Samuel D. Bernal

Detection of bone marrow metastasis in small-cell lung cancer by monoclonal antibody.

A murine monoclonal antibody against a surface antigen of small-cell carcinoma of the lung (SM1 antibody) was investigated for its use in detecting bone marrow metastasis. Bone marrow cells of healthy volunteers and of patients with small-cell carcinoma of the lung (SCCL) were examined for reactivity with SM1 antibody and indirect immunofluorescence and the results compared to conventional histochemical staining (Wright-Giemsa stain of bone marrow aspirates and hematoxylin-eosin stains of bone marrow biopsies). No SM1 reactivity was found in marrow cells of eight healthy volunteers. Thirty-six samples from 33 patients with SCCL were examined; tumor involvement was found in 69% by SM1 antibody and in 16% by histochemical stains. All bone marrow samples from patients with SCCL that were unreactive with SM1 antibody also showed no evidence of tumor involvement by histochemical stains. Samples of 29 patients were investigated at initial staging; SM1 reactive cells were found in 50% of 16 patients with limited disease and in 77% of 13 patients with extensive disease. Overall, the proportion of patients recognized to have disseminated disease at diagnosis was increased from 45% to 72% by monoclonal antibody staining. Indirect immunofluorescence with SM1 antibody allows detection of bone marrow metastasis of SCCL that cannot be seen by conventional morphology and can identify disseminated disease in patients otherwise staged limited disease.

Chromosome changes in malignant mesothelioma

Cytogenetic study was made of mesothelioma cells from 14 patients. Metaphases were obtained from 12 tumors and revealed aneuploidy and clonal abnormalities in 9 specimens. In the two remaining cases, no metaphases were obtained. The cytogenetic abnormalities were complex, and up to 12 marker chromosomes were observed in the tumors. Rearrangements of chromosomes #1, #2, #3, #6, #9, #11, #17, and #22 were most frequently observed. Chromosome markers involved diverse bands, including several that are loci of oncogenes, fragile sites, and nonrandom rearrangements in other types of cancer. This study shows that the karyotypes of malignant mesothelioma can be analyzed by standard cytogenetic techniques. Additional studies of untreated mesothelioma may help to distinguish primary cytogenetic changes from effects of prior therapy in some of our patients.

Prognostic significance of early response to a single dose of asparaginase in childhood acute lymphoblastic leukemia.

PURPOSE The in vitro and in vivo efficacy of a single dose of asparaginase in children with newly diagnosed acute lymphoblastic leukemia and the correlation between in vitro and in vivo antileukemic response and long-term outcome were prospectively evaluated. PATIENTS AND METHODS Two hundred fifty-one patients were randomized to receive 1 of 3 asparaginase preparations (Escherichia coli, Erwinia chrysanthemi [Erwinia], or pegaspargase). In vitro assessment of efficacy was expressed as the percent total cell kill (TCK), based on the number of viable cells found after 5 days of culture in the presence of asparaginase. In vivo leukemia cell kill (LCK) was calculated by comparing bone marrow cellularity and percent leukemic blasts in marrow obtained before and 5 days after treatment with a single dose of asparaginase. Acute toxicity was determined by clinical and laboratory assessment. RESULTS There was equivalent cell kill with all three types of asparaginase. The mean in vitro TCKs for E. coli, Erwinia, and pegaspargase were 31%, 39%, and 36%, respectively (P = 0.63). The mean LCKs in marrow of patients exposed to E. coli, Erwinia, and pegaspargase were 69%, 74%, and 65%, respectively (P = 0.88). The lack of response to asparaginase in vitro predicted a higher risk for clinical relapse regardless of risk assignment (12 leukemic events among 21 in vitro nonresponders; 57%, P < 0.001). There was no difference in acute toxicity among the three asparaginase preparations. CONCLUSIONS All three asparaginase preparations produced equivalent LCKs in in vitro and in vivo analyses. In vitro response to asparaginase provided a risk group-independent prognostic factor.

Cytoskeleton-associated proteins: their role as cellular integrators in the neoplastic process.

The cytoskeleton (CSK) of eukaryotic cells is composed of a complex interconnected network of filaments which is important in a wide variety of cellular functions including changes in cell shape, cell motility, mitosis, anchorage-dependent growth, and the localization of cellular organelles such as mitochondria, polyribosomes, and secretory granules. The various proteins comprising the cytoskeleton include actin in microfilaments, tubulin in microtubules, and the heterogeneous group of intermediate filament proteins that are associated with different cell types (keratin in epithelial cells, vimentin in fibroblasts, desmin in muscle cells, glial filament protein in glial cells, and the neurofilament protein subunits in neural tissue). Many other proteins in glial cells, and the neurofilament protein subunits in neural tissue). Many other proteins are closely associated with the cytoskeleton and influence its organization. In neoplastic cells, the expression of these different CSK proteins, especially the intermediate filament proteins, reflects their morphologic and functional differentiation. The carcinomas contain keratin; identification of individual keratin components may allow further sub-classification of carcinomas which is consistent with their tissue of origin. The sarcomas of muscle origin contain desmin. Vimentin is found primarily with cells of mesenchymal origin, but may coexist with other intermediate filament proteins in other tumors. One example is the coexistence of keratin and vimentin in tumors, such as mesotheliomas, which are derived from epithelial cells of embryonic origin. Glial fibrillary acidic protein is the most specific marker for glial tumors. Tumors of neural origin are characterized by the presence of neurofilament subunits. Therefore, analysis of CSK composition would be useful in diagnosis of clinical specimens and aid in studies of lineage relationships of neoplasms. Although no consistent differences in cytoskeletal structure between neoplastic and normal cells have been identified so far, the presence of more subtle biochemical alterations in the cytoskeletal structure of neoplastic cells that contributes to malignant behavior has not been ruled out. Since the cytoskeletal network plays an important role in cell shape and cell locomotion, which in turn are thought to be involved in growth control, invasion, and metastasis, further work is directed at identifying the various alterations in cytoskeletal architecture that may influence the malignant behavior of neoplastic cells.(ABSTRACT TRUNCATED AT 400 WORDS)

RHODAMINE‐123 IS SELECTIVELY TOXIC AND PREFERENTIALLY RETAINED IN CARCINOMA CELLS IN VITRO*

Rhodamine-123, a cationic fluorescent dye, has previously been shown to localize specifically in mitochondria of living ce1ls.l It has also been found to be nontoxic in a variety of cell types studied and has thus become a useful tool for probing mitochondria in situ.2 Recently, however, we have detected a toxic effect of this compound on the function and viability of cardiac muscle cells in culture. Since we have also found that cardiac muscle cells as well as carcinoma cells accumulate and retain more rhodamine-123 than do cardiacderived fibroblasts or epithelial cells, we were prompted to investigate the cytotoxic effects of this agent on carcinoma cells.

Reduced membrane protein associated with resistance of human squamous carcinoma cells to methotrexate and cis-platinum

A membrane protein recognized by monoclonal antibody SQM1 was identified in human squamous carcinomas, including those originating in the head and neck (SqCHN), lung and cervix. Cell lines derived from SqCHN of previously untreated patients expressed high amounts of this protein. In contrast, many cell lines established from SqCHN of patients previously treated with chemotherapy and/or radiation showed diminished amounts of this SQM1 protein. The expression of SQM1 antigen was determined in several SgCHN cell lines made resistant by exposure to methotrexate (MTX) in vitro. The parent cell lines all exhibited strong binding to SQM1 antibody. The MTX-resistant sublines showed much lower membrane binding of SQM1. The lowest SQM1 reactivity was found in cell lines with high resistance to MTX and with diminished rate of MTX transport. Some highly MTX-resistant cell lines which had high levels of dihydrofolate reductase, but which retained a high rate of MTX transport, also retained high levels of SQM1 binding. Reduced SQM1 protein was also found in SgCHN cells which developed resistance to the alkylating drug cis-platinum (CDDP) and which showed reduced membrane transport of CDDP. Cell growth kinetics and non-specific antigenic shifts were not responsible for the differences in SQM1 binding between the parent cell lines and their drug-resistant sublines. The finding of a novel protein which is reduced in cells resistant to MTX and CDDP could contribute to our understanding of the basic mechanisms of drug resistance. By detecting SQM1 protein in clinical specimens, it may be possible to monitor the development of drug resistance in tumors.

cDNA cloning of a novel cell adhesion protein expressed in human squamous carcinoma cells

A novel protein (SQM1 protein) present in human squamous epithelial cells has been found to be involved in cell adhesion in squamous epithelial cells, endothelial cells and extracellular matrix proteins. The corresponding cDNA that encodes a 135-residue polypeptide has been isolated. Sequence analysis indicates that the encoded polypeptide is distinct yet related to the beta subunit of integrins. A new sequence motif consisting of a heptadic repeat of positively-charged residues present in the polypeptide has been identified and is proposed to be important in protein-protein interaction. These results suggest that SQM1 protein, a new cell adhesion molecule expressed in squamous epithelial cells, may play an important role in tumor metastasis.

Effect of fluosol-DA/carbogen on etoposide/alkylating agent antitumor activity

SummaryThe tumor growth delay produced by the combination of etoposide with the alkylating agent CDDP or BCNU and Fluosol-DA with carbogen breathing in three model tumor systems was examined. The addition of Fluosol-DA to etoposide treatment increased tumor growth delay 2.8-fold, 3.3-fold and 2.2-fold in the FSaIIC fibrosarcoma, the Lewis lung carcinoma and the SW2 small-cell xenograft, respectively. In both the FSaIIC fibrosarcoma and the Lewis lung carcinoma the combination of etoposide treatment with CDDP produced an additive effect. When Fluosol-DA was added to this combination the tumor growth delay increased 1.9-fold and 1.4-fold in the FSaIIC fibrosarcoma and the Lewis lung carcinoma, respectively. Adding Fluosol-DA to a treatment regimen with etoposide and BCNU produced a 2.2-fold, 2.0-fold and 1.6-fold increase in the tumor growth delay of the FSaIIC fibrosarcoma, the Lewis lung carcinoma and the SW2 small-cell xenograft, respectively. The effect of these various treatment combinations on tumor cell survival was assessed in the FSaIIC fibrosarcoma. When the alkylating agents CDDP or BCNU were prepared in Fluosol-DA, there was an additional increase in tumor cell kill, so that with CDDP there was 2.1-fold and 4.7-fold increase in tumor cell kill and with BCNU there was 1.5-fold and 1.2-fold increase in tumor cell kill compared to the drug plus Fluosol-DA and the drug plus Fluosol-DA/carbogen breathing, respectively. The combination of etoposide and CDDP led to less than additive cell killing, and the combination of etoposide and BCNU appeared to be additive, as predicted by simple product summation, in all of the treatment conditions examined. Both etoposide + CDDP and etoposide + BCNU produced additive or less than additive toxicity to bone marrow as measured by CFU-GM.

Use of SM-1 monoclonal antibody and human complement in selective killing of small cell carcinoma of the lung.

SM-1 is a murine monoclonal antibody strongly reactive with a cell membrane antigen of small cell carcinoma (SCC) of the lung but unreactive with the membrane of most other carcinomas and normal tissues including normal bone marrow. We have found that in the presence of human complement, SM-1 antibody is highly cytotoxic to SCC cells. Using three treatments with antibody and complement, more than 99% of SCC cells in culture were lysed, as determined by the chromium release and clonogenic assays. Similar efficiency of SCC cell lysis was observed when one SM-1 antibody treatment was followed by three treatments with human complement. In contrast, there was little antibody-dependent lysis of non-small cell lung cancer cells, other carcinomas, and leukemia cell lines. The amount of chromium released from normal bone marrow cells treated with SM-1 antibody and complement was minimal and was mainly due to the effect of complement alone. Clonogenic assays, including colony-forming unit-granulocytic/monocytic, erythroid burst-forming unit, and colony-forming unit-granulocytic/erythroid/monocytic/megakaryocytic, also showed no significant SM-1 antibody-dependent cytotoxicity on normal bone marrow precursors. Since SM-1 antibody is selectively cytotoxic to SCC cells in the presence of human complement, it is a potentially useful agent for the selective eradication of tumor cell contamination in marrows of patients with metastatic small cell lung cancer and possibly for in vivo serotherapy.

Immunohistochemical identification of squamous carcinoma of the head and neck with monoclonal antibody SQM1.

Frozen tissue sections of biopsies from head and neck squamous cancer lesions were examined for immunohistochemical staining with a recently developed monoclonal antibody, designated as SQM1 antibody and directed against the surface membrane of squamous carcinoma cells. SQM1 antibody stained selectively squamous carcinoma, while normal mucosa and cells of the stroma were non-reactive. Positive staining of tumor was found in 33/35 specimens obtained from several major sites of the head and neck area and was observed in primary manifestations and lymph node metastases as well as in recurrences. The most consistent reactivity was seen with carcinomas of the tongue. Well differentiated squamous carcinomas contained a higher proportion of SQM1 positive tumor cells than poorly differentiated carcinomas. We suggest that the SQM1 antibody may aid in the immunohistochemical identification of squamous carcinoma of the head and neck area.