David H. Kern

Chemosensitivity testing of human solid tumors. A review of 1582 assays with 258 clinical correlations.

To improve clinical interpretation and use of in vitro clonogenic assay results, the authors reviewed their experience to date with chemosensitivity testing of over 1500 solid tumors. All clonogenic assays were performed using a double‐layer‐soft‐agar system with continuous exposure of cells to one concentration of standard anticancer drugs. Significant growth was defined as > 30 colonies/control plate. Clinical responses were determined according to standard criteria. Data were analyzed using two different criteria of in vitro sensitivity (>50% and >75% inhibition of colony formation) and independently for each histologic type of tumor. Overall, 68% of specimens plated produced significant growth in vitro. Cloning ability varied from 57% to 82% depending on tumor histology. The assay was 57% reliable for predicting in vivo sensitivity, and 92% reliable for in vivo resistance. Predictive accuracy for sensitivity varied from 30% to 86%, depending on the tumor histology. Use of > 50% ICF (inhibition of colony formation) as criteria for differentiating sensitivity from resistance proved most reliable, although criteria should be individualized for each tumor type to maximize predictive accuracy. Cancer 53:1240‐1245, 1984.

Chromosomal abnormalities of a mediastinal embryonal cell carcinoma in a patient with 47,XXY Klinefelter syndrome: Evidence for the premeiotic origin of a germ cell tumor

Trypsin-Giemsa banding studies were performed on 30 tumor cells from an embryonal cell carcinoma originating in the mediastinum of an 18-year-old male with the Klinefelter syndrome (47,XXY). All tumor cells revealed an XXY chromosomal pattern with the addition of extra chromosomes. Electrophoretic patterns of the patients red blood cells and tumor cells were identical. These data suggest that this germ cell tumor originated from a primordial germ cell in which oncogenesis had occurred prior to meiotic division.

Enhancement of anthracycline and alkylator cytotoxicity by ethacrynic acid in primary cultures of human tissues

SummaryEthacrynic acid [2,3-dichloro-4-(2-methylene-1-oxobutyl)phenoxyl] acetic acid, is a water-soluble diuretic agent that has been shown to potentiate the in vitro cytotoxicity of chemotherapeutic agents in established cell lines. We used the differential staining cytotoxicity (DiSC) assay to determine whether ethacrynic acid at 1 and 3.3 µM would potentiate the cytotoxicity of nitrogen mustard and/or doxorubicin in primary cultures of hematologic neoplasms from heavily pretreated patients and in normal peripheral blood lymphocytes. At 3.3 µM, ethacrynic acid was toxic to 8 of 24 (33%) tumor specimens studied. In subsequent studies, ethacrynic acid at 1 µM was toxic to only 2 of 54 (4%) tumor specimens. Significant enhancement for doxorubicin or nitrogen mustard was confined to lymphatic malignancies and to normal peripheral blood lymphocytes. Interspecimen variability was observed, with no enhancement in most individual specimens, 2-fold enhancement in some specimens, and 4-fold enhancement in occasional specimens. Clinical trials will be required to determine whether the observed in vitro activity for ethacrynic acid is associated with clinical benefit in unselected or assay-selected patients.

Breast cancers: estrogen and progesterone receptor status as a predictor of in vitro chemotherapeutic response.

Although clinical observations have shown that estrogen receptor-positive (ER+) breast tumors are more responsive to hormonal therapy than ER-negative (ER-) tumors, it remains controversial whether ER status can predict chemotherapeutic response. To determine if there was any correlation between estrogen and progesterone values and in vitro chemosensitivity to various anticancer drugs, clonogenic (CA), estrogen (ERA), and progesterone (PRA) assays on breast cancers were performed on 100 patients. Clonogenic assays were performed using the double-layer soft agar technique with continuous drug exposures. ERAs and PRAs were performed using the charcoal-coated dextran method. Chemosensitivity was defined as 50% inhibition of colony formation. ERA was considered positive if greater than or equal to 5 fmole/mg cytosol and PRA positive if greater than or equal to 10 fmole/mg cytosol. Significant tumor growth (greater than 30 colonies/plate) was achieved in 81/100 assays. ERA and PRA values were not predictive of colony formation in vitro. Of all agents or combinations of agents tested (L-PAM, 5-FU, MTX, adriamycin, vinblastine, cis-plat, FAC, CMF), only the response to 5-FU correlated significantly with ERA. Eight of 11 (73%) of the ER- tumors were sensitive to 5-FU, whereas only 6/20 (30%) of ER+ tumors were sensitive (P less than 0.05). ER- tumors were also more likely to be sensitive to CMF (P = 0.09) and adriamycin (P = 0.07) than ER+ tumors. PRA values were not predictive of chemosensitivity, nor did combining PRA and ERA enhance the predictive value of ERA alone.

Predictability of response to clinical thermochemotherapy by the clonogenic assay.

In order to assess the value of the clonogenic assay for predicting clinical response to dimethyl‐triazeno‐imidazole‐carboxamide (DTIC) plus hyperthermia (42°C), the responses of patients with measurable disease, who received combined therapy, were compared with assay results. The clonogenic assay was used independently to determine in vitro sensitivities of 53 melanomas to DTIC, with and without hyperthermia. Separate cell suspensions were incubated for 1 hour with DTIC at 37°C and at 42°C. In vitro sensitivity was determined by inhibition of colony formation in a double‐layer agar system. Three of the 53 (6%) melanomas were sensitive to DTIC at 37°C, 13 of the 53 (25%) were sensitive to 42°C hyperthermia alone, and 22 of the 53 (42%) were sensitive to DTIC at 42°C. Nine patients were treated with DTIC, plus hyperthermia, to the areas of their melanoma metastases (one pulmonary, four hepatic, and four subcutaneous). In five patients, the clonogenic assay results predicted positive tumor sensitivity to combined therapy, and 4 of the 5 had objective tumor regression. Tumors were resistant in vitro for four patients, and all had disease progression during treatment. Statistical analysis suggested that some responses were due to synergism of the combination of heat and drug, whereas others were due to an additive effect. The apparent direct correlation between in vitro tumor cell sensitivity to DTIC at 42°C and actual clinical response to chemotherapy, plus hyperthermia, in this limited trial, has been encouraging. The clonogenic assay and in vitro evaluation of drug‐heat interaction may prove helpful for selecting those patients in whom hyperthermia should be used as an adjunct to chemotherapy, and may help determine the most effective drug/heat scheduling. Further trials with other malignancies and other chemotherapeutic agents are warranted.

Immune cytolysis of human tumor cells mediated by xenogeneic "immune" RNA.

Abstract Normal, nonimmune, human peripheral blood lymphocytes, when incubated with RNA extracted from lymphoid organs of guinea pigs or sheep immunized with human tumor cells, mediated the immune cytolysis of those tumor cells in vitro . Lymphocytes incubated without RNA, or with control RNA preparations, failed to evidence cytotoxic activity. Treatment of the active RNA preparations with ribonuclease abrogated the cytotoxic activity, but treatment with deoxyribonuclease or pronase did not effect activity.

In vitro assays to predict drug sensitivity and drug resistance: a panel discussion

One of the key problems in oncology today is to try to predict which patients will respond to, or fail, a particular chemotherapy drug regimen. There are now a number of in vitro assays that claim to provide such predictive information. With such an array of assays to choose from, the practicing oncologist has been understandably cautious about accepting enthusiastic advertising claims and published reports. For many of these assays, sufficient time has elapsed to permit objective evaluation and assessment of clinical utility for routine management of cancer patients. Our panel of experts will discuss both the merits and the pitfalls of some of these assays, and try to put them in perspective for the practicing physician.

Evolution and clinical application of a rapid chemosensitivity assay

The clinical usefulness of the soft agar colony‐formation assay of in vitro chemosensitivity developed by Hamburger and Salmon is limited by long turnaround time (2‐3 weeks), low success rate for small specimens, and clumping artifacts that can lead to erroneous predictions of resistance (false‐negative errors). An improved technique was developed for measuring in vitro growth by incorporation of tritiated thymidine that can be performed in 5 days. With this rapid assay, 819 tumors were processed, with an overall success rate of 59.3%. This result compared favorably to the overall success rate of 58.2% for 1591 colony‐formation assays because more small specimens could be submitted for the rapid assay. Melanoma and ovarian cancer specimens grew particularly well (76% and 75% successful, respectfully). Sixty‐five correlations of in vitro and in vivo responses are available to date. None of 30 tumors, predicted to be resistant in vitro responded to chemotherapy clinically. Patients whose tumors were predicted to be sensitive in vitro had a 43% clinical response rate. The assay appears to be particularly accurate for predicting clinical resistance to chemotherapy, possibly because clumping artifacts do not occur in this system and peak achievable plasma concentrations of chemotherapeutic agents can be used. Optimal in vitro drug concentrations and culture conditions are still being defined, and improved success rates are being seen with more recent specimens. The introduction of this technique underscores the fact that in vitro chemosensitivity tests must continuously evolve to maximize their clinical application. Cancer 55:1367‐1371, 1985.

IMMUNOTHERAPY OF CANCER WITH IMMUNE RNA IN ANIMAL MODELS AND CANCER PATIENTS

In 1970-71, we reported that immunity to tumor isografts in inbred mice could be mediated by syngeneic spleen cells preincubated with xenogeneic immune RNA (I-RNA) extracted from the lymphoid organs of guinea pigs immunized with the specific tumor that was to be treated.1-3 This immunity was manifested when such RNA-incubated spleen cells were injected into syngeneic mice. This response was specific for the particular tumor used to immunize the guinea pig from whose lymphoid organs the I-RNA was prepared. Thus, spleen cells incubated with I-RNA against one tumor inhibited the growth of isografts of that tumor but not isografts of a syngeneic but antigenically different tumor, Administration of spleen cells incubated with I-RNA against the second tumor inhibited the growth of isografts of that tumor but not isografts of the first tumor. Nonspecific factors associated with guinea pig RNA probably did not account for these results, because RNA from guinea pigs immunized with Freunds adjuvant only was without effect. Similarly, injections of untreated syngeneic spleen cells (not incubated with RNA) did not inhibit the growth of tumor isografts. Most important was the finding that I-RNA from the lymphoid organs of guinea pigs immunized with syngeneic normal mouse tissues failed to mediate an antitumor response. This, together with the findings of the specificity of the I-RNA for the immunizing tumor, strongly suggested that the immunity expressed by the spleen cells preincubated with antitumor I-RNA was directed against tumor-associated antigens (TAA) . We then sought a more direct and simpler method of utilizing I-RNA in mediating antitumor immune responses. Such a method would be the direct

A rapid bioassay to determine stabilities of anticancer agents under conditions of the clonogenic assay

SummaryWe developed a rapid bioassay to determine in vitro drug stabilities in the clonogenic assay. The in vitro half-lives of 11 standard antitumor agents, actinomycin D, Adriamycin, bleomycin, cis-Platinum, dacarbazine, 5-fluorouracil, melphalan, mitomycin C, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), vinblastine, and vincristine, and four investigational drugs, Aziridinylbenzoquinone (AZQ) (NSC 182986), diamminecyclobutane-dicarboxylatoplatinum (CBDCA) (NSC 241240), Dihydroxyanthracenedione dihydrochloride (DHAD) (NSC 301739), and 1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl-1-nitrosourea) (PCNU) (NSC 95466), were determined under conditions of the clonogenic assay as well as under storage conditions at −40° and−196°C. BCNU and AZQ at−40°C had t1/2 of 4.7 d and 2.5 d, respectively. All other drugs were stable at −40° and −196°C with t1/2>6 wk. Under assay conditions at 37°C, actinomycin D, bleomycin, dacarbazine, 5-fluorouracil, mitomycin C, vinblastine, vincristine, and DHAD were stable, with t1/2>14 d. CBDCA, AZQ, Adriamycin, cis-Platinum, melphalan, BCNU, and PCNU had t1/2 of 94,72,29,18.5,1.8,1, and 0.5 h, respectively.