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This study evaluated the efficacy and safety of single-agent bortezomib in indolent B-cell lymphoma that had relapsed from or was refractory to rituximab. Sixty patients enrolled: 59 were treated with bortezomib 1.3 mg/m2 on days 1, 4, 8, and 11 for up to eight 21-day cycles; responders could receive 4 additional cycles; maintenance was optional. Fifty-three evaluable patients completed more than 2 cycles. The median age was 70 years, 53% female, Ann Arbor stage III-IIIE (28%) and IV (65%); 43 patients (72%) had more than 2 prior regimens; and 6 patients went on to maintenance. Overall responses are as follows: 1 complete response (1.9%), 3 unconfirmed complete response (5.7%), 3 partial response (5.7%), 34 stable disease (64.2%), and 12 progressive disease (22.6%). Median time to response = 2.2 months (range, 1.2-5.3 months); duration of response = 7.9 months (2.8-21.3 months); 1-year survival was 73% and 2-year survival was 58%; median survival = 27.7 months (range, 1.4-30.9 months); median progression-free survival = 5.1 months (range, 0.2-27.7 months), median time to progression = 5.1 months (range, 0.2-27.7 months), and median event-free survival = 1.8 months (range, 0.2-27.7 months). Treatment-related grade 3 or 4 adverse events included: thrombocytopenia (20%), fatigue (10%), neutropenia (8.5%), and neuropathy and diarrhea (6.8% each). This study demonstrates that bortezomib has modest activity against marginal zone and follicular lymphoma; it has the potential for combination with other agents in low-grade lymphomas. Maintenance therapy should be explored further.

We describe several analytical techniques for use in developing genetic models of oncogenesis including: methods for the selection of important genetic events, construction of graph models (including distance-based trees, branching trees, contingency trees and directed acyclic graph models) from these events and methods for interpretation of the resulting models. The models can be used to make predictions about: which genetic events tend to occur early, which events tend to occur together and the likely order of events. Unlike simple path models of oncogenesis, our models allow dependencies to exist between specific genetic changes and allow for multiple, divergent paths in tumor progression. A variety of genetic events can be used with the graph models including chromosome breaks, losses or gains of large DNA regions, small mutations and changes in methylation. As an application of the techniques, we use a recently published cytogenetic analysis of 206 melanoma cases [Nelson et al. (2000), Cancer Genet. Cytogenet.122, 101-109] to derive graph models for chromosome breaks in melanoma. Among our predictions are: (1) breaks in 6q1 and 1q1 are early events, with 6q1 preferentially occurring first and increasing the probability of a break in 1q1 and (2) breaks in the two sets [1p1, 1p2, 9q1] and [1q1, 7p2, 9p2] tend to occur together. This study illustrates that the application of graph models to genetic data from tumor sets provide new information on the interrelationships among genetic changes during tumor progression.

We report the cytogenetic abnormalities from a series of 206 primary malignant melanoma specimens referred to a single institution. A total of 169 out of 206 unique cases had chromosome breakpoints. A previously described statistical method was used to detect nonrandom distribution of chromosome breakpoints at the level of chromosome regions. Nonrandom occurrence of chromosome breakpoints (indicating that the observed number of breaks significantly exceeded the expected number of breaks) was detected in 28 regions, suggesting a hierarchy of genetic abnormalities in melanoma. Clinical variables and tumor characteristics were analyzed for associations with the presence of any nonrandom chromosome breakpoints; with individual, nonrandomly involved chromosome regions; and with paired, nonrandomly involved chromosome regions. No nonrandomly involved chromosome regions or pairs of regions appeared to significantly affect survival. These results identify recurring, nonrandom chromosome abnormalities in malignant melanoma. These results suggest that recurring, nonrandom chromosome alterations play a key role in the etiology and/or progression of malignant melanoma and identify targets within the genome for molecular genetic studies.

Cancer geneticists seek to identify genetic changes in tumor cells and to relate the genetic changes to tumor development. Because single changes can disrupt the cell cycle and promote other genetic changes, it is extremely hard to distinguish cause from effect. In this article we illustrate how 7 techniques from statistics, theoretical computer science, and phylogenetics can be used to infer and test possible models of tumor progression from single genome-wide descriptions of aberrations in a large sample of tumors. Specifically, we propose 4 tree models for tumor progression inferred from the large ovarian cancer data set described in the first 2 articles in this series. The models are derived from 2 different methods to select the non-random genetic aberrations and 2 different methods to infer the trees, given a set of events. Various aspects of the tree models are tested and extended by 5 methods: overall tests of independence, likelihood ratio tests, principal components analysis, directed acyclic graph modeling, and Bayesian survival analysis. All our methods lead to strikingly consistent conclusions about chromosomal breakpoints in ovarian adenocarcinoma, including (1) the non-random breakpoints in ovarian adenocarcinoma do not occur independently; (2) breakpoints in regions 1p3 and 11p1 are important early events and distinguish a class of tumors associated with poor prognosis; and (3) breakpoints in 1p1, 3p1, and 1q2 distinguish a class of ovarian tumors, and the breaks at 1p1 and 3p1 are associated with poor prognosis.

Chromosome 6 abnormalities, particularly the deletion of 6q, are among the most frequent chromosomal changes in human malignant melanoma. In this study, chromosome 6 rearrangements in 21 melanoma cell lines were identified by fluorescence in situ hybridization (FISH) with the use of chromosome 6 arm specific painting probes (CAPs). Structural abnormalities of chromosome 6 were detectable in 18 of 21 (86%) cases, including the identification of structural abnormalities that were not detectable by routine G-banding analysis. The results of this study correlate the frequency of 6q alterations and demonstrate that FISH with CAPs can be of considerable use for rapid screening of specific chromosome abnormalities in melanoma.

In a series of 128 karyotyped ovarian carcinomas, 42% of cases with chromosome 1 clonal structural abnormalities had breaks at band 1p36 (usually involving translocations of unknown material). Fluorescent in situ hybridization (FISH) studies using combinations of 1 centromere and 1p36.3-specific probes (16 cases) or 1 centromeric and 17 whole-chromosome paint probes (11 cases with 1p+) revealed a trend toward deletion of 1pter relative to 1 centromere (63%); intratumor heterogeneity; and the origin of 1p+ in 3/11 cases (27%) from chromosome 17 [t(1;17)(p36;?)]. The frequency of this specific breakpoint and its involvement in recurrent translocations suggest that these regions are loci for genes important in the pathogenesis of a subset of sporadic ovarian carcinomas.

Unlike leukemia, in which specific reciprocal translocations are frequently observed, melanomas involve complex recurring chromosome anomalies. Analysis of the constituted genome of melanoma patients should identify cancer susceptibility genes and at-risk individuals in families with a history of melanoma. The first of these genes to be cloned is the cell cycle regulatory protein inhibitor--the p16 gene-- and a second gene locus for melanoma predisposition has been linked to the chromosome 1p36 band region. Detection of the most common somatic genetic alterations in melanoma enhances our understanding of molecular mechanisms of melanoma development and may lead to genetic markers in melanoma. Some alterations may be used to identify interesting subpopulations. Others may be of prognostic value when they are considered in tandem with clinical data.

Illudin analogs are cytotoxic to a variety of multidrug resistant cell lines, and display an unusual toxicity towards DNA helicase-deficient cell lines. Earlier illudin analogs demonstrated efficacy in several xenograft models, including a metastatic MV522 lung cancer model, resistant to conventional anticancer agents. These illudin analogs prolonged life span as compared to conventional agents, but did not induce complete remission of primary tumors. In vitro screening studies identified a semisynthetic derivative, hydroxymethylacylfulvene (HMAF, MGI-114), with increased selective cytotoxicity towards carcinoma cells. The HMAF analog was markedly effective in the experimental MV 522 metastasizing lung carcinoma xenograft system, a model refractory to treatment with existing anticancer agents. Treatment with paclitaxel, doxorubicin, or cisplatin failed to significantly inhibit primary tumor growth or prolong life span of MV522 tumor-bearing animals. Treatment with mitomycin C at the LD20 increased life span in surviving animals up to 61% (p = 0.04). Treatment with HMAF induced primary tumor regression in all animals and increased life span greater than 150% (p0.001). Thus, administration of HMAF inhibited development of lung metastasis in a model refractory to treatment with conventional anticancer agents. These results support further evaluation of HMAF as a therapeutic agent for treatment of solid tumors such as adenocarcinoma of the lung.

In this study of ovarian carcinoma, we extended previous findings by performing FISH using chromosome 19 paint and microFISH probes and patient samples with and without abnormalities of chromosome 19 identified by G-banding. Karyotype interpretations of der(19) were confirmed, while additional 19 translocations were also detected by FISH with 19WCP in some cases. Similar FISH studies of ovarian carcinoma cell lines found chromosome 19 abnormalities even after extensive in vitro culture. MicroFISH probes were generated by chromosome microdissection from two cases with hsr(19) and mapped to 19q13.2 and 19q13.1-.2, respectively. FISH with these microFISH probes alone or in combination with a 19WCP probe to four patient samples and seven cell lines showed that 65% of chromosome 19 structural abnormalities contained 19q13.1-q13.2 sequences, sometimes as large hsrs. Ovarian cancer cell lines showed amplification and overexpression of the AKT2 putative oncogene, but not the ERCC-2 DNA repair gene in this chromosomal region. In addition to AKT2, amplification and overexpression of other yet-unidentified genes in the 19q13.1-q13.2 region may contribute to ovarian carcinoma pathogenesis or progression.

The EVI-1 gene was originally detected as an ectopic viral insertion site and encodes a nuclear zinc finger DNA-binding protein. Previous studies showed restricted EVI-1 RNA or protein expression during ontogeny; in a kidney and an endometrial carcinoma cell line; and in normal murine oocytes and kidney cells. EVI-1 expression was also detected in a subset of acute myeloid leukaemias (AMLs) and myelodysplasia. Because EVI-1 is expressed in the urogenital tract during development, we examined ovarian cancers and normal ovaries for EVI-1 RNA expression using reverse transcription polymerase chain reaction (RT-PCR) and RNAase protection. Chromosome abnormalities were examined using karyotypes and whole chromosome 3 and 3q26 fluorescence in situ hybridisation (FISH). RNA from six primary ovarian tumours, five normal ovaries and 47 tumour cell lines (25 ovarian, seven melanoma, three prostate, seven breast and one each of bladder, endometrial, lung, epidermoid and histiocytic lymphoma) was studied. Five of six primary ovarian tumours, three of five normal ovaries and 22 of 25 ovarian cell lines expressed EVI-1 RNA. A variety of other non-haematological cancers also expressed EVI-1 RNA. Immunostaining of ovarian cancer cell lines revealed nuclear EVI-1 protein. In contrast, normal ovary stained primarily within oocytes and faintly in stroma. Primary ovarian tumours showed nuclear and intense, diffuse cytoplasmic staining. Quantitation of EVI-1 RNA, performed using RNAase protection, showed ovarian carcinoma cells expressed 0 to 40 times the EVI-1 RNA in normal ovary, and 0-6 times the levels in leukaemia cell lines. Southern analyses of ovarian carcinoma cell lines showed no amplification or rearrangements involving EVI-1. In some acute leukaemias, activation of EVI-1 transcription is associated with translocations involving 3q26, the site of the EVI-1 gene. Ovarian carcinoma karyotypes showed one line with quadruplication 3(q24q27), but no other clonal structural rearrangements involving 3q26. However, whole chromsome 3 and 3q26 FISH performed on lines with high EVI-1 expression showed translocations involving chromosome 3q26. EVI-1 is overexpressed in ovarian cancer compared with normal ovaries, suggesting a role for EVI-1 in solid tumour carcinogenesis or progression. Mechanisms underlying EVI-1 overexpression remain unclear, but may include rearrangements involving chromosome 3q26. Images Figure 1 Figure 2 Figure 3 Figure 4

We report on the cytogenetic analyses of 158 cases of metastatic malignant melanoma, comprised of 63 cases with regional disease (RD) and 95 cases with distant (metastatic) disease (DD). Clonal structural abnormalities were identified in 126 (80%) cases and were significantly increased ( < 0.01 after adjusting for multiple comparisons) on chromosomes (in order of frequency of involvement) 1, 6, 7, 11, 9, and 3. Clustering of breakpoints occurred at 1p36, 1p22-q21, 6p11-q21, 9p, 11q23-qter, 13p (especially for cases with DD), and 19q13. The most common clonal numerical abnormalities, in a subset of 49 near-diploid cases were -10, -22, -9, +7, -19, and -Y. Analysis of chromosome segment gains and losses (CSRP) showed frequent loss of chromosomes 6 and 10, followed by equal rates of involvement of chromosomes 1, 7, and 9. Whole or segmental losses of chromosome 9 (especially 9p) correlate well with recent molecular genetic studies identifying putative suppressor genes, and are also likely important genetic abnormalities. However, based on the frequency of abnormalities in this large series of metastatic melanomas, it is likely that structural abnormalities of 1 and 6, and 10 are important in the pathogenesis of sporadic advanced melanoma.

High-dose chemotherapy regimens can cure a number of otherwise incurable diseases, such as Hodgkin's disease, non-Hodgkin's lymphoma, neuroblastoma, acute leukemia (in remission), and breast cancer. Trials of high-dose chemotherapy have generally used autologous bone marrow transplant or peripheral blood stem cell support to ensure hematologic recovery after intensive chemotherapy and/or radiation. This report describes an approach in which high-dose carboplatin chemotherapy was followed initially by granulocyte-macrophage colony-stimulating factor (GM-CSF; Escherichia coli. Sandoz-Schering, East Hanover and Kenilworth, NJ) and in subsequent patients, by both GM-CSF and repeated cycles of peripheral blood progenitor cell (PBPC) collection and administration. The addition of PBPC to this regimen led to significant reductions in the duration of neutropenia and thrombocytopenia, the requirement for erythrocyte and platelet transfusions, the length of hospital stay, and the use of intravenous antibiotics in this group relative to those patients who received GM-CSF alone. In addition, laboratory studies are presented that show a direct correlation between the number of progenitor cells reinfused and the duration of neutropenia and thrombocytopenia. The report also reviews data indicating that circulating progenitor cells are depleted by this approach. This suggests that the number of progenitor cells available for mobilization is finite. Finally, the magnitude of these effects, and their implications for future trials with repetitive cycles of dose-intensive therapy, are discussed.

A strictly factor-dependent cell line (UCSD/AML1) was established from a patient with the syndrome of multilineage acute leukemia with high platelets. The patient's cells and the cell line karyotype were 45,XX,- 7,t(3;3)(q21;q26), typical of the syndrome of acute leukemia with high platelets. The cell line expresses CD34, CD7, TdT, and myeloid (CD13, CD14, CD33) and megakaryocyte/platelet (CD36, CD41, CD42b, CDw49b) antigens. In short-term culture, UCSD/AML1 cells proliferate in response to interleukin-3 (IL-3), IL-4, IL-6, macrophage colony- stimulating factor (M-CSF), and granulocyte-macrophage CSF (GM-CSF), but not IL-1, IL-2, IL-5, or G-CSF. In long-term culture, proliferation can be sustained by GM-CSF, IL-6, or M-CSF. When maintained in GM-CSF, a small percentage of cells form multinucleated megakaryocyte-like giant cells. Culture with GM-CSF combined with IL-6, but not with IL-6 alone, increased giant cell formation fourfold to sevenfold. IL-6 alone or in combination with GM-CSF increased expression of platelet-related antigens. In contrast, culture with phorbol ester induced formation of macrophage-like cells. UCSD/AML1 is the first human acute nonlymphocytic leukemia cell line established from a patient with an acute leukemia syndrome associated with a specific chromosome abnormality.

Illudins are a novel class of agents with a chemical structure entirely different from current chemotherapeutic agents. A new semisynthetic derivative, MGI 114 (NSC 683,863, 6-hydroxymethyl-acylfulvene, HMAF), is markedly effective in a variety of lung, breast and colon carcinoma xenograft models. This analogue, MGI 114, is currently in phase I human clinical trials, and is scheduled for two different phase II trials. To determine if MGI 114 could be effective in vivo against mdr tumour cells, we generated an mdr1/gp170-positive clone of the metastatic MV522 human lung carcinoma line by transfecting a eukaryotic expression vector containing the cDNA encoding for the human gp170 protein. This MV522/mdr1 daughter line retained the metastatic ability of parental cells. The parental MV522 xenograft is mildly responsive in vivo to mitomycin C and paclitaxel, as evidenced by partial tumour growth inhibition and a small increase in life span, whereas MV522/mdr1 xenografts were resistant to these agents. In contrast to mitomycin C and paclitaxel, MGI 114 produced xenograft tumour regressions in 32 of 32 animals and completely eliminated tumours in more than 30% of MV522/mdr1 tumour-bearing mice. Thus, MGI 114 should be effective in vivo against mdr1/gp170-positive tumours.

Forty-three patients with myelodysplastic syndromes (MDS) received treatment with oral etoposide 50 mg/day for 21 consecutive days every 4 weeks. Eighteen patients (42%) experienced hematological responses, including 12 of 17 (70%) patients with chronic myelomonocytic leukemia (CMML). Three of five CMML patients who failed treatment with hydroxyurea experienced major hematological responses with oral etoposide. Median response duration exceeded 9 months (range: 4-49 + months), and one patient remains in an unmaintained complete remission for 4 years. Toxicity included nausea/vomiting in five patients, fever (four patients), infection (three patients), mucositis (two patients), and anorexia (two patients). Two patients had grade 4 neutropenia with sepsis necessitating treatment withdrawal. We conclude that low-dose oral etoposide has remitting activity in MDS and is an effective treatment alternative for patients with CMML.

The aminothiol, amifostine (Ethyol; U.S. Bioscience, West Conshohocken, PA), is a cytoprotective agent that ameliorates the toxicities of anticancer therapy. In vitro, amifostine promotes the formation and survival of primitive hematopoietic progenitors derived from myelodysplastic bone marrow (BM) specimens. To evaluate the hematological effects of amifostine, 18 patients with myelodysplastic syndrome (MDS) and one or more refractory cytopenias received treatment with amifostine in a Phase I/II study. Four cohorts received intravenous treatment with 100, 200, or 400 mg/m2 amifostine three times a week, or 740 mg/m2 weekly for three consecutive weeks followed by 2 weeks observation. Nonresponding patients received a second course of therapy at the next higher dose level depending upon drug tolerance. Bone marrow (BM) progenitor growth was assessed before treatment and after day 21. Diagnoses included refractory anemia (7), refractory anemia with ringed sideroblasts (5), refractory anemia with excess blasts (RAEB) (4), and RAEB-in transformation (RAEB-t) (2). Single- or multi-lineage hematologic responses occurred in 15 patients (83%) treated with the three-times-a-week dose schedule. Fourteen patients had a 50% or greater increase in absolute neutrophil count with amifostine treatment (range, 426 to 11,348/μL). Platelet count increased in 6 (43%) of 14 patients with thrombocytopenia (absolute increase, 16,000 to 110,000/μL), and 5 of 15 red blood cell transfusion-dependent patients had a 50% of greater reduction in transfusion needs. Assayable hematopoietic progenitors increased in 13 of 15 evaluable patients; including CFU-GEMM (12), BFU-E (8), and CFU-GM (6). Amifostine doses less than or equal to 200 mg/m2 were well tolerated, whereas grade II nausea, vomiting, and fatigue was limiting at higher doses. Three patients with excess blasts before enrollment experienced an increase in BM blast percentage and two patients had evolution to acute leukemia that persisted after treatment withdrawal. We conclude that amifostine administered at doses ≤200 mg/m2 three times a week is well tolerated and has hematologic activity in patients with MDS.

The EVI-1 gene encodes a Zn finger, DNA binding protein previously detected in some acute myelogenous leukemias (AML) and myelodysplasias (MDS), but not in normal marrow or cord blood cells. Experimental studies suggest EVI-1 blocks cellular differentiation by binding to GATA-1 or other specific DNA sequences controlling gene expression, and may be involved in the pathogenesis of some AMLs. To further define potential roles for EVI-1 in leukemia pathogenesis, we studied its regulation in acute promyelocytic leukemias (APL). Seven of 11 APL cases expressed EVI-1 RNA detected by RNA PCR at diagnosis, and expression was detected in two additional cases after treatment with all-trans retinoic acid (ATRA). Two of four cases studied at relapse also expressed EVI-1 RNA. To investigate regulation of EVI-1 expression in APL, we examined its expression in the NB4 APL cell line. NB4 cells did not express EVI-1 under basal conditions, but expressed EVI-1 after ATRA-induced differentiation. When NB4 cells were exposed to ATRA and transferred to cultures with N,N'-hexamethylene-bis-acetamide (HMBA), differentiation occurred but EVI-1 RNA was not detected, indicating that EVI-1 expression was not required for terminal, NB4 differentiation. ATRA-resistant NB4 cells were obtained by continuous culture in gradually increasing concentrations of ATRA. These cells did not express markers of differentiation but continued to express EVI-1 for several weeks even after ATRA withdrawal. To assess whether expression of the APL PML-RAR alpha fusion gene alone was sufficient for ATRA induction of EVI-1, the PML-RAR alpha gene cDNA was expressed in U937 histiocytic lymphoma cells. ATRA treatment of PML-RAR alpha-transfected or control U937 cells did not induce EVI-1 expression. In conclusion, this study demonstrates the EVI-1 gene is consistently expressed in APL cells either constitutively or after ATRA treatment. ATRA represents the first biologically active agent shown to specifically regulate EVI-1 expression in blood cells. In contrast to previous studies in AML and MDS, the pattern of EVI-1 expression suggests it may facilitate rather than inhibit myeloid differentiation during ATRA treatment. However, effects of EVI-1 expression are likely to be complex, and expression in ATRA-resistant APL cells may indicate multiple roles for this gene.

Although tissue hypoxia is the major stimulus for erythropoietin (EPO) production, serum EPO (sEPO) levels at any given Hb in iron-deficiency anaemia are relatively higher than in other anaemias. Iron chelators stimulate erythropoiesis in anaemia of chronic disease via unknown mechanisms. A recent study suggested that deferoxamine (DFO) regulates steady-state EPO RNA. Here we report that altered intracellular iron balance regulates EPO production both in vitro and in two unique clinical trials. In vitro, both iron chelation with DFO and blockade of Tf-mediated iron uptake with anti-Tf receptor antibody 42/6, stimulated EPO production in serum-deprived hepatoma cells. Conversely, iron repletion by haemin, inhibited EPO production in these cells. In clinical studies, sEPO levels rose in adult volunteers treated with DFO coupled to hydroxyethyl starch (HES-DFO) and in patients with advanced malignancy treated with anti-Tf receptor antibody 42/6, in a time- and dose-dependent manner. These studies indicate intracellular iron balance regulates EPO production in humans.

Simple near-diploid karyotypes in ovarian cancer may indicate either primary alterations related to tumor pathogenesis or abnormalities associated with early tumor progression. We have identified a series of 13 epithelial ovarian tumors with very simple karyotypes. Specifically, these karyotypes were near-diploid and displayed numeric abnormalities alone or combined with one or two structural alterations. The present series includes samples from 10 patients with newly diagnosed adenocarcinomas and 3 patients having borderline malignancies. Recurrent numeric abnormalities were identified and included 9/13 cases (69%) with + 12, eight cases (62%) with + 8, five cases (38%) with + 7, three cases (23%) each with + 3 or + 5, and two cases (15%) with -X, Five cases in this series displayed certain numeric abnormalities (+ 12, + 7, and -X) as the sole anomalies, thereby qualifying as primary karyotype changes. Of the 6 cases with structural abnormalities, 4 involved chromosome 19, 2 involved chromosome I, and the remaining abnormalities or translocation partners involved other chromosomes. These findings indicate that some numeric abnormalities are primary karyotype alterations in patients with malignant epithelial ovarian tumors and that chromosome 19 may be preferentially involved in structural rearrangements during early tumor progression. Genes Chromosom Cancer 10:262–266 (1994). © 1994 Wiley-Liss, Inc.

BACKGROUND Imexon, a 2-cyanoaziridine, is therapeutic and reverses lymphadenopathy and splenomegaly in the LP-BM5 murine retrovirus-induced immunodeficiency disease (murine AIDS). It can restore chemotherapy-induced immunosuppression. Imexon reduced the incidence of lymphoma in severe combined immune deficient mice inoculated with human lymphocytes. PURPOSE To determine its antitumor activity, we screened imexon against fresh human tumor cells and tumor cell lines. To determine the time-concentration relationships of its cytotoxicity, we studied the effects of imexon on macromolecular synthesis and on the cell cycle. METHODS Imexon was incubated at 1-200 micrograms/mL with various tumor cell lines, mitogen-stimulated peripheral blood lymphocytes, and fresh tumor cells. Cell survival, macromolecular synthesis, and cell cycle progression were studied. RESULTS The concentration of imexon that caused 50% inhibition of growth was under 10 micrograms/mL for lymphocytes stimulated with mitogens. It was about 3-10 micrograms/mL for B-cell lymphomas and both multi-drug-resistant and -sensitive myeloma cell lines. Imexon inhibited four of seven fresh lymphoma and 11 of 16 fresh myeloma biopsy specimens to less than 40% of the control. A 1-hour exposure of lymphoma cells to 50-100 microgram/mL followed by removal of drug by washing the cells and continuing culture resulted in greater than 95% inhibition during the next 48-72 hours. Imexon selectively inhibited protein synthesis during the first 24-48 hours of exposure of lymphoma and myeloma cells. Cells exposed to inhibitory concentrations of imexon were blocked in cell cycle progression. CONCLUSION Imexon may be a potentially useful agent in the treatment of malignant disease, particularly lymphoid malignancies, and should be explored further.

PURPOSE The trial was undertaken to study the effect of administering granulocyte-macrophage colony-stimulating factor (GM-CSF) with and without peripheral-blood progenitor cells (PBPC) on the hematologic and nonhematologic toxicity observed with multiple cycles of high-dose carboplatin chemotherapy. PATIENTS AND METHODS Eighteen patients with a variety of solid tumors received a total of 40 cycles of carboplatin, 1,200 mg/m2 per cycle, administered by continuous infusion over 96 hours. All 40 courses were administered with a daily 4-hour intravenous (IV) infusion of either 5 or 10 micrograms/kg/d of recombinant human Escherichia coli-derived GM-CSF. The first 20 courses were administered without PBPC support (treatment A). Because of severe neutropenia and thrombocytopenia, the next 20 courses of therapy were administered with GM-CSF, PBPC, and oral antibiotic prophylaxis (treatment B). RESULTS The addition of PBPC support led to a significant reduction in the duration of neutropenia (10.5 v 7.5 days; P = .027) and thrombocytopenia (12.4 v 5.2 days; P = .001), number of RBC transfusions (six v three; P = .01) and platelet transfusions (10.3 v 3.7; P = .013), number of hospital days (12.6 v 2.9; P = .01), and days of IV antibiotics (11.8 v 2.4; P = .007) per cycle. Significant increases in the weekly dose intensity (206 v 285 mg/m2/wk; P = .014) and total dose (2,287 v 3,600 mg/m2; P = .018) of carboplatin delivered were also observed with treatment B. The overall response rate in this study was 70%, with 11 of 16 assessable patients achieving either a complete (three patients) or partial (eight patients) remission. CONCLUSION This combination of GM-CSF and PBPC infusion represents an effective method for delivering multiple cycles of high-dose carboplatin chemotherapy and may serve as a model for the administration of high-dose chemotherapy in future trials.

The idea of attacking cancer cells at the cell surface was revived in the past decade by the availability of monospecific heteroantisera. Phase I trials using monoclonal antibodies to most tumor cell antigens have shown little anti-tumor activity (Reviewed in 1,2). The reasons for these failures are complex, but include heterogeneity of tumor antigen expression; antibodyinduced antigen modulation from the cell surface; formation of human anti-mouse antibodies; and the uncertain efficacy of the host cell anti-tumor response (1,2). Immunoconjugates employing monoclonal antibodies as carriers for toxins, drugs, or radioisotopes and chimeric human-mouse monoclonal antibodies remain under investigation (1,2).

The administration of high-dose chemotherapy has resulted in a marked increase in the frequency of both partial and complete responses in a large number of patients with solid and hematologic malignancies. This approach has led to long term remission and probable cure rates of 20–60% in patients with Hodgkin’s and non-Hodgkin’s lymphoma. Similar results have also been obtained in treating children with neuroblastoma and adults with first and second remission acute myelogenous leukemia1,2. While data on the long term results of high-dose therapy in breast cancer patients is less extensive, several reports do suggest that such regimens can prolong disease free survival for patients treated early in the course of their metastatic disease or in the adjuvant setting for women at high risk of early relapse3–6.

Cell-free extracts of bone marrow and blood cells from patients with leukemia contain an inhibitor of normal granulocyte/macrophage progenitor (CFU-GM) proliferation (leukemia-associated inhibitory activity, LIA) identified as acidic isoferritins. A comparison was made of the action of crude LIA prepared from frozen-thawed leukemic blood cells and purified spleen ferritin from a patient with chronic myelogenous leukemia, on the proliferation of blast progenitors from patients with acute myelogenous leukemia (AML), and on the promyelocytic leukemia cell line, HL-60. Crude LIA showed no inhibition of blast progenitor or HL-60 proliferation at low concentrations, but inhibited the proliferation of CFU-GM. At higher concentrations, crude LIA inhibited both blast cells and CFU-GM. Purified spleen ferritin failed to inhibit blast progenitors or HL-60 cells at any concentration tested, but inhibited both 70-day and 14-day CFU-GM. Using the thymidine “suicide„ technique, the action of LIA was confirmed as being on CFU-GM in S-phase, but it failed to affect the proliferation of blast cell in S-phase. It is concluded that acidic isoferritins inhibit normal CFU-GM but not blast cells from patients with AML. Acidic isoferritins could confer a proliferative advantage of the leukemic clone over its normal counterparts.

The peripheral blood of 2 patients with acute monocytic leukemia of the undifferentiated type was studied for the presence of leukemic progenitor cells in colony forming assays. The peripheral blood of both patients contained only one type of progenitor cell as determined with these assay systems. Daughter cells of these progenitor cells morphologically and histochemically resembled monoblasts and immature macrophages. Similar progenitor cells were not encountered in the study of 5 patients with acute myelogenous leukemia whose peripheral blood cells formed colonies in the same system. The finding of this unusual progenitor cell supports the existence of acute monocytic leukemia as a separate clinicopathologic entity, and suggests that it represents a malignant transformation of a progenitor cell of the monocyte/macrophage series. An alternative explanation of these observations would be provided by single phenotypic expressions of a multi-potent stem cell which has undergone malignant transformation.

The effect of purified human fibroblast interferon on primary and secondary colony formation by blast progenitors from the peripheral blood of patients with acute myelogenous leukemia was examined. Interferon inhibited blast progenitors and normal granulocyte/macrophage progenitors (CFU-C) in a dose-dependent manner. The magnitude of this effect on blast progenitors and CFU was similar. Interferon also inhibited secondary plating of blast progenitors (self- renewal). This effect was in marked contrast to the effect of adriamycin, which reduced primary plating efficiency of blast progenitors but did not affect self-renewal. Inhibition of blast progenitor proliferation by interferon was markedly reduced when interferon was added after 24 hr of culture and was absent when added after 72 hr. Inhibition of self-renewal was observed even when interferon was added at 72 hr. We conclude that interferon inhibits both primary proliferation and self-renewal of blast progenitors and that this effect is not due to reduction in the number of primary colonies. These experiments provide an example of how cell culture techniques may be used to test antitumor agents for effects on important cellular events other than general cytotoxicity.

Oxipurinol, the major metabolite of allopurinol, decreased the toxicity of 5-fluorouracil (5-FU) to human granulocyte colony-forming units in vitro by a factor of four. The ability of allopurinol to reduce 5-FU toxicity in vivo was studied in 23 advanced cancer patients during 42 courses of treatment. 5-FU was administered by continuous intravenous infusion for five days; allopurinol, 300 mg, po, every 8 hours was started 2 hours before and continued during and for 24 hours after 5-FU infusion. 5-FU was escalated from 1.5 to 2.25 g/m2/day on separate courses; the dose-limiting toxicity was mucositis which occurred at a level of 2.0 g/m2/day. At a 5-FU dose rate of greater than 2.0 g/m2/day 5-FU pharmacokinetics were nonlinear, reflecting saturation of catabolic pathways, and the steady-state 5-FU serum concentration was approximately 4 times that which was tolerable without allopurinol. At these concentrations of 5-FU oxipurinol significantly influenced the clearance of 5-FU. Thus concurrent allopurinol therapy permitted a doubling of the maximum tolerated dose of 5-FU and a four-fold increase in the tolerated concentration x time exposure to 5-FU.

Normal marrow granulocyte (CFU-GM) and peripheral blood T-lymphocyte (CFU-TL) colony-forming cells were studied for their sensitivity to high concentrations of thymidine (dThd) and compared to leukemic CFU from patients with acute nonlymphocytic leukemia (ANLL). The sensitivity of two ANLL cell lines was also assessed. dThd was toxic to both CFU-GM and CFU-TL at concentrations above 10(-5) M when cultured under conditions where dThd exposure was analogous to that used in clinical trials. There was little variation in the fractional colony survival between marrow samples, and the sensitivity of CFU-GM closely approximated that of CFU-TL. Thymine was not toxic at up to 10(-3) M. In liquid culture, T cells in G0 at the start of exposure were able to proliferate in the presence of 10(-3) M dThd, whereas T cells already proliferating in response to phytohemagglutinin (PHA) at the start of dThd exposure were killed. Leukemic CFU demonstrated marked variability in dThd sensitivity; blasts from some patients were resistant to dThd, while others were greater than 100-fold more sensitive than normal CFU- GM.

A case of fatal pulmonary fibrosis and atypical epithelial proliferation (AEP) in a patient with multiple myeloma treated with melphalan is presented. Review of 10 other autopsied patients with myeloma treated with melphalan but no thoracic radiation, other cytotoxic agents, or highdose oxygen therapy revealed one other patient who died with extensive pulmonary fibrosis and AEP. Four other patients with AEP not associated with pneumonitis or fibrosis were also found, while no such changes were found in 11 autopsy controls or 11 patients with myeloma who did not receive cytotoxic agents. Melphalan should be added to the growing list of agents capable of causing severe fibrotic pulmonary reactions.

Twenty-nine patients with extensive disease, small-cell carcinoma of the lung, were treated with two cycles of intensive combination chemotherapy: HexaVAC (hexamethylmelamine, vincristine, Adriamycin, cyclophosphamide). Responders received prophylactic cranial radiation (2000 rad/10 fractions) and non cross resistant chemotherapy via a schedule of alternating cycles of CMV (cyclophosphamide, methotrexate, VP-16-213) and AMV (Adriamycin, methotrexate, VP-16-213). Whenever a complete response was achieved, consolidation radiotherapy was given to the lung primary (4000 rad/20 fractions, split dose) and abdominal metastases (2000 rad/10 fractions) synchronous with CMV therapy. The complete response rate was 14% with HexaVAC, but increased to 38% during CMV/AMV. Total response rate (complete and partial) was 59% and median survival was 42 weeks. Prophylactic brain radiation prevented clinical relapse in the brain in all 14 patients who received it. However, consolidation radiotherapy failed to prevent clinical relapse in the lung and/or liver, and therapeutic brain radiation (3000 rad) failed to prevent relapse in that site. The simultaneous administration of radiotherapy and chemotherapy was well-tolerated although two patients with poor performance status died of infectious complications while leukopenic. In spite of the high response rate, durable remissions with prolonged disease free survival were rare. Further evaluation of induction, consolidation, and maintenance modes of therapy are indicated.

2-Chlorodeoxyadenosine (CdA), an adenosine-deaminase-resistant purine deoxynucleoside, is markedly toxic toward human T-lymphoblastoid cell lines in vitro and is an effective agent against L1210 leukemia in vivo. The present studies have examined the toxicity, and in some cases, metabolism, of CdA in (1) multiple established human cell lines of varying phenotype, (2) leukemia and lymphoma cells taken directly from patients, (3) normal bone marrow cells, and (4) normal peripheral blood lymphocytes. Nanomolar concentrations of CdA blocked the proliferation of lymphoblastoid cell lines with a high ratio of deoxycytidine kinase to deoxynucleotidase. The drug had virtually no effect on the growth of cell lines derived from solid tissues. The CdA inhibited the spontaneous uptake of tritiated thymidine by many T and non-T, non-B acute lymphoblastic leukemia cell specimens at concentrations less than or equal to 5 nM. The same concentrations did not impair either thymidine uptake or granulocyte-monocyte colony formation by normal bone marrow cells. In common with deoxyadenosine, but unlike several other agents affecting purine and purine metabolism, CdA was lethal to resting normal T lymphocytes and to slowly dividing malignant T cells. In both resting and proliferating lymphocytes, the CdA was phosphorylated by deoxycytidine kinase and entered a rapidly turning over nucleotide pool. Dividing lymphocytes also incorporated abundant CdA into DNA. The selective toxicity of CdA toward both dividing and resting lymphocytes may render the drug useful as an immunosuppressive or antileukemic agent.

Previous studies suggested tumor colony-forming cells (CFC) grown from xenografts might be useful as a preclinical, in vitro drug screen. To further evaluate this possibility, eight melanoma and six ovarian carcinoma xenografts were established from untreated patients and tested for in vitro CFC growth. For each tumor, linear relationships between cells plated and colony (30 cells or greater than 75 micron diameter) and cluster (10-30 cells or 50-75 micron) growth were observed. All eight melanomas grew sufficient colonies (greater than or equal to 30) for in vitro drug assessment, although four required hypoxic (pO2 = 40) incubation to reliably attain this level of growth. Only one in six of the ovary xenografts consistently grew enough colonies, and growth was not significantly improved by hypoxic incubation, or addition of luteinizing hormone, follicle-stimulating hormone, or steroid hormones. Cloning efficiencies (colonies + clusters/cells plated) for tumors demonstrating adequate growth ranged from 0.01% to 0.3%. For most tumors, no direct relationship was observed between characteristics of xenograft tumors (size) or their resulting cell suspensions (viabilities, cell yield) and CFC growth. Cell suspensions were incubated with a 3 log concentration of nine established chemotherapeutic agents. Resulting dose-effect curves were linear and showed no plateaus of drug effect. Analyzing 447 in vitro drug trials on six melanomas and one ovarian carcinoma, interexperiment variability was high. Cell lines were established from three xenografts using a low concentration of fetal bovine serum (1%), and also examined for in vitro drug sensitivity. Using both liquid culture isotope incorporation and a colony-forming assay, drug sensitivity profiles for the cell lines were nearly identical to those for parent xenograft CFC. However, assays performed using the cell lines were more reproducible than those using xenograft tissue. The authors conclude that tumor CFC can be reliably grown from melanoma xenografts, but in vitro drug assays using these xenografts are poorly reproducible. The xenografts are a resource for establishing cell lines, and drug assays performed using these lines are highly reproducible. Similarities in drug sensitivity profiles for parent xenograft CFC and derived cell lines suggest that, despite poor reproducibility, repetitive assays using melanoma CFC accurately reflect some properties of cells which sustain tumor cell growth.

The complement-dependent cytotoxicity of monoclonal T-cell antibody (T101) for normal and abnormal hemopoietic progenitors was assessed. T101 demonstrated toxicity for normal T-colony-forming cells from peripheral blood and bone marrow. Cytotoxicity was absent for normal peripheral blood and bone marrow granulocytes/macrophage (CFU-C) and erythroid (BFU-E) progenitors. The antibody was also not toxic for peripheral blood blast progenitors from patients with acute myelogenous leukemia (AML). These studies indicate the absence of the antigen defined by T101 (T65) from normal progenitor cells and from blast progenitors in patients with AML. T101 may be used in the treatment of T-cell malignancies and in the prevention of graft-versus-host disease (GVHD) without damage to normal progenitor cells.

In vitro systems were used to detect Ia-like antigens on proliferating normal myeloid and acute nonlymphocytic leukemia (ANLL) blast cells. Incubation of normal bone marrow cells with a monoclonal anti-Ia antibody and complement resulted in toxicity for both granulocyte/macrophage progenitors (CFU-GM) (toxicity 79%-100%) and cells proliferating in liquid culture in response to placenta-conditioned medium colony-stimulating factor (CSF) or medium conditioned by normal, phytohemagglutinin (PHA)-stimulated mononuclear cells. In contrast, effects of anti-Ia antibody and complement on blast colony-forming cells and 3H-TdR incorporation in liquid culture from eight patients with ANLL were variable. Colony growth with CSF after treatment was 0% to 91% of control growth and did not correlate with display of Ia-like antigens. Survival of ANLL cells growing in liquid cultures was even more variable after anti-Ia+ complement treatment (28%-227% of control). The presence of Ia-like antigens did not distinguish ANLL cells responding to PHA-conditioned medium from those responding to CSF in either colony or liquid culture. Dose-response curves for ANLL cells in liquid culture were similar before and after treatment with anti-Ia+ complement. In contrast to normal myeloid precursor cells, which show uniform display of Ia-like antigens, display of Ia antigen by proliferating leukemia cells is highly variable from patient to patient. Anti-Ia reagents such as this one would not be effective in treating ANLL marrow for autologous transplantation.

Cell lines derived from human melanoma xenografts were characterized for surface markers, karyotype abnormalities, and in vitro drug sensitivity. Xenografts were established using metastatic explants from untreated patients and passaged in nude mice. Cell lines were readily established from melanoma xenografts, and formed colonies when plated in semisolid media. The lines expressed human melanoma-associated and other surface antigens, human lactate dehydrogenase (LDH) isoenzymes, and contained only human chromosomes. They failed to express murine histocompatibility determinants and were negative for murine viruses by mouse antibody production assay. Karyotypes showed abnormalities of chromosomes 3, 6, and 7 similar to other melanomas. In vitro chemosensitivity profiles were compared using cell line and xenograft colony-forming assays. Values were similar for the original xenografts and their cell lines. Xenograft-derived human melanoma lines resemble other melanoma cell lines and primary melanomas with respect to surface antigens and karyotype abnormalities, and are appropriate models for studying in vitro drug sensitivity. When used as a model for transition from solid tumor to cell line, these studies suggest cell lines closely mirror in vitro chemosensitivities of parent tumor cells. However, occasional, unpredictable changes in sensitivity to some drugs occurs during this transition.

Thymidine (dThd) concentrations have been measured in the sera of normal subjects and solid tumor cancer patients by means of a sensitive high-pressure liquid chromatographic assay to determine whether natural and methotrexate (MTX)-induced fluctuations were large enough to alter the toxicity of MTX to marrow. The mean concentration in normal subjects with measurable levels was 1.3 X 10(-7) M (range less than 4 X 10(-8) to 6 X 10(-7) M). In cancer patients it was 2.0 X 10(-7) M (range less than 4 X 10(-8) to 8.7 X 10(-7)), and in malignant effusions 1.2 X 10(-7) M (range less than 4 X 10(-8) to 2.2 X 10(-7) M). The wide range of variation in random samples was also found when multiple samples were obtained from the same patient during a 24-h period where dThd concentration varied from a minimum of two- to greater than six-fold. Treatment with MTX 3 mg/m2 caused an average 59% reduction in serum dThd during the first 24 h after injection during nine courses of therapy. dThd was tested for its ability to modulate the toxicity of MTX to human granulocate colony-forming units in culture across the concentration range found in vivo: changes in dThd concentration equivalent to normal fluctuations in vivo altered colony survival by 31% to greater than 72%. A reduction in culture dThd equivalent to that produced in vivo by high-dose TMX increased colony kill by 25%. The results indicate that in vivo variations in serum dThd are in an appropriate range and of a sufficient magnitude to alter the toxicity of MTX to marrow, and they demonstrate that MTX can modulate its own toxicity by reducing serum dThd.

In these studies, we report adaptation of a colony-forming assay to chronic lymphocytic leukemia (CLL) peripheral blood cells. T-lymphocyte-depleted CLL peripheral blood cells were cultured with irradiated, normal T cells and media conditioned by normal, mitogen-stimulated T cells in methylcellulose. Colonies containing small and transformed lymphocytes appeared after 5-7 days incubation. The plating efficiency of CLL colonies was 0.15 +/- 0.08% (x +/- S.D.), similar to that of other colony-forming systems. The majority of CLL colony-forming cells were in S phase (50 +/- 4% x +/- S.E.) as determined by thymidine suicide and the fraction of colony-forming cells in S phase was inversely related to the WBC. Cells harvested from CLL colonies lacked surface markers for T lymphocytes and stained positively for monoclonal surface and/or cytoplasmic immunoglobulin light chains. A 1-h incubation was used to study the in vitro response of CLL colony-forming cells to adriamycin and melphalan. Preliminary studies suggest differences in patterns of in vitro sensitivity to melphalan between patients previously treated with alkylating agents and those who had not received treatment. This system can be used to study regulation of CLL cell proliferation, and may have utility in predicting response to chemotherapeutic agents.

To evaluate the usefulness of plasma calcitonin as an index of tumor burden and disease activity, we undertook a prospective study of serial calcitonin measurements in a group of patients with small cell carcinoma of the lung from diagnosis throughout a period of intensive therapy. Plasma calcitonin was significantly elevated in 84% of patients with extensive small cell carcinoma of the lung and was not elevated in patients with limited disease at the time of diagnosis. The elevated values fell significantly in response to chemotherapy and radiation therapy, and reflected regression of followable disease and improvement in clinical status. A significant correlation existed between plasma calcitonin and extent of disease. Relapse was generally associated with an increase in elevated plasma calcitonin levels, and calcitonin appeared to reflect tumor burden. The serial measurement of plasma calcitonin is useful in the management of the patient with small cell carcinoma of the lung.

In a pilot study, 21 consecutive eligible patients with extensive-stage small cell carcinoma of the lung were scheduled for treatment with combination chemotherapy followed by total-body irradiation (TBI), prophylactic cranial irradiation, and consolidative chemotherapy. Induction chemotherapy consisted of VP16-213, vincristine, cyclophosphamide, and doxorubicin (VOCA). TBI was given as 100 rads in 10 fractions over 2 wk. Consolidation chemotherapy consisted of cyclophosphamide, methotrexate, and hexamethylmelamine (CMH). VOCA chemotherapy was well tolerated, with a 79% response rate in 19 evaluable patients. TBI was successfully given after four cycles of VOCA without excessive morbidity in 11 patients, although subsequent CMH chemotherapy in 8 patients has required dose reductions and some delays in therapy. Unfortunately, TBI did not increase the degree of response, and 2 patients relapsed during this therapy. Median survival in this study was 40-44 wk. One patient has survived 78 wk and remains in remission. TBI can be safely given following induction chemotherapy in extensive-stage small cell carcinoma of the lung, but it does not appear to add to the therapeutic benefit of combination chemotherapy alone.

The effects of three monoclonal antibodies (B3/25, 43/31, and 42/6) reactive with human transferrin (Tf) receptors on growth of normal and malignant myeloid cells were examined using in vitro culture techniques. When added directly to cultures, all three antibodies caused dose-dependent inhibition of normal granulocyte/macrophage progenitor (CFU-GM) growth. Monoclonal antibody 42/6 was by far the most potent of the three, with an ID50 of less than 5 micrograms/ml. Identical effects were seen on CFU-GM from three patients with chronic myelogenous leukemia. Growth of colonies from two myeloid leukemia cells lines (KG-I, HL60) was also inhibited by all three antibodies, and these cells were generally more sensitive than normal CFU-GM. Blast colony-forming cells from three patients with acute non-lymphocytic leukemia were relatively resistant to the antibodies, and CFU-GM from a patient with myeloid metaplasia were resistant (ID50 greater than 50 micrograms/ml) to 42/6. In liquid culture, growth of the leukemic cell lines was inhibited by saturating concentrations of the three antibodies, although in both liquid and colony culture recovery was seen even after exposure to antibody for periods of up to 72 h. Analysis of the cell-cycle status of these cells showed that the antibodies did not cause accumulation of cells in any particular phase of the cell cycle. Addition to cultures of large quantities of human Tf failed to reverse the inhibitory effects of the antibodies. Competitive binding studies on the leukemia cell lines showed that only 42/6 inhibited binding of Tf to its receptor, although all three antibodies inhibited cell growth. Addition of Fe chelate (as ferric nitriloacetic acid, FeNTA) failed to reverse the inhibitory effects of the antibodies on CFU-GM and HL60 cells, but had variable effects on KG-I cell growth. FeNTA fully reversed inhibitory effects of 42/6 on KG-I cells. We conclude that monoclonal antibodies to Tf receptors can inhibit growth of both normal and malignant myeloid cells. Overall, no selectivity for malignant vs normal cells is apparent, although malignant cells from one individual were more sensitive to colony inhibition by 43/31 monoclonal antibody than normal CFU-GM.

The EVI1 gene encodes a zinc-finger, DNA-binding protein originally described as the transforming gene associated with a common ecotropic viral insertion site in myeloid leukemias. Previous studies demonstrated EVI1 expression in human leukemias in cases with 3q26 translocations, but not in normal blood or bone marrow. These studies also suggested an association between EVI1 expression and chromosome 7 deletion (del). Because of this association, we examined expression of EVI1 using RNA polymerase chain reaction (PCR) in patients with myelodysplastic syndromes (MDS) and acute leukemia with and without 3q26 translocations. EVI1 RNA was expressed in 29% of 34 (95% confidence interval, 20% to 50%) patients with the MDS subtypes refractory anemia (RA), refractory anemia with excess blasts (RAEB), or refractory anemia with excess blasts in transformation (RAEB-T). The vast majority of these cases occurred in patients with RAEB and RAEB-T. EVI1 expression was not detected in patients with chronic myelomonocytic leukemia (CMML), normal bone marrow or cord blood, or a variety of other hematologic malignancies. EVI1 RNA was detected in three of 18 patients with acute myelogenous leukemia (AML) and in two of four patients with acute promyelocytic leukemia (APL). Karyotypes showed that only one AML patient had karyotype 3q26 abnormalities, indicating that EVI1 expression is associated with cases that do not have structural abnormalities involving chromosome 3q26. These studies document for the first time the abnormal expression of EVI1 RNA by patients with MDS, and suggest an important role for EVI1 in the pathogenesis or progression of some myeloid malignancies.

Excerpt To the editor: The report by Kiang and Kennedy (1) of tamoxifen therapy in advanced breast cancer noted only two patients with hypercalcemia. Both patients had negative assays for estrogen ...