Guido Valente

Somatic mutations of the MET oncogene are selected during metastatic spread of human HNSC carcinomas

A metastatic cancer develops by accumulation of mutations in genes that control growth, survival and spreading. The latter genes have not yet been identified. In lymph node metastases of head and neck squamous cell carcinomas (HNSCC), we found mutations in the MET oncogene, which encodes the tyrosine kinase receptor for Scatter Factor, a cytokine that stimulates epithelial cell motility and invasiveness during embryogenesis and tissue remodeling. We identified two somatic mutations: the Y1230C, known as a MET germline mutation which predisposes to hereditary renal cell carcinoma, and the Y1235D that is novel and changes a critical tyrosine, known to regulate MET kinase activity. The mutated MET receptors are constitutively active and confer an invasive phenotype to transfected cells. Interestingly, cells carrying the MET mutations are selected during metastatic spread: transcripts of the mutant alleles are highly represented in metastases, but barely detectable in primary tumors. These data indicate that cells expressing mutant MET undergo clonal expansion during HNSCC progression and suggest that MET might be one of the long sought oncogenes controlling progression of primary cancers to metastasis.

Growth stimulation of colorectal carcinoma cells via the c‐kit receptor is inhibited by TGF‐β1

Activation of the receptor tyrosine kinase c‐kit by the kit‐ligand, also known as stem cell factor (SCF), is essential to melanocyte and germ cell development and during the early stages of hematopoiesis. Deregulated expression of c‐kit has been reported in malignancies affecting these lineages, i.e., myeloid leukemias, melanomas, and germ cell tumors. In addition, c‐kit and SCF are coexpressed in some breast and colorectal cancer (CRC) cells, raising the question of whether c‐kit serves an autocrine role in normal or malignant epithelial tissues. In this study, we demonstrate that human colorectal carcinomas, but not normal colorectal mucosa cells, coexpress SCF and c‐kit in situ. Expression of c‐kit was also observed in mucosa adjacent to colorectal tumor tissue. Consistent with a growth‐regulatory role of SCF in CRC cells, exogenous SCF stimulated anchorage‐dependent and anchorage‐independent growth in four out of five CRC cell lines. Exogenous transforming growth factor (TGF)‐β1 added at nanomolar concentrations to HT‐29 CRC cells, which express the type I, II, and III TGF‐β receptors, downregulated c‐kit expression to background levels and inhibited c‐kit–dependent proliferation. Similarly, TGF‐β1 inhibited SCF‐dependent proliferation of three first‐passage CRC cell lines. In summary, expression of the potential autocrine SCF/c‐kit axis is a tumor‐associated phenomenon in colorectal cancer that can be suppressed by TGF‐β1 in TGF‐β–responsive CRC cells. J. Cell. Physiol. 172:1–11, 1997.

Novel mutation in the ATP-binding site of the MET oncogene tyrosine kinase in a HPRCC family.

Germline mutations in the tyrosine‐kinase domain of the MET proto‐oncogene were found in patients suffering from the hereditary predisposition to develop multiple papillary renal‐cell carcinomas (hereditary PRCC, HPRCC). PRCCs are often multiple and bilateral even in patients without a family history. We analyzed the germline of patients carrying multiple or single papillary tumors with and without family history. One patient had a familial cancer and carried a novel (V1110I) germline MET mutation, located in MET gene exon 16. This mis‐sense mutation was found in affected members of this patients family. Interestingly, the V1110I mutation is located in the ATP‐binding site of the MET kinase and is homologous to the V157I mutation that triggers the sarcomagenic potential of the v‐erbB oncogene. The V1110I mutated MET receptor is an active kinase and transforms NIH‐3T3 fibroblasts in the in vitro assays. Patients without familiality did not show germline mutations in the MET kinase domain, showing that multiple and bilateral papillary kidney tumors develop in the absence of these mutations. In conclusion, we describe a new mutation in the MET oncogene kinase domain, associated to HPRCC, affecting an amino‐acid residue critical for kinase activation in different oncogenes. Int. J. Cancer 82:640–643, 1999.

Murine cytomegalovirus replication in salivary glands is controlled by both perforin and granzymes during acute infection.

The course of mouse cytomegalovirus (MCMV) infection was compared between wild‐type and mutant C57BL / 6 (B6) mice deficient in either RAG‐2, perforin, granzyme A, granzyme B or combinations thereof at two time points post infection (p. i.). At day 15 p. i., virus titers were similarly elevated in salivary glands of all mutant, but not wild‐type B6 mice and undetectable in lung and spleen tissues of any of the mouse strains. Significant pathological alterations were only seen in salivary glands and spleen from RAG2– / –, but not in those from other mice whereas few inflammatory foci were observed in lung tissues of all mice except B6. At day 30 p. i., elevated virus titers were observed only in salivary glands, lung and spleen from RAG2– / –, but in none of the other mice, and were accompanied by extended pathological alterations in all three organs. The data extend previous reports on the critical role of NK / CD8+ T cells in the early control of MCMV infection by showing that both perforin and granzymes A / B contribute to viral elimination in salivary glands; however, neither of the three molecules alone seem to be indispensable for the final control of infection.

Staging of head and neck squamous cell carcinoma using the MET oncogene product as marker of tumor cells in lymph node metastases.

In head and neck squamous cell carcinomas (HNSCC), metastasis to cervical lymph nodes is a major determinant of patient outcome. To detect metastases, we used the MET oncogene as marker, which encodes the receptor for hepatocyte growth factor/scatter factor, mediating epithelial cell motility and invasiveness. The MET gene is expressed in epithelia and over‐expressed in carcinomas of specific histotypes, but not in lymphatic tissue. A total of 151 lymph nodes from 20 squamous cell carcinomas were studied with both in‐depth histology and end‐point and real‐time quantitative RT‐PCR. MET‐encoded sequences were found in 61 of 151 nodes (40%), of which 24 (16%) were found metastatic by in‐depth histopathology. Parallel routine histopathologic analysis of 654 lymph nodes from the same cases identified 36 metastases (5%). Real‐time quantitative RT‐PCR was used to measure MET gene‐specific mRNA in normal tissues, primary tumors and lymphatic metastases and showed a 2–8‐fold increased expression in tumor cells which metastasize. RT‐PCR for 3 cytokeratins expressed in HNSCC (K4, K10 and K13) proved to be less sensitive in detecting occult lymphatic metastases. Western blot analysis demonstrated the presence of the full‐size MET receptor in primary tumors and lymph node metastases; immunohistochemistry showed receptor localization in tumor cells. Altogether, these data demonstrate that the MET gene product is a valuable marker with which to detect occult tumor cells in lymph nodes, thanks to its high expression in metastatic cells. After RT‐PCR analysis we were able to attribute a more advanced stage to 10 out of 20 HNSCC cases, including 5 cases classified as tumor‐free after routine histopathology. Int. J. Cancer 89:286–292, 2000.

MIB-1, Ki67, and PCNA scores and DNA flow cytometry in intermediate grade malignant lymphomas.

AIMS--To verify the correlation between MIB-1, Ki67, and proliferating cell nuclear antigen (PCNA-PC10) scores and S-phase fraction in intermediate grade non-Hodgkins lymphomas (Working Formulation F); and their reliability in differently processed tissues. METHODS--Forty one non-Hodgkins lymphomas were classified as (F) intermediate grade malignant lymphomas according to the Working Formulation; mitotic counts and percentage of large cells were assessed for each case. Sections from formalin fixed, paraffin wax embedded tissues were stained with anti MIB-1 monoclonal antibody, after microwave oven processing, and anti-PCNA (PC10) monoclonal antibody using an avidin-biotin immunoperoxidase (ABC) method. One thousand cells from 10 representative fields were scored. Frozen sections from surgical specimens were stained with Ki67 monoclonal antibody using the ABC method; the fraction of Ki67 positive cells was calculated scoring 1000 cells. Flow cytometry analysis (FCM) was performed on cell suspensions from fresh tissues. Correlations between data were estimated using linear regression. RESULTS--A linear correlation was found between MIB-1 and Ki67 scores (r = 0.92; p < 0.00001); between MIB-1 and PCNA scores (r = 0.79; p < 0.00001); and between MIB-1 score and S-phase fraction (r = 0.51; p = 0.0006). A linear correlation was also found between Ki67 and PCNA scores (r = 0.85; p < 0.00001); between Ki67 score and S-phase fraction (r = 0.6; p = 0.0002); and between PCNA score and S-phase fraction (r = 0.74; p < 0.00001). A correlation was found between mitotic counts and MIB-1 (r = 0.56; p = 0.0001), PCNA (r = 0.51; p = 0.0007), or Ki67 scores (r = 0.47; p = 0.002); between the percentage of large cells and MIB-1 (r = 0.49; p = 0.0009), PCNA (r = 0.6; p = 0.00003), and Ki67 scores (r = 0.53; p = 0.0003) and S-phase fraction (r = 0.55; p = 0.0002). CONCLUSION--MIB-1, Ki67, and PCNA (PC10) scores and S-phase fraction are highly correlated and equally well represent the proliferative activity of intermediate grade non-Hodgkins lymphomas in differently processed material. MIB-1 and PCNA stains can be applied even on small biopsy specimens. MIB-1 produces homogenous staining without background; it also strongly stains mitotic figures. It can be performed on routinely processed tissues, permitting the simultaneous evaluation of the morphology and tumour cell kinetics. The wide standard deviations of the proliferative indices found for intermediate grade NHL suggest that this category probably includes various degrees of malignancy.

Immunohistochemical Expression Analysis of the Human Interferon-Inducible Gene IFI16, a Member of the HIN200 Family, Not Restricted to Hematopoietic Cells

This is the first description of an extensive immunohistochemical analysis of interferon (IFN)-inducible gene IFI16 expression in normal tissues. Immunohistochemical detection of IFI16 in paraffin-embedded tissues is achieved by using a polyclonal antibody raised against its C-terminal fragment that recognizes its three closely migrating isoforms in Western blotting. The results clearly indicate that IFI16 expression is not restricted to the hematopoietic compartment. In normal adult human tissues, it is prominent in stratified squamous epithelia and particularly intense in parabasal cells in the proliferating compartments, but it gradually decreases in the more differentiated suprabasal layers. Understanding of IFI16 expression in vivo is essential for interpretation of the results obtained from in vitro studies and elucidation of its physiologic role. The constitutive expression and wider distribution of IFI16 in normal human tissues, not restricted to the hematopoietic compartment, strongly support the possibility of an important role in cell differentiation that can be further modulated by other stimuli, such as IFN.

Autophagy-active beclin-1 correlates with favourable clinical outcome in non-Hodgkin lymphomas

The expression of beclin-1, an oncosuppressor monoallelically deleted in >60% epithelial cancers, has been shown to be developmentally regulated in T and B lymphocytes. By interacting with either bcl-2 or class III phosphatidyl-inositol-3-phosphate kinase, beclin-1 regulates apoptosis and autophagy, two processes crucial for lymphatic tissue homeostasis. We analyzed the potential link between beclin-1-mediated autophagy and the malignant behaviour of lymphomas. The tissue expression of beclin-1 was analyzed in a large series of non-Hodgkin lymphomas and correlated with patients clinical outcome. By immunofluorescence, beclin-1 staining showed faintly detectable and diffusely distributed in the cytoplasm (regarded as negative) or confined to the perinuclear region as large and brilliant puncta suggestive of macro-aggregate reactivity (regarded as positive). The positive expression of beclin-1 well correlated with the presence of LC3-positive autophagic vacuoles and was inversely correlated with the expression of bcl-2. Non-Hodgkin lymphomas in which ⩾20% of tumour cells expressed high level of beclin-1 aggregates were associated with a complete (57%) or partial (35%) remission. The 5-year overall survival probability, calculated by the Kaplan–Meier method, was 92% and 42% in beclin-1-expressing non-Hodgkin lymphomas with ⩾20% and <20% positive cells, respectively (log-rank test, P<0.000.1). In Cox multivariate analysis, the level of beclin-1 expression, adjusted for patients age and pathologic stage, revealed to be significantly correlated with patients survival (P<0.0001). This is the first demonstration of the involvement of beclin-1 and autophagy in the clinical behaviour of non-Hodgkin lymphomas. The present data are compatible with the hypothesis that non-Hodgkin lymphomas with upregulated autophagy are more responsive to chemotherapy and indicate that beclin-1 could be a valuable independent prognostic factor in this heterogeneous group of tumours.

Detection of MET oncogene/hepatocyte growth factor receptor in lymph node metastases from head and neck squamous cell carcinomas.

The c-MET oncogene encodes the receptor for hepatocyte growth factor/scatter factor (HGF/SF), which is known to stimulate the invasive growth of epithelial cells cultured in vitro. TheMet/HGF receptor is a heterodimeric transmembrane tyrosine kinase, which is a prototype for a new family of growth factor receptors. The c-MET oncogene is expressed in several types of epithelial tissue including keratinocytes and is over-expressed in a number of human carcinomas. Studies on various carcinoma cell lines have shown that over-expression and structural alteration of the receptor result in its activation and confer tumorigenesis. We have studiedMet/HGF receptor expression in tissue specimens from 34 patients with head and neck squamous cell carcinomas (HNSCC) and in 17 regional lymph node metastases. Western blot analysis was employed, using monoclonal antibodies directed against either the intracellular or extracellular domain of the receptor. Each sample was compared to its normal counterpart. The receptor did not show any major structural alterations in HNSCC tissues, but its expression was increased from 2- to 50-fold in about 70% of tumors. Immunohistochemistry then showed that the same antibodies stained only a few cells in the basal layer of normal squamous epithelium but intensely marked tumor cells. In the lymph node metastases of Met-positive tumors, receptor expression was maintained and sometimes increased with respect to primary tumors. Immunohistochemical analysis of the metastatic lymph nodes showed that cells were negative in the normal lymphatic tissue and strongly stained in tumor cells. Over-expression of theMet/HGF receptor was found at all tumor stages but was more significant in those associated with enlarged or multiple (N2–N3) lymph node metastases. These data show that expression of theMet/HGF receptor may be involved in the progression of HNSCC towards a metastatic phenotype and may be a useful marker of head and neck tumor cell spread to regional lymph nodes.

Can Ki67 immunostaining predict response to radiotherapy in oral squamous cell carcinoma

AIMS--To determine whether immunohistochemical evaluation of the abatement of proliferating cells after a first course of radiotherapy could predict the final response to treatment in oral squamous cell carcinoma (SCC). METHODS--Frozen sections from 31 cases of histologically confirmed oral SCC were stained with the monoclonal antibody Ki67 at diagnosis and after 10 Gy of radiotherapy. The percentage difference of Ki67 positive cells among the biopsy specimens taken at the beginning and after 10 Gy was correlated with the clinical response obtained at the end of the treatment and its significance determined. RESULTS--The percentage of Ki67 positive cells at diagnosis had no significant correlation with the final therapeutic result of radiotherapy. By contrast, the 32% difference of proliferating cells after 10 Gy of radiotherapy significantly differentiated responders from non-responders (p < 0.05). Furthermore, the abatement of the growth fraction after 10 Gy of radiotherapy was significantly correlated with the complete response (p < 0.01). CONCLUSIONS--These data show that the immunohistochemical evaluation of the abatement of Ki67 positive cells after 10 Gy of radiotherapy provides an independent variable of responsiveness to radiotherapy, allowing a reliable prediction of the final therapeutic result to be made.