Clonogenic Fraction Research Articles

The influence of autologous tumor fibroblasts on the radiosensitivity of squamous cell carcinoma megacolonies

Purpose: To study the influence of tumor fibroblasts on radiosensitivity and stem cell fraction of tumor cells in squamous cell carcinoma megacolonies by determining colony cure and clonogen survival. Methods and Materials: Murine squamous cell carcinoma cells (AT478c) grown as flat but multilayered megacolonies were co-cultured with pre-irradiated tumor fibroblasts derived from the same carcinoma, and irradiated with 1, 2, 4, or 8 fractions. Recurrent clones and their growth pattern in situ were recorded. From megacolony cure data and clonogen survival data, the clonogen number and the parameters of cellular radiosensitivity were calculated. Results: The curability of the co-cultured megacolonies, as determined by TCD50 values, was significantly increased compared to the megacolonies without fibroblasts ( p < 0.01). Both the megacolony cure and clonogen survival data suggested a decrease of the clonogen fraction in the co-cultured megacolonies. Conclusion: The presence of tumor fibroblasts increases megacolony radiosensitivity. This is due to a decrease in the fraction of clonogens in the tumor megacolony, apparently caused by a downregulation of the stem cell fraction of the tumor cells.

Acute myeloid leukemia cells with low P-glycoprotein expression and high rhodamine 123 efflux capacity display high clonogenicity.

This study was designed to correlate the clonogenic capacity of acute myeloid leukemia (AML) cells with P-glycoprotein (P-gp) expression level and P-gp-mediated efflux capacity. Fifty AML cell samples were tested for P-gp expression using MRK16 monoclonal antibody and flow cytometry. Among them, 12 samples were selected for sorting experiments according to the following two criteria: their clonogenic capacity in methylcellulose in the presence of 5637 conditioned medium, and the heterogeneity of P-gp distribution in leukemic cells. For each of these 12 samples, leukemic cells which displayed the highest P-gp expression level (P-gp++) and P-gp- leukemic cells were sorted after MRK16 staining and seeded into methylcellulose for primary clonogenic assay. In each case, the number of CFU-L in the P-gp fraction was significantly higher than that of the P-gp++ fraction (P < 0.01); the median number of CFU-L for 10(5) seeded cells being 147 (range 3-1855) and 495 (range 60-4100) for P-gp++ and P-gp- populations, respectively. Furthermore, in order to correlate clonogenic capacity and P-gp function, AML cells were stained with rhodamine 123 (Rh 123), washed and then sorted after 4 h incubation at 37 degrees C in Rh 123-free media on the basis of their residual fluorescence intensity before plating. For each of six samples, we found that the number of CFU-L in the AML cell fraction which displayed the most efficient Rh 123 efflux capacity (Rh 123dull) was significantly higher compared to that of the AML cell fraction which displayed high residual fluorescence signal (Rh 123bright) (P = 0.05); the median number of CFU-L for 10(5) seeded cells being 1025 (range 250-2240) and 296 (range 11-838) for Rh 123dull and Rh 123bright populations, respectively. Altogether this study suggests that, for an individual AML cell population, the clonogenic fraction is preferentially recruited in AML cells which display low P-gp expression and high P-gp-mediated efflux capacity.

Transfection and expression of MnSOD cDNA decreases tumor malignancy of human oral squamous carcinoma SCC-25 cells.

Overexpression of human manganese-containing superoxide dismutase (MnSOD) activity has been demonstrated to suppress malignancy in human melanoma and breast carcinoma cells in vitro and in vivo. To study its effects on human oral squamous carcinoma cells, stable transfection and expression of MnSOD in SCC-25 cells have been conducted. The MnSOD-overexpressing cell clones were shown to have approximately two- to five-fold increased MnSOD activity compared to the wild-type parental- or vector control-transfected cell clones, respectively. Plating efficiency with different concentrations of serum was decreased in the high MnSOD activity cell clones. Soft agar assays demonstrated that the clonogenic fractions of high-expressing MnSOD clones were dramatically reduced. When inoculated in nude mice, tumor growth was markedly inhibited in MnSOD overexpressing cell clones compared with the wild-type or vector control transfected cell lines. Thus, gene therapy of human oral cancer by increasing the expression of MnSOD activity in target cells might be used to prevent or reduce human oral tumor malignancy.

Reporting and analyzing dose distributions: a concept of equivalent uniform dose.

Modern treatment planning systems for three-dimensional treatment planning provide three-dimensionally accurate dose distributions for each individual patient. These data open up new possibilities for more precise reporting and analysis of doses actually delivered to irradiated organs and volumes of interest. A new method of summarizing and reporting inhomogeneous dose distributions is reported here. The concept of equivalent uniform dose (EUD) assumes that any two dose distributions are equivalent if they cause the same radiobiological effect. In this paper the EUD concept for tumors is presented, for which the probability of local control is assumed to be determined by the expected number of surviving clonogens, according to Poisson statistics. The EUD can be calculated directly from the dose calculation points or, from the corresponding dose-volume distributions (histograms). The fraction of clonogens surviving a dose of 2 Gy (SF2) is chosen to be the primary operational parameter characterizing radiosensitivity of clonogens. The application of the EUD concept is demonstrated on a clinical dataset. The causes of flattening of the observed dose-response curves become apparent since the EUD concept reveals the finer structure of the analyzed group of patients in respect to the irradiated volumes and doses actually received. Extensions of the basic EUD concept to include nonuniform density of clonogens, dose per fraction effects, repopulation of clonogens, and inhomogeneity of patient population are discussed and compared with the basic formula.

Multivariate determinants of radiocurability I: Prediction of single fraction tumor control doses

Purpose : The relationship between various laboratory determinants of radiocurability considered alone and in combination, and the observed 50% tumor control dose, has been examined in rodent and xenografted human tumors. Methods and Materials : The single fraction 50% tumor control dose (TCD 50) under normal and clamp hypoxic conditions, 50% tumor cell transplant dose (Td 50), and in vitro estimated tumor cell radiosensitivity parameters, were determined in each of six tumor types (four isografted murine and two xenografted human tumors). Subcutaneous transplant sites and identical or similar tumor generations were used for both the (Td 50), and (TCD 50) studies. Radiosensitivity parameters were obtained using the clonogenic assay, after allowing cells to enter the active growth phase to recover from trypsin induced alterations of cell radiosensitivity. Both control and irradiated cells were multiplicity corrected. Results : No single parameter (InTd 50, hypoxic fraction, or intrinsic radiosensitivity) correlated with the observed tumor control doses under aerobic or hypoxic conditions. However, when considered in combination, clonogenic fraction (estimated by (Td 50), and intrinsic radiosensitivity, predicted the rank-order of tumor control doses with a significant degree of accuracy, and tumor hypoxia influenced the value of the control dose. All parameters were demonstrated to be significant determinants of radiocurability, with substantial tumor to tumor variation in the relative importance of each. For the six tumor types, the combined laboratory determinants predicted 50% tumor control doses which differed from the observed TCD 50s by an average of approximately 9 Gy under hypoxic conditions. Conclusion : The results obtained demonstrate: (a) the necessity of simultaneously considering all determinants of radiocurability if the role of a single determinant is to be assessed; (b) laboratory determinants may accurately predict tumor radiocurability.

Interstitial hyperthermia and interstitial radiotherapy of a rat rhabdomyosarcoma; effects of sequential treatment and consequences for clonogenic repopulation.

Animal tumour experiments have been performed to elucidate the interactions between interstitial hyperthermia (IHT) and interstitial radiotherapy (IRT), and to obtain information about the most effective sequence of these treatment modalities. Experimental tumours, transplanted in the flank of Wag/Rij rats, were treated with IHT for 0.5 h at 44 degrees C, and with IRT using low dose-rate (LDR) iridium-192 sources. Both tumour cure probability and the fraction of clonogenic cells in vitro after different IHT and IRT treatments in vivo, were used as endpoints. The sequence of a short (0.5 h) IHT treatment followed by an extended LDR-IRT treatment lasting up to 10 days appeared to be very effective, and resulted in a significant thermal enhancement ratio of 1.34 at the 50% tumour cure probability level. A not significantly increased thermal enhancement of 1.06 was found when the same IHT treatment followed IRT. The level of clonogenic cell survival after IHT alone is high (0.24 +/- 0.08) compared with that after an IRT dose of 20 Gy (0.017 +/- 0.004). Clonogenic cell repopulation started 2-4 days after the in vivo treatment irrespective of the type of treatment. The in vivo combination of IHT and LDR-IRT resulted in lower surviving fractions compared with IRT alone, regardless of the time interval between the end of treatment and in vitro clonogenic assay. IHT followed by LDR-IRT appeared to be the most effective treatment in terms of tumour cure. The in vivo/in vitro studies indicated that the effect of hyperthermia is mainly attributed to radiosensitization, possibly by partial inhibition of sublethal damage repair processes during the subsequent irradiation. The hyperthermia-induced cytotoxicity was of minor importance as estimated from the surviving clonogenic fraction.

Dose, volume, and tumor-control predictions in radiotherapy

Purpose : Tumor volume has a profound influence on the dose required to control a given type of tumor. The most obvious explanation for this is related to the larger number of stem cells which must be sterilized, leading to a more stringent requirement on cell survival. There are, however, other mechanisms by which volume may influence tumor control, such as clonogenic fraction, oxygenation or inter-cellular communication. We investigate the question of whether the effect of volume on tumor control is, in general, predictable on the basis solely of the differing number of stem cells. Methods and Materials : We investigate whether the effect of volume on tumor control in four sites can be predicted, using the linear-quadratic formalism, based on the assumption that the number of cells that must be sterilized is directly proportional to the tumor volume. We require that the biological parameters in the model should have plausible values. Results : We find that the results of four clinical data sets, exhibiting a wide range of doses, volumes, and tumor control rates, are consistent with the hypothesis that the number of potential stem cells which must be sterilized is proportional to the tumor volume. Conclusions : If these considerations are correct, the potential exists that realistic radiobiologically-based dose corrections for tumor size could be routinely made. This applies both to an entire treatment, and also between fractions, as the tumor shrinks. Such an approach may contribute towards optimized radiotherapy.

Similar Heat-Induced Cell Cycle Delays in the Clonogenic and Nonclonogenic Fractions of a Thermotolerant Population

We have tested the theory that a faster rate of recovery from thermal damage in thermotolerant cells relative to nontolerant cells is the critical factor which confers heat resistance. We measured the rate of recovery from the heat-induced cell cycle delay in Chinese hamster ovary cells, since this delay is shortened in thermotolerant cells (i.e., exhibits thermotolerance) and can be measured in individual cells. Individual thermotolerant cells were followed by discontinuous microscopic observation from shortly after heating, through the first mitosis after heating, until a colony formed or failed to form by 8 days. The heat-induced delay in the clonogenic and nonclonogenic fractions was the same. This shows that the rate of recovery from the cell cycle delay was not the determining factor as to whether or not a cell survived to form a colony. Either additional factors are involved or the rate of recovery from the cell cycle delay plays no role in cell survival. These data show the importance of determining whether a faster rate of repair of thermal damage is specific for the clonogenic fraction, since most if not all types of thermal damage are likely to be repaired more quickly when thermotolerant and nontolerant cells are compared at isodose.

Simultaneous evaluation of determinants of radiocurability: Intrinsic cell radiosensitivity,, clonogenic fraction, and hypoxic fraction

Simultaneous evaluation of determinants of radiocurability: Intrinsic cell radiosensitivity,, clonogenic fraction, and hypoxic fraction

Multiple independent immunoglobulin class-switch recombinations occurring within the same clone in myeloma.

Multiple myeloma (MM) is characterized by the expansion of terminally differentiated plasma cells. It is still uncertain whether the clonogenic fraction is confined to the plasma cell or pre-plasma cell compartment. We examined the immunoglobulin (Ig) rearrangement of myeloma heavily infiltrated bone marrow cells with a probe from the heavy chain J region (JH) and the BamHI, EcoRI and HindIII restriction enzymes which are appropriate for the detection of clonal VDJ recombination. In 23/39 MMs, clonal Ig gene rearrangement was detected with BamHI, EcoRI and HindIII enzymes. Unexpectedly, in 14/39 patients both BamHI and EcoRI failed to detect Ig rearrangement, whereas HindIII consistently demonstrated VDJ recombination. The 5' sites of BamHI, EcoRI and HindIII restriction fragments are precisely defined by the VDJ rearrangement. Since the 3' ends of BamHI and EcoRI restriction fragments are downstream from the switch mu region and change in size during switch recombination, the absence of rearranged bands is determined by several autonomous recombinations affecting the switch region. By contrast, the 3' ends of HindIII restriction fragments are upstream, their size does not vary during isotype switch allowing the constant detection of clonality. Accordingly, in 35% of patients the clonogenic fraction seems to originate from a pre-switch B cell. This B cell will differentiate to a mature plasma cell developing multiple independent switch recombinations, as the variable mechanism of switch recombination suggests.

The effects of goitrogenesis, involution, and goitrogenic rechallenge on the clonogenic cell content of the rat thyroid.

A fraction of enzymatically monodispersed rat thyrocytes from untreated animals clonally proliferate into thyroid follicular units following transplantation into the subcutaneous fat pads of syngeneic recipients. During the induction of experimental goiters in rats either with 3-amino-1,2,4-triazole/iodine sufficient diet or KClO4/Remington low iodine diet, the clonogenic fractions of cells from aminotriazole goiters decreased to 1.9 x 10(-4) and KClO4 goiters to 9.8 x 10(-5) as compared to 5.8 x 10(-3) for cells from age-matched controls during the growth phase of goitrogenesis. With continued aminotriazole treatment after thyroid hyperplasia had ceased, the clonogenic fraction increased to 2.0 x 10(-3) while continued KClO4 treatment had little further effect. The changes in the clonogenic fraction induced by both regimens were reversed during involution; goitrogenic rechallenge of involuted glands led to changes in the clonogenic fraction similar to that noted during the initial challenge. The clonogenic fractions of cells from aminotriazole goiters were greater than that of cells from KClO4 goiters at all time points examined despite similar TSH levels in situ. We conclude that the rat thyroid contains a hierarchy of cells with different proliferative capacities and that the clonogenic thyrocytes possess many of the attributes of a stem-cell.

The plateau phase rat goiter contains a sub-population of TSH-responsive follicular cells capable of proliferation following transplantation

In the rat, chronic TSH stimulation leads to self-limited thyroid hyperplasia, goitrogenesis, and TSH-responsive thyroid tumors. The current studies were aimed at clarifying the mechanism by which hormone-responsive, proliferating follicular cells arise in quiescent plateau phase rat goiters. Enzymatically monodispersed rat thyrocytes from early plateau phase and involuting goiters were analyzed for the capacity to form thyroid follicular units after transplantation into syngeneic recipients. Goiters induced with two different regimens contained substantial numbers of cells capable of proliferating into functioning thyroid follicular tissue after transplantation. The clonogenic fractions of cells from goiters induced by 3-amino-1,2,4-triazole or KClO4/Remington low iodine diet were significantly lower than that of cells from control glands. Furthermore, the clonogenic fraction of cells from the KClO4 goiters was also found to be significantly less than that of cells from aminotriazole goiters despite similar TSH levels in the cell donors. The hormone responsiveness of the clonogenic goiter cells and the histological appearance and functional capacity of the thyroid tissue which arose after grafting were indistinguishable from controls. We conclude that the capacity to clonally proliferate into follicular units is a specific trait which characterizes a unique subset of follicular cells and speculate that the hormone-responsive tumors which typically appear in the chronically stimulated rat thyroid originate from cells within this subset.

Kinetics of depopulation, repopulation and host cell infiltration in the rhabdomyosarcoma R1H after 14 MeV neutron irradiation.

The kinetics of depopulation and repopulation of the solid transplantable rhabdomyosarcoma R1H in the rat was studied following irradiation with 5 Gy of 14 MeV neutrons. Several parameters were sequentially measured over a time period of 4 weeks after irradiation: the tumour volume was assessed by in situ caliper measurements; the numerical density of tumour cells was obtained by morphometry; the clonogenic fraction of tumour cells was derived from in vitro colony assay; and the numerical ratio of host to tumour cells was determined by flow cytometry. From these primary parameters the number of clonogenic tumour cells, non-clonogenic tumour cells, and nucleated host cells per tumour, as well as their variation with time, were derived. The results were compared with two sets of data obtained previously for the same tumour exposed to 15 Gy of 200 kVp X-rays. Survival of tumour cells was reduced to 5.5 +/- 0.5% by 5 Gy neutrons and to 4.5 +/- 0.5% by 15 Gy X-rays, i.e. an RBE of close to 3. There was a lag period before the onset of repopulation (4.9 +/- 0.4 days and 4.9 +/- 0.5 days, respectively), followed by a high initial rate of repopulation corresponding to a doubling time of 2.0 +/- 0.2 days for neutrons and 2.1 +/- 0.2 days for X-rays. The rate of depopulation was significantly different for the two treatment modalities; the halving time for the number of non-clonogenic tumour cells was 11 +/- 4 days for neutrons and 2.8 +/- 0.5 days for X-rays.(ABSTRACT TRUNCATED AT 250 WORDS)

Protection of cultured malignant cells from mitoxantrone cytotoxicity by low extracellular pH: A possible mechanism for chemoresistance in vivo

In malignant tumors the distribution of pH values is shifted to lower values (range, pH 5.8–7.4) as compared to normal tissues (range, pH 6.9–7.4) or peripheral blood (pH 7.35–7.45). We have investigated whether the cytotoxic effect of the anthracenedione anti-cancer drug mitoxantrone (MX) on malignant cells in culture is dependent on changes of extracellular pH. The clonogenic fraction of M1R rat mammary carcinoma cells was measured after exposure to MX at an extracellular pH (pH e) of 6.5–7.4. At pH e 6.8 (approximately the average pH measured in a number of malignant tumors in vivo) the clonogenic fraction of M1R cells exposed to MX (0.1 μ g/ml) only decreased to 1 × 10 −1 as compared to 2.5 × 10 −4 at pH e 7.4, corresponding to a 400-fold inhibition of MX cytotoxicity at reduced environmental pH. The H + ion-mediated resistance of M1R cells to MX could be partially reversed by verapamil, suggesting that a reduced microenvironmental pH possibly interferes with intracellular MX accumulation. Therefore, drugs like MX may not be effective in the elimination of cells in acidic tumor areas. Moreover, investigations on anti-cancer drug activity in vitro at what is frequently referred to as ‘physiological pH’ may be irrelevant in terms of the cytotoxic effects of the respective agents at the pH values prevailing in malignant tissues in vivo.

Cell Population Kinetics of the Rhabdomyosarcoma R1H of the Rat after Single Doses of X-rays

The kinetics of depopulation and repopulation of the solid transplantable rhabdomyosarcoma R1H of the rat following local irradiation with single subcurative X-ray doses of 7.5, 15 and 30 Gy was studied. Several parameters were sequentially measured over a time interval of 4 weeks after irradiation: the ratio of the number of tumour to host cells, and the cellular DNA content of tumour and host cells, were determined by flow cytometry; the amount of DNA per gram of tumour tissue was determined biochemically; the clonogenic fraction of tumour cells was obtained from in vitro colony assay; and the tumour volume was assessed by in situ caliper measurements. From the amount of DNA per gram and the average DNA content per cell, the total number of cells per gram of tumour tissue was obtained. From this and the other parameters measured, the number of clonogenic tumour cells, non-clonogenic tumour cells and nucleated host cells per tumour, as well as their variation with time and dose, could be derived. The results showed that there was a lag period prior to depopulation amounting to 3.8 +/- 1.4, 1.4 +/- 0.8 or 0 +/- 0.7 days for 7.5, 15 or 30 Gy, respectively. The rate of depopulation of non-clonogenic tumour cells increased with dose; the halving times of non-clonogens were 4.7 +/- 1.8, 2.6 +/- 0.7 or 2.1 +/- 0.4 days for the three doses applied. There were no indications that proliferation of doomed cells contributed significantly to tumour growth after irradiation. After lag periods that were similar in length to those prior to depopulation, a massive immigration of host cells was observed. Under certain conditions more than 97 per cent of the cells present in irradiated tumours were found to be of host origin. There was a lag period before the onset of repopulation by clonogenic tumour cells, the length of which increased from 2.7 +/- 0.7 to 5.0 +/- 0.8 or 6.3 +/- 1.0 days for 7.5, 15 or 30 Gy, respectively. The initial rate of repopulation increased with radiation dose; after the end of the lag period the doubling time of clonogenic tumour cells (in controls amounting to 3.7 +/- 0.2 days) was 3.1 +/- 0.1, 2.1 +/- 0.1 and 1.1 +/- 0.1 days for the three doses applied.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunophenotypic analysis of clonogenic cells in acute lymphoblastic leukemia using an in vitro colony assay

A culture system was used to analyze the expression of membrane differentiation antigens on the proliferative or clonogenic fraction of cells from cases of common (precursor B) acute lymphoblastic leukemia (c-ALL). Colonies of leukemic cells were obtained in 18 of 20 cases after 1 week in culture in a liquid layer containing recombinant interleukin-2 (IL-2), phytohemagglutinin (PHA), and B-cell growth factor over an agar feeder layer containing irradiated peripheral blood mononuclear cells plus fetal calf serum (FCS) and horse serum. Cultured cells expressed HLA-DR, CD-9, CD-10, CD-20, and CD-34 antigens, indicating conservation of precursor B phenotype. Differentiation antigen expression on the clonogenic subpopulation giving rise to leukemic colonies was assessed by treating cells prior to culture with selected cytotoxic monoclonal antibodies (MoAbs) and complement or by fluorescence-activated cell sorting. Lytic treatment with HLA-DR, CD- 10, CD-9, and CD-20 antibodies produced median reductions in colony formation of 93%, 81%, 73%, and 58% respectively. Combined treatment with CD-9 and CD-10 antibodies produced complete inhibition in 11 of 13 cases. Cell-sorting experiments after CD-10 staining indicated a good correlation with complement lysis studies and also demonstrated that the CD-19 antigen is expressed on a high proportion of clonogenic cells. Colony formation was also significantly reduced in 13 of 17 cases by lytic treatment of cells with the CD-33 antibody MY-9, suggesting expression of this “myeloid” lineage antigen on ALL clonogenic cells. The substantial case-to-case variation in expression of individual differentiation antigens indicates that leukemic cellular heterogeneity is a major consideration in ex vivo bone marrow purging using MoAbs and emphasizes the need for more critical evaluation of purging techniques.

Immunophenotypic Analysis of Clonogenic Cells in Acute Lymphoblastic Leukemia Using an In Vitro Colony Assay

A culture system was used to analyze the expression of membrane differentiation antigens on the proliferative or clonogenic fraction of cells from cases of common (precursor B) acute lymphoblastic leukemia (c-ALL). Colonies of leukemic cells were obtained in 18 of 20 cases after 1 week in culture in a liquid layer containing recombinant interleukin-2 (IL-2), phytohemagglutinin (PHA), and B-cell growth factor over an agar feeder layer containing irradiated peripheral blood mononuclear cells plus fetal calf serum (FCS) and horse serum. Cultured cells expressed HLA-DR, CD-9, CD-10, CD-20, and CD-34 antigens, indicating conservation of precursor B phenotype. Differentiation antigen expression on the clonogenic subpopulation giving rise to leukemic colonies was assessed by treating cells prior to culture with selected cytotoxic monoclonal antibodies (MoAbs) and complement or by fluorescence-activated cell sorting. Lytic treatment with HLA-DR, CD-10, CD-9, and CD-20 antibodies produced median reductions in colony formation of 93%, 81%, 73%, and 58% respectively. Combined treatment with CD-9 and CD-10 antibodies produced complete inhibition in 11 of 13 cases. Cell-sorting experiments after CD-10 staining indicated a good correlation with complement lysis studies and also demonstrated that the CD-19 antigen is expressed on a high proportion of clonogenic cells. Colony formation was also significantly reduced in 13 of 17 cases by lytic treatment of cells with the CD-33 antibody MY-9, suggesting expression of this "myeloid" lineage antigen on ALL clonogenic cells. The substantial case-to-case variation in expression of individual differentiation antigens indicates that leukemic cellular heterogeneity is a major consideration in ex vivo bone marrow purging using MoAbs and emphasizes the need for more critical evaluation of purging techniques.

Immunophenotype of clonogenic cells in myeloid leukaemia

Leukaemic clonogenic cells, capable of forming colonies of blast cells in an in-vitro assay, were examined for surface antigen expression using a panel of monoclonal antibodies (Mabs) to stem cell and myeloid differentiation antigens in nine cases of acute myeloid leukaemia (AML) and four cases of chronic myeloid leukaemia in myeloid blast crisis (CML-MBC). Clonogenic cells were found to be most frequently positive with anti-HLA-DR (positive in 100% cases) and RFB-1 (71%) Mabs, with significant reactivity also being seen with CD-33 (69%) and CD-13 (61%) myeloid specific antibodies. CD-11b and CD-15 antigens, expressed predominantly on mature leucocytes, were not significantly expressed on the clonogenic population. Interestingly, the CD-34 antigen, detected by MY-10 Mab on normal myeloid progenitor cells, was demonstrated on the clonogenic fraction of only one of seven cases tested. A discrepancy between antigen expression of clonogenic cells and immunophenotype of the total leukaemic population was frequently seen, with “early” markers (CD-33, HLA-DR, RFB-1) expressed on a higher proportion of the clonogenic fraction than the overall population, while the converse was the case for the “later” marker, CD-11b. Based on the known normal distribution of differentiation antigens, particularly the CD-13 antigen, cases could be ranked according to clonogenic phenotype into immature (CD-13 - HLA-DR + CD-33 + or CD-33 -; five cases), and mature (CD-13 + HLA-DR + CD-33 +; eight cases), levels. However, there was no correlation between these maturation levels and the morphology according to the FAB classification. Of note, the mature group included three CML-MBC, as well as two AML cases with a history of myelodysplasia or myeloproliferative disorder. These immunophenotypic findings indicate a heterogeneity in the level of maturation of the clonogenic population, not only in cases of de-novo AML, but also in AML thought to derive from multipotential stem cells.

Tumor radioresponsiveness versus fractionation sensitivity

Since the introduction of mammalian cell survival curves, the parameters D 0 and N have been used as quantitative measures of inherent radiation sensitivity, as was the shoulder width Dq. These parameters are more generally applicable at high doses. We propose to introduce a measure of tumor radioresponsitivity that is more applicable to the clinical treatment schedules that employ small fractional doses (1–2 Gy), the ratio α/E, derived from the linear quadratic model for cell inactivation as the intercept on the reciprocal-dose plot. For tumor-control experiments this ratio is the reciprocal of the TCD 50 when radiation is given in very small fractions or at low dose rates (assuming negligible clonogen proliferation). The rationales for this choice are: (1) α is a measure of the steepness of the initial linear segment of the dose-survival curve. Accordingly, at doses per fraction of 1–2 Gy the observed effect increases with α. (2) E is by definition a positive measure of the clonogen kill required for a specified tumor response, e.g., E = −log (surviving fraction of clonogens at the 50% control level). Therefore it is also a measure of the number of clonogens present at the time of inception of treatment, which for a given dose is a prime determinant of the probability of tumor control. This measure of radioresponsitivity is to be distinguished from the ratio α/ β, which is a measure of fractionation sensitivity. A survey of the literature indicates that these do not correlate, except in highly hypoxic tumors (e.g., clamped); such tumors are characterized by low radioresponsitivity as well as low fractionation sensitivity (high α/ β ratio). There are at present only limited data for determination of this ratio, however, since reciprocal-dose analysis requires tumor control doses for several different sizes of dose per fraction.

Dose-response curve and split-dose recovery in human skin cancer

The data of 946 skin cancer patients treated with single doses or with 4, 8, 17, 40 or 47 fractions of X-rays were analysed using Strandquist's formalism and the linear-quadratic model. The analysis of tumour control in relation to tumour size and fractionation schedule shows a strong correlation between tumour size and tumour control rate. For small tumours, single dose irradiation was as effective as fractionated treatment. For large tumours, a straight line was easily fitted to the TCD50 and TCD90 values plotted logarithmically against time or number of fractions; the exponent for overall treatment time was 0.27 and for number of fractions it was 0.31. With the linear-quadratic model, an alpha/beta ratio of 13.8 Gy was calculated. On the assumption that the number of clonogenic cells in the tumour increases in proportion to tumour volume, Do values between 0.76 and 1.33 Gy were calculated and cellular survival curves were constructed. The estimated clonogenic fraction of tumour cells is about 10(-3); no influence of hypoxic clonogenic cells on local tumour control with single doses could be demonstrated.