Hypoxia and vascular endothelial growth factor expression in human squamous cell carcinomas using pimonidazole as a hypoxia marker.

Hypoxia in human tumors is associated with poor prognosis, but the molecular mechanisms underlying this association are poorly understood. One possibility is that hypoxia is linked to malignant progression through vascular endothelial growth factor (VEGF) induction and the associated angiogenesis and metastasis. The present clinical study measures hypoxia and VEGF expression on a cell-by-cell basis in human squamous cell carcinomas to test the hypothesis that hypoxia and VEGF protein expression are coupled in human tumors. Eighteen patients with invasive squamous cell carcinoma of the uterine cervix and head and neck have been investigated by a quantitative image analysis of immunostained sections from their tumors. The hypoxia marker pimonidazole was used to measure tumor hypoxia, and a commercially available antibody was used to measure VEGF protein expression. A quantitative immunohistochemical comparison of hypoxia and VEGF protein expression revealed no correlation between the two factors.

A clinical study of hypoxia and metallothionein protein expression in squamous cell carcinomas.

The objective was to discover whether the oxygen-regulated protein, metallothionein, is expressed in the hypoxic cells of squamous cell carcinomas. Twenty patients with squamous cell carcinoma of the uterine cervix or head and neck were infused with a solution of the hypoxia marker, pimonidazole hydrochloride, at a dose of 0.5 g/m2. The following day, biopsies were collected, formalin fixed, paraffin embedded, and sectioned at 4 microm. Sections from each biopsy were immunostained for pimonidazole binding, metallothioneins I and II, involucrin, and proliferating cell nuclear antigen. A total of 84 biopsies were analyzed. Sixty-four of 84 biopsy sections contained hypoxia. Of the hypoxia-containing sections, 43 of 64 or 67% showed no microregional overlap between hypoxia and metallothionein; 7 of 64 showed overlap; and 14 of 64 showed a combination of overlap and no overlap. On a tumor-by-tumor basis, 5 of 7 head and neck and 7 of 13 cervix tumors showed no overlap between metallothionein and hypoxia at the microregional level. Ranges for the percentage of the area of hypoxia in head and neck (<0.9 to 17%) and cervix (<0.1 to 14%) tumors were similar. In the hypoxia-containing sections, immunostaining for involucrin, a molecular marker for differentiation, overlapped with that for hypoxia in 82% of the cases. The majority of hypoxic cells in squamous cell carcinomas do not express metallothionein protein, although metallothionein is induced by hypoxia in human tumor cells in vitro. Hypoxic cells in human tumors tend to be in regions immunostaining for involucrin, and it seems possible that differentiation of hypoxic cells in squamous cell carcinomas might affect metallothionein I and II expression.

A spreadsheet program for brachytherapy planning.

A computer program (brachy-spread) which allows spreadsheet-like interactive adjustment of the loading of any brachytherapy application has been implemented. Sources are collected into objects, each assigned an activity and duration of implant. Activities and times may be adjusted by moving a cursor to the datum to be edited and entering a new value from the keyboard. Alternatively, the desired total dose to a given calculation point may be edited resulting in a recalculation of the time for all objects. For each of a set of calculation points, dose rates, total doses, and the percent contribution of each object to the point are displayed and instantly updated as the times and activities are adjusted. The program design includes rapidly updated display of isodose curves in previously selected arbitrary planes. A strategy for providing rapid dose display involving precalculation of fractional dose tables is used. The program has significantly reduced the time required to determine the appropriate loading of GYN applications and of implants which involve a combination of line sources and seeds.

Semiquantitative immunohistochemical analysis for hypoxia in human tumors.

OBJECTIVE: The goal of this study was to develop a semiquantitative scoring system for measuring hypoxia in human tumors by an immunohistochemical marker approach. METHODS AND MATERIALS: Eighteen patients diagnosed with squamous cell carcinoma of the uterine cervix or head and neck were infused intravenously with a solution of pimonidazole hydrochloride at a dose of 0.5 gm/m2. Twenty-four hours later, four biopsies on average from each tumor were fixed in formalin, processed into paraffin blocks, and sectioned. Tissue sections were immunostained for the presence of pimonidazole adducts. Microscopic images (x200) of immunostaining were captured and quantitated by standard image analysis. Images with known amounts of hypoxia spanning ranges of > 0% to 5%, > 5% to 15%, > 15% to 30%, and >30% were assigned scores of +1, +2, +3, and +4, respectively. Three observers then used this calibrated scoring system to analyze hypoxia in tumor sections in a blinded fashion. RESULTS: Excellent interobserver reproducibility was obtained with the calibrated, semiquantitative, immunohistochemical assay for hypoxia in squamous cell carcinomas. CONCLUSION: The calibrated, semiquantitative assay shows promise as an approach to simplifying the quantitation of human tumor hypoxia by immunohistochemical techniques.

Relationship of hypoxia to metallothionein expression in murine tumors.

PURPOSE: To investigate if metallothionein, an endogenous chemo- and radioprotectant, is expressed in hypoxic cells in mouse C3H mammary carcinomas and if that expression responds to acute changes in tumor hypoxia. METHODS AND MATERIALS: C3H mammary tumors were established in the hind legs of female CDF1 mice. The mice were then subjected to air breathing (chronic hypoxia), carbogen breathing (acute decrease in hypoxia), or hydralazine injection (acute increase in hypoxia). Ninety minutes after the start of the experiment, tumors were excised, fixed in formalin, and sectioned. Hypoxic cells and metallothionein-containing cells were quantitated by image analysis. Pimonidazole hydrochloride and an IgG1 mouse monoclonal antibody were used to detect hypoxia, and a mouse antimetallothionein monoclonal antibody (DAKO) was used to detect Type I and II metallothionein in sets of contiguous tissue sections. RESULTS: The distribution of immunostaining intensity for metallothionein was the same in all three groups-heavy in hypoxic cells and light in other regions of the tumors. The acute increase in hypoxia caused by hydralazine injection was accompanied by an increase in metallothionein expression (p = 0.04). Carbogen breathing largely eliminated pimonidazole binding, but metallothionein expression persisted in the tumors of carbogen-breathing mice. CONCLUSIONS: Hypoxic cells in C3H mammary carcinomas strongly express metallothionein. Metallothionein expression is responsive to acute increases in hypoxia brought about by hydralazine injection. The effectiveness of hydralazine in enhancing the activation of bioreductive cytotoxins might be offset by the increased expression of metallothionein. The persistence of metallothionein in tumors of carbogen-breathing mice might contribute to a residual radioresistance in the tumors.

Invasive carcinoma of the uterine cervix in women age 25 or less.

The incidence of cervix cancer in young women appears to be increasing. However, the influence of young age on prognosis remains unknown. There is almost no information on the prognosis of very young women, age 25 years or less, with invasive cervical carcinoma. From April 1969 to June 1987, 40/2195 (1.8%) patients, age 25 years or less, with invasive carcinoma of the uterine cervix were diagnosed, staged, and treated at our institution. Median age was 24.7 years (range 20.7 to 25.9 years). Distribution by FIGO stage was: Stage IA 7 (18%), Stage IB 23 (58%), Stage II 4 (10%), Stage III 4 (10%), and Stage IVA 2 (4%). Thirty-four (85%) patients had squamous cell carcinoma and six (15%) had adenocarcinoma. Treatment consisted of radical hysterectomy for all Stage IA patients, radical hysterectomy with or without bilateral pelvic node dissection for the 12 early Stage IB patients, and radiation with or without surgery for the remaining 11 Stage IB patients and all Stage II-IVA patients. Median follow-up was 122 months (range 13.2-190.6 months). Five-year disease-free survival rates were: Stage IA 100%; Stage IB 54.8%; and Stage II-IVA 13.7%. Five-year disease-free survival for the Stage IB patients with squamous cell carcinoma age 25 years or less was 64.7%, compared with 83% for women of all ages with Stage IB squamous histology treated at our institution. Seven of 23 Stage IB patients suffered regional recurrence only, one a local recurrence only, one a distant recurrence only, and one a combined recurrence. Seventy-five percent of these patients presented with Stage I disease; however, one-third died from their disease. The major site of failure was in the pelvis only. This, coupled with the low risk of long-term serious complications, suggests that more aggressive pelvic therapy may result in improved disease-free survival.

An integrated system for interstitial 192Ir implants.

An efficient system for preparing, afterloading, and removing interstitial 192Ir strands has been developed. Use of the system reduces the risk of personnel exposure and eliminates some patient discomfort. The system is "integrated" in that all aspects of the implantation process are considered, from source preparation to source removal. Strand preparation is facilitated by an "assembly line" process using shielded equipment. Components include a handling block for measuring and cutting active strands, a mirror, and a transport container. Afterloading and removal techniques use quick release devices and several forms of afterloading tubing and catheters, each terminated by a Luer lock adapter. Both blind-end and through-and-through implants are possible. Each 192Ir strand, threaded through an injection cap that mates with the Luer lock adapter, is quickly inserted into its tubing or catheter and locked into place. No crimping is required and no additional positioning of the sources is needed. Strand removal is easily accomplished by unlocking and removing the injection cap. The strands receive no mechanical damage and can be reused after appropriate cleaning. More than 100 cases have been performed without incident. Applications include head/neck, breast, and template and non-template vaginal wall treatments.

1000 cGy single dose palliation for advanced carcinoma of the cervix or endometrium.

Between January 1980 and the present, 42 patients with symptomatic, incurable gynecologic malignancies were treated at the University of North Carolina with 1000 cGy in a single fraction to the pelvis, repeated once or twice at monthly intervals as necessary. Of patients with adequate follow-up, total cessation of bleeding was seen in 18 of 30 (60%), complete pain relief in 2/9 (22%), and complete tumor eradication in 7/28 (25%). These palliative benefits were permanent in approximately half of the patients. Five serious treatment complications have been documented, four occurred more than 10 months after treatment. We conclude that 1000 cGy single-fraction whole pelvis treatment can be an effective means of palliating advanced gynecologic cancer provided the patient has a life expectancy of less than 1 year. Patients with a longer life expectancy are at risk for both recurrence of symptoms and for treatment related complications.

Carcinoma of the cervix stage IB: results of treatment with radiation therapy.

An analysis has been made of 101 patients treated with radiation therapy for epidermoid carcinoma of the cervix Stage IB (FIGO) from January 1970 through December 1976. The patients were treated with a combination of intracavitary therapy and external beam therapy delivering a total combined dose of 8000 rad to the paracervical areas (Points AR and AL) and 5500 rad to the pelvic lymph nodes (Points IR and IL). The cumulative, disease-free survival at 2, 3 and 5 years was 89%, 87% and 84%. Sixteen failures were recorded in this group of patients, of which 3 were a result of loco-regional disease, 5 loco-regional disease plus distant metastasis and 8 distant metastasis only. The failure rate was greater among the patients who had lesions 4 or more cm in diameter and in patients who received doses of external beam therapy to the whole pelvis of 4000 rad or more. Eighteen patients developed complications; however, one patient had a recto-vaginal and a vesico-vaginal fistula; thus 19 complications were recorded. The complications were divided according to their severity into three groups: Grade I (mild), Grade II (moderate) and Grade III (severe). There were 10 Grade I, 4 Grade II and 5 Grade III complications.

Cervical carcinoma metastatic to para-aortic nodes: extended field radiation therapy with concomitant 5-fluorouracil and cisplatin chemotherapy: a Gynecologic Oncology Group study.

PURPOSE: A multicenter trial of chemoradiation therapy to evaluate the feasibility of extended field radiation therapy (ERT) with 5-fluorouracil (5-FU) and cisplatin, and to determine the progression-free interval (PFI), overall survival (OS), and recurrence sites in patients with biopsy-confirmed para-aortic node metastases (PAN) from cervical carcinoma. METHODS AND MATERIALS: Ninety-five patients with cervical carcinoma and PAN metastases were entered and 86 were evaluable: Stage I--14, Stage II--40, Stage III--27, Stage IVA--5. Seventy-nine percent of the patients were followed for 5 or more years or died. ERT doses were 4500 cGy (PAN), 3960 cGy to the pelvis (Stages IB/IIB), and 4860 cGy to the pelvis (Stages IIIB/IVA). Point A intracavitary (IC) doses were 4000 cGy (Stages IB/IIB), and 3000 cGy (Stages IIIB/IVA). Point B doses were raised to 6000 cGy (ERT + IC) with parametrial boost. Concomitant chemotherapy consisted of 5-FU 1000 mg/m2/day for 96 hours and cisplatin 50 mg/m2 in weeks 1 and 5. RESULTS: Eighty-five of 86 patients completed radiation therapy and 90% of patients completed both courses of chemotherapy. Gynecologic Oncology Group (GOG) grade 3-4 acute toxicity were gastrointestinal (18.6%) and hematologic (15.1%). Late morbidity actuarial risk of 14% at 4 years primarily involved the rectum. Initial sites of recurrence were pelvis alone, 20.9%; distant metastases only, 31.4%; and pelvic plus distant metastases, 10.5%. The 3-year OS and PFI rate were 39% and 34%, respectively, for the entire group. OS was Stage I--50%, Stage II--39%, and Stage III/IVA--38%. CONCLUSIONS: Extended field radiation therapy with 5-FU and cisplatin chemotherapy was feasible in a multicenter clinical trial. PFI of 33% at 3 years suggests that a proportion of patients achieve control of advanced pelvic disease and that not all patients with PAN metastases have systemic disease. This points to the importance of assessment and treatment of PAN metastases.

Proliferation and hypoxia in human squamous cell carcinoma of the cervix: first report of combined immunohistochemical assays.

PURPOSE: To characterize the distribution of hypoxia and proliferation in human squamous cell carcinoma of the cervix via an immunohistochemical approach prior to initiation of therapy. METHODS AND MATERIALS: Patients with primary squamous cell carcinoma of the cervix uteri received a single infusion of the 2-nitroimidazole, pimonidazole (0.5 g/m2 i.v.), and 24 h later punch biopsies of the primary tumor were taken. Tissue was formalin fixed, paraffin embedded, and sectioned for immunohistochemistry. Hypoxia was detected by monoclonal antibody binding to adducts of reductively activated pimonidazole in malignant cells. Staining for endogenous MIB-1 and PCNA was detected in tumor cells via commercially available monoclonal antibodies. Point counting was used to quantitate the fraction of tumor cells immunostained for MIB-1, PCNA, and hypoxia marker binding. RESULTS: Immunostaining for pimonidazole binding was distant from blood vessels. There was no staining in necrotic regions, and only minimal nonspecific staining, mostly in keratin. In general, cells immunostaining for MIB-1 and PCNA did not immunostain for pimonidazole binding. Cells immunostaining for MIB-1 and PCNA showed no obvious geographic predilection such as proximity to vasculature. Quantitative comparison showed an inverse relationship between hypoxia marker binding and proliferation. CONCLUSIONS: Immunohistochemical staining for pimonidazole binding is consistent with the presence of hypoxic cells in human tumors and may be useful for estimating tumor hypoxia prior to radiation therapy. Immunostaining for pimonidazole binding is an ideal complement to immunohistochemical assays for endogenous proliferation markers allowing for comparisons of tumor hypoxia with other physiological parameters. These parameters might be used to select patients for radiation protocols specifically designed to offset the negative impact of hypoxia and/or proliferation on therapy. The inverse relationship between pimonidazole binding and proliferation markers is a preliminary result requiring verification.

A controlled study of efficacy of interstitial or external irradiation in a virus-induced brain-tumor model in rats.

In a controlled study of interstitial radiotherapy in the avian sarcoma virus (ASV)-induced glioma model in rats, prolongation of survival was demonstrated (p = 0.08 in Experiment 1 and p = 0.03 in Experiment 2) following mean dosages of 7582 to 9902 cGy 125I, when compared to nontreatment or to control studies with implantation of nonradioactive seeds. More significant (p = 0.02) prolongation of survival was demonstrated following external beam whole-head radiotherapy with nine fractions of 333 cGy, three times weekly over 3 weeks (total dose 3000 cGy). Survival was more prolonged when whole-head radiotherapy was begun 35 days following virus inoculation rather than at 71 days, probably reflecting a greater efficacy with smaller tumor targets.

Treatment of autochthonous rat brain tumors with fractionated radiotherapy. The effects of graded radiation doses and of combined therapy with BCNU or steroids.

The avian sarcoma virus-induced glioma model in rats was used to evaluate radiation dose response (survival curves), giving fractionated treatments to the whole head. Groups of 14 to 20 rats received total doses of 2300, 4600, or 5750 rads over 2, 4, or 5 weeks, respectively. Median group survival times were compared to controls and to each other. All doses of radiation significantly prolonged survival with reference to control. The 4600- and 5750-rad doses were significantly more effective than the 2300-rad dose. In another series of experiments, BCNU chemotherapy (10 mg/kg) was combined with 2300 and 4600 rads radiation therapy. Synergism of therapies was demonstrated. Methylprednisolone acetate (2 mg/kg twice weekly over 4 weeks) alone did not affect survival curves, and its combination with 4600 rads radiation therapy negated the prolongation of survival achieved with 4600 rads alone.

A role for radiation in the treatment of hairy cell leukemia complicated by massive lymphadenopathy: a case report.

A patient with leukemic reticuloendotheliosis is described who two years following splenectomy developed massive retroperitoneal, abdominal, and mediastinal lymphadenopathy. Therapeutic intervention was necessary because the bulky tumor masses produced disabling pain in the back and chest regions. Low-dose radiation to the involved fields produced prompt regression of the adenopathy along with dramatic pain relief. This case suggests that hairy cells may be quite sensitive to radiation, and this therapeutic modality should be considered, especially when local problems are present.

Evidence that involucrin, a marker for differentiation, is oxygen regulated in human squamous cell carcinomas.

The majority of hypoxic cells in squamous cell carcinomas of the head and neck and cervix express involucrin, a molecular marker for differentiation. This raises the question of whether involucrin is an oxygen-regulated protein and, if so, whether it could serve as an endogenous marker for tumour hypoxia. Consistent with oxygen regulation, involucrin protein was found to increase with increasing hypoxia in confluent cultures of moderately differentiated human SCC9 cells. Cells harvested at the point of confluence and exposed to graded concentrations of oxygen revealed a K(m) of approximately 15 mmHg for involucrin induction. This is similar to K(m)s for HIF-1alpha, CAIX and VEGF. Involucrin induction showed a steep dependence on pO(2) with a transition from minimum to maximum expression occurring over less than an order of magnitude change in pO(2). In contrast to SCC9 cells, involucrin was not induced by hypoxia in poorly differentiated SCC4 cells. It is concluded that involucrin is an oxygen-regulated protein, but that differentiation modulates its transcription status with respect to hypoxia induction.

Primary squamous cell carcinoma of the endometrium: a first report of adjuvant chemoradiation.

Primary squamous cell carcinoma of the endometrium is very rare, with only 49 cases reported in the literature. In 1928 Fluhmann proposed three criteria to establish the diagnosis: (1) no coexisting endometrial adenocarcinoma, (2) no connection between the endometrial tumor and the squamous epithelium of the cervix, and (3) no squamous cell carcinoma of the cervix present. The median age at presentation is similar to adenocarcinoma of the endometrium. Although most patients have presented with FIGO stage I disease, long-term survival has been dismal despite surgery and radiation therapy. We report a new case treated with postoperative radiation and cisplatin and review the literature.

Analysis of results of radiation therapy for stage IB carcinoma of the cervix.

From April of 1969 through December of 1980, 197 patients with Stage IB, invasive, epidermoid carcinoma of the cervix received radical radiation therapy. The treatment consisted of external beam and intracavitary therapy designed to deliver 7000 to 8000 rad to Point A and 5000 to 5500 rad to the pelvic lymph nodes. The 2-, 5-, and 10-year, disease-free survival rates were 87%, 83%, and 81%, respectively. Thirty-six patients developed recurrent and/or metastatic disease. The sites that failed to remain disease-free were: locoregional in five patients (3%), locoregional with distant metastases in 15 patients (8%), and distant metastases only in 14 patients (7%). In addition, there were two patients (1%) who are considered to have died of disease but the site or sites of recurrence could not be determined. Thirty-nine patients developed complications. The complications were mild and self-limiting--Grade I--in 24 patients (12%) and of moderate severity--Grade II--in eight patients (4%). Seven patients (4%) developed severe--Grade III--complications. A correlation was found between the dose to Point A, the bladder, and the rectum and the complications. The mean dose to Point A for patients without and with complications were 7453 rad (SE +/- 91) and 7737 (SE +/- 124), respectively, with a P value of .05. The mean dose to the bladder for patients without and with urinary complications was 5590 rad (SE +/- 103) and 6335 rad (SE +/- 411), respectively, with a P value of 0.14. The mean dose to the rectum for patients without and with intestinal complications was 5837 rad (SE +/- 103) and 6810 rad (SE +/- 236), respectively, with a P value of 0.001. No correlation was found between the dose to Point A and locoregional recurrences.

Analysis of results of radiation therapy for Stage II carcinoma of the cervix.

From April 1969 through December 1980, 251 patients with invasive, epidermoid carcinoma of the cervix received radical radiation therapy consisting of a combination of external beam and intracavitary therapy designed to deliver 7000 to 8000 rad to Point A and 6000 to 6500 rad to the pelvic lymph nodes. The disease-free survival at 2, 5, and 10 years for patients with Stage IIA disease was 90%, 76%, and 76%, respectively, whereas for patients with Stage IIB disease it was 77%, 62%, and 59%, respectively. The survival for the entire group at 2, 5, and 10 years was 80%, 65%, and 62%, respectively. Sixty-eight patients had a recurrence within the irradiated volume, for a locoregional recurrence rate of 27% (68/251). In 49 patients complications developed for an overall complication rate of 19.5% (49/251). An analysis of the complications and their degree of severity revealed a correlation with the dose of intracavitary plus external beam therapy given to Point A and to the rectum. The mean dose to Point A for patients with and without complications were 7877 rad (standard error [SE] +/- 95) and 7593 rad (SE +/- 67), respectively. The mean rectal dose for patients with and without intestinal complications were 6767 rad (SE +/- 157) and 6426 rad (SE +/- 78), respectively. The dose difference between patients with and without complications was statistically significant for Point A (P = to 0.0163) but not for the rectal dose (P = to 0.0887). There was no correlation between the bladder dose and urinary complications. Other treatment methods as well as patient and tumor parameters, are being currently analyzed to identify which factors, singly or in combination, may contribute to the development of treatment failures or complications.

Carcinoma of the cervix, stage III. Results of radiation therapy.

From April 1969 through December 1980, 203 patients with Stage III epidermoid carcinoma of the cervix were treated with radiation therapy with curative intent. The disease-free survival at 2, 5, and 10 years was 50%, 33%, and 27%, respectively. The survival was better for patients with Stage IIIB disease than for those with Stage IIIA disease. Eighty-eight patients were treated with external beam therapy only, and 115 received external beam and brachytherapy. The disease-free survival was better for the combination therapy group initially, but this difference was not sustained beyond 5 years. One hundred eight patients experienced recurrence within the irradiated field, for a locoregional recurrence rate of 53%. Twenty-seven patients had complications (13%). The complications were mild in 13 patients, moderate in 4 patients, and severe in 10 patients. A study was made of the relationship of the dose to Point A and the occurrence of complications. Similar analyses were made of the bladder and rectal doses and the subsequent occurrence of urinary and intestinal complications. In these analyses, the mean dose to Point A and the critical organs was higher for the groups of patients with complications than for those patients without complications. This relationship was also observed when the patients were stratified for treatment with either external beam plus brachytherapy or external beam therapy alone.