CCNU Treatment Research Articles

ZEB1 Is a Transcription Factor That Is Prognostic and Predictive in Diffuse Gliomas.

Objective: To address the unmet medical need to better prognosticate patients with diffuse gliomas and to predict responses to chemotherapy regimens.Methods: ZEB1 alterations were retrospectively identified from a cohort of 1,160 diffuse glioma patients. Epigenome-wide association scans (EWAS) were performed on available data. We determined the utility of ZEB1 as a prognostic indicator of patient survival in diffuse gliomas and assessed the value of ZEB1 to predict the efficacy of treating diffuse glioma patients with procarbazine, CCNU, and vincristine along with radiation at diagnosis. Decision curve analysis (DCA) was used to determine if ZEB1 added benefit to clinical decision-making over and above conventional methods.Results: Fifteen percent of diffuse glioma patients had a ZEB1 deletion. ZEB1 deletion was associated with poor overall survival (OS) with and without adjustment for age and tumor grade (adjusted HR: 4.25; 95% CI: 2.35 to 7.66; P < 0.001). Decision curve analysis confirmed that ZEB1 status with or without IDH1 was more beneficial to clinical decision making than conventional information such as age and tumor grade. We showed that ZEB1 regulates TERT expression, and patients with ZEB1 deletions likely subsume patients with mutant TERT expression in diffuse gliomas. ZEB1 influenced clinical decision making to initiate procarbazine, CCNU, and vincristine treatment.Conclusion: We demonstrate the prognostic value of ZEB1 in diffuse glioma patients. We further determine ZEB1 to be a vital and influential molecular marker in clinical decisions that exceed conventional methods regarding whether to treat or not treat patients with diffuse glioma.

Is CCNU (lomustine) valuable for treatment of cutaneous epitheliotropic lymphoma in dogs? A critically appraised topic.

BackgroundCCNU and other treatment protocols are commonly offered to owners for the treatment of dogs diagnosed with cutaneous (epitheliotropic) T-cell lymphoma (CTCL). Chemotherapy protocols provide variable benefits; they have different side-effects, and they typically require monitoring to detect drug toxicity at a non-negligible cost to the owner. At this time, even though CCNU is most often recommended to treat dogs with CTCL, there is no clear consensus on the benefit of this drug. Knowing which chemotherapy protocol yields the highest rate of complete remission and longest survival times would help veterinarians and pet owners select treatment options based on the best evidence available. Our objective was to review the literature to compare the complete remission rates and survival times of CCNU-based protocols to those of other interventions. We critically assessed the data included in articles reporting treatment outcome in at least five dogs with CTCL. Single case reports and case series with less than five patients were not reviewed to avoid anecdotal evidence of lower quality.ResultsThe search for, and review and analysis of, the best evidence available as of February 8, 2017, suggests that CCNU and pegylated liposomal doxorubicin appear to yield the highest rate of complete remission in approximately one-third of dogs with CTCL. Other treatment protocols did not report usable information on remission rates. Without any treatment, the mean/median survival time in dogs with CTCL varied between 3 and 5 months. With CCNU protocols, the median survival time was 6 months and the one with retinoids (isotretinoin and/or etretinate), PEG L-asparaginase or prednisolone monotherapy was 11, 9 and 4 months, respectively; all these durations were obtained from small numbers of dogs, however.ConclusionsCCNU leads to a complete remission of signs in approximately one-third of dogs with CTCL, but such remissions are of short duration. The median survival time after CCNU appears longer than that without treatment, but other drugs appear to provide a better long-term prognosis. Further studies are required to investigate the effect of CCNU, alone or in combination, on remission rates, survival times and impact on quality of life.

Kinetic evaluation of low-grade gliomas in adults before and after treatment with CCNU alone.

The aim of this study was to evaluate the impact of CCNU chemotherapy alone on low-grade glioma (LGG) growth dynamics. The authors measured the evolution of the mean tumor diameter (MTD) in adult patients with LGG before (n=28 patients) and after (n=38 patients) CCNU administration. Natural (spontaneous) growth of LGG in the present study was 4.3 mm/year (range 2.1-6.6 mm/year). The median MTD decrease after CCNU was 5.1 mm/year (range 1-8.9 mm/year). MTD decrease was noted in 30 patients (late decrease in 4 patients, and ongoing decrease in 24 patients with oligodendroglial tumors and 2 with astrocytic tumors). The median duration it took for the MTD to decrease after initiation of CCNU treatment was 619 days (1038 days for oligodendroglial tumors vs 377 days for astrocytic tumors; p=0.003). These results show that CCNU as a single agent has a significant impact on LGG tumor growth. The impact of CCNU seems to be comparable to the previously reported impact of temozolomide therapy and of combined procarbazine, CCNU, and vincristine chemotherapy.

GE-10 * IDENTIFICATION OF PATIENTS WITH RECURRENT GBM THAT BENEFIT FROM BEVACIZUMAB

INTRODUCTION: To assess the effects of bevacizumab (BEV) in recurrent GBMs, a three-arm randomized phase II study was initiated (the BELOB trial) comparing BEV monotherapy, CCNU monotherapy and BEV + CCNU combinationtherapy. Primary endpoint was 9 months OS and was 38% [25,51], 43% [29,57] and 59% [43,72] in the BEV, lomustine and combination arms respectively. The BELOB trial results therefore provide evidence for clinical efficacy of BEV, at least in a recurrent GBM setting and when given in combination with lomustine. However, not all patients in this trial appeared to benefit equally from the combination treatment. In this study, we therefore aimed to identify patients that benefit from BEV. METHODS: RNA expression profiling was performed using DASL (Illumina, n = 118) and RNA sequencing (n = 78) of samples. RESULTS: The DASL and RNA-seq platform both show a high concordance between technical and biological replicates and with expression profiling on snap frozen tissue samples. Sample assignment to one of seven ‘intrinsic glioma subtypes’ (IGS; molecular subtypes of glioma based on similarities in gene-expression profiles [PMID: 19920198]) was also highly similar between the different platforms. Analysis of BELOB samples shows that most are assigned to poor prognostic subtypes (IGS-18 "EGFR-amplified, classical" n = 73 and IGS-23 "mesenchymal" n = 28). Nine patients were assigned to prognostically more favourable subtypes (IGS-9 and IGS-17), but these do not explain the more favourable outcome in the BEV + CCNU arm. Patients with tumors assigned to IGS-18 show benefit from combined treatment with bevacizumab + CCNU. In contrast, tumors assigned to IGS-23 may have worse performance in this arm, though patient numbers are small. CONCLUSIONS: DASL and RNA-seq perform well on RNA isolated from FFPE tissues stored (up to 20 years) in paraffin. Specific molecular subtypes of recurrent GBM show benefit from BEV + CCNU treatment.

IDENTIFICATION OF PATIENTS WITH RECURRENT GBM THAT BENEFIT FROM BEVACIZUMAB

INTRODUCTION: To assess the effects of bevacizumab (BEV) in recurrent GBMs, a three-arm randomized phase II study was initiated (the BELOB trial) comparing BEV monotherapy, CCNU monotherapy and BEV + CCNU combinationtherapy. Primary endpoint was 9 months OS and was 38% [25,51], 43% [29,57] and 59% [43,72] in the BEV, lomustine and combination arms respectively. The BELOB trial results therefore provide evidence for clinical efficacy of BEV, at least in a recurrent GBM setting and when given in combination with lomustine. However, not all patients in this trial appeared to benefit equally from the combination treatment. In this study, we therefore aimed to identify patients that benefit from BEV. METHODS: RNA expression profiling was performed using DASL (Illumina, n = 118) and RNA sequencing (n = 78) of samples. RESULTS: The DASL and RNA-seq platform both show a high concordance between technical and biological replicates and with expression profiling on snap frozen tissue samples. Sample assignment to one of seven ‘intrinsic glioma subtypes’ (IGS; molecular subtypes of glioma based on similarities in gene-expression profiles [PMID: 19920198]) was also highly similar between the different platforms. Analysis of BELOB samples shows that most are assigned to poor prognostic subtypes (IGS-18 EGFR-amplified, classical n = 73 and IGS-23 mesenchymal n = 28). Nine patients were assigned to prognostically more favourable subtypes (IGS-9 and IGS-17), but these do not explain the more favourable outcome in the BEV + CCNU arm. Patients with tumors assigned to IGS-18 show benefit from combined treatment with bevacizumab + CCNU. In contrast, tumors assigned to IGS-23 may have worse performance in this arm, though patient numbers are small. CONCLUSIONS: DASL and RNA-seq perform well on RNA isolated from FFPE tissues stored (up to 20 years) in paraffin. Specific molecular subtypes of recurrent GBM show benefit from BEV + CCNU treatment.

Abstract C201: The combination of the small molecule TGFβR1 inhibitor LY2157299 monohydrate with CCNU substantially blocks SMAD phosphorylation and significantly suppresses human glioblastoma xenograft growth.

Abstract TGF-β signals through the type I and type II TGF-β receptors (TGFβRI and TGFβRII) to phosphorylate the SMAD proteins. In normal tissues, this signaling axis provides a growth inhibitory signal. However, in some cancers, TGF-β is often secreted at high levels and has been implicated in tumor invasion, angiogenesis and decreased immune surveillance. Recent work in GBM indicates that TGFβ may promote aggressive proliferation if the tumor cells are able to express Platelet Derived Growth Factor in response to TGFβ. Indeed, in glioblastoma patient samples, high pSmad2 levels are related to increased proliferation as well as reduced progression-free and overall patient survival. These data strongly implicate aberrant TGFβ signaling in human glioblastoma (GBM) and provide strong rationale to target this pathway for therapeutic intervention. Indeed, phase 1 and 2 clinical trials in GBM patients have begun using the recently developed small molecule inhibitor of TGFβR1 (LY2157299 monohydrate) alone and in combination with lomustine (CCNU). Herein, we show, in GBM cell lines, that LY2157299 monohydrate blocks signaling through the heteromeric TGFβ receptor complex to reduce levels of active, phosphorylated SMAD. Similarly, CCNU treatment of these same GBM cell lines also blocks Smad phosphorylation. In combination, CCNU and LY2157299 monohydrate more effectively block Smad phosphorylation than either agent alone. Furthermore, in U87MG and CRL-2611 human glioblastoma xenografts, oral dosing of LY2157299 monohydrate significantly enhanced the efficacy of CCNU (lomustine). Taken together, these preclinical data support the exploration of LY2157299 monohydrate in combination with CCNU for glioblastoma therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C201.

Combination of 6-thioguanine, capecitabine, and celecoxib with temozolomide or lomustine for recurrent high-grade glioma

We evaluated the efficacy of temozolomide (TMZ) or lomustine (CCNU) in combination with 6-thioguanine, capecitabine, and celecoxib for the treatment of recurrent high-grade glioma. Forty-three patients with recurrent glioblastoma and 31 patients with recurrent anaplastic glioma (AG) were enrolled in this open-label, non-comparative study. Patients previously treated with TMZ received CCNU while all others received TMZ; all patients received 6-thioguanine, capecitabine, and celecoxib. Endpoints were 12-month progression-free survival (PFS) for patients with AG, 6-month PFS for patients with glioblastoma, duration of PFS, and MRI-based objective response rates. Results from the TMZ and CCNU treatment arms were combined in the final analysis because there was no statistically significant difference between them. Thirty-eight patients with glioblastoma were treated with the lomustine-based regimen, and five received the TMZ-based regimen. For the 43 glioblastoma patients, the objective response rate was 12 and 33% had stable disease; the 6-month PFS was 14% and median overall survival 32weeks. For the 31 AG patients, the combined objective response rate was 26 and 42% had stable disease; the 12month PFS was 44%. Treatment was reasonably well tolerated with hematological toxicity common and more frequent with CCNU than TMZ. The combination therapy with 6-thioguanine, capecitabine and celecoxib plus CCNU or TMZ does not appear to be more effective than other alkylating agent schedules for patients with recurrent glioblastoma. The combination, however, is promising for patients with recurrent high-grade AG.

Combination of 6-thioguanine, capecitabine, and celecoxib with temozolomide or lomustine for recurrent high-grade glioma.

2057 Background: There is currently no standardized therapy for high-grade gliomas after recurrence. Since chemotherapy for these tumors is still dominated by alkylating agents, we evaluated the efficacy of combination chemotherapy with temozolomide (TMZ) or lomustine (CCNU) and 6-thioguanine (6-TG), capecitabine (CAP), and celecoxib (CEL). Methods: Forty-three patients with recurrent glioblastoma (GBM) and 31 patients with recurrent anaplastic glioma (AG) were enrolled in this open-label, non-comparative, study. Patients previously treated with TMZ received CCNU while all others received TMZ; all patients received 6-TG, CAP, and CEL. Endpoints were 12-month progression-free survival (PFS) for patients with AG and 6-month PFS for patients with GBM, duration of PFS, and MRI- based objective response rates (OR). Results: Results from the TMZ and CCNU treatment arms were combined in the final analysis because there was no statistically significant difference between them. 38 patients with GBM were treated with the CCNU-based regimen, and 5 received the TMZ-based regimen. For the 43 GBM patients, the OR rate was 12% and 33% had stable disease. For GBM, the 6 month PFS was 14% and median overall survival 32 weeks. For the 31 AG patients, the combined OR rate was 26% and 42% had stable disease; the 12 month PFS was 44%. Treatment was reasonably well tolerated; hematologic toxicity was most common and more frequent with CCNU. Conclusions: The combination therapy with 6-TG, CAP, and CEL plus either CCNU or TMZ does not appear to be more effective than conventional alkylating agents for patients with recurrent GBM. The combination, however, is promising for patients with recurrent AG. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Bayer, Castle Biosciences, Genentech, Merck, Novartis, Pfizer, Schering-Plough Bayer, IMEDEX, Merck, Novartis, Schering-Plough Castle Biosciences, Celgene, Genentech, Lilly, MGI Pharma, Novartis, Pfizer, Schering-Plough Schering-Plough

Diagnosis and treatment of a feline oral mast cell tumor

A cat was diagnosed with an oral mast cell tumor following incisional biopsy. The location of the tumor, possible metastasis, financial restraint and patient disposition severely limited therapeutic options. The patient was treated with six doses of 1-(2-chloroethyl)3-cyclohexyl-1-nitrosurea (CCNU) and methylprednisolone acetate. Complete remission was obtained after the third dosing regimen. This is the first documented case of feline oral mast cell tumor and one of a small group of cats with various cancers to be responsive to CCNU treatment.

Effects of lipid association on lomustine (CCNU) administered intracerebrally to syngeneic 36B-10 rat brain tumors

A syngeneic, intracerebral rat brain tumor model was developed and characterized and then used to evaluate the therapeutic enhancement of lipid-associated 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU). The Fisher rat glioma cell 36B-10 (100,000–500,000 cells) was implanted intracranially to Fisher F-344 rats into the caudate nucleus. Animals treated with lipid-associated CCNU showed a 2- to 10-fold decrease in tumor size compared with animals treated with free CCNU, indicating that lipid association increases the therapeutic index of intracerebral CCNU treatment. Moreover, the syngeneic rat brain tumor model may be useful for evaluation of other therapeutic modalities.

Phase I Evaluation of CCNU (Lomustine) in Tumor‐Bearing Cats

l‐(2‐Chloroethyl)3‐cyclohexyl‐l‐nitrosourea (CCNU) is an alkylating agent in the nitrosourea subclass. A prospective evaluation of CCNU was done to determine the maximally tolerated dosage of CCNU in tumor‐bearing cats. Response data were obtained when available. Twenty‐five cats were treated with CCNU at a dosage of 50–60 mg/m2 body surface area. Complete hematologic data were available for 13 cats. Neutropenia was the acute dose‐limiting toxicity. The median neutrophil count at the nadir was 1,000 cells/μL (mean, 2,433 cells/μL; range, 0–9,694 cells/μL). The time of neutrophil nadir was variable, occurring 7–28 days after treatment, and counts sometimes did not return to normal for up to 14 days after the nadir. Based on these findings, a 6‐week dosing interval and weekly hematologic monitoring after the 1st treatment with CCNU are recommended. The nadir of the platelet count may occur 14–21 days after treatment. The median platelet count at the nadir was 43,500 cells/μL. No gastrointestinal, renal, or hepatic toxicities were observed after a single CCNU treatment, and additional studies to evaluate the potential for cumulative toxicity should be performed. Five cats with lymphoma and 1 cat with mast cell tumor had measurable responses to CCNU. Phase II studies to evaluate antitumor activity should be completed with a dosing regimen of 50–60 mg/m2 every 6 weeks.

Lomustine (CCNU) for the Treatment of Resistant Lymphoma in Dogs

Forty‐three dogs with lymphoma that had relapsed or had failed to achieve complete remission to previous chemotherapy were treated with lomustine (1‐(2‐chloroethyl)‐3‐cyclohexyl‐l‐nitrosourea [CCNU]) at a dosage of 90–100 mg/m2 body surface area PO every 3 weeks. Durable complete or partial responses occurred in 11 dogs for a median of 86 days. The acutely dose‐limiting toxicosis was neutropenia 7 days after administration, resulting in a recommended dosage of 90 mg/m2. Cumulative thrombocy‐topenia occurred in dogs receiving continued CCNU treatment, and a dose interval of 3 weeks may be too short for continued administration of this drug. Toxicoses evident as fever or central nervous system signs or renal damage were uncommon or rare. CCNU is effective in the treatment of relapsed lymphoma.

Reirradiation and lomustine in patients with relapsed high-grade gliomas

Purpose: The aim of this study was to evaluate the toxicity, response, and survival of patients with relapsed high-grade gliomas after radiation therapy (RT) combined with lomustine (CCNU).Methods and Materials: Thirty-one patients with relapsed gliomas at least 6 months after completion of RT were reirradiated. Twenty-four patients had a pathological diagnosis of high-grade gliomas, whereas 7 had a radiological diagnosis of relapsed malignant gliomas. The study focused on patients with high-grade relapsed gliomas. A total dose of 34.5 Gy was delivered in 23 fractions over 4.5 weeks. Oral administration of CCNU (130 mg/m2) was begun at the same time as RT, and was repeated every 6 weeks until disease progression, or up to 12 courses.Results: Twelve of 24 patients had surgery before RT plus CCNU treatment. Median interval between RT courses was 14 months (range 6–73). All patients received a complete course of RT, and 22 of 24 patients received at least one course of CCNU. Objective responses were seen in 14 evaluable patients: 3 with partial response, 5 with stable disease, and 6 with progressive disease. Duration of partial response was 20, 9, and 8 months. Median time to progression and overall survival from the onset of retreatment were 8.4 months (range 1–22) and 13.7 months (range 1–63+), respectively. One case of G4 thrombocytopenia was observed. Five patients had G1 or G2 leucopenia and 3 patients had G3 leucopenia. Moderate nausea and vomiting were reported in 4 patients. One patient, after one course of CCNU, refused further chemotherapy. No significant difference in survival from relapse was found between patients who underwent surgery before RT plus CCNU and those who received only RT plus CCNU (p = 0.74).Conclusion: Overall, the acute toxicity was moderate, and patient compliance was good. Reirradiation of high-grade glioma was associated with modest subjective and objective response rates. It is remarkable that median overall survival from relapse was 13.7 months.

Lomustine (CCNU) for the treatment of resistant lymphoma in dogs.

Forty-three dogs with lymphoma that had relapsed or had failed to achieve complete remission to previous chemotherapy were treated with lomustine (1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea [CCNU]) at a dosage of 90-100 mg/m2 body surface area p.o. every 3 weeks. Durable complete or partial responses occurred in 11 dogs for a median of 86 days. The acutely dose-limiting toxicosis was neutropenia 7 days after administration, resulting in a recommended dosage of 90 mg/m2. Cumulative thrombocytopenia occurred in dogs receiving continued CCNU treatment, and a dose interval of 3 weeks may be too short for continued administration of this drug. Toxicoses evident as fever or central nervous system signs or renal damage were uncommon or rare. CCNU is effective in the treatment of relapsed lymphoma.

The effect of topical CCNU(1-(2-Chloroethyl)-3 cyclohexyl 1 nitrosurea) treatment on lipid peroxidation of glial tumours transplanted on rat brain

1. 1. The effect of topical CCNU treatment on lipid peroxidation of glial tumors transplanted on rat brain has been investigated in 22 rats. 2. 2. Four groups have been selected as normal brain tissue, normal brain tissue + CCNU, tumour tissue, tumour tissue + CCNU. In these groups malondialdehyde (MDA) levels have been measured as a marker of lipid peroxidation. 3. 3. Lipid peroxidation was high in tumour tissue in respect to controls ( P < 0.05). In normal tissues topical CCNU treatment caused increase in lipid peroxidation ( P < 0.05). In tumor tissues, topical CCNU decreased lipid peroxidation ( P < 0.05). It is concluded that further studies have to be performed in order to determine whether increases in lipid peroxidation on CCNU treated normal brain tissues is a result of the inhibitory effect on cellular defence systems or promotion of free radical production.

Chemosensitization of CCNU in KHT murine tumor cells in vivo and in vitro by the agent RB 6145 and its isomer PD 144872

The cytotoxicity and chemosensitizing potential of the nitrohetercyclic agent RB 6145 and its R enantiomer PD 144872 were determined in rodent tumor cells grown in tissue culture or as solid tumors. Using a clonogenic cell survival assay the degree of selective cytotoxicity of these bioreductive drugs was first determined in KHT/iv cells. Cells treated under hypoxic conditions were observed to be ~50–80-fold more susceptible to the action of RB 6145 or PD 144872 than were cells exposed under air. To assess the in vitro chemosensitizing potential of RB 6145 and PD 144872, doses of these agents which led to survival values between 0.5 and 1.0 under hypoxic conditions were administered, and were then combined concomitantly with variable doses of the nitrosourea CCNU. Exposure to the nitrosourea was for 1 h. The results showed that inclusion of either sensitizer enhanced the cell killing of the chemotherapeutic agent 2.4-2.6-fold. Subsequent experiments evaluated the therapeutic benefit of combining these bioreductive agents with CCNU in KHT sarcoma-bearing C3H/HeJ mice. When given at times ranging from 90 min before to 60 min after CCNU exposure, these bioreductive drugs increased the tumoricidal activity of the chemotherapeutic agent. Complete dose response curves combining RB 6145 and PD 144872 and a range of CCNU doses also were evaluated. The sensitizers (0.75 mmol/kg) were administered 30 min prior to the chemotherapeutic agent and survival of clonogenic tumor cells 22–24 h after treatment was used to assay tumor response. Normal tissue toxicity under similar treatment conditions was assessed using a bone marrow stem cell (CFU-GM) assay. When combined with CCNU, the bioreductive agents increased tumor cell kill by a factor of ~1.7. Bone marrow toxicity was not, however, enhanced by the addition of either agent to the CCNU treatment. These data suggest that further consideration should be given to the clinical application of these bioreductive agents.

Studies on high grade cerebral gliomas

A brief review of attempts in the United Kingdom to improve the results of treatment of high grade (grade 3, 4) supra-tentorial astrocytomas is presented. The radiosensitizer misonidazole failed to improve the results of postsurgical radiotherapy, however, multivariate analysis of data from these patients has provided a prognostic index of use in defining good and poor prognosis patients. An overview study of adjuvant nitrosourea therapy trials has shown a small significant advantage for the chemotherapy. A study of chemosensitization by benznidazole of CCNU treatment of patients in relapse failed to demonstrate any effect.

The chemosensitizing and cytotoxic effects of RSU 1164 and RSU 1165 in a murine tumor model

RSU 1069, the lead compound in a series of nitroimidazoles containing an alkylating aziridine function, has been shown to be a potent radiosensitizer and chemopotentiator both in vitro and in vivo. However, this agent also demonstrates significant in situ toxicity. Recently it has been shown that less toxic analogues of RSU 1069 can be produced by the introduction of alkyl substituents to moderate the reactivity of the aziridine function. The present investigations were undertaken to evaluate the in vivo cytotoxicity and chemosensitizing efficacy of two such analogues, RSU 1164 and RSU 1165. All experiments were performed with KHT sarcomas grown intra-muscularly. In the cytotoxicity studies, a range of sensitizer doses was utilized whereas in the chemopotentiation investigations a fixed sensitizer exposure was combined simultaneously with a range of doses of the nitrosourea CCNU. In both studies, tumor cell survival was determined 22–24 hr after treatment using a soft agar clonogenic assay. Normal tissue toxicity in the chemopotentiation studies was assessed by bone marrow CFU-S assay. Both analogues were found to be significantly :less cytotoxic to KHT sarcoma cells than RSU 1169 (a factor of 4–6 in dose at 50% cell survival). Combining a 1.0 to 2.0 mmol/kg dose of RSU 1164 or RSU 1165 with a range of doses of CCNU increased tumor cell killing by a factor of 1.5–1.6 but did not enhance bone marrow stem cell toxicity. The addition of either sensitizer to CCNU treatment therefore led to a significant therapeutic benefit.

Effects of CCNU therapy on human chromosomes

Peripheral blood lymphocytes of 9 patients under CCNU therapy were examined for frequency of sister-chromatid exchanges (SCEs) and chromosomal aberrations (CAs). 7 out of 9 patients were treated with only CCNU, whereas the remaining 2 were treated with other chemotherapeutic agents in combination with CCNU. Compared to normal individuals, a significantly increased frequency of SCE was observed in the patients before starting anticancer therapy ( P < 0.001). Increased incidences of structural changes in chromosomes were observed in cells from all the treated patients. The most frequent aberrations were of chromatid type. After administration of a single dose of CCNU, an increase in SCE frequencies was observed which remained elevated even after 6 weeks. It was concluded that increases in SCEs and CAs in lymphocytes were caused by CCNU treatment. Further studies are needed to elucidate whether any CAs observed in the present study could participate in the induction of second neoplasm.

Potentiation of CCNU activity by misonidazole in metastases.

The KHT sarcoma is a model system in which metastases can be studied in multiple organs without prior clonal selection. The present series of experiments were designed to evaluate and compare the extent of potentiation of chemotherapeutic agent activity by radiosensitizers when KHT sarcoma cells were grown in different anatomical sites. In these studies the effect of combining misonidazole (MISO) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) on KHT lung nodules and ovarian metastases was determined. Ovarian metastases were a consequence of the direct spread of tumor cells growing as lung nodules. Once established, KHT cells in the lungs and ovaries grew with a similar doubling time (1-2 days). Response of tumors at either site to chemotherapy in the presence or absence of a sensitizer was assessed using an in vivo to in vitro excision assay. MISO (1.0 mmol/kg) was administered simultaneously with a range of CCNU doses and survival of clonogenic tumor cells was measured 24 h after treatment. The results demonstrate that the addition of MISO to CCNU treatment potentiated the action of this chemotherapeutic agent at both tumor sites although greater cell kill enhancement occurred in the ovarian metastases. In the lung nodules, when combined with CCNU, a 1.0 mmol/kg dose of MISO was found to yield a dose modifying factor (DMF) of approximately 1.3. The same combination resulted in a DMF of approximately 1.8 in the ovarian metastases. This difference in DMF was not a result of an intrinsic difference in sensitivity to CCNU since cells grown at either site gave rise to the same dose response curve. Rather the difference in dose modification by MISO appears to be a consequence of the larger fraction of radiobiologically hypoxic cells in the ovarian tumors (approximately 50 per cent) than in the lung nodules (approximately 5 per cent) at the time of treatment. These findings suggest the use of drug-sensitizer combinations to treat disseminated disease and provide further evidence for the requirement of hypoxia in chemopotentiation by radiosensitizers.