the median latency may be around 6 years. In adenocarcinomas of the kidney local re- currences after post-operative radiotherapy are also occasionally found after very long latency periods, although we do not know of any systematic study on this problem. Thus, it may be stated that those adenocarcinomas which, autoradiographically,

3

years

median ~otency to [ocot 2 recurrence

f J

J f

cot0~ectQ~, / ~ breast

. i �9 safivary gland

~ �9 [eft

bladder

f J

f t ~

~- prostate

0 10 20 30 days Ootentia[ doubling hme

Fig. 5. The correlation of the calculated potential doubling times and the observed median latency times to local recurrences for different turnout types.

have low labelling indices tend to have very long latency times to local recurrence after definitive radiotherapy. There is a good correlation (Fig. 5) between the potential doubling times and the laten- cy times to local recurrence, which supports the clinical importance of the pre-treatment labelling index as a prognosticator of the repopulation po- tential of the individual tumour.

Conclusions

There is no doubt that repopulation is one of the crucial factors determining cure in radiotherapy. Clinical data suggest that in some tumours more than 50% of the radiation dose given on Monday is wasted in compensating the repopulation which took place in the tumour during the treatment-free week-end. We have only few data on which to base the selection of tumour types or individual patients for accelerated fractionation schedules. Yet the pre- treatment labelling index of tumours or equivalent kinetic data obtained more quickly by more so- phisticated new techniques are probably the best information we can obtain. According to the data of Table I, large squamous cell carcinomas of the head and neck and of the cervix, bladder cancer and colorectal carcinomas are most promising candi- dates for accelerated fractionation. On the other hand, in tumours with a long potential doubling

time, overall treatment time might not be critical. In these tumours hyperfractionation may be a way to increase the therapeutic ratio.

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