Effects of tumor necrosis factor on sensitive and multidrug resistant human leukemia and myeloma cell lines.

Two human tumor cell lines exhibiting acquired multidrug resistance (MDR) with increased expression of a cell surface glycoprotein (GP-170) were tested for their sensitivity to human recombinant tumor necrosis factor (rTNF). The drug resistant mutant lines (CEM/V, a T-cell leukemia line resistant to vinblastine, and 8226/D, a multiple myeloma line resistant to doxorubicin), were markedly more sensitive to rTNF in clonogenic assay than were their drug-sensitive parental lines (CEM, 8226). As determined by radioreceptor assay, the number of cell surface receptors for rTNF did not differ on the parental and drug-resistant lines. During the first 24 hours after addition of rTNF, there was a decrease in intracellular ATP content in the CEM/V line but not in the CEM line. No differential effect of rTNF on ATP content was observed between 8226 and 8226/D. As determined by RNA dot-blot analysis, total cellular RNA for GP-170 was increased in the 8226/D cells. After rTNF exposure, expression of total cellular RNA for GP-170 was not altered. Accumulation of radiolabeled doxorubicin by 8226/D cells was not altered by previous or coincubation with rTNF. These findings suggest that the effects of rTNF on MDR cells is not related to TNF receptor number and is mediated at a step subsequent to rTNF binding and not by either inhibition of synthesis of GP-170 or by alteration in the function of the GP-170 efflux pump.

Antitumor activity of bovine cartilage extract (Catrix-S) in the human tumor stem cell assay.

Catrix is an acidic mucopolysaccharide complex derived from bovine tracheal cartilage. The antitumor efficacy of Catrix has been evaluated in the human tumor stem cell assay system using three human tumor cell lines and fresh biopsy specimens from 22 patients with malignant tumors. In vitro efficacy has been demonstrated with high dose continuous exposure Catrix, particularly against the 8226 human myeloma cell line as well as ovarian, pancreatic, colon, testicular, and sarcoma biopsy specimens. The level of sensitivity was at less than or equal to 30% survival of colony growth in vitro. Since the in vitro concentrations may be achievable in vivo, the results justify more detailed in vitro evaluation as well as potential clinical trials.

Antitumor activity of esorubicin in human tumor clonogenic assay with comparisons to doxorubicin.

The new anthracycline analog, esorubicin (4'deoxy-doxorubicin, ESO), was tested against fresh biopsies of human solid tumors in vitro in clonogenic assay and the results were contrasted to those obtained with doxorubicin (DOX). ESO appeared to be significantly more potent on a weight basis than DOX in these studies, and exhibited a spectrum of antitumor activity in vitro that was in general qualitatively similar to that observed with DOX. In vitro antitumor activity was observed in a wide variety of human cancers including anthracycline-sensitive tumor types. ESO has previously been reported to have decreased cardiac toxicity in preclinical models as compared to DOX. Comparative testing of these anthracyclines on granulocyte-macrophage colony-forming units (GM-CFUs) and tumor colony forming units (TCFUs) indicated that the in vitro GM-CFU assay is more sensitive to these myelosuppressive drugs than are TCFUs, and underscores the need for in vivo studies to determine normal tissue toxicity and the therapeutic index of a drug. Early results of phase I studies suggest that with respect to myelosuppression, the maximally tolerated dose of ESO will be about half that of DOX. The increased in vitro antitumor potency observed for ESO and a spectrum of activity (even at one half the dose of DOX) supports the broad testing of ESO in the clinic to determine whether it will prove to be a more effective and less toxic anthracycline.

Immunocompetence and malignant lymphoma: immunologic status before therapy.

One-hundred-eighty-six previously untreated patients with malignant lymphoma were evaluated for immunocompetence by means of several tests of immune function: total circulating lymphocytes, T cells (E-rosettes), B cells (EAC-rosettes), delayed hypersensitivity to six recall antigens, serum immunoglobulins, mixed lymphocyte culture, and lymphocyte mitogenic response to phytohemagglutinin and pokeweed mitogen. The results were correlated with histology, stage, and clinical features. Diffuse lymphomas, especially diffuse histiocytic (large cell) (DHL), were associated with decreased absolute lymphocytes and E-rosette forming cells. Skin test reactivity varied with both histology and stage. For example, only one of six tests was impaired in diffuse lymphocytic well differentiated (DLWD) lymphoma in contrast to two of six in localized DHL and five of six in advanced DHL. Patients with nodular lymphoma exhibited depressed mean levels of IgA and IgG, while only IgA was significantly decreased in diffuse lymphoma. Mitogen stimulation was depressed in all groups, although mixed lymphocyte cultures did not differ significantly from controls. In summary, there is a spectrum of immunodeficiency of both B and T cell type in patients with malignant lymphoma that correlated with histology and stage. Implications and possible mechanisms of these observations are discussed.

New drugs in ovarian cancer and malignant melanoma: in vitro phase II screening with the human tumor stem cell assay.

The successful development of a soft agar clonogenic assay for human tumor stem cells provides an in vitro technique with a high degree of accuracy for predicting in vivo clinical response to standard anticancer drugs. We used this system to conduct an "in vitro phase II trial" in human ovarian cancer and melanoma. This approach can potentially identify active phase I--II drugs suitable for treatment of given tumor types for specific patients and eliminates the need to subject patients (who would be predicted not to respond) to toxic side effects. In vitro sensitivity for new agents was operationally defined as at least a 70% reduction of tumor colony-forming units (TCFU) at concentrations which are readily achievable pharmacologically. The new agents AMSA and vindesine (as well as vinblastine) appeared to have activity in melanoma, while PALA and thymidine were inactive. Pentamethylmelamine, mitomycin C, methyl-GAG, and AMSA were relatively ineffective in ovarian cancer. Vinblastine and vindesine had definite activity. The human tumor stem cell assay may thus provide the basis for a useful alternative to the current clinical phase II testing approach for identifying antitumor activity of new agents. Validation of this concept with correlative in vitro and in vivo phase II trials of new agents in patients with tumor types predicted to be sensitive is clearly warranted.

In vitro clonal assay for human metastatic melanoma cells.

An in vitro assay for clonogenic tumor cells was applied to human metastatic melanomas. Melanoma colony formation was observed in 22 of 33 samples obtained from a variety of involved tissues. The melanocytic tumor origin of colonies was established by serial observations by inverted light microscopy, staining of fixed colonies for melanin, and cytological and karyotypic analysis of cells within colonies. Two morphologically distinct types of colonies were identified, one consisting of light large cells and the second of dark small cells. Investigations of factors which modulate growth and differentiation of clonogenic melanoma cells may provide a clearer understanding of this neoplasm.

In-vitro clonogenic assay for predicting response of ovarian cancer to chemotherapy.

95 clinical trials of anticancer drugs were done in 40 patients with advanced ovarian cancer who had undergone an in-vitro soft-agar human tumour stem-cell assay for drug sensitivity. In the 95 clinical trials, 13 of 21 patients responded clinically to drugs predicted to be effective in vitro, while in 73 of 74 instances in which drug resistance had been predicted in vitro there was no response to treatment. 2 patients achieved a complete remission of 1 and 12+ months and 11 had partial remissions lasting a median of four months. Prediction of in-vitro resistance was 99% accurate. The 62% complete or partial remission rate to agents with proven in-vitro efficacy was achieved in extensively pretreated ovarian cancer patients who were unlikely to respond to empirically chosen secondary agents. The human tumour stem-cell assay, in which ovarian tumour colony-forming units are grown in a two-layer agar-culture system incorporating tests for drug sensitivity or resistance, can be effectively used to plan a suitable chemotherapy regimen for ovarian cancer patients.

Clinical correlations of drug sensitivity in the human tumor stem cell assay.

We have applied an in vitro soft-agar tumor-colony assay (which is now applicable to a variety of human cancers) to measurement of in vitro sensitivity to drugs and prediction of clinical response to cancer chemotherapy. The assay predicts drug resistance with 96% accuracy and sensitivity (in healthy pretreated patients) with 62% accuracy. On a pharmacokinetic basis the zone in vitro sensitivity for any given drug was only 5%-10% of the clinical concentration-time product (Cxt) achievable. This suggests that intratumoral drug concentrations in vivo may be lower than those in the plasma, and/or that > 2 log kills of tumor stem cells (not measurable in the assay) are required for clinical response. Serial in vitro studies showed that acquisition of drug resistance is a common clinical phenomenon which can be directly detected and quantitated in vitro.

Quantitation of differential sensitivity of human-tumor stem cells to anticancer drugs.

With a direct in vitro tumor-colony assay developed to measure sensitity of human-tumor stem cells to anticancer drugs, we performed 32 retrospective or prospective clinical studies in nine patients with myeloma and nine with ovarian cancer treated with standard agents that were tested in vitro. The results were clearly correlated (P is less than 0.00001). Unique patterns of sensitivity and resistance to the six drugs tested were observed for individual patients. In eight cases of myeloma and three of obarian carcinoma in vitro sensitivity corresponded with in vivo sensitivity whereas in one case of myeloma it did not. In vitro resistance correlated with clinical resistance in all five comparisons in myeloma and all 15 in ovarian cancer. We conclude that this assay shows sufficient promise to warrant larger-scale testing to determine its efficacy for selection of new agents and individualized cancer chemotherapy regimens.

Inhibition of tumor-induced lymphocyte blastogenesis by a factor or factors associated with peripheral leukocytes.

A factor or factors concentrated on leukocytes of cancer patients depress the proliferative response of lymphocytes to stimulation with autologous tumor. Inhibitory activity with material eluted from cells resides in neither high nor lower molecular weight fractions but in a combination of the two. The finding suggests that in vitro inhibition of lymphocyte proliferation to autochthonous tumor occurs because of antigen-antibody complexes.