The effect of transportation and lairage on faecal shedding and carcass contamination with Escherichia coli O157 and O26 in very young calves in New Zealand.

The effect of transportation and lairage on the faecal shedding and post-slaughter contamination of carcasses with Escherichia coli O157 and O26 in young calves (4-7-day-old) was assessed in a cohort study at a regional calf-processing plant in the North Island of New Zealand, following 60 calves as cohorts from six dairy farms to slaughter. Multiple samples from each animal at pre-slaughter (recto-anal mucosal swab) and carcass at post-slaughter (sponge swab) were collected and screened using real-time PCR and culture isolation methods for the presence of E. coli O157 and O26 (Shiga toxin-producing E. coli (STEC) and non-STEC). Genotype analysis of E. coli O157 and O26 isolates provided little evidence of faecal-oral transmission of infection between calves during transportation and lairage. Increased cross-contamination of hides and carcasses with E. coli O157 and O26 between co-transported calves was confirmed at pre-hide removal and post-evisceration stages but not at pre-boning (at the end of dressing prior to chilling), indicating that good hygiene practices and application of an approved intervention effectively controlled carcass contamination. This study was the first of its kind to assess the impact of transportation and lairage on the faecal carriage and post-harvest contamination of carcasses with E. coli O157 and O26 in very young calves.

Hypoxia and the metabolic phenotype of prostate cancer cells.

Many cancer cells have an unusual ability to grow in hypoxia, but the origins of this metabolic phenotype remain unclear. We compared the metabolic phenotypes of three common prostate cancer cell models (LNCaP, DU145, PC3), assessing energy metabolism, metabolic gene expression, and the response to various culture contexts (in vitro and xenografts). LNCaP cells had a more oxidative phenotype than PC3 and DU145 cells based upon respiration, lactate production, [ATP], metabolic gene expression, and sensitivity of these parameters to hypoxia. PC3 and DU145 cells possessed similar Complex II and mtDNA levels, but lower Complex III and IV activities, and were unresponsive to dinitrophenol or dichloroacetate, suggesting that their glycolytic phenotype is due to mitochondrial dysfunction rather than regulation. High passage under normoxia converted LNCaP from oxidative to glycolytic cells (based on respiration and lactate production), and altered metabolic gene expression. Though LNCaP-derived cells differed from the parental line in mitochondrial enzyme activities, none differed in mitochondrial content (assessed as cardiolipin levels). When LNCaP-derived cells were grown as xenografts in immunodeficient mice, there were elements of a hypoxic response (e.g., elevated VEGF mRNA) but line-specific changes in expression of select glycolytic, mitochondrial and fatty acid metabolic genes. Low oxygen in vitro did not influence the mRNA levels of SREBP axis, nor did it significantly alter triglyceride production in any of the cell lines suggesting that the pathway of de novo fatty acid synthesis is not directly upregulated by hypoxic conditions. Collectively, these studies demonstrate important differences in the metabolism of these prostate cancer models. Such metabolic differences would have important ramifications for therapeutic strategies involving metabolic targets.

Exponential decrease during aging and random lifetime of mouse spermatogonial stem cells.

Variation in the number of spermatogonial stem cells during the lifespan of the mouse was examined by assaying the number of clonogenic cells, that is, spermatogenic stem cells, surviving known doses of radiation. The results indicated that the stem cell number decreased exponentially with age.

Modeling growth kinetics and statistical distribution of oligometastases.

The kinetics of development of micrometastases, and especially of small numbers of metastases (oligometastases), was explored by using simple assumptions to develop concepts that may be useful for framing future research. The conclusions depend on the assumptions and hence must be considered speculative. It is assumed that beyond a threshold size for initiation of metastatic spread, which varies widely from tumor to tumor, the rate at which a primary tumor sheds new metastases increases exponentially, in parallel with its exponential growth. This increasing rate of release of new metastatic clonogens from the primary tumor is accompanied by a similar exponential growth of each of the micrometastases newly established at a secondary site. This creates a log-log linear relationship between the volume distribution of metastases and number of metastases, there being one largest metastasis followed by an exponentially expanding number of logarithmically smaller micrometastases. For example, if the micrometastases and the primary tumor grew at the same rate for 6 doublings after initiation of the first metastasis, then the primary tumor would have increased its volume by a factor of 64 (2(6)) and would be shedding metastatic clonogens at 64 times the initial rate. The first metastasis would undergo 6 doublings and contain 64 cells; the succeeding 2 metastases, released as the primary doubled in volume, would undergo 5 doublings and each would contain 32 cells; and so forth down to the 64 most recently developed single-cell metastases. However, the growth rate of metastases is expected to be faster than that of the primary tumor so that the rate of increase in volume of the micrometastases would be faster than the rate of increase in their numbers (through release of new metastases from the primary). Thus, although the log-log linear relationship is maintained, the slope of the volume frequency curve is changed; if the micrometastases grew 5 times faster than the primary, the slope would change by a factor of 5. Removal of the primary tumor as a source of new metastases truncates the expansion in numbers of metastases without affecting the growth rate of existing micrometastases, with the result that the volume-frequency relationship is maintained but the whole curve is shifted to larger volumes as micrometastases grow toward clinical detectability. The development of an oligometastatic distribution requires that the exponential expansion in the number of new metastases be stopped by eliminating the primary tumor soon after the first metastasis is shed. A cell destined to become part of an oligometastatic distribution had just been newly deposited at its metastatic site at the time the primary tumor was removed and must undergo about 30 doublings to become clinically detectable as an overt metastasis (2(30) or 10(9) cells). Thus, the time interval between removal of the primary and subsequent appearance of oligometastases will be toward the upper end of a distribution of "metastasis-free" intervals for its particular class of tumor. The actual time to appearance of a solitary metastasis, or of oligometastases, in any particular patient will depend on the growth rate of the metastases in that individual but will always require about 30 volume doublings. An apparently solitary metastasis appearing synchronously with the primary tumor is unlikely to be solitary because, to do so, it would have to have undergone about 30 doublings without further release of metastatic clonogens from the primary that is, in our model, within 1 doubling in volume of the primary tumor. For the same reason, a synchronous or early appearing oligometastatic distribution is unlikely, but if it were to exist, there would be a steep gradient between the volumes of largest and smallest metastases because the growth rate of the micrometastases to produce synchronous metastases, without having further metastases shed from the primary, would have to be fast (up to 30x) relative to the growth rate of the primary. Conversely, a steep gradient in volumes of successive echelons of metastases reflects fast growth of metastases relative to the primary and favors the possibility of an oligometastatic distribution. This ratio of growth rates of metastases to primary is defined by the slope of the log-log curve for the volume-frequency distribution of metastases, which, in clinical practice, is difficult to determine over a wide range and is, by definition, essentially impossible for oligometastases. However, the volume-frequency relationship, measured over a wide range, is the same as the ratio of the volume of the largest to second-largest metastases in an oligometastatic situation. For example, if the metastasis doubled 5 times faster than the primary, the largest metastasis would be larger by 5 doublings than its closest follower(s), that is, by a factor of 2(5) or 32, equivalent to a 3.2-fold difference in diameter if the metastases were spherical. Alternatively, if an initially solitary and measurable metastasis is subsequently joined by measurable followers, the number of volume doublings separating successive echelons in the series can be determined directly, and the larger the difference (measured in doublings), the greater the probability that there will be a limited, oligometastatic condition (ie, in clinical terms, subsequent metastases will stop appearing after the large leader metastasis and a short succession of followers have been removed at 1 or more operations). In summary, the probability of there being an oligometastatic distribution is increased as the interval between removal of the primary tumor and appearance of metastases lengthens. It is also more likely the faster the metastases are growing relative to the growth rate of the primary tumor before its removal. Effective systemic cytotoxic treatment (eg, chemotherapy, hormonal manipulation, biological agents) given in the perioperative period, or concomitantly with radiation therapy for the primary tumor, would truncate the volume-frequency distribution toward an oligometastatic one by eliminating the smallest, most recently formed "tail-ender" metastases. That process, which only occurs at the threshold volume of the primary at which metastases are first initiated, would not be influenced by whether surgery or radiation therapy was chosen to treat the primary tumor, regardless of the overall duration of radiation therapy. Chemotherapy adjuvant to surgery is not usually indicated in the curative treatment of solitary or oligometastases because they represent a truncated distribution with few or no stragglers. If subclinical stragglers exist, they would usually be relatively large and, even though subclinical, too large to be cured by chemotherapy. Exceptions would be early rapidly growing oligometastases, especially from a slowly growing primary, or solitary metastases from an unknown primary where second echelon metastases, if they exist, may still be small. Otherwise chemotherapy could be postponed and used for palliative growth restraint of unusually large and/or numerous stragglers.

The biological effectiveness of antiproton irradiation.

BACKGROUND AND PURPOSE: Antiprotons travel through tissue in a manner similar to that for protons until they reach the end of their range where they annihilate and deposit additional energy. This makes them potentially interesting for radiotherapy. The aim of this study was to conduct the first ever measurements of the biological effectiveness of antiprotons. MATERIALS AND METHODS: V79 cells were suspended in a semi-solid matrix and irradiated with 46.7MeV antiprotons, 48MeV protons, or (60)Co gamma-rays. Clonogenic survival was determined as a function of depth along the particle beams. Dose and particle fluence response relationships were constructed from data in the plateau and Bragg peak regions of the beams and used to assess the biological effectiveness. RESULTS: Due to uncertainties in antiproton dosimetry we defined a new term, called the biologically effective dose ratio (BEDR), which compares the response in a minimally spread out Bragg peak (SOBP) to that in the plateau as a function of particle fluence. This value was approximately 3.75 times larger for antiprotons than for protons. This increase arises due to the increased dose deposited in the Bragg peak by annihilation and because this dose has a higher relative biological effectiveness (RBE). CONCLUSION: We have produced the first measurements of the biological consequences of antiproton irradiation. These data substantiate theoretical predictions of the biological effects of antiproton annihilation within the Bragg peak, and suggest antiprotons warrant further investigation.

Quorum sensing as an integral component of gene regulatory networks in Gram-negative bacteria.

Bacterial cell-to-cell communication (quorum sensing) relies upon the interaction of a small diffusible signal molecule with a sensor or transcriptional activator to couple gene expression with cell population density. In Gram-negative bacteria, it is now clear that N-acylhomoserine lactones bind directly to LuxR homologues and can be synthesized via one of three unrelated bacterial protein families and by transgenic plants. New chemical classes of signal molecules have been identified, some of which exhibit crosstalk with N-acylhomoserine-lactone-mediated quorum sensing. As the determinant of cell population density, quorum sensing is emerging as an integral component of bacterial global gene regulatory networks responsible for facilitating bacterial adaptation to environmental stress. N-acylhomoserine lactones are produced during experimental animal and human infections, and a function beyond quorum sensing has been suggested by their intrinsic immunomodulatory and pharmacological activities.

Isolation from a murine fibrosarcoma of cell lines with enhanced plating efficiency in vitro.

The technique for the isolation of mutants was applied to establish highly clonogenic cells from a fibrosarcoma (FSA) that had an extremely poor growth capacity in vitro (10(-6)--10(-7) of surviving fraction of cells). After consecutive clonings, the surviving fraction of cells increased to 1--5 X 10(-1), whereas that of the ordinarily maintained culture remained at a low level. Selected clones were analyzed in vitro and/or in vivo. The results indicated that the FSA was composed of heterogeneous cells or cells having a potential variability in cloning ability in vitro, metastatic ability in vivo [lung colony-forming efficiency (LCFE)], and DNA content. The relatively high DNA content of one of the clones, FSA 1233, continued after growth in vivo or in vitro, indicating its hereditary nature. FSA 1233 was also demonstrated to have a lower LCFE when the cell suspension was made from a tumor rather than from a culture in vitro. The difference of surviving fractions between the cells in the two conditions was not enough to explain the difference in LCFE's between them. These cells could grow under either in vitro or in vivo conditions and could be made readily into single-cell suspensions. The cells are, therefore, available as a system for analysis of the response of cells in vitro after in vivo treatment with various agents.

Metronidazole: effect on radiosensitivity of tumor and normal tissues in mice.

Local control of a mammary carcinoma in air-breathing C3H mice was enhanced by giving metronidazole before radiotherapy; When 0.5-1.0 mg metronidazole/g body weight was given ip 10 minutes before irradiation, the dose required for local control of 50% of the tumors (TCD50) was reduced by a factor of 1.37-1.38. The TCD50 was reduced by a factor of 1.48-1.50 when tumors were irradiated 30-60 minutes after 1.0 mg/g. Metronidazole alone, at a dose of 1.0 mg/g, only slightly inhibited tumor growth. The acute skin response of the leg, as determined by the radiation dose required to produce complete moist desquamation in 50% of the animals (DD50), was enhanced by a factor of 1.26 when mice were irradiated 10 minutes after a dose of 1.0 mg metronidazole/g. Clonogenic cells in the jejunal epithelium were not sensitized after metronidazole administration in either air-breathing mice or those irradiated in O2 30 pounds per square inch, but the drug appeared to exert a slight protective effect at higher doses of radiation.

Nonspecific Immunotherapy of Murine Solid Tumors With Corynebacterium granulosum.

A single intraperitoneal (ip) or intravenous (iv) injection of Corynebacterium granulosum into C3Hf/Bu mice shortly after subcutaneous (sc) injection of cells from a strongly antigenic syngeneic fibrosarcoma induced by 3-methylcholanthrene caused complete and lasting regressions of 100 and 70% of resulting tumors, respectively. Treatment with this bacterium sc only slightly inhibited the growth of some tumors. C. granulosum given iv to mice 3 days after the sc injection of fibrosarcoma cells caused complete regressions of 39 of 45 tumors; two iv injections with this immunostimulant given 1 month apart were no more effective than a single injection. Intralesional treatment of fibrosarcomas 8 mm in diameter induced complete regressions of tumors in 30% of the animals, whereas sc treatment contralateral to the growing tumor only slightly reduced tumor growth. Intraperitoneal growth of a fibrosarcoma was efficiently controlled (58-80% survival of mice) if C. granulosum was given ip, but not iv, 3 days after inoculation with tumor cells. Again, two injections of C. granulosum (given ip 4 days apart) were only as effective as a single injection. Treatment with C. granulosum iv at 3, 7, 14, or 21 days after sc inoculation of a weakly antigenic, spontaneously arising mammary carcinoma (MC-1) strongly inhibited tumor growth. Three complete but temporary tumor regressions were observed. The subcutaneous growth of another spontaneous mammary carcinoma (MC-2), which contained fairly strong tumor-specific antigen(s), was also significantly inhibited if C. granulosum was given 3,7, or 14 days after, but not 7 days before, tumor cell inoculation. However, pretreatment of mice with the immunostimulant significantly protected the mice against artifically induced pulmonary metastases of this tumor.

In vitro destruction of tumor cells by macrophages from mice treated with Corynebacterium granulosum.

Peritoneal macrophages from C3Hf/Bu mice treated with killed Corynebacterium granulosum bacteria were tested for their effect on in vitro growth of syngeneic fibrosarcoma cells, tumorigenic mouse L-P59 cells, human malignant melanoma cells, allogeneic fibroblasts, erythrocytes, and epithelial kidney cells. Only the cell cultures having neoplastic properties were destroyed by stimulated macrophages; the rate of tumor cell destruction was greater as the ratio of effector to target cells was increased. Neither irradiation nor trypsinization of macrophage monolayers altered the cytotoxicity of stimulated macrophages. The results indicated that C. granulosum activated macrophages to destroy tumor cells in an immunologically nonspecific manner but had no cytotoxic effect on normal allogeneic cells.

Heterogeneity and variability of artificial lung colony-forming ability among clones from mouse fibrosarcoma.

Lung colony-forming ability of clones isolated from a fibrosarcoma was examined by i.v. injection of the cells cultured in vitro. The clones were heterogeneous in their lung colony-forming ability, demonstrating variability of this malignant characteristic.

Effects of hyperthermia and radiation on mouse testis stem cells.

The response of mouse testis stem cells to hyperthermia and combined hyperthermia-radiation treatments was assayed by spermatogenic colony regrowth, sperm head counts, testis weight loss, and fertility. With the use of spermatogenic colony assay, thermal enhancement ratios at an isosurvival level of 0.1 were 1.27 at 41 degrees, 1.80 at 42 degrees, and 3.97 at 43 degrees for testes exposed to heat for 30 min prior to irradiation. Sperm head counts were reduced by heat alone from a surviving fraction of 0.58 at 41 degrees to 0.003 at 42.5-43.5 degrees. Curves for sperm head survival measured 56 days after the testes had been heated for 30 min prior to irradiation were biphasic and showed a progressive downward displacement to lower survival with increasing temperature. The 41, 42, and 43 degrees curves were displaced downward by factors of 2, 58, and 175, respectively. The proportion of animals remaining sterile after 30 min of heat (41-43 degrees) and the median sterility period in days increased with increasing temperature. The minimum sperm count necessary to regain fertility was 13% of the normal mouse level.

Effect of interleukin 1, inflammation, and surgery on the incidence of adhesion formation and death after abdominal irradiation in mice.

There is clinical evidence that prior surgery and inflammation can increase the risk of the chronic complications of radiotherapy delivered to the pelvic/abdominal region. We have established a murine model to study this interaction using as end points mortality and late gut-associated peritoneal adhesion formation. A single dose of 16 Gy of total abdominal irradiation (TAI) was used. This gave no early deaths (less than 1 mo) and a relatively low mortality over the period 1 to 6 mo after TAI. The incidence of adhesions, which is the most serious complication 2 to 6 mo after TAI, was also low. Injection of lipopolysaccharide (50 micrograms, i.p.) or human recombinant interleukin 1 (IL-1) in doses as low as 100 units prior to TAI greatly enhanced both radiation-induced adhesion formation and death. Prior surgery also increased radiation-induced mortality, so much so that adhesions could not be accurately quantified. The timing of administration of lipopolysaccharide and IL-1 and of surgery relative to TAI was important in determining the outcome. For example, IL-1 enhanced adhesion formation and death if given from 3 days before to 1 day after, but not 4 days or 4 wk after, TAI. If given 20 h or less before TAI, there was a dramatic increase in early mortality 1 to 3 wk later, which was not seen if IL-1 was given at other times. These early deaths were not caused by bone marrow or gut stem cell depletion and may be a result of fluid leakage. We propose that surgery, bacterial invasion, or other inflammatory signals might act through a common mechanism of stimulating IL-1 production to enhance radiation-induced adhesion formation and the early and late morbidity and mortality associated with abdominal irradiation. If this is the case, blocking IL-1 production might inhibit the development of these late complications.

Effects of Corynebacterium granulosum on weight and histology of lymphoid organs, response to mitogens, skin allografts, and a syngeneic fibrosarcoma in mice.

We studied the effect of single and multiple injections of Corynebacterium granulosum on weight and histology of lymph nodes and spleen, on peripheral white blood cell count, response of peripheral blood lymphocytes, lymph node, and spleen cells to phytohemagglutinin and pokeweed mitogen, survival of skin allografts, and lung metastases of a syngeneic fibrosarcoma in C3Hf/Bu mice. Corynebacterium parvum was used in some studies on antitumor activity. The weight of lymph nodes and spleen was markedly increased by single and multiple i.p. injections of C. granulosum, the peak enlargement occurring at Day 7 in lymph nodes and at Day 16 in spleen. Histologically, there was an extensive proliferation of nucleated cells in the enlarged organs. C. granulosum did not change the total white blood cell count but caused a temporary lymphopenia. In general, in vitro response to phytohemagglutinin and pokeweed mitogen of blood lymphocytes and spleen cells was decreased. Lymph node cell response to phytohemagglutinin was increased by small doses (0.025 mg) of C. granulosum, was not altered by a single large dose (0.5 mg), and was decreased by multiple doses. The response of lymph node cells to pokeweed mitogen was increased by all treatments. These changes in response to mitogens were demonstrable for about 2 months after treatment. Treatment i.v. with 0.1 or 0.25 mg of C. granulosum given before but not after grafting significantly prolonged the survival of grafted BALB/c skin. Smaller doses of this bacterium were not effective. Splenectomy of skin graft recipients did not prevent the effect of C. granulosum. Treatment i.p. or i.v. with this bacterium significantly decreased the number of lung metastases from i.v.-injected fibrosarcoma cells, even if the cells were injected 3 to 4 months later. The magnitude of this effect varied with the dose and frequency of injection of C. granulosum and C. parvum.

Cell cycle dependency of metastatic lung colony formation.

Exponentially growing FSA 1233 cells, one of the clones isolated from a mouse fibrosarcoma, were synchronized by fractionation according to cell size by centrifugal elutriation. Cells from each fraction were analyzed by flow microfluorometry to determine the stages of the cell cycle and were injected i.v. to determine lung colony-forming efficiency. In vitro plating efficiency of these cells was similar throughout the cell cycle except for a slight reduction at G2 + M. On the other hand, lung colony-forming efficiency showed marked cell cycle and cell size dependencies, being lowest at G1, highest at S, and declining slightly at G2.

Cytochrome pools in membranes of Escherichia coli grown aerobically on L-proline.

The cytochromes of membranes of the cydA mutant Escherichia coli GR19N grown on a proline-amino acid medium were examined. Reduced minus oxidized difference spectra (including fourth-order finite difference spectra) showed that cytochromes with absorption maxima at 554-555, 556-557, 560-561.5 and 563.5-564.5 nm were present. In addition, there were two components with absorption maxima at 548.5 and 551.5 nm which made a minor contribution to the alpha-band absorbance. These were not examined further. Two pools within the cytochromes were detected. One pool, which was reduced rapidly by the substrates NADH, formate and succinate, consisted of cytochromes of the cytochrome o complex. These cytochromes had absorption maxima at 555, 557 and 563.5 nm. In addition, the low-potential cytochrome associated with formate dehydrogenase was reduced rapidly by formate, and a component absorbing at 560-561.5 nm was also present in this pool. The second pool of cytochromes was reduced more slowly by substrate, although the rate was accelerated greatly in the presence of the electron mediator phenazine methosulfate. These cytochromes absorbed maximally at about 556.5 nm. A portion of the cytochrome in this pool was reoxidized by fumarate. This cytochrome may be a component of the fumarate reductase pathway, since the membranes showed high NADH-fumarate reductase activity. The respiratory chain inhibitor 2-n-heptyl-4-hydroxyquinoline N-oxide appeared to act at two sites. One site of inhibition was between the dehydrogenases and the cytochromes. A second site of inhibition was located in the cytochrome o complex between cytochrome b-564 and oxygen.

Radiation dose response for subclinical metastases.

The development of adjuvant therapies for subclinical metastases has been empiric. Decades of experience showed that 45 to 50 Gy resulted in high control rates for subclinical lymph node involvement.1 By analogy with the response of macroscopic tumors, in which doses below a threshold yield no benefit, it was commonly believed that doses lower than 40 to 50 Gy would not be useful in elective treatment of subclinical disease. Biological phenomena, such as presumed metastatic tumor cell burden and growth rate of micrometastases, which could guide the oncologist, had little or no role in the empirical development of adjuvant cytotoxic therapies. In this article, some assumptions regarding the biology of subclinical metastases are discussed and examined in the light of treatment responses reported from clinical experience. In particular, the importance of early initiation of adjuvant therapy can be appreciated as well as the significant reductions in the incidence of metastases that can be achieved even when doses less than 45 to 50 Gy have to be accepted if necessitated by normal tissue tolerance.

Predicting late normal tissue responses.

One goal of radiation research is to predict, in an individual patient, the response of the cancer and relevant normal tissues to a course of radiotherapy and to use such information in adjusting the individual's risk-benefit ratio. There are four potential types of predictors of radiation response: physical dose, biological dose, risk factors and realtime assays. Biological dose, which is derived from physical dose and tissue radiobiology, is likely to be the most useful predictor for late normal tissue injury, although risk factors are also important. For any real-time assay to be useful in predicting ultimate response to a conventional regimen, for example, of 30 fractions, it must be both accurate and precise, especially if used after only one dose fraction. No sufficiently accurate such assays exist and, in any event, they are less critical for normal tissues than for tumors, because normal tissues are less heterogeneous in their cellular composition and radiation responses than tumors. In order to apply recent concepts in biological dosimetry to late normal tissue responses, it is important to determine "tolerance" doses of human tissues more accurately; to understand and quantify the volume effect; and to determine isoeffect relationships for all tissues at doses per fraction less than 2 Gy.

Where next with preoperative radiation therapy for rectal cancer?

PURPOSE: The basic question for radiation oncologists is what we hope to achieve from treatments that are adjuvant to surgery: better local (pelvic) control and, hopefully, because of that, fewer metastases. Chemotherapy could add to the local effect of irradiation and may also decrease distant metastases directly. Selection criteria for individual treatment could enhance the therapeutic index. LOCAL CONTROL: Total mesorectal excision reduces the incidence of local recurrence, but preoperative (chemo) radiation is still indicated for more advanced tumors (T3-T4) and for lymph node involvement. Pelvic recurrences arise from tumor clonogens residual beyond the surgical margins. Thus, the practice of shrinking fields to boost the dose to the primary tumor makes no sense, except for tumors that invade residual structures, such as the sacrum. Subclinical disease beyond the future surgical margins grows more quickly than the primary tumor, and hence treatment should be as intense as tolerable. A short treatment course (e.g. 5 x 5 Gy) is desirable, but this regimen, which is currently the gold standard, should be compared (as in the recently closed randomized Polish trial) with higher-dose, longer-duration chemoradiotherapy regimens. The recently closed EORTC trial 22921 examines the benefit of pre- and postoperative chemotherapy combined with a long schedule of radiation. Likewise, continuous infusion of a cycle-active agent rather than bolus administration is a logical addition to radiation therapy in the treatment of fast-growing subclinical tumor extensions. SYSTEMIC DISEASE: The reduction in distant metastasis rates attributable to adjuvant chemotherapy varies greatly among reports. If the reduction is of the order of 10-25%, the efficacy of chemotherapy equates to as little as about 5 to 12.5 Gy and not more than 20 Gy of total body irradiation. INTERVAL BETWEEN RADIATION THERAPY AND POSTRADIATION SURGERY: Early excision after preoperative irradiation would be desirable if the primary tumor were still disseminating viable metastatic clonogens. Most tumors do not metastasize until they contain enough viable clonogens to render them clinically detectable. A dose of 10 Gy in 2 Gy fractions reduces at least 30-fold the absolute number of viable clonogens in the primary tumor, to levels that do not yield metastases from the untreated tumor. After a dose of 44-50 Gy in 2 Gy fractions, there is little chance that the surviving tumor clonogens could regrow to a metastasis-yielding volume in any reasonable radiation-surgery interval. Thus there is no tumor-related necessity for early postradiation surgery. The importance of the interval between radiation and surgery is currently being addressed in a Swedish randomized trial. PROGNOSTIC AND PREDICTIVE CHARACTERIZATION: Tumor volume should be included in the staging system. There are many tumor- and host-related characteristics that can be used to fingerprint the tumor to help select appropriate individual treatment.

Applying radiobiological principles to combined modality treatment of head and neck cancer--the time factor.

PURPOSE: Combined modality treatment is indicated for most advanced stage head and neck cancers. It is postulated that the efficacy of combined modality regimens could be enhanced by applying principles derived from radiotherapy fractionation studies to optimize the time factor in treatment scheduling. METHODS AND MATERIALS: The premise that tumor clonogens surviving a therapeutic intervention undergo accelerated repopulation in a time-dependent fashion as their numbers are depleted is used as a model to interpret the results of various chemoradiotherapy and postsurgical radiotherapy protocols and to suggest ways in which future combined modality regimens can be more rationally designed. RESULTS: Meta-analyses of chemoradiotherapy trials show the general superiority of concomitant vs. neoadjuvant sequential protocols. There is also emerging evidence that both the duration of postoperative radiotherapy and the delay in its instigation affect treatment outcome. These results are compatible with the hypothesis that the overall duration of the "package deal" of combined modality treatment is an important determinant of outcome. However, a large decrease in duration of the "package deal" does not necessarily translate into a therapeutic gain because the total dose has to be lowered to prevent intolerable acute reactions. In these circumstances tumor control will improve only if the reduced treatment time circumvents more tumor cell regeneration than the cytoreduction that could be achieved by the extra dose tolerable in a longer time period. More modest reductions in treatment time can be accomplished without dose reduction and so avoid this risk. The design of new protocols should take account of the fact that regeneration of tumor clonogens can be predicted to be nonuniform with time. Thus, the greatest therapeutic gain should be achieved by targeting periods of maximal regenerative capacity for shortening or, alternatively, for intensification of treatment. These periods are the latter part of a course of radiotherapy or chemotherapy and the early postoperative phase after surgery. CONCLUSIONS: The rational design of combined modality protocols should include principles concerning the time factor derived from radiotherapy fractionation studies. Periods of maximal tumor cell regeneration should be targeted for shortening or for treatment intensification. Any dose sacrifice necessitated by reducing treatment duration must be less than the dose equivalent of regeneration during the same time period.