Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
FIELD OF THE INVENTION
The present invention is in the field of radiation treatment for cancer. More particularly, the present invention describes a novel radiation resistance assay which quickly and reliably predicts patient outcome and response to radiation treatment of tumors. The novel assay can be used in conjunction with other radio-sensitive or chemotherapeutic agents to assay synergistic effects and to determine an effective course of treatment.
Radiation therapy is commonly used to treat various forms of cancers, either alone or in combination with chemotherapeutic agents. However, the effectiveness of radiation therapy varies depending on the nature of the cancer, the individual patient and whether radiation is used in combination with other treatments. Lokeshwar et al (1995)
15(1):93-98. For example, Hennequin et al. (1996)
56(8):1842-50 have shown that chemical agents such as paclitaxel (Taxol) or docetaxel (Taxotere) can either reduce or enhance radiation sensitivity of cancer cell lines depending on the drug concentration. Edelstein et al. (1996)
23(2 Suppl. 5):41-7 report that the chemotherapeutic agent vinorelbine can be used to potentiate the antitumor effects of radiation in cycling cells. Where cancers develop resistance to chemotherapeutic agents, Siler et al. (1996)
77(9):1850-1853 report that successful clinical outcomes may be obtained by combining chemotherapy with external beam radiation. Thus, it would be useful to have a fast, reliable in vitro assay which could accurately predict an individual's response to radiation treatment and the effect of radiation in combination with other chemotherapeutic or radiation-sensitizing agents.
In cervical cancer, for instance, the majority of patients are diagnosed with early stage disease. Among 13,458 staged patients with cervical carcinoma registered by the Surveillance, Epidemiology and End Results (SEER) program between 1973 and 1987, 71% were diagnosed with the International Federation of Gynecology and Obstetrics (FIGO) stage I-IIA tumors. However, patients with more advanced lesions accounted for the majority of cervical cancer deaths during the same time period (Kosary (1994)
10:31-46). These deaths occurred despite current radiotherapy protocols, often as a direct result of clinical treatment failure. Indeed, the 1973 and 1978 Patterns of Care Studies reported the 4-year in-field treatment failure rates to range from 20% in women with stage IIB cancers to 47% in those with stage IIIB lesions. In these studies, FIGO stage and laterality of disease were the only significant pre-treatment predictors of in-field control and survival (Lanciano et al. (1991)
International J. Radiation Oncology,
20:667-76). The most current FIGO staging for cervical cancer is shown in Table 1.
Carcinoma in situ, intraepithelial carcinoma (cases of stage 0
should not be included in any therapeutic statistics for
Carcinoma strictly confined to the cervix; extension to the corpus
should be disregarded
a. Preclinical carcinomas of the cervix; that is, those diagnosed
only by microscopy
Ia1. Minimal microscopically evident stromal invasion
Ia2. Lesions detected microscopically that can be measured;
the upper limit of the measurement should not show a
depth of invasion of more than 5 mm taken from the base
of the epithelium, either surface or glandular, from which it
originates, and a second dimension, the horizontal spread
must not exceed 7 mm; larger lesions should be staged as Ib
Ib. Lesions of greater dimension than stage Ia2, whether seen
clinically or not; space involvement should not alter the
staging, but should be specifically recorded so as to
determine whether it should affect treatment decisions in
Carcinoma extending beyond the cervix, but not onto the pelvic
wall; involves the vagina, but not the lower one-third
a. No obvious parametrial involvement
b. Obvious parametrial involvement
Carcinoma extending onto the pelvic wall; (on rectal examination,
there is no cancer-free space between the tumor and the pelvic
wall; the tumor involves the lower one-third of the vagina; all
cases with a hydronephrosis or nonfunctioning kidney)
a. No extension onto the pelvic wall
b. Extension onto the pelvic wall; urinary obstruction of one or
both ureters on intravenous pyelogram (IVP) without the other
criteria for stage III disease
Carcinoma extending beyond the true pelvis or clinically
involving the mucosa of bladder or rectum (a bullous edema, as
such, does not permit a case to be allotted to stage IV)
a. Spread to adjacent organs
b. Spread to distant organs
Presumably, control of tumors in the pelvic region in patients with advanced cervical cancer depends not only on stage and tumor volume but also on intrinsic biologic radiation sensitivity. Studies assessing the intrinsic radiation sensitivity of various cancer cell lines are known in the art Ruka et al. (1996)
J. Surg. Oncol.
61(4):290-294 report that human soft tissue sarcoma cell lines do not show unusual radiation resistance when compared to human breast carcinomas or glioblastoma cell lines. Ma et al (1996)
Cell Biol. Int.
20(4):289-292 describe how human diploid skin fibroblast cells exhibit heterogeneity in their response to radiation. None of these studies, however, indicate how in vitro radiation sensitivity could be used to predict clinical outcome.
To date, several trials investigating the utility of concomitant chemotherapy to improve radiation sensitivity have been inconclusive or have indicated that in vitro sensitivity is not predictive of clinical outcome. Ramsay et al. (1994)
Int. J. Radiation Oncology,
31(2):339-344 describe how a tetrazolium-based colorimetric assay (MTT) is not a useful predictor of radiosensitivity of lymphocytes derived from breast cancer patients. Similarly, Taghian et al. (1995)
Int. J. Radiat. Oncol. Biol. Phys.
32(1):99-104, report that in vitro radiation sensitivity of human glioblastoma, squamous cell carcinoma, soft tissue sarcoma and cancer colon samples does not correlate with either in vivo radiation sensitivity or clinical outcome.
Immunohistochemical in vitro assays have also shown no correlation between the expression of genes involved in cancer and radiation response. Zaffaroni et al. (1995)
13:77-85 report that p53 expression does not correlate with in vitro response to gamma irradiation in primary cultures of human ovarian cancers and cutaneous melanomas. Bristow et al. (1996)
Int. J. Radiat. Oncol. Biol. Phys.
34(2):341-355 also found no relationship between radiation resistance and metastatic potential in cells transfected with the p53 gene.
Another assay described by Griffon et al. (1995)
European J. Cancer
31A(1):85-91, uses a multicellular tumor spheroid (MTS) three-dimensional model to determine radiosensitivity. MTS cultures exhibit characteristic phenotypes, including having a spheroid, three-dimensional shape. Griffon measured the doubling time and DNA ploidy of MTS in response to radiation treatments. Notably, this study does not indicate if radiosensitivity in vitro is predictive of clinical outcome. In addition, the assay described by Griffon is both labor and time intensive. Although established tumor cell lines often produce MTS, primary tumor specimens do so only occasionally. Furthermore, the specimens obtained from patients must be grown for 4 to 5 days to develop spheroids which can be exposed to radiation. After radiation, the MTS must be cultured for at least 7 days before radiosensitivity can be measured. Thus, even if an MTS culture can be established from a patient sample, radiation response results are not available for a minimum of 11 days.
Chromosomal painting methods have also been used to try and predict radiation sensitivity. Dunst et al. (1995)
171(10):581-586 describe how patients with abnormal and extreme radiosensitivity could possibly be identified by in-vitro testing of
Fruehauf John P.
Parker Ricardo J.
Campbell Eggerton A.
Morrison & Foerster / LLP
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