Prognostic importance of paratracheal lymph node metastases.

OBJECTIVES: The aim of this retrospective study was to evaluate the prognostic significance of paratracheal lymph node (PTLN) metastases for tumor recurrence and survival for patients treated with total laryngectomy (TL) and PTLN dissection. STUDY DESIGN: Records from 85 patients who underwent TL combined with PTLN dissection for laryngeal or hypopharyngeal carcinomas were reviewed. RESULTS: In 20 of 85 (24%) patients, PTLN metastases were found, and in 7 patients, extranodal spread (ENS) was present in these metastases. The incidence of PTLN metastases was high in patients with hypopharyngeal or cervical esophageal (35%) and laryngeal carcinoma with subglottic extension (27%). Multivariate analysis shows that the most important prognostic factor for overall survival is the presence of PTLN metastases with ENS (P < .0005). CONCLUSIONS: Because PTLN metastases with ENS is an important prognostic factor and can only be assessed by histopathologic examination, PTLN dissection in patients with a laryngeal or hypopharyngeal tumor is important for prognostication.

Histopathologic validation of the sentinel node concept in oral and oropharyngeal squamous cell carcinoma.

BACKGROUND: In patients with head and neck squamous cell carcinoma (HNSCC), the presence of lymph node metastases is the most important prognosticator. Sentinel node (SN) biopsy has been shown to be an accurate staging technique for patients with breast cancer and melanoma and might also be suited for patients with HNSCC. This study was undertaken to determine whether the SN concept holds true for HNSCC and could be exploited for SN biopsy. METHODS: In 22 patients with T2 to T4 N0 oral or oropharyngeal squamous cell carcinoma (SCC) who were scheduled to undergo combined primary tumor excision and elective unilateral (n = 17) or bilateral (n = 5) neck dissection, SN identification was performed the day before surgery by use of lymphoscintigraphy after peritumoral injections of 99mTc-labeled colloidal albumin. After the neck dissection specimens were removed, all SNs, all other radioactive lymph nodes, and all nonradioactive lymph nodes were retrieved for histopathologic analysis, including serial sectioning at 250-microm intervals and immunohistochemical analysis (IHC). RESULTS: Overall, in 21 (78%) of 27 neck sides, an SN was identified by scintigraphy. Of the six neck sides in which SNs were not identified by scintigraphy, four were from three patients who underwent bilateral neck dissection. In another patient treated by bilateral neck dissection, the SN identified by scintigraphy could not be found in the specimen. In the remaining 20 neck dissection specimens, 23 SNs and 30 additional radioactive lymph nodes could be found. At histologic examination of the 20 neck specimens in which the SN was found, at least one SN was tumor positive in eight cases. In one neck specimen, a metastasis was detected in a nonradioactive lymph node, whereas the SN was tumor free, also at serial sectioning and IHC. In the remaining 11 neck sides in which the SN was tumor negative, none of the other radioactive (n = 13) and none of the nonradioactive (n = 279) lymph nodes contained tumor at histopathologic analysis, including serial sectioning and IHC. The sensitivity of the SN procedure for predicting lymph node metastases, therefore, was 89% (eight of nine neck specimens) when an SN was identified by scintigraphy and found in the specimen. The overall accuracy of the SN procedure for predicting the presence or absence of lymph node metastases in the neck was 95% (19 of 20 neck specimens). CONCLUSIONS: Our study seems to validate the SN hypothesis for oral and oropharyngeal cancer. The role of SN biopsy in the management of the N0 neck in such patients has yet to be established through prospective trials. SN identification (and thus biopsy) does not seem to be reliable in patients with tumors located in or close to the midline.

Clinical significance of micrometastatic cells detected by E48 (Ly-6D) reverse transcription-polymerase chain reaction in bone marrow of head and neck cancer patients.

PURPOSE: Despite improvements in locoregional treatment of head and neck squamous cell carcinoma (HNSCC), local and distant failure rates remain high. The strongest prognostic indicator of HNSCC is the presence of lymph node metastases in the neck, but the value of this indicator has limitations when using for the individual patient. The presence of micrometastatic cells in bone marrow has been shown to be a putative prognostic indicator in HNSCC and other epithelial malignancies, which might allow more accurate staging and selection of patients for whom adjuvant or experimental therapy is recommended. The gene encoding the E48 antigen is selectively expressed by HNSCC, and the detection of E48 transcripts in bone marrow by reverse transcription-polymerase chain reaction (RT-PCR) presumably represents the presence of micrometastatic cells. The purpose of this study was to determine the association between the presence of micrometastatic cells in bone marrow of HNSCC patients and clinical outcome. EXPERIMENTAL DESIGN: A total of 162 patients treated surgically for primary HNSCC underwent a single bone marrow aspiration from the upper iliac crest for detection of micrometastatic cells using E48 RT-PCR. In total, 139 patients were evaluable. The primary statistical endpoints were disease-free survival and distant metastasis-free survival. In addition, bone marrow samples of 30 noncancer controls were evaluated. RESULTS: E48 RT-PCR indicated the presence of micrometastatic cells in the bone marrow in 56 of 139 (40%) of the HNSCC patients and 0 of 30 of the noncancer controls (P < 0.0001). The presence of micrometastatic cells had no significant influence on disease-free survival or distant metastasis-free survival for the whole group of HNSCC patients (P = 0.1460 and P = 0.2912, respectively). For patients with >or=2 lymph node metastases, however, the presence of micrometastatic cells was associated with a poor distant metastasis-free survival (P = 0.0210). CONCLUSIONS: The presence of micrometastatic cells in bone marrow of HNSCC patients with >or=2 lymph node metastases is correlated with a poor distant metastasis-free survival. In this subgroup of HNSCC patients, E48 RT-PCR seems to be a valuable tool to identify patients who are at increased risk for development of distant metastases and therefore might benefit from experimental adjuvant systemic therapy.

Molecular diagnosis of surgical margins and local recurrence in head and neck cancer patients: a prospective study.

PURPOSE: Approximately 10-30% of surgically treated head and neck cancer patients develop local recurrences while the resection margins are histologically tumor free. These recurrences may arise from cancer cells left behind but not detected by the pathologist, or they may develop from precursor lesions adjacent to the tumor that were not completely resected. We have investigated whether TP53-mutated DNA in the surgical margins is suitable to identify patients with head and neck squamous cell carcinoma at risk for local and locoregional recurrence. EXPERIMENTAL DESIGN: In a prospective cohort study of 76 patients with histologically tumor-free margins, the presence of TP53-mutated DNA was determined in the surgical margins using the phage plaque assay and correlated to clinical outcome. Immunostaining of the molecular-positive margins for mutated p53 protein was used to identify whether unresected precursor lesions or residual tumor cells were left behind. RESULTS: The absence of TP53-mutated DNA in surgical margins was significantly associated with remaining free of local and locoregional recurrence (P = 0.027 and P = 0.028, respectively). Moreover, the presence of TP53-mutated DNA in the surgical margins was an independent prognosticator for locoregional recurrence (relative risk = 7.1; P = 0.021; 95% confidence interval, 0.9-56). In 20% of the cases, the presence of TP53-mutated DNA in the surgical margins was found to be related to the presence of tumor-related precursor lesions. CONCLUSIONS: This study shows the value of TP53-mutated DNA as a molecular marker to predict locally recurrent head and neck squamous cell carcinoma. The observation that all patients who were negative for TP53-mutated DNA in the surgical margins remained free of local recurrence raises hope that molecular analysis of histologically tumor-free surgical margins can be exploited to decide on postoperative radiotherapy. Furthermore, our data provide evidence that local recurrences originate mainly from tumor cells left behind but also originate, in part, from unresected precursor lesions.

Safety, biodistribution, pharmacokinetics, and immunogenicity of 99mTc-labeled humanized monoclonal antibody BIWA 4 (bivatuzumab) in patients with squamous cell carcinoma of the head and neck.

UNLABELLED: Previous studies have shown the potential of murine and chimeric anti-CD44v6 monoclonal antibodies (MAbs) for radioimmunotherapy (RIT) of head and neck squamous cell carcinoma (HNSCC). A limitation of these MAbs, however, appeared to be their immunogenicity. Therefore, humanized monoclonal antibody BIWA 4 (bivatuzumab), with an intermediate affinity for CD44v6, was recently selected. As a prelude to RIT, we evaluated the safety, tumor-targeting potential, pharmacokinetics, and immunogenicity of technetium-99m-labeled BIWA 4 in patients undergoing operations for primary HNSCC in this study. Ten patients were treated at BIWA 4 dose levels of 25 mg (n=3), 50 mg (n=4), and 100 mg (n=3). Patients received 2 mg of 750 MBq 99mTc-BIWA 4, together with 23-, 48-, and 98-mg unlabeled BIWA 4, respectively. Radioimmunoscintigraphy (RIS) was performed within 1 h and after 21 h, and patients underwent surgery at 48 h after injection. Biodistribution of 99mTc-BIWA 4 was evaluated by radioactivity measurements in blood, bone marrow, and in biopsies of a surgical specimen obtained 48 h after injection. BIWA 4 concentration in blood was assessed by ELISA and high performance liquid chromatography and related to soluble CD44v6 levels in serum samples. The development of human anti-human antibody (HAHA) responses was determined. Administration of 99mTc-BIWA 4 was well tolerated by all patients and no HAHA responses were observed. A mean t1/2 in plasma of 54.8 +/- 11.5 h, 76.1 +/- 21.8 h, and 68.5 +/- 21.2 h was found for the 25-, 50-, and 100-mg dose group, respectively. No complex formation of BIWA 4 with soluble CD44v6 in blood was observed. RIS showed targeting of primary tumors and lymph node metastases in 8 of 10 and 1 of 5 patients, respectively. The highest tumor uptake and tumor to nontumor ratios were observed for the 50-mg dose group. Tumor uptake was 12.9 +/- 5.9, 26.2 +/- 3.1, and 15.4 +/- 1.9% of the injected dose (ID)/kg for the 25-, 50-, and 100-mg dose group, respectively, while the tumor to bone marrow ratios for these groups were 1.7 +/- 0.5, 3.2 +/- 1.1, and 2.0 +/- 0.6, respectively. CONCLUSION: 99mTc-BIWA 4 can safely be administered to patients with HNSCC, with absence of detectable HAHA responses. The 50-mg dose level showed the highest tumor uptake and tumor to nontumor ratios. These findings support the use of BIWA 4 for RIT studies in patients with HNSCC.

Quantitative 89Zr immuno-PET for in vivo scouting of 90Y-labeled monoclonal antibodies in xenograft-bearing nude mice.

UNLABELLED: Immuno-PET as a scouting procedure before radioimmunotherapy (RIT) aims at the confirmation of tumor targeting and the accurate estimation of radiation dose delivery to both tumor and normal tissues. Immuno-PET with (89)Zr-labeled monoclonal antibodies (mAbs) and (90)Y-mAb RIT might form such a valuable combination. In this study, the biodistribution of (89)Zr-labeled and (88)Y-labeled mAb ((88)Y as substitute for (90)Y) was compared and the quantitative imaging performance of (89)Zr immuno-PET was evaluated. METHODS: Chimeric mAb (cmAb) U36, directed against an antigen preferentially expressed in head and neck cancer, was labeled with (89)Zr using the bifunctional chelate N-succinyldesferrioxamine B (N-sucDf) and with (88)Y using the bifunctional chelate p-isothiocyanatobenzyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (p-SCN-Bz-DOTA). The radioimmunoconjugates were coinjected in xenograft-bearing nude mice, and biodistribution was determined at 3, 24, 48, 72, and 144 h after injection. (89)Zr was evaluated and compared with (18)F in phantom studies to determine linearity, resolution, and recovery coefficients, using a high-resolution research tomograph PET scanner. The potential of PET to quantify cmAb U36-N-sucDf-(89)Zr was evaluated by relating image-derived tumor uptake data (noninvasive method) to (89)Zr uptake data derived from excised tumors (invasive method). RESULTS: (89)Zr-N-sucDf-labeled and (88)Y-p-SCN-Bz-DOTA-labeled cmAb U36 showed a highly similar biodistribution, except for sternum and thigh bone at later time points (72 and 144 h after injection). Small differences were found in kidney and liver. Imaging performance of (89)Zr approximates that of (18)F, whereas millimeter-sized (19-154 mg) tumors were visualized in xenograft-bearing mice after injection of cmAb U36-N-sucDf-(89)Zr. After correction for partial-volume effects, an excellent correlation was found between image-derived (89)Zr tumor radioactivity and gamma-counter (89)Zr values of excised tumors (R(2) = 0.79). CONCLUSION: The similar biodistribution and the favorable imaging characteristics make (89)Zr a promising candidate for use as a positron-emitting surrogate for (90)Y.

Comorbid condition as a prognostic factor for complications in major surgery of the oral cavity and oropharynx with microvascular soft tissue reconstruction.

BACKGROUND: Identification of factors, especially comorbidity, that affect the incidence and severity of complications in head and neck cancer patients. METHODS: One hundred patients with an oral/oropharynx carcinoma undergoing composite resection and microvascular soft tissue transfer were analyzed. Patient data and tumor and treatment factors were recorded. Comorbidity was graded by an Adult Comorbidity Evaluation 27 (ACE-27) test. Postoperative complications were scored according to their severity. RESULTS: Comorbidity score ACE-27 grade 2 or higher was present in 47% of patients, whereas 33% had a clinically important complication develop. A comorbidity score of ACE-27 grade > or =2 was a strong predictor for complications (p <.001). There were no other predictors for postoperative complications. CONCLUSIONS: Comorbidity is of great importance for prediction of postoperative complications in head and neck cancer patients, especially an ACE-27 grade > or =2. It may be concluded from these results that prevention of complications should focus on comorbidities.

Phase I therapy study with (186)Re-labeled humanized monoclonal antibody BIWA 4 (bivatuzumab) in patients with head and neck squamous cell carcinoma.

PURPOSE: In previous studies, we have shown the potential of radioimmunotherapy (RIT) with (186)Re-labeled chimeric monoclonal antibody (MAb) U36 for treatment of head and neck cancer. A limitation of this anti-CD44v6 MAb, however, appeared to be its immunogenicity, resulting in human antichimeric antibodies in 40% of the patients. Aiming for a less immunogenic anti-CD44v6 MAb, the humanized MAb BIWA 4 (bivatuzumab) was introduced. In the present Phase I RIT study, we determined the safety, maximum tolerated dose (MTD), pharmacokinetics, immunogenicity, and therapeutic potential of (186)Re-labeled BIWA 4 in patients with squamous cell carcinoma of the head and neck. EXPERIMENTAL DESIGN: Twenty patients with inoperable recurrent and/or metastatic head and neck squamous cell carcinoma received a single dose of (186)Re-labeled BIWA 4 in radiation dose-escalation steps of 20, 30, 40, 50, and 60 mCi/m(2). Three patients received a second dose at least 3 months after the initial dose. After each administration, whole-body images as well as planar and tomographic images of the head and neck region were obtained, and the pharmacokinetics and the development of human antihuman antibody responses were determined. Radiation absorbed doses were calculated for whole body, red marrow, organs, and tumor. RESULTS: First and second administrations were all well tolerated, and targeting of tumor lesions proved to be excellent. The only significant manifestations of toxicity were dose-limiting myelotoxicity consisting of thrombo- and leukocytopenia and, to a lesser extent, oral mucositis (grade 2). Grade 4 myelotoxicity was seen in two patients treated with 60 mCi/m(2). The MTD was established at 50 mCi/m(2), at which level dose-limiting myelotoxicity was seen in one of six patients. Stable disease, varying between 6 and 21 weeks, was observed in three of six patients treated at the MTD level. The median tumor dose, recalculated to MTD level, was 12.4 Gy. The absorbed dose in red marrow was 1.82 +/- 0.11 cGy/mCi for males and 2.35 +/- 0.10 for females. Two patients experienced a human antihuman antibody response. Pharmacokinetics showed consistency across patients and within the three patients receiving (186)Re-BIWA 4 on two occasions. CONCLUSIONS: This study shows that (186)Re-labeled BIWA 4 can safely be administered, also in a repeated way. The MTD was established at 50 mCi/m(2). In comparison with the previously described anti-CD44v6 MAb U36, the humanized MAb BIWA 4 seems to be less immunogenic. The fact that antitumor effects were seen in incurable patients with bulky disease justifies the evaluation of RIT with (186)Re-labeled BIWA 4 in an adjuvant setting.

Long-lived positron emitters zirconium-89 and iodine-124 for scouting of therapeutic radioimmunoconjugates with PET.

Antibody-PET imaging might be of value for the selection of radioimmunotherapy (RIT) candidates to confirm tumor targeting and to estimate radiation doses to tumor and normal tissues. One of the requirements to be set for such a scouting procedure is that the biodistributions of the diagnostic and therapeutic radioimmunoconjugates should be similar. In the present study we evaluated the potential of the positron emitters zirconium-89 ((89)Zr) and iodine-124 ((124)I) for this approach, as these radionuclides have a relatively long half-life that matches with the kinetics of MAbs in vivo (t(1/2) 3.27 and 4.18 days, respectively). After radiolabeling of the head and neck squamous cell carcinoma (HNSCC)-selective chimeric antibody (cMAb) U36, the biodistribution of two diagnostic (cMAb U36-N-sucDf-(89)Zr and cMAb U36-(124)I) and three therapeutic radioimmunoconjugates (cMAb U36-p-SCN-Bz-DOTA-(88)Y-with (88)Y being substitute for (90)Y, cMAb U36-(131)I, and cMAb U36-MAG3-(186)Re) was assessed in mice with HNSCC-xenografts, at 24, 48, and 72 hours after injection. Two patterns of biodistribution were observed, one pattern matching for (89)Zr- and (88)Y-labeled cMAb U36 and one pattern matching for (124)I-, (131)I-, and (186)Re-cMAb U36. The most remarkable differences between both patterns were observed for uptake in tumor and liver. Tumor uptake levels were 23.2 +/- 0.5 and 24.1 +/- 0.7%ID/g for the (89)Zr- and (88)Y-cMAb U36 and 16.0 +/- 0.8, 15.7 +/- 0.79 and 17.1 +/- 1.6%ID/g for (124)I-, (131)I-, and (186)Re-cMAb U36-conjugates, respectively, at 72 hours after injection. For liver these values were 6.9 +/- 0.8 ((89)Zr), 6.2 +/- 0.8 ((88)Y), 1.7 +/- 0.1 ((124)I), 1.6 +/- 0.1 ((131)I), and 2.3 +/- 0.1 ((186)Re), respectively. These preliminary data justify the further development of antibody-PET with (89)Zr-labeled MAbs for scouting of therapeutic doses of (90)Y-labeled MAbs. In such approach (124)I-labeled MAbs are most suitable for scouting of (131)I- and (186)Re-labeled MAbs.

Assessment and clinical significance of micrometastases in lymph nodes of head and neck cancer patients detected by E48 (Ly-6D) quantitative reverse transcription-polymerase chain reaction.

The presence of lymph node metastases is the major determinant for prognosis in head and neck squamous cell carcinoma (HNSCC). It is at present unknown whether the same holds true for the presence of histologically undetectable micrometastases. We analyzed 456 histologically tumor-negative lymph nodes of 23 HNSCC patients without (pN0) and 18 patients with one or two tumor-positive lymph nodes (pN+) in their neck dissection specimens at histopathologic examination. To detect the presence of disseminated tumor cells and micrometastases in these lymph nodes, we used real-time quantitative RT-PCR with E48 (Ly-6D) transcripts as a squamous cell-specific molecular marker. The results were compared with histopathologic examination and clinical outcome. E48 transcripts were detected in lymph nodes of 5 (22%) of 23 patients in the pN0 group, and in histologically negative lymph nodes of 10 (56%) of 18 patients in the pN+ group. In the pN0 group, the presence of E48-positive lymph nodes was significantly associated with a distinctly poor cause-specific survival as compared with those with E48-negative lymph nodes. Our results indicate that E48 real-time quantitative RT-PCR is a suitable method for the detection of micrometastases in lymph nodes of patients with HNSCC. Moreover, detection of micrometastases seems clinically relevant but should be confirmed in a large multicenter trial.

89Zr immuno-PET: comprehensive procedures for the production of 89Zr-labeled monoclonal antibodies.

UNLABELLED: The use of immuno-PET, the combination of PET with monoclonal antibodies (mAbs), is an attractive option to improve tumor detection and mAb quantification. The long-lived positron emitter (89)Zr has ideal physical characteristics for immuno-PET, such as a half-life of 3.27 d, which is compatible with the time needed for intact mAbs to achieve optimal tumor-to-nontumor ratios. Thus far, a major limitation in the use of (89)Zr has been the lack of suitable methods for its stable coupling to mAbs. In this article, practical protocols for reproducible isolation of highly pure (89)Zr and the production of optimal-quality mAb-(89)Zr conjugates are provided. METHODS: (89)Zr was produced by a (p,n) reaction on natural yttrium ((89)Y), isolated with a hydroxamate column, and used for labeling of premodified mAbs. mAbs were premodified with a novel bifunctional derivative of the chelate desferrioxamine B (Df) via a new linker chemistry. To this end, Df was initially succinylated (N-sucDf), temporarily filled with Fe(III), esterified by use of tetrafluorophenol, and then directly coupled to mAbs. Chimeric mAb (cmAb) U36, directed against head and neck cancer, was used for in vitro and in vivo evaluation. The in vitro stability of cmAb U36-N-sucDf-(89)Zr was assessed in human serum, and its in vivo behavior was evaluated by biodistribution and PET imaging studies in tumor-bearing nude mice. A cmAb U36-Df-(89)Zr conjugate containing a previously described succinimide ring-thioether unit in the linker was used as a reference. RESULTS: (89)Zr was produced in large batches (6.5-13.5 GBq) and isolated with improved radionuclidic purity (>99.99%) and high yield (>94%). The Df-premodified mAbs gave (89)Zr-labeling efficiencies of 80% within 30 min, resulting in conjugates with preserved integrity and immunoreactivity. With respect to stability, the novel cmAb U36-N-sucDf-(89)Zr conjugate appeared to be superior to the reference conjugate. In vivo, the novel conjugate demonstrated selective tumor targeting, and on PET images obtained at 24, 48, and 72 h after injection of this conjugate, small tumors in the range of 19-154 mg were readily visualized. CONCLUSION: Methods were developed for improved purification of the long-lived positron emitter (89)Zr. Moreover, a novel bifunctional Df chelate was synthesized for the reproducible coupling of (89)Zr to mAbs. The suitability of such conjugates to detect millimeter-sized tumors in xenograft-bearing nude mice was demonstrated.

Basic principles, applications in oncology and improved selectivity of photodynamic therapy.

Photodynamic therapy (PDT) is a promising approach for the treatment of superficially localized tumors. This review starts with a summary of the basic principles of PDT, in which the current practice, the photochemical mechanisms, cellular and subcellular targets, as well as the most prominent photosensitizers are discussed. Next, the clinical results obtained with PDT for the treatment of a variety of tumor types are outlined. Unfortunately, most of these studies revealed a lack of tumor selectivity of the photosensitizers, which resulted in prolonged skin photosensitivity and severe normal tissue toxicity. The last section of this review, therefore, focuses on novel strategies designed to improve the tumor selectivity of photosensitizers.

Quantitative molecular detection of minimal residual head and neck cancer in lymph node aspirates.

PURPOSE: Staging of the clinically N(0) neck in patients with head and neck squamous cell carcinoma (HNSCC) using ultrasound-guided, fine needle aspiration cytology (USgFNAC) has a false-negative rate of approximately 20% that might be caused by inaccurate cytology. Molecular analysis of aspirate residues might reduce the false-negative rate, and we therefore set up a quantitative reverse transcription-PCR (Q-RT-PCR) assay based on TaqMan technology using the squamous cell-specific antigen E48 (Ly-6D) as molecular marker. EXPERIMENTAL DESIGN: The detection limit of the assay was determined in reconstruction experiments. The sensitivity of the assay was tested on cytological tumor-positive aspirate residues and the specificity on lymph node aspirate residues of noncancer controls. Subsequently, 235 lymph node aspirate residues of 64 HNSCC patients staged with USgFNAC were examined for the presence of E48 mRNA. E48 Q-RT-PCR results of the aspirated lymph nodes were compared with cytology and clinical outcome. RESULTS: The detection limit of E48 Q-RT-PCR was a single tumor cell in a background of 10(6) peripheral blood mononuclear cells. From the 41 aspirates that were not evaluable at cytology, 24 (59%) could be diagnosed with E48 Q-RT-PCR. In the 191 aspirates that were tumor negative or not evaluable at cytology, 8 samples from 6 patients were E48 positive. These results were confirmed by histology or clinical outcome in 3 of 6 patients. E48 Q-RT-PCR showed an increase in sensitivity from 56 to 67% and an increase in frequency of reached diagnosis from 97 to 100% compared with cytology. The specificity decreased from 100 to 92%. CONCLUSIONS: Real-time E48 Q-RT-PCR is an accurate technique for squamous cell detection in lymph node aspirates of HNSCC patients. The assay shows an increase in sensitivity and frequency of reached diagnosis compared with cytology. The test could be implemented routinely in USgFNAC to diagnose cases for which cytological examination is not conclusive.

Characterization of the human Ly-6 antigens, the newly annotated member Ly-6K included, as molecular markers for head-and-neck squamous cell carcinoma.

The E48 antigen is a successfully explored molecular marker for the diagnosis and therapy of HNSCC. The applicability of E48 as an HNSCC-associated antigen, however, is restricted due to its heterogeneous expression in 30% of tumors; and identification of additional target antigens is therefore desired. E48 belongs to the Ly-6 antigen family, comprising a group of highly homologous, low m.w., GPI-anchored surface proteins, of which some show tissue-restricted expression patterns. To identify novel human HNSCC-associated Ly-6 members with squamous cell-associated expression patterns, we performed comprehensive gene-screening consisting of BLAST searches within GenBank databases, followed by expression analysis. Using this approach, the Ly-6K gene could be annotated as a novel member of the human Ly-6 family. Expression of the human Ly-6 genes E48, Ly-6K, PSCA, GML, RIG-E, G6C and Ly-6H was prescreened by qualitative RT-PCR and subsequently analyzed by quantitative RT-PCR in normal keratinocytes, HNSCC cell lines, normal mucosa, HNSCC tumors as well as normal peripheral blood and bone marrow cells. PSCA was highly expressed in normal mucosa, but 100-fold decreased expression was seen in HNSCC. For Ly-6H, GML and G6C, no or very low expression was observed in keratinocytes and HNSCC. Expression of RIG-E was high in normal and malignant squamous cells and in peripheral blood and bone marrow cells, thus limiting its applicability as an HNSCC-associated marker. In contrast, besides the E48 gene, the Ly-6K gene also appeared to be selectively expressed in HNSCC and normal squamous cells. Moreover, expression of Ly-6K was shown in HNSCC cell lines, in which no E48 expression could be detected. These data justify further evaluation of Ly-6K as potential target antigen for the diagnosis and therapy of HNSCC.

Mutated p53 as a molecular marker for the diagnosis of head and neck cancer.

In total, 10-30% of patients with head and neck squamous cell carcinoma (HNSCC) develop local recurrences despite seemingly adequate tumour resection. This may result from minimal residual cancer (MRC): small numbers of tumour cells left behind in the surgical margins, undetectable by routine histopathology. In recent studies, p53 mutations have been considered as selective and sensitive DNA markers of cancer cells. There are two potential problems in using mutated-p53 DNA as a marker. Firstly, p53 mutations occur early in progression and might therefore detect unresected precursor lesions besides tumour cells. Secondly, DNA is a very stable biomolecule that might lead to false-positive results. These two potential problems have been evaluated in this study. Fifty patients with a radical tumour resection were included, of whom 30 showed a p53 mutation in the primary tumour. Histopathologically tumour-free surgical margins were quantitatively analysed for mutated p53 by molecular diagnosis (plaque assay) and subsequent (immuno)histopathology. p53 mutated DNA was detected in the surgical margins of 19/30 patients. Immunohistochemistry confirmed the presence of small tumour foci in 2/19 mutated p53-positive cases. In 7/19 cases, the tumour-specific p53 mutation was found in unresected dysplastic mucosal precursor lesions. Moreover, in a number of cases small p53-immunostained patches were detected, but the mutations found were never tumour-related. By screening contralateral exfoliated cells and plaque assays on RNA it was shown that detection of mutated-p53 DNA is prone to false-positive results. In conclusion, using p53 mutations as a marker, both MRC and unresected mutated p53-positive mucosal precursor lesions are detected within surgical margins. Molecular assessment of surgical margins using p53 mutations enables the selection of HNSCC patients at high risk for tumour recurrence, but tumour RNA seems at present to be a more specific biomolecule for analysis than tumour DNA.

Reinfusion of unprocessed, granulocyte colony-stimulating factor-stimulated whole blood allows dose escalation of 186Relabeled chimeric monoclonal antibody U36 radioimmunotherapy in a phase I dose escalation study.

PURPOSE: In an earlier Phase I radioimmunotherapy (RIT) study with rhenium-186-labeled chimeric monoclonal antibody (cMAb) U36 in patients with refractory head and neck squamous cell carcinoma, the maximum tolerated activity was established at 1.0 GBq/m2, at which bone marrow doses ranged from 0.7 to 1.1 Gy. In the present study, further dose escalation in RIT was evaluated using a facile method of reinfusion of granulocyte colony-stimulating factor (G-CSF)-stimulated unprocessed whole blood. EXPERIMENTAL DESIGN: Nine patients with recurrent or metastatic head and neck squamous cell carcinoma were treated at radiation dose levels of 1.0, 1.5, and 2.0 GBq/m2. Before RIT, G-CSF (10 microg/kg/day) was administered s.c. at home during 5 days. On day 6, just before administration of 186Relabeled cMAb U36, 1 liter of whole blood was harvested and kept unprocessed at 4 degrees C until reinfusion after 72 h. Blood samples were taken for analysis of pharmacokinetics and bone marrow dosimetry. Patients were evaluated for myelotoxicity and tumor response. RESULTS: Blood harvesting, RIT, and reinfusion of whole blood were well tolerated by all patients. G-CSF stimulation resulted in a mean of 0.41 x 10(6) CD34+ cells/kg (range, 0.15-0.83 x 10(6) CD34+cells/kg) and a mean committed colony-forming units granulocyte macrophage count of 5.62 x 10(4)/kg (range, 0.62-13.37 x 10(4)/kg). The mean biological half-life of 186Relabeled cMAb U36 in blood was 72.6 +/- 16.0 h, and bone marrow doses ranged from 2.1 to 2.8 Gy at the highest dose level. Myelotoxicity exceeding grade 3 was not observed. Stable disease was observed in five of nine patients, ranging from 3 to 5 months, and was still ongoing in one of these patients. CONCLUSIONS: This study indicates that a doubling of the maximum tolerated activity and bone marrow dose of 186Relabeled cMAb U36 can be achieved using reinfusion of G-CSF-stimulated unprocessed whole blood.

Assessment of patency of the internal jugular vein following neck dissection and microvascular flap reconstruction by power Doppler ultrasound.

The objective of this study was to assess patency of the internal jugular vein following modified radical or selective neck dissection and microvascular flap reconstruction by power Doppler ultrasound and its impact on free flap survival. In 23 patients who underwent selective or modified radical neck dissection and microvascular flap reconstruction the patency of the internal jugular vein was examined by power Doppler ultrasound on the first post-operative day and after follow-up of at least four months. On the first post-operative day in one patient partial thrombosis was found, while in the other 22 patients the internal jugular vein was normal patent. During follow-up in 17 (74 per cent) patients a normal patent internal jugular vein was found, while partial and complete thrombosis were found in three (13 per cent) patients each. On the first post-operative day 22 of the 23 (96 per cent) free flap veins were visualized. There was no free flap loss during follow-up. Power Doppler ultrasound is a valuable diagnostic technique for determination of internal jugular vein patency and may be useful as screening method or in case of clinical suspicion of thrombosis to determine internal jugular vein patency. Late internal jugular vein thrombosis may probably not effect free flap survival due to neovascularization.

Multiple head and neck tumors frequently originate from a single preneoplastic lesion.

The development of second primary tumors has a negative impact on the prognosis of head and neck squamous cell carcinoma. Previously, we detected genetically altered and tumor-related mucosal lesions in the resection margins in 25% of unselected head and neck squamous cell carcinoma patients (Tabor MP, Brakenhoff RH, van Houten VMM, Kummer JA, Snel MHJ, Snijders PJF, Snow GB, Leemans CR, Braakhuis BJM: Persistence of genetically altered fields in head and neck cancer patients: biological and clinical implications. Clin Cancer Res 2001, 7: 1523-1532). The aim of this study was to determine whether first and second primary tumors are clonally related and originate from a single genetically altered field. From 10 patients we analyzed the first tumor of the oral cavity or oropharynx, the >3-cm remote second primary tumor, and the mucosa from the tumor-free margins from both resection specimens. We compared TP53 mutations and loss of heterozygosity profiles using 19 microsatellite markers at chromosomes 3p, 9p, 13q, and 17p. In all patients, genetically altered mucosal lesions were detected in at least one resection margin from both first and second primary tumor. Evidence for a common clonal origin of the first tumor, second primary tumor, and the intervening mucosa was found for at least 6 of 10 patients. Our results indicate that a proportion of multiple primary tumors have developed within a single preneoplastic field. Based on different etiology and clinical consequences, we propose that independent second primary tumors should be distinguished from second field tumors, that arise from the same genetically altered field the first tumor has developed from.

Nonmalignant oral keratinocytes from patients with head and neck squamous cell carcinoma show enhanced metabolism of retinoic acid.

BACKGROUND AND OBJECTIVE: Retinoids show promise in the treatment of various (pre)malignancies, including head and neck squamous cell carcinoma (HNSCC). It has been shown that metabolic pathways of retinoids are important in their anticancer effect and that these pathways may change during HNSCC carcinogenesis. We have previously reported that HNSCC cells have a 17-fold greater turnover rate of retinoic acid (RA) than normal oral keratinocytes from noncancer controls, and that the formation of polar metabolites such as 4-oxo-RA and 4-hydroxy-RA is only seen in HNSCC cell lines. We aimed to establish whether this altered retinoid metabolism is an intrinsic characteristic of HNSCC patients. METHODS: The normal mucosa of cancer and noncancer patients was the source of keratinocyte cultures. The cells were exposed to RA for various time periods, and the levels of various retinoids were measured in the culture medium and cell pellets with reverse-phase liquid chromatography. RESULTS: Cells from cancer patients were morphologically normal and showed no genetic aberrations (i.e. loss of heterozygosity). The RA turnover rate in normal oral keratinocytes of cancer patients was 15 times higher (p = 0.003) than that in normal oral keratinocytes of noncancer controls, with average turnover rates of 218.6 and 14.8 pmol/mg protein/h, respectively. Specific profiles of RA metabolites were similar. CONCLUSION: The observed higher RA metabolism in noncancer cells of HNSCC patients suggests that individuals with a relatively high RA turnover have an increased risk of developing HNSCC.

Distant metastases of adenoid cystic carcinoma of the salivary glands and the value of diagnostic examinations during follow-up.

BACKGROUND: Adenoid cystic carcinoma (ACC) accounts for approximately 10% of all neoplasms of the salivary glands. Late distant metastases and local recurrences are responsible for a rather low long-term survival rate. AIM: To study the metastasizing pattern of ACC of the salivary glands in 51 patients. METHOD: Fifty-one patients with an ACC in the intraoral/sublingual salivary glands (n = 30), parotid gland (n = 8), submandibular gland (n = 2), maxillary sinus (n = 6), and nasal cavity (n = 5) have been studied. The male-female ratio was 1:1, the mean age was 54.3 (range, 19-81) years. In 30 cases treatment consisted of surgery and radiotherapy. In 13 cases surgery alone was carried out. The average follow-up time was 117.8 (range, 1-171) months. RESULTS: In 28 cases (54.9%) distant metastases occurred, and in 3 patients (5.8%) regional lymph node metastases occurred. In 12 of the 28 patients with distant metastases (42.8%), only the lungs were involved, whereas in 5 of the 28 patients (17.8%), the distant metastases occurred in other organs (eg, bones and brain). In 11 of the 28 patients (39.2%) metastases were found both in the lungs and other organs. Twenty-four of the 51 patients (47.0%) died because of their tumor. The average time between the occurrence of lung metastases and death was 32.3 months and between the occurrence of metastases elsewhere and death 20.6 months. CONCLUSION: Distant metastases of ACC of the salivary glands occur most often in the lungs. Although these lung metastases are the first to occur, these patients die later than patients with distant metastases in other organs. The value of annual chest films or other tests for the presence of distant metastases during follow-up after surgical removal of an ACC seems rather questionable.