The human E48 antigen, highly homologous to the murine Ly-6 antigen ThB, is a GPI-anchored molecule apparently involved in keratinocyte cell-cell adhesion.

The E48 antigen, a putative human homologue of the 20-kD protein present in desmosomal preparations of bovine muzzle, and formerly called desmoglein III (dg4), is a promising target antigen for antibody-based therapy of squamous cell carcinoma in man. To anticipate the effect of high antibody dose treatment, and to evaluate the possible biological involvement of the antigen in carcinogenesis, we set out to molecularly characterize the antigen. A cDNA clone encoding the E48 antigen was isolated by expression cloning in COS cells. Sequence analysis revealed that the clone contained an open reading frame of 128 amino acids, encoding a core protein of 13,286 kD. Database searching showed that the E48 antigen has a high level of sequence similarity with the mouse ThB antigen, a member of the Ly-6 antigen family. Phosphatidylinositol-specific (PI-specific) phospholipase-C treatment indicated that the E48 antigen is glycosylphosphatidylinositol-anchored (GPI-anchored) to the plasma membrane. The gene encoding the E48 antigen is a single copy gene, located on human chromosome 8 in the 8q24-qter region. The expression of the gene is confined to keratinocytes and squamous tumor cells. The putative mouse homologue, the ThB antigen, originally identified as an antigen on cells of the lymphocyte lineage, was shown to be highly expressed in squamous mouse epithelia. Moreover, the ThB expression level is in keratinocytes, in contrast to that in lymphocytes, not mouse strain related. Transfection of mouse SV40-polyoma transformed mouse NIH/3T3 cells with the E48 cDNA confirmed that the antigen is likely to be involved in cell-cell adhesion.

Malignancy grading is no better than conventional histopathological grading in small squamous cell carcinoma of tongue and floor of mouth: retrospective study in 128 patients.

There is an ongoing debate about the predictive value of histopathological parameters in oral cancer. In the past decades, the emphasis was on the possible added value of the so-called malignancy grading system. In a retrospective study on 128 previously untreated patients with a T1 or T2 squamous cell carcinoma of the tongue and the floor of the mouth, the value of the classical Broders' grading system and the malignancy grading system were compared with regard to various outcome measures such as regional metastasis, local recurrence and 5-year survival. The results show that neither of the histological grading systems has a strong predictive value and that none is superior to the other.

Inherited susceptibility to bleomycin-induced chromatid breaks in cultured peripheral blood lymphocytes.

BACKGROUND: Susceptibility to bleomycin-induced chromatid breaks in cultured peripheral blood lymphocytes may reflect the way a person deals with carcinogenic challenges. This susceptibility (also referred to as mutagen sensitivity) has been found to be increased in patients with environmentally related cancers, including cancers of the head and neck, lung, and colon, and, in combination with carcinogenic exposure, this susceptibility can greatly influence cancer risk. The purpose of this study was to assess the heritability of mutagen sensitivity. METHODS: Heritability was determined by use of a maximum likelihood method that employed the FISHER package of pedigree analysis. Bleomycin-induced breaks per cell values for 135 healthy volunteers without cancer were determined. These individuals were from 53 different pedigrees and included 25 monozygotic twin pairs (n = 50), 14 pairs of dizygotes (twin pairs and siblings, n = 28), and 14 families selected on the basis of a first-degree relative who was successfully treated for head and neck cancer and who had no sign of recurrence for at least 1 year. All data were analyzed simultaneously, and different models of familial resemblance were fitted to the data. All P values are two-sided. RESULTS: Our results showed no evidence for the influence of a shared family environment on bleomycin-induced chromatid breaks. Genetic influences, however, were statistically significant (P =. 036) and accounted for 75% of the total variance. CONCLUSIONS: The high heritability estimate of the susceptibility to bleomycin-induced chromatid breaks indicates a clear genetic basis. The findings of this study support the notion that a common genetic susceptibility to DNA damage--and thereby a susceptibility to cancer--may exist in the general population.

Genetic susceptibility to head and neck squamous cell carcinoma.

BACKGROUND: In addition to influences of exposure to carcinogenic compounds, the development of cancer may depend on an individual intrinsic cancer susceptibility. Biomarkers for cancer susceptibility can be powerful additions to epidemiologic analyses. PURPOSE: This multicenter, case-control analysis combines previously published data and new data to substantiate the value of mutagen sensitivity as a biomarker of susceptibility to head and neck squamous cell carcinoma and, more importantly, to gain insight into the interaction between susceptibility and exposure to carcinogens. METHODS: Mutagen sensitivity (mean number of chromatid breaks per cell of cultured lymphocytes treated with bleomycin in the late S-G2 phase of the cell cycle) was determined in 313 patients with head and neck cancer and in 334 control subjects at two major U.S. medical institutions and one European institution, yielding a unique study population. The ages of the case and control subjects, as well as their history of use of tobacco and alcohol, were also recorded. The relationships between variables were analyzed by use of Student's t tests, Spearman's rank correlations, and multiple linear regression. For estimation of cancer risk, crude odds rations (ORs) were measured and multiple logistic regression was performed. All P values were based on two-sided tests. RESULTS: There were no differences across institutions in the distribution of mutagen sensitivity (Kruskal-Wallis test) for both case subjects and control subjects. Values for case subjects were consistently and significantly (P<.0001) higher than values for control subjects in the overall analyses. Age and tobacco or alcohol use did not influence the outcome in terms of mutagen-sensitivity values for either the case or the control subjects. A mean number of breaks per cell dichotomized at 1.0 was found to be the best predictor of a hypersensitive phenotype. For nonsensitive, heavy smokers, the OR was 11.5 (95% confidence interval [CI] = 5.0-26.6). This risk increased dramatically in mutagen-hypersensitive, heavy smokers to 44.5 (95% CI = 17.4-114.0). Multiple logistic regression analysis confirmed these results, and a significant trend was found (P<.01) for the dose-dependent increase in cancer risk by smoking. The consumption of alcohol potentiated the effects of smoking, resulting in an OR of 57.5 (95% CI = 17.5-188.0) in hypersensitive persons. CONCLUSIONS: Mutagen sensitivity was found to be a biomarker of cancer susceptibility. This study underscores the importance of utilizing both susceptibility markers and the exposure data for the identification of persons at high risk of developing cancer. IMPLICATIONS: More accurate risk estimation can define susceptible subgroups who might be targeted for intensive behavioral interventions, surveillance through screening, and enrollment in chemoprevention programs.

Preclinical in vivo activity of 2',2'-difluorodeoxycytidine (Gemcitabine) against human head and neck cancer.

2',2'-Difluorodeoxycytidine (dFdCyd, Gemcitabine) is a new deoxycytidine analogue with striking preclinical antitumor activity in solid tumors from murine and human origin. In this study, dFdCyd was tested for its antitumor effect in human tumor xenografts derived from squamous cell carcinoma of the head and neck (SCCHN). NMRI nude mice bearing s.c. growing tumors with a volume of 50 to 150 mm3 were given i.p. injections of a maximum tolerated dose of 120 mg/kg dFdCyd, every 3 days for four injections. A significant antitumor effect was observed in all five tested SCCHN tumor lines; in four of these lines the median tumor volume doubling time increased more than a 3-fold upon dFdCyd treatment. In two lines dFdCyd was curative (no tumor regrowth 90 days after treatment) in one of six and two of eight xenografts, respectively. Schedule dependency was investigated in three SCCHN lines, showing, in the two most sensitive lines, that treatment with a 3-day interval was superior to the schedules with daily or weekly injections. At equitoxic doses, dFdCyd was more active in this model than the drugs that are clinically used in SCCHN, i.e., cisplatin, methotrexate, 5-fluorouracil, and cyclophosphamide. dFdCyd is a good candidate for clinical trials with SCCHN patients.

Targeting of aluminum (III) phthalocyanine tetrasulfonate by use of internalizing monoclonal antibodies: improved efficacy in photodynamic therapy.

The use of monoclonal antibodies (MAbs) directed against tumor-associated antigens for targeting of photosensitizers is an interesting option to improve the selectivity of photodynamic therapy (PDT). Hydrophilic photosensitizers are most suitable for conjugation to MAbs because of their water solubility. The photosensitizer aluminum (III) phthalocyanine tetrasulfonate [AlPc(SO3H)4] has many ideal photochemical properties; however, because of its hydrophilicity, the free form of this sensitizer does not readily reach the critical intracellular target and, therefore, is ineffective in PDT. On the basis of our previous studies, we hypothesized that AlPc(SO3H)4 might be suitable for PDT when coupled to internalizing tumor-selective MAbs. In this study, a reproducible procedure is presented for coupling of AlPc(SO3H)4 to MAbs via the tetra-glycine derivative AlPc(SO2Ngly)4. Conjugation was performed to chimeric MAb (cMAb) U36 and murine MAbs (mMAb) E48 and 425 using a labile ester. Conjugates showed preservation of integrity and immunoreactivity and full stability in serum in vitro. At molar ratios >4, the solubility of the conjugates decreased. Data on the in vitro efficacy of PDT showed that in the chosen experimental setup the internalizing AlPc(SO2Ngly)4-mMAb 425 conjugate was about 7500 times more toxic to A431 cells than the free sensitizer (IC50s, 0.12 nM versus 900 nM). The AlPc(SO2Ngly)4-mMAb 425 conjugate was also more toxic than meta-tetrahydroxyphenylchlorin-mMAb 425 conjugates and free meta-tetrahydroxyphenylchlorin that had been tested previously (M. B. Vrouenraets et al., Cancer Res., 59: 1505-1513, 1999) in the same system (IC50s, 7.3 nm and 2.0 nM, respectively). Biodistribution analysis of AlPc(SO2Ngly)4-125I-labeled cMAb U36 conjugates with different sensitizer:MAb ratios in squamous cell carcinoma-bearing nude mice revealed selective accumulation in the tumor, although to a lesser extent than for the unconjugated 125I-labeled cMAb U36, whereas tumor:blood ratios were similar. These findings indicate that AlPc(SO3H)4 has high potential for use in PDT when coupled to internalizing tumor-selective MAbs.

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.

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.

Development of meta-tetrahydroxyphenylchlorin-monoclonal antibody conjugates for photoimmunotherapy.

A limitation of photodynamic therapy is the lack of tumor selectivity of the photosensitizer. To overcome this problem, a protocol was developed for coupling of meta-tetrahydroxyphenylchlorin (mTHPC), one of the most promising photosensitizers, to tumor-selective monoclonal antibodies (MAbs). mTHPC was radiolabeled with 131I to facilitate the assessment of the in vitro and in vivo behavior. After the modification to 131I-mTHPC-(CH2COOH)4, thus increasing the water solubility and creating a functional moiety suitable for coupling, conjugation was performed using a labile ester. Insoluble aggregates were not formed when mTHPC-MAb conjugates with a molar ratio of up to 4 were prepared. These conjugates showed a minimal impairment of the integrity on SDS-PAGE, full stability in serum in vitro, and an optimal immunoreactivity. To test the in vivo behavior of the mTHPC-MAb conjugates, the head and neck squamous cell carcinoma-selective chimeric MAb U36 was used in head and neck squamous cell carcinoma-bearing nude mice. Biodistribution data showed that the tumor selectivity of cMAb U36-conjugated mTHPC was increased in comparison with free mTHPC, despite the fact that conjugates with a higher mTHPC:MAb ratio were more rapidly cleared from the blood. Preliminary results on the in vitro efficacy of photodynamic therapy with MAb-conjugated mTHPC showed that mTHPC coupled to the internalizing murine MAb 425 exhibited more phototoxicity than when coupled to the noninternalizing chimeric MAb U36.

Considerations for in vitro retinoid experiments: importance of protein interaction.

Retinoids, natural and synthetic substances structurally related to vitamin A, are important modulators of cell proliferation and differentiation, and have proven activity in cancer therapy. Experiments to reveal the mechanism of action of retinoids are routinely performed in in vitro models. As retinoids are relatively hydrophobic and unstable, we hypothesized that the composition of culture media is of critical importance for the stability and bioavailability of these compounds. Various culture media were incubated with all-trans-, 13-cis- and 9-cis-retinoic acid (RA). Without fetal calf serum (FCS) or bovine serum albumin (BSA) in the medium, the concentration of these retinoids was found to decrease to considerably low levels. This excessive loss of retinoids was due to absorption to culture plates, reaction tubes and pipet tips. Binding of retinoids to BSA was demonstrated to have attenuating effects on uptake and metabolism of all-trans-RA, as studied in oral keratinocytes and head and neck cancer cells, indicating that a balance exists between the bioavailability and the aspecific loss of retinoids. In this study we demonstrate that the type of culture medium and especially the presence of protein in the medium is of paramount importance to perform reproducible experiments with retinoids.

Effect of chemotherapy on survival of craniofacial osteosarcoma: a systematic review of 201 patients.

PURPOSE: To evaluate the possible value of chemotherapy in the treatment of craniofacial osteosarcoma (CFOS). DESIGN: In a systematic review, data of 201 patients (age, 36.6 +/- 19.0 years [mean +/- SD]) from 20 uncontrolled series on CFOS indexed in Medline and Excerpta Medica between 1974 and 1994 were pooled; 180 patients had undergone surgery. Various chemotherapy regimens had been given to 71 patients. Radiotherapy was used in 69 patients. All patients had data for overall survival, and 182 had data for disease-free survival analysis. Cumulative survival distributions were estimated by the product-limit method, and a multiple regression analysis was performed. RESULTS: Patients' overall and disease-free survival rates were significantly improved by treatment with chemotherapy. This was confirmed for patients with complete surgical removal, as well as for those with incomplete removal of tumor. In a proportional hazards model, complete surgical removal and chemotherapy were independent significant factors for overall and disease-free survival. The effect of radiotherapy was insignificant. No prognostic factors could be identified among age groups, sex, and subsites. CONCLUSION: Evidence exists that chemotherapy improves survival in CFOS. We advocate the adoption of the chemotherapy protocols used for OS of the long bones for CFOS.

Enhanced turnover of all-trans-retinoic acid and increased formation of polar metabolites in head and neck squamous cell carcinoma lines compared with normal oral keratinocytes.

Retinoids show promise in the treatment of various (pre)malignancies, including head and neck squamous cell carcinoma (HNSCC). Previous studies have shown that the metabolic pathways of retinoids are important in the anticancer effect of retinoids, and that these pathways may change during carcinogenesis. In the present study, we analyzed HNSCC cell lines (n = 11) and normal oral keratinocyte cultures (n = 11) by reverse-phase high-performance liquid chromatography and conducted growth inhibition assays. We demonstrate here that in contrast to normal oral keratinocytes, HNSCC cell lines: (a) had averaged a 17-fold greater turnover rate of all-trans-retinoic acid (RA); (b) had a 1.9-fold less RA-induced growth inhibition; (c) were able to form polar metabolites; and (d) were able to catabolize 4-oxo-RA. Furthermore, the mRNA expression of the RA-specific 4-hydroxylase, CYP26A1, was dramatically increased after RA-induction in the two HNSCC cell lines with the highest metabolism, was undetectable in normal keratinocytes, and was not inducible by RA. Next, introduction of CYP26A1 cDNA in a low-metabolizing HNSCC cell line resulted in an 11-fold higher turnover rate of RA and a 12-fold increase in the amount of polar metabolites, but it did not change sensitivity to RA. These observations point to fundamental changes in RA metabolism pathways during HNSCC carcinogenesis and may provide clues to a more rational approach for RA-mediated intervention.

Biodistribution of radiolabeled monoclonal antibody E48 IgG and F(ab')2 in patients with head and neck cancer.

Biodistribution and pharmacokinetics of radiolabeled mAb E48 IgG and E48 F(ab')2 were analyzed and compared in 39 patients with histologically proven squamous cell carcinoma of the head and neck who were included in a radioimmunoscintigraphy study and underwent surgery 44 h after injection. Three groups of patients were distinguished: group 1 (n = 19) received technetium-99m (99mTc)-labeled E48 F(ab')2, group 2 (n = 9) received 99mTc-labeled E48 IgG, and group 3 (n = 11) received 99mTc- and 131I-labeled E48 IgG as well as 125I-labeled F(ab')2. Two patients in group 1 and four patients in group 3 received a high mAb dose (10-50 mg), while all other patients received a low mAb dose (1-4 mg). From all patients in groups 2 and 3 biopsies from the surgical specimen were obtained 44 h postinjection. Tumor uptake of 99mTc-labeled E48 IgG was high, ranging from 0.007 to 0.082% of the injected dose/g, with a mean of 0.031 +/- 0.020% of the injected dose/g. The mean tumor:nontumor ratio of this conjugate was 2.8 for mucosa, 4.6 for bone marrow aspirate, 4.1 for blood, 20.3 for fat, and 21.0 for muscle. Activity uptake in tumor positive lymph nodes was 4.7 times higher as compared to negative lymph nodes. Sixteen h postinjection radioimmunoscintigraphy revealed activity uptake in the primary tumor, lymph node metastases, oral cavity, and adrenal glands. Using regions of interest, the uptake in the adrenal glands was estimated to be 0.050% of the injected dose/g. If a high mAb dose was used, no adrenal glands were visualized and the uptake in the oral cavity was clearly diminished, while the tumor uptake and tumor:nontumor ratios were increased. The mean elimination half-lifes t1/2 alpha and t1/2 in plasma were: for E48 IgG (n = 20) 6.6 +/- 2.6 and 54.1 +/- 24.3 h and for E48 F(ab')2 (n = 19) 2.3 +/- 0.4 and 19.9 +/- 4.6 h, respectively. Tumor uptake of 131I-labeled E48 IgG was 49% higher than of 125I-labeled F(ab')2. For most tissues except normal oral mucosa, tumor:nontumor ratios were slightly higher for F(ab')2 than for IgG. The present study shows that mAb E48 accumulates selectively and to a high level in head and neck squamous cell carcinoma. Although no definite conclusions can be drawn as to which mAb form is more suitable, IgG or F(ab')2, mAb E48 seems to have potential for radioimmunotherapy in head and neck squamous cell carcinoma patients.

Radioimmunoscintigraphy and biodistribution of technetium-99m-labeled monoclonal antibody U36 in patients with head and neck cancer.

So far, mAb E48 is the most promising antibody described for specific targeting of head and neck squamous cell carcinoma (HNSCC) in patients. On the basis of its more homogeneous reactivity pattern on HNSCC, the novel mAb U36 may be even better suited for targeting. In this study the biodistribution of mAb U36 was evaluated by radioimmunoscintigraphy (RIS) and biopsy measurements in 10 patients who were suspected of having neck lymph node metastases from a histologically proven HNSCC and who had been scheduled to undergo resection of the primary tumor and neck dissection. Patients received 1.8-53.0 mg mAb U36 IgG labeled with 756 +/- 95 MBq technetium-99m i.v. Preoperatively, palpation, computerized tomography, magnetic resonance imaging, and RIS were performed. RIS images included planar and single-photon emission computerized tomography images of the head and neck and planar images of the whole body. The diagnostic findings were recorded per side as well as per lymph node level of the neck and compared to the histopathological outcome. Radioactivity in blood samples and biopsies from the surgical specimens were measured. All 10 primary tumors were visualized by RIS. All diagnostic modalities were correct in 7 of 14 tumor-involved lymph node levels. The missed lymph node metastases comprised micrometastases, small tumor-involved nodes (<9 mm), and tumor-involved nodes with much necrosis, keratin, or fibrin. There were no false-positive observations with mAb U36. Besides activity uptake in tumor tissue, only a slight accumulation of activity was observed in the mouth, lungs, liver, spleen, kidneys, and scrotal area. Biopsies from the surgical specimen showed a high tumor uptake of 20.4 +/- 12.4% of the injected dose/kg (range, 8.0-43.0% of injected dose/kg), 44 h postinjection. An increase in the mAb dose did not influence uptake of activity in tumor tissue. The mean tumor:nontumor ratio at this time point was 2.3 for mucosa, 2.8 for blood, 3.0 for bone marrow aspirate, 12.9 for fat, and 13.0 for muscle tissue. The present clinical study shows that technetium-99m-labeled U36 IgG accumulates selectively and to a high level in HNSCC. The tumor-targeting results for U36 IgG are comparable to those previously described for E48 IgG. On the basis of the results of ongoing biodistribution studies in which both mAbs E48 and U36, labeled with different iodine isotopes, are simultaneously evaluated for tumor uptake and retention in HNSCC patients, one of these mAbs will be selected for future adjuvant radioimmunotherapy trials.

Sensitive detection of squamous cells in bone marrow and blood of head and neck cancer patients by E48 reverse transcriptase-polymerase chain reaction.

In previous studies, we described the selective reactivity of monoclonal antibody E48 with normal squamous and transitional epithelia and their malignant counterparts and the capacity of monoclonal antibody E48 for selective tumor targeting in head and neck cancer patients. Cloning of the E48 encoding cDNA and elucidation of the gene structure enabled the selection of an intron-spanning primer set for the detection of circulating tumor cells in blood and bone marrow of head and neck cancer patients. Extensive optimizations led to a reproducible reverse transcriptase-PCR assay with an internal standard for RNA quality control and an external standard for sensitivity control. In reconstruction experiments, we were able to reach a reproducible sensitivity of one single tumor cell per 7 ml of blood (2 x 10(7) nucleated cells). When applying this method to patient material, we were able to detect positive signal in 35% of the bone marrow samples (0 of 2 stage II, 0 of 4 stage III, 4 of 11 stage IV, and 4 of 6 recurrences) and 10% of the blood samples (2 of 21) of patients with squamous cell carcinoma of the head and neck. The specificity of the method was demonstrated on 29 blood and bone marrow samples of noncancer controls, which were all negative. Our study shows the feasibility of E48 reverse transcriptase-PCR for the detection of squamous cells in nonsquamous tissues.

Evaluation of soluble CD44v6 as a potential serum marker for head and neck squamous cell carcinoma.

In recent years, the measurement of soluble CD44 levels in the circulation of patients with malignant diseases has been introduced as a new and simple diagnostic tool for the detection of human cancer. The high CD44v6 expression in head and neck squamous cell carcinoma (HNSCC) would enable the use of soluble CD44v6 proteins present in the circulation of HNSCC patients as a marker of disease. In the present study, we determined CD44v6 plasma levels using a domain-specific ELISA in healthy volunteers, non-cancer patients, and HNSCC patients before and after surgical removal of the tumor. A difference between the CD44v6 plasma levels of HNSCC patients and controls could not be observed. Moreover, surgical removal of the tumor did not result in a reduction of the CD44v6 plasma level in the HNSCC patients. In addition, the spectrum of soluble v6-containing CD44 proteins present in the plasma of HNSCC patients and controls was determined by immunoprecipitation experiments, but again, tumor-related isoforms could not be distinguished in patient samples. Additional experiments to unravel the biological source of these circulating proteins indicated surprisingly that the v6-containing proteins present in the circulation of healthy individuals are only released in part, if at all, by activated lymphocytes or other nucleated blood cells. Most circulating CD44v6 proteins seem to be derived from the normal epithelial cell compartments, including breast cells, colon cells, and squamous cells. Taken together, these data do not support the use of soluble CD44v6 as a tumor marker in HNSCC or any other tumor type that has developed from tissues producing soluble isoforms.

Molecular assays for the diagnosis of minimal residual head-and-neck cancer: methods, reliability, pitfalls, and solutions.

The prognosis of cancer patients is determined by the radicalness of treatment: residual tumor cells will grow out and develop in manifest local recurrences, regional recurrences, and distant metastases. Classical diagnostic methods such as radiology and histopathology have limited sensitivities, and only by molecular techniques can minimal residual disease be detected. In tissue samples containing the normal tissue counterpart of a tumor, only tumor-specific markers can be exploited, whereas in other samples, tissue-specific markers can be used. At present, there are two main methodologies in use, one based on antigen-antibody interaction and the other based on amplified nucleic acids. The most commonly used nucleic acid markers are mutations or alterations in tumor DNA (tumor-specific markers) or differentially expressed mRNA (tissue-specific markers). Many reports and reviews have been published on the assessment of minimal residual disease by molecular markers, showing either positive or negative clinical correlations. One of the main reasons for these contradictory findings is the technical difficulty in finding the small numbers of tumor cells in the large number of normal cells, which necessitates sensitivities of the assays up to 1 tumor cell in 2 x 10(7) normal cells. These assays often are complex, demand considerable experience, and usually are laborious. In this review, we will address a number of the technical issues related to molecular assays for tumor cell detection that make use of nucleic acids as markers. Many difficulties in data interpretation are at least in part because of technical details that might have been solved by the incorporation of one or more appropriate controls. We hope that this review clarifies a number of these issues and help clinicians and investigators interested in this field to understand and weigh the contradictory findings in the published studies. This will help move the field forward and facilitate clinical implementation.

Persistence of genetically altered fields in head and neck cancer patients: biological and clinical implications.

In 1953, Slaughter et al. [D. P. Slaughter et al., Cancer (Phila.), 6: 963-968, 1953] proposed the concept of field cancerization in patients with squamous cell carcinoma of the head and neck (HNSCC) and discussed its clinical significance for the development of second primary tumors and local recurrences. To define the process of field cancerization and its putative clinical implications, we analyzed genetic aberrations in HNSCC and the accompanying macroscopically normal mucosa. In 28 HNSCC patients, loss of heterozygosity was determined in tumor and five noncontiguous mucosal biopsies using eight microsatellite markers at 9p, 3p, and 17p. For patients who showed loss of heterozygosity in their mucosal biopsies, all margins of the surgical specimen were subsequently analyzed to determine the extension of the field. In these cases, additional markers at 8p, 13q, and 18q as well as p53 mutations were included to determine subclonal differences between field and tumor. Genetically altered fields were detected in 36% (10 of 28) of the HNSCC patients. The field varied in size between patients and consisted of genetically different subclones. In 7 of 10 cases, the field extended into the surgical margins. One particular patient with a genetically altered field in a surgical margin developed a local recurrence after 28 months of follow-up. Microsatellite analysis showed that this recurrence had more molecular markers in common with the nonresected premalignant field than with the original tumor, suggesting that this persistent field has progressed further into a new malignancy. Our data show that genetically altered mucosa remains after treatment in a significant proportion of HNSCC patients, which may explain in part the high frequency of local recurrences and second primary tumors. Adequate identification and risk assessment of these genetically altered fields may have profound implications for future patient management.

Safety and biodistribution of 99mTechnetium-labeled anti-CD44v6 monoclonal antibody BIWA 1 in head and neck cancer patients.

The CD44 protein family consists of isoforms, encoded by standard exons and up to nine alternatively spliced variant exons (v2-v10), which are expressed in a tissue-specific way. Expression of v6-containing variants (CD44v6) has been related to aggressive behavior of various tumor types and was shown to be particularly high in squamous cell carcinoma (SCC). Therefore, CD44v6 might be a suitable target for radioimmunoscintigraphy (RIS) and therapy. The present study evaluates the novel high-affinity murine anti-CD44v6 monoclonal antibody (MAb) BIWA 1 for its safety and targeting potential in patients with SCC of the head and neck (HNSCC). Twelve HNSCC patients, who had planned to undergo resection of the primary tumor and neck dissection, were included. Preoperatively, 2, 12, or 52 mg of 99nTc-labeled MAb BIWA 1 was administered. RIS results obtained 21 h after injection were compared with palpation, computed tomography, and magnetic resonance imaging, with histopathology as the gold standard. Moreover, biodistribution of BIWA 1 was evaluated by radioactivity measurement in blood and bone marrow and in biopsies from the surgical specimen obtained 40 h after injection. The distribution of BIWA 1 in tumor biopsies was analyzed by immunohistochemistry. BIWA 1 integrity in the blood was assessed by high-performance liquid chromatography and related to soluble CD44v6 levels in serum samples. No drug-related adverse events were observed. Human antimouse antibody responses were observed in 11 patients. The diagnostic efficacy of RIS appeared to be comparable for the three BIWA 1 dose levels and for the four diagnostic methods. Besides activity uptake in tumor tissue, minimal accumulation of activity was observed in mouth, lungs, spleen, kidney, bone marrow, and scrotal area. Analysis of tissue biopsies revealed high uptake in tumors, with a mean value of 14.2+/-8.4% of the injected dose/kg tumor tissue and a mean tumor:blood ratio of 2.0+/-1.4 at 40 h after injection. Differences among the three dose groups were not statistically significant, although a trend toward lower uptake in the highest dose group was noted. Distribution of BIWA 1 throughout the tumor was heterogeneous for all dose groups, which might be related to the high affinity of the MAb. The mean biological half-life in blood (34.5+/-6.1 h) was not dose dependent. Extensive complex formation of BIWA 1 was observed in the 2-mg group, most probably with soluble CD44v6 present in the blood, and complex formation relatively diminished upon increase of the MAb dose. BIWA 1 is a promising MAb for targeting HNSCC because it can be safely administered to HNSCC patients, while it shows high and selective tumor uptake. However, BIWA 1 is immunogenic, and therefore a chimerized or humanized derivative of BIWA 1 with intermediate affinity will be used in future clinical trials.

Effect of perioperative nutrition, with and without arginine supplementation, on nutritional status, immune function, postoperative morbidity, and survival in severely malnourished head and neck cancer patients.

BACKGROUND: Malnourished head and neck cancer patients are at increased risk of postoperative complications. OBJECTIVE: We studied the effect of perioperative, arginine-supplemented nutritional support on nutritional status, immune status, postoperative outcome, and survival in severely malnourished (weight loss >10% of body weight) head and neck cancer patients undergoing major surgery. DESIGN: Forty-nine patients were randomly assigned to receive 1) no preoperative and standard postoperative tube feeding, 2) standard preoperative and postoperative tube feeding, or 3) arginine-supplemented preoperative and postoperative tube feeding. RESULTS: Patients in both prefed groups received approximately 9 d of preoperative tube feeding, resulting in energy intakes of 110% and 113% of calculated needs (compared with 79% in the control group; P = 0.007). Compared with no preoperative feeding, preoperative enteral nutrition did not significantly improve nutritional status or any of the studied biochemical or immunologic indexes. Major postoperative complications occurred in 53%, 47%, and 59% of patients in study groups 1, 2, and 3 (NS). A trend was seen toward better survival in the arginine-supplemented group (P = 0.15). Secondary analysis showed that survivors had better human leukocyte antigen-DR expression on monocytes (P = 0.05) and higher endotoxin-induced cytokine production (P = 0.010 for tumor necrosis factor alpha and P = 0.042 for interleukin 6) at the start of the study than did patients who died. CONCLUSIONS: Nine days of preoperative tube feeding, with or without arginine, did not significantly improve nutritional status, reduce the surgery-induced immune suppression, or affect clinical outcome in severely malnourished head and neck cancer patients. Patients supplemented with arginine-enriched nutrition tended to live longer. Some markers of immune function may distinguish patients with good or bad prognoses.