J. Alain Kummer

Notch1 functions as a tumor suppressor in mouse skin

Notch proteins are important in binary cell-fate decisions and inhibiting differentiation in many developmental systems, and aberrant Notch signaling is associated with tumorigenesis. The role of Notch signaling in mammalian skin is less well characterized and is mainly based on in vitro studies, which suggest that Notch signaling induces differentiation in mammalian skin. Conventional gene targeting is not applicable to establishing the role of Notch receptors or ligands in the skin because Notch1−/− embryos die during gestation. Therefore, we used a tissue-specific inducible gene-targeting approach to study the physiological role of the Notch1 receptor in the mouse epidermis and the corneal epithelium of adult mice. Unexpectedly, ablation of Notch1 results in epidermal and corneal hyperplasia followed by the development of skin tumors and facilitated chemical-induced skin carcinogenesis. Notch1 deficiency in skin and in primary keratinocytes results in increased and sustained expression of Gli2, causing the development of basal-cell carcinoma–like tumors. Furthermore, Notch1 inactivation in the epidermis results in derepressed β-catenin signaling in cells that should normally undergo differentiation. Enhanced β-catenin signaling can be reversed by re-introduction of a dominant active form of the Notch1 receptor. This leads to a reduction in the signaling-competent pool of β-catenin, indicating that Notch1 can inhibit β-catenin-mediated signaling. Our results indicate that Notch1 functions as a tumor-suppressor gene in mammalian skin.

Performance of Immuno–Positron Emission Tomography with Zirconium-89-Labeled Chimeric Monoclonal Antibody U36 in the Detection of Lymph Node Metastases in Head and Neck Cancer Patients

Purpose: Immuno–positron emission tomography (PET), the combination of PET with monoclonal antibodies (mAb), is an attractive option to improve tumor detection and to guide mAb-based therapy. The long-lived positron emitter zirconium-89 (89Zr) has ideal physical characteristics for immuno-PET with intact mAbs but has never been used in a clinical setting. In the present feasibility study, we aimed to evaluate the diagnostic imaging performance of immuno-PET with 89Zr-labeled-chimeric mAb (cmAb) U36 in patients with squamous cell carcinoma of the head and neck (HNSCC), who were at high risk of having neck lymph node metastases. Experimental Design: Twenty HNSCC patients, scheduled to undergo neck dissection with or without resection of the primary tumor, received 75 MBq 89Zr coupled to the anti-CD44v6 cmAb U36 (10 mg). All patients were examined by computed tomography (CT) and/or magnetic resonance imaging (MRI) and immuno-PET before surgery. Six patients also underwent PET with 18F-fluoro-2-deoxy-d-glucose. Immuno-PET scans were acquired up to 144 hours after injection. Diagnostic findings were recorded per neck side (left or right) as well as per lymph node level (six levels per side), and compared with histopathologic outcome. For this purpose, the CT/MRI scores were combined and the best of both scores was used for analysis. Results: Immuno-PET detected all primary tumors (n = 17) as well as lymph node metastases in 18 of 25 positive levels (sensitivity 72%) and in 11 of 15 positive sides (sensitivity 73%). Interpretation of immuno-PET was correct in 112 of 121 operated levels (accuracy 93%) and in 19 of 25 operated sides (accuracy 76%). For CT/MRI, sensitivities of 60% and 73% and accuracies of 90% and 80% were found per level and side, respectively. In the six patients with seven tumor-involved neck levels and sides, immuno-PET and 18F-fluoro-2-deoxy-d-glucose PET gave comparable diagnostic results. Conclusion: In this study, immuno-PET with 89Zr-cmAb U36 performed at least as good as CT/MRI for detection of HNSCC lymph node metastases.

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.

Comparative molecular and histological grading of epithelial dysplasia of the oral cavity and the oropharynx.

Histological grading of epithelial dysplasia in the oral cavity and oropharynx is used to predict the risk for cancer and to determine the treatment strategy. This grading, however, is subjective and not well reproducible. Recent publications have shown that molecular markers are promising in cancer risk assessment. The aim of the present study was to compare classical histological and molecular grading and to relate these to the proliferation rate by quantitative assessment of Ki‐67 staining. Forty‐three samples were analysed from the margins of patients who had undergone resection of their squamous cell carcinoma in the oral cavity/oropharynx. Three experienced pathologists performed the histological grading. With the consensus score, 12 samples were classified as normal and 31 as dysplastic (21 mild, six moderate, and four severe). Loss of heterozygosity (LOH) was assessed in the same samples with 15 microsatellite markers at chromosomes 3p, 9p, 17p, 8p, 13q, and 18q, and was present in 28 of the 43 samples. Twenty‐four of the 28 cases (86%) with LOH were classified as dysplastic and four as normal. All ten samples with moderate and severe dysplasia and 14 of 21 samples with mild dysplasia contained LOH. In four of 12 biopsies classified as normal, LOH was found. A very striking and significant difference of the Ki‐67 index was observed between LOH‐positive and LOH‐negative cases, 36.6 ± 11.1% versus 19.4 ± 2.8% positive cells, respectively. In mild dysplasia, 13 of 14 lesions containing LOH had a higher Ki‐67 index than all seven lesions without LOH. Thus, in the oral cavity/oropharynx, LOH is more frequently found in the histologically higher‐grade lesions (moderate dysplasia or worse) and in the lower grade lesions when a high proliferation rate is present. Assessment of proliferation with Ki‐67 is a better surrogate for LOH than histological grading. Copyright

The estimation of tumor cell percentage for molecular testing by pathologists is not accurate

Molecular pathology is becoming more and more important in present day pathology. A major challenge for any molecular test is its ability to reliably detect mutations in samples consisting of mixtures of tumor cells and normal cells, especially when the tumor content is low. The minimum percentage of tumor cells required to detect genetic abnormalities is a major variable. Information on tumor cell percentage is essential for a correct interpretation of the result. In daily practice, the percentage of tumor cells is estimated by pathologists on hematoxylin and eosin (H&E)-stained slides, the reliability of which has been questioned. This study aimed to determine the reliability of estimated tumor cell percentages in tissue samples by pathologists. On 47 H&E-stained slides of lung tumors a tumor area was marked. The percentage of tumor cells within this area was estimated independently by nine pathologists, using categories of 0–5%, 6–10%, 11–20%, 21–30%, and so on, until 91–100%. As gold standard, the percentage of tumor cells was counted manually. On average, the range between the lowest and the highest estimate per sample was 6.3 categories. In 33% of estimates, the deviation from the gold standard was at least three categories. The mean absolute deviation was 2.0 categories (range between observers 1.5–3.1 categories). There was a significant difference between the observers (P<0.001). If 20% of tumor cells were considered the lower limit to detect a mutation, samples with an insufficient tumor cell percentage (<20%) would have been estimated to contain enough tumor cells in 27/72 (38%) observations, possibly causing false negative results. In conclusion, estimates of tumor cell percentages on H&E-stained slides are not accurate, which could result in misinterpretation of test results. Reliability could possibly be improved by using a training set with feedback.

Maintenance of Head and Neck Tumor Gene Expression Profiles upon Lymph Node Metastasis

Spread of cancer and development of solid metastases at distant sites is the main cause of cancer-related deaths. To understand and treat metastases, it is important to determine at which stages the most pivotal steps for development of metastases occur. In head and neck squamous cell carcinoma (HNSCC), metastasis nearly always occurs first in local lymph nodes before development of distant metastasis. Here, we have investigated gene expression patterns in HNSCC lymph node metastases using DNA microarrays. Several types of analyses show that the gene expression patterns in lymph node metastases are most similar to the corresponding primary tumors from which they arose, as long as samples contain sufficient proportions of tumor cells. Strikingly, gene expression patterns of metastatic primary HNSCC are largely maintained upon spread to the lymph node. Only a single gene, metastasis-associated gene 1 (MTA1), was found to show consistently changed expression between a large number of matched primary tumor-lymph node metastasis pairs. The maintained expression pattern includes the predictive signature for HNSCC lymph node metastasis. These results underscore the importance of the primary tumor gene expression profile for development and treatment of metastasis. The findings also agree with the concept that disseminated cancer cells alter the surrounding tissue into a metastatic environment that resembles the primary tumor microenvironment.

High numbers of granzyme B/CD8-positive tumour-infiltrating lymphocytes in nasopharyngeal carcinoma biopsies predict rapid fatal outcome in patients treated with curative intent.

This study determined whether tumour‐infiltrating lymphocytes (TILs) in nasopharyngeal carcinomas (NPCs) include activated cytotoxic T lymphocytes (CTLs) and whether the numbers of activated CTLs in these biopsies are related to clinical outcome. Moreover, the study investigated whether the numbers of activated CTLs are associated with the expression of MHC class I proteins and the granzyme B antagonist PI‐9 in the tumour cells. Forty‐three Indonesian NPC patients (T1–3, N1–3, M0), who were treated with curative intent by radiotherapy only, were studied. Tumour‐infiltrating activated CTLs were detected using antibodies against granzyme B, CD8, and CD56. Expression of MHC class I proteins and PI‐9 was also determined by immunohistochemistry. Granzyme B‐positive TILs were detected in all NPC biopsies. The presence of a high percentage (>25%) of granzyme B‐positive TILs appeared to be a very strong predictor of a rapid fatal clinical outcome, independent of stage. Complete absence of MHC class I heavy chain expression in tumour cells was observed in 11 of 31 evaluable cases and low levels were observed in seven additional cases. No association between MHC class I expression and the numbers of granzyme B‐positive TILs was observed. Expression of the granzyme B antagonist PI‐9 in tumour cells was detected in three cases. It is concluded that the presence of many granzyme B‐positive TILs in a selected group of Indonesian NPC patients is a strong and stage‐independent marker for a rapid fatal clinical outcome. Copyright

Validation of a Gene Expression Signature for Assessment of Lymph Node Metastasis in Oral Squamous Cell Carcinoma

PURPOSE Current assessment of lymph node metastasis in patients with head and neck squamous cell carcinoma is not accurate enough to prevent overtreatment. The aim of this study was validation of a gene expression signature for distinguishing metastasizing (N+) from nonmetastasizing (N0) squamous cell carcinoma of the oral cavity (OSCC) and oropharynx (OPSCC) in a large multicenter cohort, using a diagnostic DNA microarray in a Clinical Laboratory Improvement Amendments/International Organization for Standardization-approved laboratory. METHODS A multigene signature, previously reported as predictive for the presence of lymph node metastases in OSCC and OPSCC, was first re-evaluated and trained on 94 samples using generic, whole-genome, DNA microarrays. Signature genes were then transferred to a dedicated diagnostic microarray using the same technology platform. Additional samples (n=222) were collected from all head and neck oncologic centers in the Netherlands and analyzed with the diagnostic microarray. Human papillomavirus status was determined by real-time quantitative polymerase chain reaction. RESULTS The negative predictive value (NPV) of the diagnostic signature on the entire validation cohort (n=222) was 72%. The signature performed well on the most relevant subset of early-stage (cT1-T2N0) OSCC (n=101), with an NPV of 89%. CONCLUSION Combining current clinical assessment with the expression signature would decrease the rate of undetected nodal metastases from 28% to 11% in early-stage OSCC. This should be sufficient to enable clinicians to refrain from elective neck treatment. A new clinical decision model that incorporates the expression signature is therefore proposed for testing in a prospective study, which could substantially improve treatment for this group of patients.

Expression of Cellular FLICE Inhibitory Protein, Caspase-8, and Protease Inhibitor-9 in Ewing Sarcoma and Implications for Susceptibility to Cytotoxic Pathways

Purpose: Ewing sarcoma is a common pediatric bone tumor with an unfavorable prognosis for metastatic or recurrent disease. Cellular immunotherapy may provide new treatment options and depends on the cytolytic death receptor and perforin/granzyme pathways. Expression of death receptor pathway inhibitor cellular FLICE inhibitory protein (cFLIP), initiator caspase-8, and granzyme B inhibitor protease inhibitor-9 (PI-9) have been reported to determine susceptibility to cell- and chemotherapy-mediated killing in several tumor types. Here, we have studied their in vitro and in vivo expression in Ewing sarcoma and the implications for susceptibility to cytotoxicity. Experimental Design: Ewing sarcoma cell lines (n = 8) were tested for cFLIP, PI-9, and caspase-8 expression. Functional significance was tested by anti-Fas antibody (death receptor pathway) or natural killer cell (perforin/granzyme pathway) treatment. Immunohistochemistry was done on 28 sections from 18 patients. In half of the cases, sequential material, including metastases, was available. Results: Although all tested Ewing sarcoma cell lines expressed cFLIP, resistance to CD95/Fas–mediated apoptosis was only observed in two cell lines lacking caspase-8 expression. PI-9 was expressed at low levels in four of eight Ewing sarcoma cell lines, but positive cell lines remained susceptible to perforin/granzyme–mediated killing. In primary Ewing sarcoma, including metastases, cFLIP was abundantly expressed in 18 of 18 patients. Caspase-8 was expressed in all patients but showed more intertumoral and intratumoral variation in both intensity and heterogeneity of staining. PI-9, in contrast, was undetectable. Conclusions: The expression patterns of cFLIP, caspase-8, and the absence of PI-9 provide a rationale to preferentially exploit the perforin/granzyme pathway in cytotoxic therapies against Ewing sarcoma.

Spatial Differences in the Presence of FOXP3+ and GranzymeB+ T Cells between the Intra- and Extravascular Compartments in Renal Allograft Vasculopathy

Background Allograft vasculopathy (AV) and native atherosclerosis (NA) share the presence of a T-cell mediated inflammatory response, but differ in overall plaque morphology and growth rate. We studied the distribution and frequency of regulatory- and cytotoxic T cells in the arterial intima lesions in both conditions. Methodology/Principal Findings The study is based on vessels of 15 explanted human renal allografts with AV and 10 carotid artery plaques obtained at surgery. Distribution and frequency of cytotoxic- and regulatory T cells, as identified by the expression of Granzyme B (GrB) and FOXP3 was established in NA and AV. Furthermore, we compared the distribution of these cells in AV with the perivascular, interstitial renal tissue using immunohistochemistry. The total number of T cells was much higher in AV than in NA lesions (711±135 and 37±8 CD3/mm2 respectively, p<0.005, mean, ± SEM). Total numbers of FOXP3+ regulatory cells were also significantly increased in AV (36±10 and 0.9±0.3 FOXP3+/mm2 p<0.05), but relative numbers, expressed as a percentage of the total number of CD3+ T cells ((FOXP3+/CD3+) ×100), were not significantly different (4.6%±0.9 and 2.7%±0.6). GrB+ cells were rare in NA, but significantly increased numbers of GrB+ cells were found in AV lesions (85±24 and 0.2±0.1 GrB+/mm2, p<0.05). Perivascular tissues in the allografts showed a higher relative frequency of FOXP3+ cells than adjacent intimal lesions (14.0%±2.7 and 4.6%±0.9, respectively, p<0.05), but a lower frequency of GrB+ cytotoxic T cells (16.1%±2.7 and 22.6%±3.6, p<0.05). Conclusions Similar to NA, AV is characterized by a low frequency of intimal FOXP3+ regulatory T cells. Moreover, significant spatial differences exist in the distribution of functional T cell subsets between the intra- and extravascular micro-environments of the graft.