Intratumor genetic heterogeneity and head and neck cancer relapse.

BACKGROUND: Head and neck squamous cell carcinomas are treated by surgery, radiotherapy (RT), chemoradiotherapy (CRT) or combinations thereof, but locoregional recurrences (LRs) occur in 30-40% of treated patients. We have previously shown that in approximately half of the LRs after CRT, cancer driver mutations are not shared with the index tumor. AIM: To investigate two possible explanations for these genetically unrelated relapses, treatment-induced genetic changes and intratumor genetic heterogeneity. METHODS: To investigate treatment-induced clonal DNA changes, we compared copy number alterations (CNAs) and mutations between primary and recurrent xenografted tumors after treatment with (C)RT. Intratumor genetic heterogeneity was studied by multi-region sequencing on DNA from 31 biopsies of 11 surgically treated tumors. RESULTS: Induction of clonal DNA changes by (C)RT was not observed in the xenograft models. Multi-region sequencing demonstrated variations in CNA profiles between paired biopsies of individual tumors, with copy number heterogeneity scores varying from 0.027 to 0.333. In total, 32 cancer driver mutations could be identified and were shared in all biopsies of each tumor. Remarkably, multi-clonal mutations in these same cancer driver genes were observed in 6 of 11 tumors. Genetically distinct heterogeneous cell cultures could also be established from single tumors, with different biomarker profiles and drug sensitivities. CONCLUSION: Intratumor genetic heterogeneity at the level of the cancer driver mutations might explain the discordant mutational profiles in LRs after CRT, while there are no indications in xenograft models that these changes are induced by CRT.

[The formation of infliximab and anti-infliximab immune complexes as an explanation for non-responding to infliximab treatment of rheumatoid arthritis: observational study in 4 patients]. / Het ontstaan van immuuncomplexen van infliximab en anti-infliximab als een verklaring voor het falen van infliximabtherapie bij reumatoïde artritis: observationeel onderzoek bij 4 patiënten.

OBJECTIVE: To investigate the in vivo mechanism of non-responding to infliximab treatment of patients with rheumatoid arthritis (RA) and the role of anti-infliximab antibodies by using radiolabeled infliximab. DESIGN: Descriptive and comparative study. METHOD: Two responding and two non-responding RA patients were infused with radiolabeled infliximab. Subsequently imaging investigations and serum analysis were performed at set times. RESULTS: The scintigrams showed that the labelled infliximab was mainly present in the blood until 24 h after infusion. There was a trend of faster blood clearance and higher liver and spleen uptake of 99mTc-infliximab in one non-responding patient. Labelled infliximab was taken up by inflamed joints. The anti-infliximab level was high (1008 and 1641 U/ml) in the non-responders and low or not detectable in the responders. Sucrose gradients of serum revealed antibody complexes in both non-responders. Various sizes of antibody complexes, including very large ones, were observed in one non-responder who developed a serious infusion reaction. CONCLUSION: Infliximab-anti-infliximab immune complexes were found to form in RA non-responders due to the presence of significant quantities of anti-infliximab. This finding may partly explain the failure of the infliximab treatment.

Imaging and serum analysis of immune complex formation of radiolabelled infliximab and anti-infliximab in responders and non-responders to therapy for rheumatoid arthritis.

BACKGROUND: Many patients with rheumatoid arthritis are currently successfully treated with infliximab (anti-tumour necrosis factor); however, about 30% of the patients do not respond to infliximab. One of the postulated hypotheses of not responding is the fast clearance of infliximab due to the development of infliximab-anti-infliximab complexes. OBJECTIVE: To investigate the in vivo mechanism of not responding and the role of human anti-chimeric antibodies (HACAs) by using radiolabelled infliximab. METHODS: Two responding and two non-responding patients with rheumatoid arthritis, infused with radiolabelled infliximab, were investigated by both imaging and serum analysis. RESULTS: Images showed predominant presence of infliximab in blood up to 24 h, with a trend of faster blood clearance and of higher liver/spleen uptake in a non-responding patient. Clinically inflamed joints showed uptake of the drug. The HACA level in the non-responders was high (1641 and 1008 U/ml), but low or not detectable in responders. Sucrose gradients of serum showed antibody complexes in both non-responders. Various sizes of antibody complexes, including very large ones, were observed in a non-responder who developed a serious infusion reaction. CONCLUSION: Formation of infliximab-anti-infliximab complexes were found in non-responders due to the presence of large amounts of HACA. This finding, supported by both imaging and serum analysis data, may explain failure of infliximab treatment.

Preparation and evaluation of (89)Zr-Zevalin for monitoring of (90)Y-Zevalin biodistribution with positron emission tomography.

PURPOSE: To evaluate whether (89)Zr can be used as a PET surrogate label for quantification of (90)Y-ibritumomab tiuxetan ((90)Y-Zevalin) biodistribution and dosimetry before myeloablative radioimmunotherapy. METHODS: Zevalin was labelled with (89)Zr by introducing N-succinyldesferal (N-sucDf) as a second chelate. For comparison of the in vitro stability of (89)Zr-Zevalin and (88)Y-Zevalin (as a substitute for (90)Y), samples were incubated in human serum at 37 degrees C up to 6 days. Biodistribution of (89)Zr-Zevalin and (88)Y-Zevalin was assessed at 24, 48, 72 and 144 h p.i. by co-injection in nude mice bearing the non-Hodgkin's lymphoma (NHL) xenograft line Ramos. The clinical performance of (89)Zr-Zevalin-PET was evaluated via a pilot imaging study in a patient with NHL, who had undergone [(18)F]FDG-PET 2 weeks previously. RESULTS: Modification of Zevalin with N-sucDf resulted in an N-sucDf-to-antibody molar ratio of 0.83+/-0.04. After radiolabelling and purification, the radiochemical purity and immunoreactivity of (89)Zr-Zevalin always exceeded 95% and 80%, respectively. (89)Zr-Zevalin showed the same stability in serum as (88)Y-Zevalin, with a radiochemical purity >95% during a period of 6 days. The co-injected (89)Zr-Zevalin and (88)Y-Zevalin conjugates showed a very similar biodistribution, except for liver and bone accumulation at 72 and 144 h p.i., which was significantly higher for (89)Zr than for (88)Y. PET images obtained after injection of (89)Zr-Zevalin showed clear targeting of all known tumour lesions. CONCLUSION: (89)Zr-Zevalin and (88)Y-Zevalin showed a very similar biodistribution in mice, implying that (89)Zr-Zevalin-PET might be well suited for prediction of (90)Y-Zevalin biodistribution in a myeloablative setting.

Characterization of CD44v6 isoforms in head-and-neck squamous-cell carcinoma.

CD44 splice variants, especially those containing the v6 domain, are assumed to play a critical role in the malignant progression of many human tumors. This concept was based on (i) the aberrant expression of CD44v6 in malignant cells, often encoded by alternatively spliced transcripts, and (ii) the absence of CD44v6 expression in corresponding normal tissues. Remarkably, data on CD44v6 expression in squamous cells do not support this hypothesis: the v6 domain is highly expressed in normal squamous tissues and down-regulation has been described in the majority of squamous-cell carcinomas of the head and neck (HNSCC). In this study, we have compared the expression of v6 in normal oral mucosa and HNSCC in a qualitative and quantitative way. Immuno-histochemistry was performed with 3 different anti-v6 antibodies (U36, U39 and VFF18) on a large panel of HNSCC cell lines and tumors. The v6-encoding splice variants were characterized by screening a cDNA library of a human HNSCC cell line and by RT-PCR on HNSCC cell lines, microdissected normal mucosa and primary as well as metastatic HNSCC tissue. The results revealed that there is no, or only marginal, down-regulation of CD44v6 in HNSCC. About 97% of the primary HNSCC tumors exhibited a high and homogeneous staining pattern (U36, 270/277; U39, 268/277 tumors with more than 50% positive cells). Furthermore, the v6-containing CD44 splice variants present in HNSCC primary tumors and metastases were identical to those expressed in normal mucosa. Our data indicate that v6-containing CD44 splice variants do not play a role in the malignant progression of HNSCC.

Perspectives of combined radioimmunotherapy and anti-EGFR antibody therapy for the treatment of residual head and neck cancer.

Rhenium-186 based radioimmunotherapy (RIT) may have potential for the treatment of minimal residual disease in patients with squamous cell carcinoma of head and neck (HNSCC). In an effort to enhance the efficacy of RIT, we evaluated the combination of RIT and anti-epidermal growth factor receptor (EGFR) therapy in nude mice bearing established HNSCC s.c. xenografts. For this purpose we used the EGFR-blocking monoclonal antibody (MAb) 425. Treatment of HNSCC-bearing mice with the combination of a single administration of 200 microCi 186Re-labeled MAb U36 as well as 1.1 mg unlabeled MAb 425 showed an enhanced efficacy in comparison to the single treatments. When 500 microCi 186Re-labeled MAb U36 were administered, all tumors eventually regressed completely. The combination of this RIT treatment with multiple injections of MAb 425 significantly increased the rate of tumor regression. Although RIT with 186Re-labeled MAbs appears to be very efficient on HNSCC xenografts, the combination with anti-EGFR MAb 425 may enhance the efficacy.

Construction and characterization of the chimeric monoclonal antibody E48 for therapy of head and neck cancer.

Data from an ongoing clinical radioimmunoscintigraphy trial indicate that 99mTc-labeled monoclonal antibody (mAb) E48 is highly capable of selectively targeting squamous cell carcinoma of the head and neck (HNSCC). The percentage of the injected dose per gram of tumor tissue was found to be high, rendering mAb E48 a promising candidate mAb for therapeutic purposes. We now describe the construction of a chimeric (mouse/human) mAb E48 by recombinant DNA technology. The genes encoding the variable domains of the heavy and light chain were cloned and ligated into expression vectors containing the human gamma 1 heavy-chain gene and the human kappa light-chain gene respectively. Biological properties of the resulting chimeric mAb E48 were compared to the murine form in vitro and in vivo. The reactivities of chimeric (c)mAb and murine (m)mAb E48 with HNSCC, as assessed by immunohistochemical staining as well as immuno-blotting were shown to be similar. The affinity constant appeared to be 0.9 x 10(10) M-1 and 1.6 x 10(10) M-1 for the mmAb and cmAb respectively. The biodistribution of both antibodies was tested by simultaneous injection into nude mice bearing human HNSCC xenografts. cmAb E48 was found to be cleared more rapidly from the blood than mmAb E48, resulting in a 30% lower tumor uptake but similar tumor to non-tumor ratios, 3 days after injection. Moreover, it was shown that cmAb E48 is highly capable of lysing HNSCC targets in ADCC assays in vitro, whereas the mmAb appeared to be almost inactive. These data indicate that cmAb E48 has potential as a targeting agent for the eradication of HNSCC in man.

Complete ablation of small squamous cell carcinoma xenografts with 186Re-labeled monoclonal antibody E48.

In previous studies we have shown that in mice bearing head and neck squamous cell carcinoma (HNSCC) xenografts, radioimmunotherapy (RIT) with 186Re-labeled MAb E48 resulted in complete regressions in one-third of the tumors (followup > 150 d). MAb E48 was labeled with 186Re following a novel labeling procedure developed at our institute. The injected dose was 600 microCi, which was the maximum tolerated dose (MTD; < 15% wt loss) in these studies. The mean size of the tumors was 140 +/- 60 mm3. To investigate whether the therapeutic efficacy of RIT in our xenograft model would be improved when treating smaller xenografts, mice bearing 2 HNSCC xenografts with a vol of 75 +/- 17 mm3 (number of mice, n = 6; number of tumors, t = 12) were treated with 600 microCi of 186Re-labeled MAb E48 IgG. All tumors completely regressed and did not regrow during followup (> 150 d). In all mice, weight loss did not exceed 10%. To obtain biodistribution data, mice bearing two xenografts with a vol of 58 +/- 31 mm3 were injected with 100 microCi of 186Re-labeled MAb E48 IgG. The maximum uptake in blood was 26.4% injected dose/g (%ID.g-1) at 2 h pi and was 53.1%ID.g-1 in the tumor at d 7 pi. In normal tissues, no nonspecific accumulation was observed. Based on these biodistribution data, the absorbed cumulative radiation dose was calculated. The accumulated dose in blood and tumor was 2004 cGy and 8580 cGy, respectively. In other tissues, the dose was less than 8.1% of the dose delivered to tumor.(ABSTRACT TRUNCATED AT 250 WORDS)

MAb U36, a novel monoclonal antibody successful in immunotargeting of squamous cell carcinoma of the head and neck.

Using viable cells of a human head and neck squamous cell carcinoma (HNSCC) cell line as immunogen, we developed monoclonal antibody (MAb) U36. Immunohistochemical examination revealed distinct surface labeling of MAb U36 with normal human squamous epithelium and squamous cell carcinomas of distinct sites of origin; head and neck, lung, esophagus, cervix, and epidermis. MAb U36 shows high affinity binding (affinity constant, 3.5 x 10(10)/M) with a cell surface antigen expressed by in vitro cultured HNSCC cell lines. Similarity of the reactivity profiles of MAb U36 and MAb E48, currently the most promising antibody described for specific targeting of HNSCC in patients, warranted further comparison of these MAbs. MAb U36 recognizes a M(r) 200,000 antigen, which is different from the MAb E48 defined antigen. Furthermore, comparison of immunohistochemical staining patterns of MAb U36 and MAb E48 on a broad panel of primary HNSCC sections revealed more extensive staining for MAb U36: more tumors showed reactivity with MAb U36 and more tumor cells per tumor showed positive reaction, and staining was found to be more intense. MAb U36 does not show cross-reactivity with mouse, rat, pig, sheep, or bovine tongue epithelium. As a first approach to evaluate the suitability of MAb U36 for tumor targeting in vivo, radiolabeled MAb U36 was administered to athymic nude mice bearing human HNSCC xenografts on both flanks. Selective tumor accumulation of the radioimmunoconjugate was observed. Mean tumor uptake (in percent injected dose/g wet-weight of tissue) of MAb U36 at days 1, 2, 3, 5, 7, and 12 was 15.1, 17.9, 24.0, 21.0, 25.8, and 16.0%, respectively. The tumor to blood ratio at day 1 was 0.9 and increased up to 3.8 at day 12. The tumor uptake at day 12 was at least 10 times higher when compared to other tissues. The corollary of these findings is that MAb U36 harbors high potential for specific targeting of HNSCC.

186Re-labeled monoclonal antibody E48 immunoglobulin G-mediated therapy of human head and neck squamous cell carcinoma xenografts.

In our laboratory, a solid-phase synthesis of 186Re-mercaptoacetyltriglycine for reproducible and aseptic production of stable 186Re-monoclonal antibody conjugates was recently developed. Monoclonal antibody (MAb) E48 IgG, when labeled with 99mTc according to the same labeling procedure, was recently shown to be highly capable of detecting recurrent and metastatic disease in patients with head and neck squamous cell carcinoma. In the present study, MAb E48 was labeled with 186Re and tested for its capacity to eradicate established human head and neck squamous cell carcinoma xenografts growing s.c. in nude mice. Experimental groups received a single bolus injection of 200 [number of mice (n) = 6, number of tumors (t) = 11], 400 (n = 6, t = 11), 500 (n = 6, t = 12), or 600 (n = 5, t = 9) microCi 186Re-labeled MAb E48 IgG; control animals were given diluent (n = 4, t = 8). In the 200 microCi group, 5 of 11 tumors showed regression while the remaining tumors showed a decreased growth rate. In the other treatment groups, all tumors regressed. In all treatment groups, remissions were observed (no regrowth within 4 months after injection). The number of remissions in the 200, 400, 500, and 600 microCi group were 2 of 11 (18.2%), 3 of 11 (27.3%), 6 of 12 (50%), and 3 of 9 tumors (33.3%), respectively. In comparison with the median tumor volume doubling time of the controls, the tumor volume doubling time in the remaining tumors in the groups receiving 200, 400, 500, or 600 microCi was increased 5.5-, 7.8-, 8.7-, and 11.3-fold, respectively. Dosimetry was based on the biodistribution of 200 microCi 186Re-labeled MAb E48 IgG. In the group receiving 600 microCi, the absorbed cumulative radiation dose was 3432 cGy for tumor and 1356 cGy for blood. In other tissues, the accumulated dose was < 17% of the dose delivered to tumor. The whole-body dose was 11-fold lower than the dose delivered to tumor. Apparent toxicity was limited to weight loss, which did not exceed 12% and which returned to control levels within 2 weeks. No treatment-related deaths occurred. These data suggest radioimmunotherapy with 186Re-labeled MAb E48 IgG to be a feasible approach for the treatment of head and neck cancer.

Radioimmunotherapy of human head and neck squamous cell carcinoma xenografts with 131I-labelled monoclonal antibody E48 IgG.

Monoclonal antibody (MAb) E48 reacts with a 22 kD antigen exclusively expressed in squamous and transitional epithelia and their neoplastic counterparts. Radiolabelled with 99mTc, MAb E48 is capable of targeting metastatic and recurrent disease in patients with head and neck cancer. In this study, the capacity of 131I-labelled MAb E48 to eradicate xenografts of human squamous cell carcinoma of the head and neck (HNSCC) in nude mice was examined. Experimental groups received a single i.v. bolus injection of 400 microCi MAb E48 IgG (number of mice (n = 6, number of tumours (t) = 9) or 800 microCi MAb E48 IgG (n) = 5,t = 7), whereas control groups received either diluent (n = 3,t = 5), unlabelled MAb E48 IgG (n = 4,t = 5) or 800 microCi 131I-labelled isotype-matched control MAb (n = 6,t = 9). A 4.1-fold increase in the median tumour volume doubling time and regression of two out of ten tumours (20%) was observed in mice treated with 400 microCi. In mice treated with 800 microCi. In mice treated with 800 microCi, two out of seven tumours (29%) showed complete remission without regrowth during follow-up (greater than 3 months). Median tumour volume doubling time in the remaining five tumours was increased 7.8-fold. No antitumour effects were observed in mice injected with diluent, unlabelled MAb E48 or 131I-labelled control MAb. In the same xenograft model, chemotherapy with doxorubicin, 5-fluorouracil, cisplatin, bleomycin, methotrexate or 2',2'-difluorodeoxycytidine yielded a less profound effect on tumour volume doubling time. Increases in tumour volume doubling time with these chemotherapeutic agents were 4, 2.2, 2.1, 1.7, 0, and 2.6 respectively. Moreover, no cures were observed with any of these chemotherapeutic agents. From the tissue distribution of 800 microCi MAb E48, the absorbed cumulative radiation doses of tumour and various organs were calculated using the trapezoid integration method for the area under the curve. To tumour xenografts, 12,170 cGy was delivered, blood received 2,984 cGy, whereas in every other tissue the accumulated dose was less than 6% of the dose delivered to tumour. These data, describing the first radiolabelled MAb with therapeutic efficacy against HNSCC, suggest radioimmunotherapy with MAb E48 to be a potential therapeutic modality for the treatment of head and neck cancer.

Potential for targeting head and neck squamous cell carcinoma with monoclonal antibody K984.

In previous reports we have described the development of mAb K984, reactive with an epitope expressed on the outer cell surface of undifferentiated, proliferating cells in normal stratified squamous epithelia and their neoplastic counterparts. The K984 antigen was also found to be homogeneously expressed by in vitro cultured squamous cell carcinoma (SCC) cell lines. In the present study we demonstrate that mAb K984 induces a significant, dose-dependent growth inhibition when SCC cells are grown in vitro as monolayer cultures in the presence of mAb K984. These data seem to indicate that mAb K984 has potential for tumour targeting, especially in a therapeutic setting. As a first approach to evaluate the suitability of mAb K984 for tumour targeting in vivo, radiolabelled mAb K984 was administered to SCC-xenografted nude mice. Selective tumour accumulation of mAb K984 was observed. Tumour to blood ratios and tumour to non-tumour ratios, as based on the biodistribution data, were at least ten times higher in case of the specific mAb K984 when compared to another non-specific, isotype-matched control antibody. mAb K984 was also capable of visualizing tumour deposits in xenografted nude mice. The corollary of these findings is that the mAb K984-defined antigen probably is involved in the regulation of proliferation of stratified squamous epithelium and squamous cell carcinoma and that mAb K984 has potential for specific tumour targeting.

Evidence for a role of the monoclonal antibody E48 defined antigen in cell-cell adhesion in squamous epithelia and head and neck squamous cell carcinoma.

Monoclonal antibody (MAb) E48 recognizes a 20- to 22-kDa antigen expressed by human squamous and transitional epithelia and their neoplastic counterparts. Histochemical examination of these tissues revealed distinct surface labeling with MAb E48. To investigate the subcellular localization of the E48 antigen we have performed electron microscopical analysis. In cells of normal oral mucosa, the E48 antigen was expressed on the plasmalemma, particularly associated with desmosomes, suggesting involvement of the E48 antigen in intercellular adhesion. Furthermore, the level of expression of the E48 antigen appeared to be influenced by the cellular organization. In squamous cell carcinoma (SCC) cell lines grown in vitro as subconfluent monolayer cultures, the E48 antigen expression was low. However, E48 antigen expression increased when SCC cells were grown to confluency. E48 antigen expression was similarly high when SCC cell lines were cultured under conditions promoting three-dimensional growth either as colonies within floating collagen gels or as xenograft in tumor-bearing nude mice. Further evidence for the involvement of the E48 antigen in cell-cell adhesion was found when SCC cells were grown within collagen gels in the presence of MAb E48: no spherical colonies were formed, but cells grew out to colonies composed of single cells. Moreover, in this culture system the percentage of SCC cells growing out to colonies was decreased by the presence of MAb E48. These findings indicate that the E48 antigen is involved in the structural organization of squamous tissue and might have a role in intercellular adhesion.

Superior localisation and imaging of radiolabelled monoclonal antibody E48 F(ab')2 fragment in xenografts of human squamous cell carcinoma of the head and neck and of the vulva as compared to monoclonal antibody E48 IgG.

Monoclonal antibody (MAb) E48 and its F(ab')2 fragment, radiolabelled with 131I, were tested for tumour localisation and imaging in nude mice bearing a squamous cell carcinoma xenograft line derived from a head and neck carcinoma (HNX-HN) or from a vulva carcinoma (VX-A431). MAb IgG or F(ab')2 fragments were injected in parallel and at day 1, 2, 3 and 6 or 7, mice were either scanned with a gamma camera or dissected for determination of isotope biodistribution. In HNX-HN bearing mice, E48 IgG as well as F(ab')2 showed highly specific localisation in tumour tissue. The mean tumour uptake (n = 4) expressed as the percentage of the injected dose per gram of tumour tissue (percentage ID/g) of IgG was 11.9% at day 1 and increased to 14.6% at day 6 whereas percentage ID/g of F(ab')2 was 7.2% at day 1 and decreased during subsequent days. Tumour to blood ratios (T/B) at day 1 were 1.2 for IgG and 13.6 for F(ab')2 and reached a maximum at day 6 with values of 6.4 and 54.2 respectively. In VX-A431 bearing mice, only E48 F(ab')2 showed preferential localisation in tumour tissue. At day 1, Percentage ID/g of IgG was 3.7 and T/B was 0.3, while percentage ID/g of F(ab')2 was 2.4 and T/B was 3.2. Percentage ID/g decreased after day 1 while T/B increased. In these experiments no preferential localisation of either isotype matched 125I-labelled control IgG or F(ab')2 was observed. In F(ab')2 injected HNX-HN bearing mice as well as VX-A431 bearing mice, tumours could be visualised at day 1 and 2 without any appreciable background activity. With MAb IgG this was also possible in HNX-HN bearing mice (but not in VX-A431 bearing mice) but only at day 3 and 6. These findings suggest that the superior tumour to non-tumour ratios render the E48 F(ab')2 fragment more qualified for specific targeting of radioisotopes to tumour xenografts in this experimental setting.

Antitumor activity of brequinar sodium (Dup-785) against human head and neck squamous cell carcinoma xenografts.

The effect of Brequinar sodium on the growth of xenografts established from head and neck squamous cell carcinomas (HNSCC) was assessed. Brequinar sodium is a novel drug, known to inhibit dihydroorotic acid dehydrogenase (DHO-DH), resulting in a decrease of the pyrimidine de novo synthesis. The drug was administered i.p. to tumor-bearing nude mice, once a day, during 5 days at a maximum tolerated dose of 50 mg/kg/day. Statistically significant growth delaying effects were observed in 4 out of 5 lines tested. In 3 of these lines the effect was moderate and short lasting, whereas in one line (HNX-LP) tumor growth rate was totally inhibited for a 17-day period. In this line, Brequinar sodium was superior to 5 drugs known to be active in HNSCC patients. In two tumor lines DHO-DH activity could be measured and the results are in agreement with the concept that there is a relation between Brequinar sodium sensitivity and enzyme activity.

Preclinical chemotherapy on human head and neck cancer xenografts grown in athymic nude mice.

This study was undertaken to investigate the potential role of xenografts established from human head and neck squamous cell carcinoma (HNSCC) in the selection of new anticancer agents for phase II clinical trials. Eight HNSCC tumor lines were established in NMRI nude mice. The tumor-bearing animals were then treated with drugs at the maximum tolerated dose level. Treatment with drugs known for their activity in 15%-30% of HNSCC patients [cisplatin (CDDP), bleomycin (BLEO), 5-fluorouracil (5-Fu), cyclophosphamide (CY), and doxorubicin (DOX)] caused strong responses in up to 38% and moderate responses in 50%-67% of the HNSCC tumor lines. Methotrexate (MTX), known to cause remissions in about 40% of HNSCC patients, was only minimally active in this model system. A clinically ineffective drug, amsacrine (m-AMSA), was included as a negative control and showed no or minimal activity in all four HNSCC lines tested. A number of experimental drugs that have promising preclinical activity were also tested. Brequinar sodium (Dup 785) and 10-ethyl, 10-deaza-aminopterin (10-EdAM) showed activity in three of five, and two of the four tested tumor lines respectively. N,N-dimethylformamide (DMF) and 5-aza-2'-deoxycytidine (5-aza-dCyd), agents with the capacity to induce differentiation in in vitro systems, showed moderate activity in 43% and 40%, and strong activity in 14% and 40% of the lines, respectively. Our results indicate that the nude mouse xenograft model may play a role in the screening of new drugs, and in particular, it could be of help in the selection of drugs to be tested in phase II HNSCC clinical trials.

Activity of the folate analog 10-ethyl, 10-deaza-aminopterin (10-EdAM) against human head and neck cancer xenografts.

In a previous study we established a markedly increased antitumour activity of 10-ethyl, 10-deaza-aminopterin (10-EdAM) as compared to methotrexate (MTX) when tested in vitro in squamous cell carcinoma cell lines from the head and neck (HNSCC). In this paper we describe the antitumour activity of these drugs in vivo in athymic nude mice bearing HNSCC xenografts. Using a schedule of 125 mg/kg i.p. for both drugs, injected on day 0 and 7, 10-EdAM caused a significant response in 2 out of 5 tumour lines, whereas MTX was completely inactive. These two lines moderately sensitive to 10-EdAM were not affected when the drug was given daily times 5 at an equitoxic dose of 0.75 mg/kg, indicating that the effect of the drug may be schedule dependent.

Transplantability of human head and neck squamous cell carcinomas in athymic nude mice.

Tumor material from 91 patients with squamous cell carcinoma of the head and neck was transplanted subcutaneously in athymic nude mice. In the first (man to mouse) passage, the calculated mean probability of tumor take in a single mouse was 11%. The probability of growth in the first passage was significantly better for moderately and poorly differentiated tumors than for well-differentiated tumors. Also, the implantation of lymph node material resulted in a significantly better tumor take rate than material taken from a primary tumor. Transplantability was not dependent on the following characteristics: localization, T or N stage of the tumor, or the sex of the patients. Once growth was established, all variables studied had no influence on the probability of growth in the subsequent mouse passages. A relationship between tumor growth in nude mice and patient prognosis could not be found. When transplanting head and neck squamous cell carcinoma in nude mice, it has to be recognized that some tumor characteristics will influence the success of tumor growth.

Enhanced therapeutic efficacy of 5'deoxy-5-fluorouridine in 5-fluorouracil resistant head and neck tumours in relation to 5-fluorouracil metabolising enzymes.

Four human head and neck xenograft (HNX) tumour lines grown in nude mice and two murine colon carcinomas (Colon 26 and 38) were tested for their sensitivity to 5-fluorouracil (5-FU) and its prodrug 5'deoxy-5-fluorouridine (Doxifluridine, 5'd-FUR). 5-FU sensitivity at the maximum tolerated dose (MTD) showed the following pattern; HNX-DU less than HNX-KE = HNX-E = HNX-G less than Colon 26 much less than Colon 38. The sensitivity pattern to 5'd-FUR was: HNX-DU less than HNX-G less than HNX-E less than HNX-KE less than Colon 38 less than Colon 26. For HNX-KE, HNX-E and Colon 26 an increase in therapeutic efficacy was observed with 5'd-FUR as compared to 5-FU; Colon 38 was as sensitive to 5'd-FUR as to 5-FU. Plasma pharmacokinetics of 5'd-FUR and 5-FU were comparable in normal and nude mice. Metabolism of 5-FU and 5'd-FUR was studied in the tumours. Conversion of 5'd-FUR to 5-FU was highest in Colon 26 and 15-20 times lower in HNX-DU, HNX-KE and Colon 38. The Km for 5'd-FUR in all tumours was 1-2 mM. Further anabolism of 5-FU to fluorouridine (FUR) was 5-10 times higher than that of 5-FU to FUR in HNX tumours and 3 times in the colon tumours. 5-FU conversion to FUMP via FUR had the following pattern: Colon 26 much greater than HNX-DU greater than HNX-G greater than HNX-E greater than HNX-KE much greater than Colon 38; of 5-FU to FdUMP via FUdR: Colon 26 greater than HNX-DU = HNX-KE greater than HNX-E greater than HNX-G = Colon 38; and that of 5-FU to FUMP catalysed by orotate phosphoribosyl transferase (OPRT); Colon 26 greater than or equal to Colon 38 greater than HNX-KE greater than HNX-E = HNX-DU = HNX-G. Only those tumours with a relatively high activity of OPRT were sensitive to 5'd-FUR. Colon 26, which has a very high rate of pyrimidine nucleoside phosphorylase, showed a relatively high increase in the therapeutic efficacy. It is concluded that a low rate of pyrimidine nucleoside phosphorylase is enough to convert 5'd-FUR to 5-FU; further anabolism of 5-FU catalysed by OPRT may be limiting and explain the differential sensitivity.

Preclinical antitumor activity of 5-aza-2'-deoxycytidine against human and neck cancer xenografts [corrected].

The antitumor activity of 5-aza-2'-deoxycytidine (5-aza-dCyd), a nucleoside analog, was established in human head and neck cancer xenografts, transplanted in nude mice. A significant response was noted in 3 of 5 lines, when the drug was injected intraperitoneally at a maximum tolerated dose of 2 mg/kg every four days for three doses. In two most sensitive lines 1 out of 6 tumors regressed completely. The antitumor activity of the drug may depend on the schedule used, as illustrated by the fact that just one of these two lines appeared to be sensitive when treated with low daily doses (0.25 mg/kg). In two lines, 5-aza-dCyd showed equal or better antitumor activity when compared to the conventional drugs known to produce remissions in patients with head and neck cancer (cisplatin, methotrexate, bleomycin, 5-fluorouracil and cyclophosphamide). 5-Aza-dCyd is a drug with potential value in the chemotherapeutic treatment of patients with head and neck cancer [corrected].