Correlation between c-erbB-4 receptor expression and response to gemcitabine-cisplatin chemotherapy in non-small-cell lung cancer.

BACKGROUND: While the overexpression of c-erbB gene family in several malignancies is associated with poorer prognosis, the association between the expression of the cellular markers and the response to chemotherapy is not yet clear. In this study we investigated the expression of c-erbB-4 receptor in NSCLC and correlated it with the response to gemcitabine-cisplatin combination chemotherapy. PATIENTS AND METHODS: Forty-three NSCLC patients with histologically or cytologically proven disease were treated with gemcitabine-cisplatin combination chemotherapy. Immunohistochemical stains for c-erbB-4 receptor were performed in 20 cases on paraffin sections using the avidin-biotin-peroxidase method. RESULTS: Two patients achieved complete response (5%), and 16 achieved partial response (37%) yielding an overall objective response rate of 42%. Minimal response was observed in seven patients (16%) and disease stabilization in 7%. Immunohistochemical stain was positive for the presence of c-erbB-4 receptor in 25% of patients, and negative in 75%. No response was documented in c-erbB-4 positive patients (0 of 5) while an objective response (complete, partial or minimal) was seen in 11 of 15 (73%) c-erbB-4 negative patients. Negative stain for c-erbB-4 significantly favored response to gemcitabine-cisplatin combination (P < 0.01). CONCLUSION: C-erbB-4 expression status showed no correlation with survival and cannot be accepted at this time as a guiding factor for therapeutic management. These interesting results deserve further evaluation in a large-scale prospective trial before treatment recommendations on the basis of c-erbB-4 presence can be finally made.

Correlative study of preoperative transthoracic core cutting needle biopsy of focal thoracic lesions and thoracotomy findings.

We conducted a retrospective study to determine the clinical utility of percutaneous core needle biopsy (PCNBx) in 36 patients with peripheral focal chest lesions who later underwent thoracic surgery for diagnostic or therapeutic purposes. PCNBx provided adequate material in 31/36 cases, giving an overall sample yield of 86.1%. PCNBx diagnosis was confirmed by surgery in 27/31 patients, giving a sensitivity of 91.6% and a specificity of 87.5%. In 4 patients, the lesions were misdiagnosed by PCNBx. In 5 patients with benign processes, surgical intervention could have been avoided, according to PCNBx results. The rate of PCNBx-induced pneumothorax was 11%. Radiologically guided PCNBx is an easy and safe procedure that can provide important preoperative diagnostic information and can circumvent the need for exploratory diagnostic surgery in cases of benign lesions. PCNBx also allows better preoperative planning in cases of malignancy.

Percutaneous core needle biopsy vs. fine needle aspiration in diagnosing benign lung lesions.

OBJECTIVE: To determine the diagnostic value of percutaneous core needle biopsy (PCNB) in comparison with fine needle aspiration (FNA) in patients with benign pulmonary lesions. STUDY DESIGN: A retrospective review was undertaken of computed tomography-guided PCNBs and FNAs performed between 1988 and 1997. Both FNA and PCNB biopsies were carried out sequentially at the same visit in every patient. RESULTS: A specific benign diagnosis was made in 10/60 cases (16.7%) by FNA and in 49/60 (81.7%) by PCNB. PCNB findings resulted in significant modification of the diagnosis established by FNA. The only significant complication encountered was pneumothorax, at a rate of 11.7%, which is compatible with that reported in the literature for complications induced by FNA alone. CONCLUSION: Radiologically guided PCNB is a safe procedure, can provide sufficient histologic material for a specific diagnosis of peripheral lung disease and can avoid more-invasive surgical procedures in many cases. Our experience demonstrated that the histologic analysis provided by PCNB can greatly increase the diagnostic accuracy in benign pulmonary diseases as compared with the yield of FNA.

Percutaneous core needle biopsy in the diagnosis of mediastinal tumors.

OBJECTIVE: to determine the contribution of percutaneous core cutting needle biopsy (PCNB) in the diagnosis of mediastinal tumors. DESIGN: retrospective review of 70 patients with mediastinal lesions who underwent CT-guided PCNB between 1988 and 1996. RESULTS: PCNB provided adequate material in 62/70 cases, giving a total sample rate of 88.6%. Of these 62 patients, 57 were diagnosed correctly by PCNB whereas 5/62 were misdiagnosed as nonspecific inflammation, providing an overall sensitivity of 91.9%. PCNB established a specific histologic diagnosis in 90.3% of the patients, mainly in cases of lymphoma, bronchogenic carcinoma, and thymoma. Pneumothorax was the most commonly encountered complication (11%). Hemoptysis (30-50 ml) occurred in only one (1.6%) of the patients. CONCLUSION: CT guided PCNB is an easy and safe procedure which can provide a precise diagnosis in the majority of mediastinal tumors and can obviate the need for exploratory thoracic surgery in cases which are medically treatable or non-resectable.

Percutaneous core cutting needle biopsy compared with fine-needle aspiration in the diagnosis of peripheral lung malignant lesions: results in 156 patients.

BACKGROUND: The authors attempted to determine the utility of percutaneous core needle biopsy (PCNB) compared with fine-needle aspiration (FNA) in the diagnosis of peripheral lung carcinoma. METHODS: A retrospective review was undertaken of 156 computed tomography (CT)-guided PCNBs and FNAs of malignant lung lesions between 1988-1996. Both CT-guided FNA and PCNB biopsies were performed sequentially at the same visit for each subject. RESULTS: The authors reviewed 156 malignant lesions whose specific diagnosis was obtained by FNA in 133 cases (85.3%) and by PCNB in 121 cases (77.6%) (P < 0.05). PCNB confirmed the FNA diagnosis in 90 patients (57.7%), provided additional information in 17 patients (10.9%), and was less informative than FNA in 35 patients (22.4%), mostly those with nonsmall cell carcinoma. The PCNB was marginally superior to FNA only in cases of metastatic carcinoma. The only significant complication encountered was a 24% rate of pneumothorax, which is comparable to the reported rate for FNA alone-induced complications. CONCLUSIONS: PCNB offers no substantial advantage over FNA in the evaluation of peripheral malignant lung lesions. Therefore, the authors recommend the use of FNA biopsy as the initial diagnostic procedure in all cases of suspected malignancy. The use of the PCNB technique is recommended when the diagnosis of malignancy by FNA is uncertain, or when a more detailed characterization of the lesion is required.

Additional information from percutaneous cutting needle biopsy following fine-needle aspiration in the diagnosis of chest lesions.

OBJECTIVE: To determine the contribution of percutaneous cutting needle biopsy (PNB) subsequent to fine-needle aspiration (FNA) in the diagnosis of chest lesions. DESIGN: A retrospective review of 220 patients who underwent CT-guided FNA followed immediately by PNB performed at our center between 1988 and 1995 was undertaken. Thirty-eight patients were excluded because FNA and/or PNB specimens were nondiagnostic, yielding a study group of 182 patients. RESULTS: A diagnosis of malignancy was made in 141 (77.5%) and nonmalignancy in 41 (22.5%) cases. The yield of histospecific diagnosis due to FNA was marginally higher than PNB in malignant lesions (86.5% vs 78%, respectively). In contrast, PNB was superior to FNA for the histospecific diagnosis of benign lesions (87.8% for PNB vs 31.7% for FNA, p<0.00001) and lymphomas (88% for PNB vs 56% for FNA, p<0.05). In 58.8% of the patients with benign lesions and in 37.5% of the patients with lymphoma, PNB performances altered clinical management, either by avoiding further surgery or allowing specific medical treatment. Pneumothorax occurred in 24.7% of the cases but only five patients (2.7%) required hospitalization. CONCLUSION: PNB is extremely effective for making a specific diagnosis in benign lesions compared with FNA. PNB does not increase the yield of histospecific diagnosis for malignant lesions except for the subset of lymphoma, where it seems to provide important additional information in many instances. We recommend that FNA be performed as the initial procedure, followed by PNB in cases of equivocal diagnosis of carcinoma, for lymphoma and for suspected benign lesions.

Biological behavior and cell properties of new AKR lymphoma malignancy variants.

The AKR lymphoma-leukemia is a T lymphocyte neoplasm, most suitable as a model for human T cell malignancies. We have been interested in the process of tumor progression in the AKR lymphoma system. In the present study, two newly isolated variants, the TAU-42 and TAU-44, were characterized with respect to their biological behavior, by comparing them to a previously studied low-malignancy variant, the TAU-39. While the TAU-44 variant formed large s.c. local tumors, the TAU-42 variant formed only small growths or none at all. The TAU-42 lymphoma was found to have the highest malignant potential: it displayed very marked dissemination to spleen, lymph nodes, liver and lungs. The TAU-44 variant had an intermediate degree of metastatic potential but presented a predilection for spread to lymph nodes and spleen and was sometimes found to metastasize to peculiar organs, such as heart and pancreas. Cells derived from the different lymphoma variants varied in their immunophenotype: the highly malignant variant cells expressed more CD4 antigen than the low-malignancy one. The opposite was observed with regard to CD8. The variant cells also differed in their migrating capacity, the more malignant one exhibiting a higher motile activity. Studies on the tumor progression model of AKR lymphoma might contribute to the elucidation of the features determining the aggressiveness of T lymphocytic malignancies.

Comparison of growth rate of two B16 melanomas differing in metastatic potential in young versus middle-aged mice.

The rise of cancer frequency as a function of age is a well-established fact. The aspect of the host age-tumor progression relationship, namely the slower metastatic spread in aged patients, has been investigated to a lesser extent. In the present study, we examined whether host-age-dependent growth rate varies with metastatic capacity of the tumor. The parental B16 and the B16/Col/R, a highly metastatic variant, were employed. A more pronounced growth of both tumors in young as compared to middle-aged mice was found. However, the differential growth in middle-aged versus young mice was more evident in the highly metastatic variant. According to the tumor size data, a sixfold growth reduction in middle-aged mice was observed with B16/Col/R and an only twofold growth reduction was seen with the B16 melanoma. The data might eventually contribute to the finding of more appropriate treatment modalities for the middle-aged cancer patient.

Metastatic potential and multidrug resistance correlation in the B16 melanoma system.

The question of whether metastatic potential and drug resistance are related phenotypes was addressed by comparing the biological behavior of the parental B16 melanoma and a multidrug resistant variant derived from it, the B16/Col/R. A more pronounced metastatic spread to lungs was observed in mice inoculated i.v. with the B16/Col/R variant than in those bearing the parental line. In addition, in the mice injected with the drug resistant melanoma, unusual tumor masses were observed. Large abdominal and spinal cord growths were seen with the MDR variant but not encountered in mice inoculated with the original B16 melanoma. We further attempted to test the capacity of the two cell types to perform several cellular functions relevant to the metastatic process. The B16/Col/R cells displayed a higher aggregability and cell motility than did the B16 cells. Adherence to endothelial cells was higher in the parental line than in the B16/Col/R, possibly supporting a more efficient extravasation of the variant cells. The drug resistant variant displayed a higher capacity to grow locally in kidney, spleen, cecum and peritoneum, as compared to the parental melanoma, indicating a higher ability of homing and growth in these potential target organs for metastasis. A correlation between metastatic potential and multidrug resistance appears therefore to exist in the system examined.

Metastasis-related cell functions in primary and metastatic tumor cells of AKR lymphoma.

The metastatic phenotype is of extreme complexity. To complete all the stages of metastasis, the tumor cell must possess a whole series of functional abilities. Multiple biological markers are therefore needed to achieve a deeper understanding of the metastatic phenotype. In the present study we compared primary (PT) to metastatic tumor (MT) cells of two AKR lymphoma variants with respect to several cellular functions relevant to various steps of tumor dissemination. The MT cells of the TAU-44 variant had a higher capacity than the PT cells to attach to endothelial monolayers and ECM, exhibited a more elevated motility and a higher capacity to grow in the spleen as a metastatic target organ. However, the TAU-44-MT cells had a lower ability to grow in the kidney than the PT cells. The TAU-33-MT cells had a higher ability to attach to endothelial cells and to grow in both spleen and kidney but were less motile compared to PT cells. Metastatic cells showed, on the whole, higher ability to perform in most, but not all, stage-specific models than primary tumor cells.

Predominance of the metastatic phenotype in hybrids formed by fusion of mouse and human melanoma clones.

The fusion of mouse and human melanoma cells that were tumorigenic but had different metastatic capabilities resulted in hybrids that were metastatic when injected intravenously or subcutaneously into nude mice, regardless of whether it was the mouse or the human melanoma clone that was metastatic. The H7 hybrid line, formed by fusing murine nonmetastatic K1735 C19 cells with human metastatic A375 C15 cells retained high metastatic potential over more than 50 sub-culture passages, suggesting that the dominant metastatic phenotype in these hybrid cells was stable. Using fluorescent in situ hybridization (FISH), human chromosome 17 was consistently identified as the predominant human chromosome in the majority of H7 cells tested between passages 20 and 60. Western blot analysis showed that the hybrid cells expressed human nm23 protein, indicating that at least one gene on the human chromosome 17 was functional. Immunocytochemistry and immunoprecipitation showed that the metastatic A375 C15 and H7 cells expressed p53 protein, but that the nonmetastatic K1735 C19 melanoma cells did not. Sequencing the human p53 gene in A375 C15N and H7 showed mutations in exon 7. Using a bioassay technique, we showed that K1735 C19 cells can spread from subcutaneous tumors to the lungs of nude mice yet fail to form metastases. With the addition of human chromosome 17 from A375 C15 cells, which carries a mutant p53 gene, the cells readily formed lung metastases. In this melanoma hybrid, a mutant p53 gene appears to confer a survival advantage on cells arrested in the lungs of nude mice and thus contributes to the growth of metastatic cells.

Metastasis-associated cell functions in AKR lymphoma malignancy variants.

Multiple structural and functional cell properties are responsible for the complex phenotype of the metastatic cell. Cells derived from AKR lymphoma malignancy variants were compared with regard to several cellular functions relevant to the metastatic behavior. Metastatic behavior correlated with relevant in vitro cell activities, such as cell motility, homotypic and heterotypic adhesion as well as proteolytic activity. The Variant displaying the highest degree of malignancy, TAU-42, exhibited the most elevated ability to perform almost each of the cell functions examined: motility, aggregability, capacity to adhere to endothelium and extracellular matrix, heparanase and protease activity and ability to grow in the spleen. The TAU-33 variant was second in rank in the performance of the above activities. The TAU-44 variant cells, the third in rank of malignancy, displayed some peculiar functional behavior: while manifesting the lowest homotypic adherence and heparanase activity, they had a high predilection for growth in kidney.

Modulation of tumor cell response to chemotherapy by the organ environment.

The outcome of cancer metastasis depends on the interaction of metastatic cells with various host factors. The implantation of human cancer cells into anatomically correct (orthotopic) sites in nude mice can be used to ascertain their metastatic potential. While it is clear that vascularity and local immunity can retard or facilitate tumor growth, we have found that the organ environment also influences tumor cell functions such as production of degradative enzymes. The organ microenvironment can also influence the response of metastases to chemotherapy. It is not uncommon to observe the regression of cancer metastases in one organ and their continued growth in other sites after systemic chemotherapy. We demonstrated this effect in a series of experiments using a murine fibrosarcoma, a murine colon carcinoma, and a human colon carcinoma. The tumor cells were implanted subcutaneously or into different visceral organs. Subcutaneous tumors were sensitive to doxorubicin (DXR), whereas lung or liver metastases were not. In contrast, sensitivity to 5-FU did not differ between these sites of growth. The differences in response to DXR between s.c. tumors (sensitive) and lung or liver tumors (resistant) were not due to variations in DXR potency or DXR distribution. The expression of the multidrug resistance-associated P-glycoprotein as determined by flow cytometric analysis of tumor cells harvested from lesions in different organs correlated inversely with their sensitivity to DXR: increased P-glycoprotein was associated with overexpression of mdr1 mRNA. However, the organ-specific mechanism for upregulating mdr1 and P-glycoprotein has yet to be elucidated.

Inverse correlation between expression of inducible nitric oxide synthase activity and production of metastasis in K-1735 murine melanoma cells.

The purpose of these studies was to determine whether the induction of NO synthase activity in murine K-1735 melanoma cells correlated with their metastatic potential. Nonmetastatic, metastatic, and somatic cell hybrids (produced by fusion of nonmetastatic and metastatic cells) were injected i.v. into syngeneic C3H/HeN mice. Metastatic cells survived to produce experimental lung metastases, whereas nonmetastatic cells did not. The various clones and somatic cell hybrids were incubated in vitro with combinations of tumor necrosis factor, interleukin 1, gamma-interferon, and lipopolysaccharide. Nonmetastatic cells exhibited high levels of inducible NO synthase activity and NO, whereas metastatic cells did not. Both the cytotoxic effects of the cytokines and NO production were inhibited by the addition of NG-monomethyl-L-arginine, a specific inhibitor of NO synthase. These data demonstrate an inverse correlation between production of endogenous NO and the ability of K-1735 cells to survive in syngeneic mice to produce lung metastases.

Critical factors regulating site-specific brain metastasis of murine melanomas.

The intracarotid injection of B16 melanoma cells syngeneic to C57BL/6 mice and K-1735 melanoma cells syngeneic to C3H/HeN mice results in site-specific brain metastasis in C57BL/6 x C3H/HeN F1 mice. The K-1735 cells produce lesions only in the brain parenchyma, whereas the B16 cells produce lesions only in the meninges and ventricles. To determine the mechanisms that regulate this site-specific brain metastasis, we transfected the melanoma cells with DNA from plasmids pSV2neo or pSV2hvgro, which confer resistance to the drugs neomycin and hygromycin, respectively. Hybrids between the B16 and K-1735 cells were obtained by fusion. Cells of the K-1735 x K-1735 hybrid produced lesions only in the brain parenchyma of C57BL/6 x C3H/HeN F1 mice, whereas all B16 x K-1735 hybrids produced lesions only in the meninges and the ventricles. Initial cell arrest in the meninges or the brain parenchyma, production of collagenolytic activity, motility, and expression of CD44 did not predict or correlate with site-specific brain metastasis. The response of the different melanomas and hybrid cells to transforming growth factor-beta (TGF-beta) correlated with growth in the brain parenchyma. B16 cells and B16 x K-1735 hybrids bound more TGF-beta than K-1735 cells. The in vitro growth of B16 cells and all B16 x K-1735 hybrid cells was significantly inhibited by TGF-beta1 and TGF-beta2, whereas the growth of K-1735 cells and K-1735 x K-1735 hybrids was enhanced. Since TGF-beta is abundant in brain tissue, the results suggest that the ability of melanoma cells to proliferate in the brain parenchyma determines the production of site-specific brain metastasis.

Expression level of the nm23 gene in clonal populations of metastatic murine and human neoplasms.

The purpose of this study was to determine whether nm23 steady-state mRNA expression levels correlate with metastatic potential of mouse K-1735 melanoma cells, human KM12 colon cancer cells, and human SN12 renal cancer cells. Since neoplasms are heterogeneous and contain subpopulations of cells with different metastatic potentials, we analyzed multiple sets of nonmetastatic and metastatic clones isolated from each neoplasm. In addition, we also examined nine somatic cell hybrids produced by the fusion of nonmetastatic and metastatic K-1735 clones. In the mouse melanoma, we found heterogeneity in nm23-1 steady-state expression levels among the clones and hybrids that did not correlate with their metastatic phenotype. Clones isolated from human colon or renal carcinomas expressed similar levels of nm23-HI regardless of metastatic potential in nude mice. All of the human tumor cells were heterozygous for the nm23-HI-specific allelic DNA fragments, with no allelic deletions or gross alterations detected. Since the failure of tumor cells to produce metastasis can be due to multiple deficiencies, these data stress the importance of using independent clones with different metastatic potentials for the analysis of gene regulation of this process.

The use of molecular genetic markers to demonstrate the effect of organ environment on clonal dominance in a human renal-cell carcinoma grown in nude mice.

Transduction of the SN12C human renal-cell carcinoma line with the neoR gene produces a genetically "tagged" cell population within which individual clones can be identified if they dominate the tumor during its growth in vivo. We used this technique to determine whether the clones that dominate the primary local tumor and its metastases are the same or different when the tumor is growing in different organ sites in nude mice. The results show that clonal dominance is influenced by the organ environment in which the primary tumor grows, i.e., distinct clones dominated in the kidney, colon and subcutaneous sites. In addition, tumors grown in the orthotopic site (kidney) were all populated by the same dominant clones, and each distant visceral metastasis retained the same clonality. SN12C neoR-cells grown in an epithelial, ectopic site (colon) produced tumors with uniquely different dominant clones, and their visceral metastases retained the dominant pattern expressed by the parent tumor from which they were derived. In contrast, SN12C tumors growing subcutaneously showed a random pattern of clonal dominance in both their primary and metastatic sites. Parallel cytogenetic analyses could not demonstrate these patterns. We conclude that the organ environment significantly influences clonal dominance of human renal carcinoma and that tumor injection into orthotopic sites may produce a more reproducible selection of dominant clones.

Cellular functions related to metastasis differing between low- and high-malignancy variants of AKR lymphoma.

Two AKR lymphoma variants of low (LM) and high (HM) malignancy were characterized for cell properties important for metastatic capacity. Previous data suggested that these murine lymphoma variants differed in the late phase of metastasis. In the present study we attempted to determine more accurately the stage in which they differ. The HM cells attached less efficiently than the LM ones to both endothelial cells (5 times) and extracellular matrix (twice), possibly indicating a more efficient extravasation capacity of the HM cells. The level of heparinase was low in the two variant cells. Attachment to liver sections was, however, more efficient with the HM than with the LM cells.

Predominance of the metastatic phenotype in somatic cell hybrids of the K-1735 murine melanoma.

The purpose of these studies was to determine whether the metastatic phenotype will dominate when metastatic and nonmetastatic clones of the K-1735 mouse melanoma are hybridized by somatic cell fusion. Three nonmetastatic and three metastatic clones were transfected with DNA from plasmids pSV2neo or pSV2hygro, which confer resistance to the drugs neomycin or hygromycin, respectively. The metastatic properties of the six clones were not altered by these transfections. The tumorigenicity and metastatic capacity of cell hybrids formed by somatic cell fusion of nonmetastatic and metastatic clones were examined. To do so, near-tetraploid hybrids containing a nearly complete chromosomal complement from both parental cells were injected i.v. into syngeneic mice, and the number of metastatic nodules in the lung was determined at 45 days or when the mice became moribund. Seven of nine hybrids produced from the fusion of metastatic and nonmetastatic clones exhibited a highly metastatic phenotype, although in most cases the metastatic potential of the hybrids was lower than that of the metastatic parent cells. Very similar results were obtained in athymic nude mice. The metastatic potential of the hybrids was directly correlated with their growth in the subcutis of nude mice. These results indicate that the metastatic capacity of K-1735 cells predominates in somatic cell hybrids between nonmetastatic and metastatic cells. When fusion of nonmetastatic and metastatic cells yields a hybrid with nonmetastatic properties, it may be due to suppression of growth.

Specific chromosomal defects associated with metastatic potential in K-1735 melanoma clones. Involvement of chromosomes 4 and 14.

In this study we sought to identify specific cytogenetic defects associated with the metastatic phenotypes in clones isolated from the parental K-1735 murine melanoma. All nonmetastatic clones (C-3, C-10, and C-19) exhibited trisomy of chromosomes 1, 3, 12, and 15. The only structural defect present in these clones was an interstitial deletion in a chromosome 4. In contrast, the highly metastatic clones (C-4, M-2, BB1, and X-21) exhibited trisomy of chromosomes 1, 3, 12, and 15, plus structural abnormalities of chromosomes 4 and 14, with the net result of a deletion in both. Parental K-1735 cells and clone C-16 cells, which are intermediate in their metastatic potential, had some cells with 4 and 14 alterations and others with only a deletion of chromosome 4. Clone C-16 revealed other non-clonal structural abnormalities. Our results indicate that structural anomaly of chromosome 4 and numerical alterations of certain autosomes may be associated with tumorigenic properties. In addition, structural defect in chromosome 14 is associated with high metastatic potential of K-1735 melanoma cells.