The prognosis of patients with cancer is largely determined by the specific histological diagnosis, tumor mass stage, and host performance status. The management of neoplastic disease with the currently available treatment armamentarium may be further advanced if individual patients' risk factors could be better defined. Some of the determinants of tumor response seem to be expressed at the cellular level in terms of degree of tumor cell differentiation, growth kinetics, and hormone receptor expression, which are not readily appreciated by descriptive morphology. Quantitative cytology in the form of flow cytometry has greatly advanced the objective elucidation of tumor cell heterogeneity by using probes that discriminate tumor and normal cells and assess differentiative as well as proliferative tumor cell properties. Abnormal nuclear DNA content is a conclusive marker of malignancy and is found with increasing frequency in leukemia (23% among 793 patients), in lymphoma (53% among 360 patients), and in myeloma (76% among 177 patients), as well as in solid tumors (75% among 3611 patients), for an overall incidence of 67% in 4941 patients. The degree of DNA content abnormality varies according to disease type, with a predominant excess of 10 to 20% in the hematological cancers, whereas ploidy levels in solid-tissue neoplasms span the entire range from almost haploid to hyperoctaploid abnormalities, with near-triploid mean and median DNA content values. The proportion of cells in the S phase of the cell cycle increases with increasing DNA excess in a number of different solid tumors and in acute leukemia. This cytokinetic parameter permits discrimination of low- and high-grade malignant lymphomas. Several reports demonstrate increasing morphological immaturity to be associated with increasing DNA content abnormality and increasing S percentage, all of which adversely affect prognosis. Among phenotypic tumor cell markers, surface membrane antigens have been extensively studied in lymphoid and myeloid neoplasms by the use of hybridoma-generated monoclonal antibodies, which have recently also found in vitro and in vivo therapeutic application. Cellular RNA content is useful for the objective discrimination of acute leukemias and of multiple myeloma. Newer applications of flow cytometry concern studies in the areas of cytoenzymology and cellular pharmacology. Current research is dedicated to the identification of neoplastic marker probes for DNA-diploid disease ( e.g. , nucleolar antigen) and additional phenotypic ( e.g. , hormone receptors) and cytokinetic ( e.g. , cycle traverse rate, growth fraction) parameters.

From a patient management perspective, a role for flow cytometry is emerging as a tool for diagnosis of cancer (abnormal DNA content), specific histopathological diagnosis (RNA for hematological cancers; surface markers for lymphoid and myeloid neoplasms), prognosis (adverse impact of aneuploidy and high S percentage), and treatment (cytokinetically oriented, monoclonal antibodies, drug pharmacology). The pace of past progress justifies the hope that cytometry may soon provide “fingerprinttype” information of an individual patient's tumor which, if proven prognostically relevant, may provide the basis for treatment selection in the future.

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Flow Cytometry in Clinical Cancer Research

Clinical development of platinum complexes in cancer therapy: an historical perspective and an update

The demonstration in the 1960's that cis-diamminedichloroplatinum(II), or cisplatin, inhibits cellular division of Escherichia coli1 led to the subsequent discovery that this simple coordination compound is also an effective antitumor agent in mouse models.2 Subsequent studies validated cisplatin as an effective anticancer agent in humans as well,3–7 and FDA approval of cisplatin for the treatment of metastatic ovarian and testicular cancers was granted in 1978.8 Its introduction as a chemotherapeutic agent significantly improved the survival outlook for many cancer patients; the cure rate for testicular cancer before the approval of cisplatin was less than 10%, significantly lower than the 90% cure rate attained with modern platinum chemotherapy.9,10

Cisplatin kills cancer cells primarily by cross-linking DNA and inhibiting transcription.11 The chemical origin of this process begins when cisplatin enters the cell and undergoes aquation involving loss of one or both chloride ligands. The resulting platinum(II) aqua complexes are potent electrophiles that readily react with a number of biological ligands with loss of the bound water molecules. The purine bases of nucleic acids are strongly nucleophilic at the N7 position. Thus, cisplatin binds readily to DNA, forming primarily bifunctional adducts with loss of both chloride ligands. The major cisplatin-DNA adduct is the intrastrand 1,2-d(GpG) cross-link, which accounts for 60–65% of the bound platinum.12 The resulting Pt-DNA adducts, which distort and bend the DNA structure,13–15 impede transcription.16 The downstream effects of transcription inhibition ultimately lead to cell death.

Despite its great curative success in testicular cancer, cisplatin is not universally effective in other cancer types and induces a number of toxic side effects.17–19 Additionally, certain cancers are resistant to cisplatin therapy. This resistance is either intrinsic or developed during prolonged treatment.20,21 To circumvent these problems, new platinum complexes have been pursued and investigated for their antitumor properties. Although well over a thousand complexes have been prepared and tested thus far,22 only two other platinum drugs are approved for clinical use worldwide, and three additional compounds are approved for regional use in Asia.23 These complexes, displayed in Chart 1, operate with a mechanism of action similar to that of cisplatin, which involves DNA binding and transcription inhibition.






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Chart 1


Chemical structures of the clinically used platinum-based anticancer drugs. The top three complexes, cisplatin, carboplatin, and oxaliplatin, are approved for use worldwide. The bottom three complexes, nedaplatin, lobaplatin, and heptaplatin, are approved for use in Japan, China, and Korea, respectively.

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Synthetic Methods for the Preparation of Platinum Anticancer Complexes

Summary In this essay we have considered possible relationships between errors in DNA replication and malignant progression. Theoretical and experimental studies designed to determine the energy of interaction between different nucleotides have been reviewed. The difference of the energy of interaction between complementary and noncomplementary base pairs (1 to 3 kcal) is not sufficient to account for the exactness of DNA replication that is observed in vivo or in vitro. Thus, DNA polymerases must play a role in base selection. This point has been established by the detection of polymerases that copy polynucleotide templates with few errors and the detection of others that make many errors. Different mechanisms for base selection by polymerases have been considered. Our findings that both extracts of human leukemic lymphocytes and a purified DNA polymerase from avian myeloblastosis virus have exceptionally high error rates have been considered in relationship to oncogenesis. A hypothesis is presented that relates mistakes in DNA replication, as promoted by error-prone DNA polymerases, to tumor progression. We have also considered a more general concept encompassing early cellular changes that lead to oncogenesis. The justification for this essay is that both of these hypotheses are open to immediate experimental analysis.

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Errors in DNA Replication as a Basis of Malignant Changes

cis-Diammine-1,1-cyclobutane dicarboxylate platinum II (CBDCA, JM8), an analogue of cisplatin showing reduced toxicity in preclinical studies, was evaluated in 60 patients. Doses were given initially every 3 weeks and escalated from 20 to 520 mg/m2. Following this, doses were given every 4 weeks and escalated from 300 to 500 mg/m2. The dose-limiting toxicity, thrombocytopoenia, occurred in four-fifths of patients treated at 520 mg/m2, with the nadir occurring 3 weeks after treatment. Leucopoenia and anaemia also occurred but were less severe. Vomiting occurred in all patients receiving over 120 mg/m2 but seldom persisted beyond 24 h. Serial measurements of 51Cr-EDTA clearances, urinary N-acetylglucosaminidase, urinary leucine aminopeptidase, and β2-microglobulin did not reveal significant evidence of nephrotoxicity. Detriment to the audiogram has not been seen in the first 13 patients studied. Pharmacological studies showed that most of the dose of platinum was excreted in the urine, and that impairment of renal function may be associated with drug retention and an increased risk of myelosuppression. The previous therapy and age of the patient also affected the tolerance of the drug. Clinical responses were seen in patients with ovarian carcinoma receiving >120 mg/m2. A further dose escalation was performed on a 4-week schedule in patients under 65 with good renal function. The maximum dose it was possible to administer repeatedly without incurring myelosuppression was in the range 400–500 mg/m2. JM8 is not significantly nephrotoxic and is less emetic than cisplatin. It has antitumour activity in man and deserves wider evaluation, along with the other analogues under study in various centres, as an alternative to cisplatin.

Early clinical studies with cis-diammine-1,1-cyclobutane dicarboxylate platinum II

Diamminodichloroplatinum in the Chemotherapy of Testicular Tumors

Cis-diamminedichloroplatinum (NSC-119875): a phase I study.

The discovery in human leukemic cells of particulate elements encapsulating 70S RNA and RNA-directed DNA polymerase made possible the synthesis of a [3H]DNA probe that could detect leukemia-specific sequences in the DNA of normal and leukemic individuals. In an earlier study of a series of unrelated leukemic patients, we established that the nuclear DNA of their leukemic cells contain particle-related sequences that cannot be detected in leukocytes of normal individuals. This result is inconsistent with the virogene concept that demands the inclusion of one complete copy of oncogenic information in the genome of every normal cell. 

The present study carries this analysis one step further by showing, with two sets of identical twins, that the leukemic member contains particle-related sequences in the DNA of his leukocytes that cannot be detected in the leukocytes of his healthy identical sibling. This finding implies that the additional leukemia-specific information found in the DNA of the leukemic individuals must have been inserted subsequent to fertilization. This outcome argues against the virogene hypothesis or any other etiologic concept that invokes vertical transmission through the germ line of the particle-related information found uniquely in the DNA of leukemic cells.

Leukemia-Specific DNA Sequences in Leukocytes of the Leukemic Member of Identical Twins

cis-Dichlorodiammineplatinum(II) (DDP) was studied in 16 children with far-advanced malignancies. Three dosage schedules were tried: regimen A, 20 mg/m2/day x 5 days for 3-4 weeks (11 patients); regimen B, 50 mg/m2 once a week (four patients); and regimen C, 60 mg/m2/day x 2 days every 3-4 weeks (one patient). Four of 16 patients (25%) showed partial response, including one with osteogenic sarcoma, one with neuroblastoma, one with seminoma, and one with medullary carcinoma of the thyroid. Two patients showed clinical improvement. The major toxic manifestations included nausea and vomiting (16 of 16), renal toxicity (three of 16), transient pancytopenia (six of 12), and hearing loss (two of 16). It is apparent that DDP has activity in pediatric tumors; however, a more precise response rate must be delineated in a larger series of patients.

Clinical response and toxicity with cis-dichlorodiammineplatinum(II) in children.

Patients with a wide variety of previously treated, advanced solid tumors were assigned to receive either adriamycin-cis-dichlorodiammineplatinum(II) (cis-platinum) or adriamycin-cyclophosphamide as combination chemotherapy. Hematologic toxicity was prominent but short-lived in most patients; renal toxicity occurred in 14 of 23 patients receiving cis-platinum and frequently precluded further administration of that agent. Of the patients receiving adriamycin-cis-platinum, 37.5% had useful responses, with four of six patients with bronchogenic carcinomas showing partial regression of disease and two patients with embryonal cell carcinoma of the testes showing complete response. In the patients receiving adriamycin-cyclophosphamide, one complete response occurred in a patient with a malignant lymphoma, and the overall response rate was 18%. The combination of adriamycin-cis-platinum merits further study, especially in bronchogenic carcinoma.

Wallace Hj

Papers

Diamminodichloroplatinum in the Chemotherapy of Testicular Tumors

Cis-diamminedichloroplatinum (NSC-119875): a phase I study.

Leukemia-Specific DNA Sequences in Leukocytes of the Leukemic Member of Identical Twins

Clinical response and toxicity with cis-dichlorodiammineplatinum(II) in children.

Adriamycin-cyclophosphamide and adriamycin-cis-dichloro-diammineplatinum(II) combination chemotherapy in patients with advanced cancer.