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Since the translocation breakpoint t(15;17) (q22;q21) in acute promyelocytic leukemia (APL) occurs within the retinoic acid receptor- alpha (RARA) gene, the expression of many genes normally regulated by RARA may be affected by this translocation. To identify genes that may be aberrantly expressed in APL, a subtraction cDNA library of an APL patient with t(15;17) was constructed. A cDNA, pRD1, specifically expressed in APL was identified. DNA sequence analysis of pRD1 showed that this gene is similar to the DNA sequence of annexin VIII, a gene which encodes a vascular anticoagulant. The annexin VIII gene was assigned to chromosome 10, which indicates that specific expression of this gene in APL is not directly involved in the t(15;17) breakpoint region. We have analyzed the expression of annexin VIII gene in nine t(15;17)-positive APL patients and one APL patient with a chromosome 17q-abnormality. We found that all APL samples expressed high levels of the annexin VIII gene. Expression of the annexin VIII gene in all other leukemias, including acute myelogenous leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, and acute lymphoblastic leukemia, was undetectable, except in one patient with acute myelogenous leukemia in which a very low level of expression was detected. Annexin VIII is highly expressed in the APL cell line, NB4. Its expression was significantly reduced after 8 hours of all-trans retinoic acid (ATRA) treatment, whereas the expression of RARA increased several-fold within 4 hours postinduction. Thus, increased expression of RARA preceded the downregulation of annexin VIII after ATRA induction, suggesting an inverse relationship between RARA and annexin VIII expression. Since increased expression of the fusion transcript was seen after ATRA induction and an APL without a t(15;17) translocation expressed high levels of annexin VIII, it appears that increased expression of annexin VIII in APL is not related to the fusion transcript. Therefore, dysregulation of the RARA gene may be related to the overexpression of annexin VIII in APL.

Vascular endothelial growth factor receptor-2 (VEGFR-2), also termed KDR, is a high-affinity vascular endothelial growth factor (VEGF) receptor. VEGFR-2 plays a role in de novo blood vessel formation and hematopoietic cell development. Recently, we found that chronic lymphocytic leukemia (CLL) cells express high levels of VEGF. Therefore, we sought to investigate the role of VEGFR-2 in CLL. Using Western blot analysis, we first determined that VEGFR-2 is present in peripheral blood CLL cells. We then quantified the cellular levels of VEGFR-2 protein using a solid-phase radioimmunoanalysis in peripheral blood cells from 216 patients with CLL. As control, we used peripheral blood mononuclear cells (PBMNCs) from 31 hematologically normal individuals. The median of VEGFR-2 levels detected in the control samples was assigned a value of 1.0, and VEGFR-2 protein levels were normalized to the control median value. The median level of VEGFR-2 in CLL cells was 1.57. Patients with VEGFR-2 levels higher than 1.57 had elevated lymphocyte counts, severe anemia, elevated beta(2)-microglobulin and advanced-stage disease. Elevated VEGFR-2 levels were also associated with statistically significantly shorter survival (35.4 versus 60.1 months; P0.01). Our data indicate that cellular VEGFR-2 levels may serve as a prognostic factor in CLL. Further studies should investigate the biological implications of these findings and the effect of the interaction between VEGF and VEGFR-2 on CLL cell proliferation.

The objectives of this study were to describe cases of avascular necrosis of the femoral head (ANFH) observed in chronic myeloid leukemia (CML) patients who were treated with interferon-alpha and to review the literature.The authors undertook a case review of the M. D. Anderson experience with ANFH occurring in CML patients who were managed with interferon-alpha-based therapy. MEDLINE (from 1966 to November 1999) and CancerLit (from 1983 to November 1999) searches were conducted to identify cases of avascular necrosis (AVN) associated with either CML or interferon-alpha.Three patients with ANFH were identified from the authors' experience. No common features related to the disease or therapy were seen among them, except for the presence of thrombocytosis and loss of response. A literature review revealed seven cases of ANFH associated with CML with or without interferon-alpha-based therapy. ANFH was not reported in association with interferon-alpha use for indications other than the treatment of patients with CML.ANFH may be the result of an interaction between CML and interferon-alpha therapy. ANFH that occurs in patients with CML who are treated with interferon-alpha should be recognized for treatment implications. Thrombocytosis with consequent microvascular thrombi and avascular necrosis manifesting in susceptible vascular or weight-bearing areas (e.g., the femoral head) may be an associated finding along with loss of response to interferon-alpha therapy.

The molecular mechanism for the occurrence of leukemia in multiple members of a family has not been fully elucidated but data support the contribution of highly penetrant mutations in leukemia susceptibility genes. We have investigated the genetic etiology of an unusual three-generation family with apparent autosomal dominant transmission of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) accompanied by somatic loss of the long arm of chromosome 5 and/or loss of heterozygosity (LOH) analysis and fluorescence in situ hybridization (FISH) of leukemia cells have been performed, confirming acquired hemi- and homozygous deletion of the long arm of chromosome 5. However, the chromosome lost in the observed LOH event is from the affected parent, in contradiction to the expectation for a two-hit hypothesis involving a tumor suppressor gene. Furthermore, genetic linkage has been performed at 5q31-33 as well as other loci (21q22 and 16q21-23.2) previously implicated in familial leukemia. In this family, linkage analysis excludes loci at 5q31-33 and 21q22, but localization to 16q21-23.2 cannot be excluded. We observed a maximum multipoint LOD score of 1.19 between marker D16S265 and D16S503 at 16q22 (P = 0.03), suggesting possible linkage to this locus. Considering this family and the previous 16q-linked family together, the linkage of a leukemia susceptibility gene to 16q22 achieved an LOD score of 3.63 at D16S265 with theta = 0. Thus, somatic deletion of the long arm of chromosome 5 appears as a necessary but surprisingly noncausative event for onset of AML and MDS in this family, thereby confirming a multistep etiology in which chromosome 5 plays an important secondary role.

All-trans retinoic acid administered orally (oral ATRA) may not regularly lead to either molecular complete remissions (CRs) or prolonged hematologic CRs (HCR) unless combined with chemotherapy. Because serum tretinoin concentrations are higher, and maintained longer, after use of liposomal-encapsulated ATRA (lipoATRA) rather than oral ATRA, we investigated lipoATRA monotherapy in newly diagnosed acute promyelocytic leukemia (APL). Patients received lipoATRA 90 mg/m(2) every other day for remission induction. The same dose was given 3 times a week until 9 months had elapsed from HCR date. Treatment then stopped. Chemotherapy (idarubicin 12 mg/m(2) daily days 1-2 for 2 courses) was to be added only if 2 polymerase chain reaction (PCR) tests, performed 2 weeks apart, were positive at 3, 6, or 9 months from HCR date. The sensitivity level of the PCR was 10(-4). We treated 18 patients (median age, 54 years; median white blood cell [WBC] count 4,500/microL). The HCR rate was 12/18 (67%, 95% confidence interval [CI], 41% to 87%). This rate was similar to that we observed in a previous study using oral ATRA + idarubicin. Nine of 10 patients studied at HCR date were PCR-positive. Subsequently, however, overall (+/- idarubicin) rates of PCR positivity were 0/12 at 3 months, 1/10 at 6 months, 1/7 at 9 and 12 months, and 0/4 at 15 to 17 months. Idarubicin has been added in 3 patients, with this addition occurring at 6 months in 2 patients and at 9 months in 1 patient. Among patients who had not received idarubicin when the PCR was evaluated, 0 of 12 were PCR-positive at 3 months, 1 of 10 was positive at 6 months, 1 of 6 was positive at 9 months, 0 of 4 were positive at 12 months, and 0 of 3 were positive at 15 to 17 months. Morphologic APL has recurred in 1 patient, with a median follow-up time of 13 months in the 11 patients remaining in first CR. The median follow-up time is 91/2 months (range, 3 to 17) in the 9 patients who have received only lipoATRA and who remain PCR-negative and in first CR. Our data suggest that lipoATRA is an effective means of producing molecular CR in newly diagnosed APL.

Interleukin-2 (IL-2) has immunomodulatory effects, including stimulating the activity of cytotoxic T cells and natural killer cells, and inducing the generation of lymphokine-activated killer cells. The authors investigated whether IL-2 may improve the duration of complete remission (CR) and survival in acute myelogenous leukemia (AML) patients in first CR.Eighteen patients were included after achieving a CR and receiving at least two courses of consolidation chemotherapy. Therapy was comprised of IL-2 4.5 x 10(5) U/m2 daily by continuous infusion (CI) for 12 weeks, plus boluses of 1 x 10(6) U/m2 on Day 8 and weekly thereafter while continuing the CI. No further chemotherapy was given after the administration of IL-2 was started.The median age of the patients was 50 years (range, 18-73 years), and 7 patients (39%) had an antecedent hematologic disorder (AHD). The median CR duration was 12 months, with 6 patients still alive in CR at a median follow-up of 64 months (range, 50-82 months). Long term CR by cytogenetics occurred in 2 of 5 patients with a normal karyotype (CR duration of 68+ months and 72+ months, respectively), 1 of 3 patients with t(8;21) (CR duration of 82+ months), 1 patient with inv(16) (CR duration of 67+ months), none of 2 patients with -5/-7 (1 patient died in CR after 10 months), 1 of 2 patients with abnormalities in chromosome 11 (CR duration of 60+ months), and 1 of 4 patients with miscellaneous abnormalities (CR duration of 74+ months). The median survival was 47 months. To assess the significance of these results, the authors selected two historic controls receiving long term postremission chemotherapy per each IL-2 case. The controls had remained in CR for at least as long as the cases when the latter underwent treatment initiation with IL-2 and were matched for the number of induction courses required to achieve CR, AHD, cytogenetic abnormalities, and age. Six of 18 IL-2 patients (33%) were alive in CR at 3 years compared with 7 of 36 controls (19%) (P = 0.31). Nine IL-2 patients (50%) were alive at 3 years compared with 10 controls (28%) (P = 0.13).These results suggest that IL-2 is tolerable in AML patients in first CR and should be studied further in future studies as a therapeutic strategy to prolong remission duration.

The development of chemotherapy for patients with the major cancers progressed from the initial success attained in the treatment of acute leukemias and choriocarcinoma. Many of the principles of therapy were based on the concepts developed in the experimental laboratories and early clinical studies done at the NIH Clinical Center and other centers around the country. The purpose of this review is to describe some of the early advances in cancer therapy and show how many are based on the efforts of Dr. Emil J Freireich. Over his career, Dr. Freireich has published more than 500 papers and worked on more than 70 different drugs and combinations. The principles defined by Dr. Freireich, namely, the use of intermittent intensive chemotherapy to induce complete remissions (CRs), intensification of therapy in remission, and the use of unmaintained remissions to assess cure, have been important in developing curative chemotherapy programs in patients with acute leukemias. These same principles were applied to combination therapy of Hodgkin's disease as the nitrogen mustard, vincristine, procarbazine, and prednisone combination was developed. This led to the high CR and cure rate for this disease. The treatment of metastatic breast cancer does not produce a high proportion of CRs, and cures of metastatic disease are unlikely with chemotherapy alone. But adjuvant chemotherapy after surgery has resulted in a significant reduction in cancer mortality. Many challenges remain in increasing the cure rate for the major solid tumors. New avenues of controlling cell growth and metastases need to be explored. One approach that is exploitable is the use of drugs or nutrients to prevent cancer. Laboratory approaches are now becoming a clinical reality.

Dr. Emil J Freireich is a pioneer in the rational treatment of cancer in general and leukemia in particular. This essay in his honor suggests that the cell kill concept of chemotherapy of acute myeloblastic leukemia be extended to include two additional ideas. The first concept is that leukemic blasts, like normal hemopoietic cells, are organized in hierarchies, headed by stem cells. In both normal and leukemic hemopoiesis, killing stem cells will destroy the system; furthermore, both normal and leukemic cells respond to regulators. It follows that acute myelogenous leukemia should be considered as a dependent neoplasm. The second concept is that cell/drug interaction should be considered as two phases. The first, or proximal phase, consists of the events that lead up to injury; the second, or distal phase, comprises the responses of the cell that contribute to either progression to apoptosis or recovery. Distal responses are described briefly. Regulated drug sensitivity is presented as an example of how distal responses might be used to improve treatment.

The purpose of this study was to review the progress in clinical and translational research in chronic myelogenous leukemia (CML) over the past 20 years at M.D. Anderson Cancer Center. The CML database updating the clinical and basic research investigations was reviewed as the source of this report. Publications resulting from these investigations were summarized. The long-term results with intensive chemotherapy, IFN-alpha therapy alone or in combination, autologous stem cell transplantation, and new agents such as homoharringtonine and decitabine showed encouraging results. Biological studies related to the BCR-ABL molecular abnormality, other molecular events, and the detection of minimal residual disease were detailed. Future strategies with potential promise in CML were outlined. Significant progress in understanding CML biology and in treating patients afflicted with the disease has occurred. Several therapeutic and research tools are currently investigated, which should hopefully improve further the prognosis of patients with CML.

A review of the last four decades of leukemia research and treatment provides a view toward the next four decades. What is learned in leukemia research may apply to the treatment of other cancers. The biology of cancer, the importance of chromosomes, and the suppressor genes may hold the answers to future treatments.

The recently characterized immunotoxin HuM195-gelonin consists of a humanized anti-CD33 monoclonal antibody conjugated to the single-chain plant toxin gelonin. Binding of the immunotoxin to hematopoietic cells that express the CD33 differentiation antigen has been demonstrated and results in cytotoxicity due to ribosomal inactivation by gelonin. Blast cells from most patients with acute myelogenous leukemia express CD33, whereas normal stem cells necessary for maintenance of hematopoiesis do not. We asked whether an immunoconjugate using recombinant gelonin rather than plant gelonin is toxic to acute myelogenous leukemia (AML) cell lines and primary AML blasts obtained from patients and exposed to the immunotoxin in vitro. The CD33pos cell lines HL60, OCI/AML2, and OCI/ AML5 showed decreased proliferation when exposed to immunotoxin for 24-72 h. The CD33neg cell line OCI/AML3 was relatively resistant to HuM195, and all cell lines were resistant to equimolar concentrations of unconjugated antibody and gelonin. Primary blast cultures from seven patients with AML had CD33 detectable on 75.7-99.8% of cells by flow cytometry, and all showed dose-dependent decreases in clonogenic cell survival during 24-h incubation with the immunotoxin. Cells selected for low CD33 expression by cell sorting or by prolonged incubation with immunotoxin could reexpress CD33 at baseline levels and remained sensitive to immunotoxin. We conclude that humanized M195 conjugated to recombinant gelonin has antileukemic activity and should be considered for clinical testing in Phase I trials.

One hundred seventy-four patients with progressive or advanced chronic lymphocytic leukemia (CLL) have received initial therapy with fludarabine as a single agent or fludarabine combined with prednisone. The overall response rate was 78% and the median survival was 63 months. No difference in response rate or survival was noted in the 71 patients receiving fludarabine as a single agent compared with the 103 patients who received prednisone in addition. The median time to progression of responders was 31 months and the overall median survival was 74 months. Patients over the age of 70 years had shorter survivals. Patients with advanced stage disease (Rai III and IV) had a somewhat shorter survival than earlier stage patients. More than half the patients who relapsed after fludarabine therapy responded to salvage treatment, usually with fludarabine-based regimens. Second remissions were more common in patients who had achieved a complete remission on their initial treatment. The CD4 and CD8 T-lymphocyte subpopulations decreased to levels in the range of 150 to 200/microL after the first 3 courses of treatment. Although recovery towards normal levels was slow, the incidence of infections was low in patients in remission (1 episode of infection for every 3.33 patient years at risk) and decreased with time off treatment. There was no association of infections or febrile episodes with the use of corticosteroids or the CD4 count at the end of treatment and a poor correlation with the increase in CD4 counts during remission. Infectious episodes were less common in patients who had a complete response compared with partial responders. Richter's transformation occurred in 9 patients and Hodgkin's disease occurred in 4 patients. Five other patients died from other second malignancies. Fludarabine appears to be an effective initial induction therapy with a reasonable safety profile for patients with CLL.

Chemotherapy is known to cure a small minority of patients with acute myeloid leukemia (AML). Less is known about the risk of such patients developing subsequent cancers or about their ability to return to work.The authors analyzed outcomes among 1892 patients who received treatment for newly diagnosed AML at the University of Texas M. D. Anderson Cancer Center from 1965 to May 1995.Because failure rates declined to relatively low levels after a first or later complete remission ofor = 3 years' duration, such patients comprised a "potentially cured" cohort. The criterion for entry into this cohort was fulfilled by 215 patients (10.7%; 203 in first complete remission and 12 in second remission). At a median of 6.2 years after entry into the cohort (i.e., 9.2 years from complete remission), 163 patients (76%) remain alive and in complete remission. Approximately 9% and 5% of the 1892 patients have been in complete remission for5 years and10 years, respectively. The pretreatment prognostic importance of cytogenetics is still apparent even after 5 years in complete remission. On average, members of the potentially cured cohort were not observed to be at increased risk of subsequent invasive malignancies compared with a normal population. Furthermore, two-thirds of those in the potentially cured cohort who were working full time before diagnosis of AML claimed to have returned to full-time work. Of those not working, only 10% cited physical limitation as the reason.The major threat to the life and well-being of the patient with AML is clearly the disease and not its treatment.

Recent work has demonstrated that glucocorticoids, nucleoside analogues, and other cancer chemotherapeutics induce apoptosis in chronic lymphocytic leukemia (CLL) cells. In this study, we investigated the involvement of protease activation in these responses using selective peptide inhibitors of the interleukin-1beta converting enzyme (ICE)/caspase family and a Ca2+-activated protease we recently implicated in thymocyte apoptosis. Apoptosis was associated with proteolytic cleavage of poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) and increased caspase protease activity, and cell-permeant caspase antagonists [zVAD(OMe)fmk and Boc-D(OBzl)cmk] blocked apoptosis in response to the glucocorticoid methylprednisolone or the nucleoside analogue fludarabine, indicating that caspase activation was required for these responses. However, a peptide-based inhibitor of the Ca2+-dependent lamin protease (zAPFcmk) also completely suppressed DNA fragmentation and the cleavage of lamin B1 . Strikingly, treatment of cells with zAPFcmk alone led to characteristic PARP cleavage, depletion of the precursor forms of two ICE family proteases (CPP32 and ICH-1), and phosphatidylserine exposure, suggesting that blockade of the lamin protease led to activation of the ICE family. Our results implicate the lamin protease as a target for Ca2+ during chemotherapy-induced apoptosis in CLL lymphocytes, and they identify a novel functional interaction between the protease and members of the ICE family.

We analyzed the 67 of 278 patients with newly-diagnosed AML or 'high-risk' MDS, treated in 1994 and 1995, who developed pneumonia during course 1 of their induction therapy. Pneumonia responded to treatment in 66%, but outcome depended on when pneumonia was diagnosed. Patients with pneumonia diagnosed during week 1 or 2 (group 2 patients) had the lowest response rate (43%). Patients who developed pneumonia in the 3rd week after treatment initiation had the best outcome with all 16 patients recovering. Patients presenting with pneumonia had an intermediate response rate (75%). The different patient groups were comparable with regard to age, underlying disease, prophylactic therapy, and G-CSF application. Although a lower CR rate was not entirely responsible for the lower response rate in group 2, failure to achieve CR predicted unsuccessful treatment of pneumonia in all groups. Fungal pathogens appeared more common in group 2 patients. However, in these patients, administration of amphotericin B was associated with a significantly higher failure rate (15/21 failures vs 2/9 who received no amphotericin B). We conclude that patients who develop pneumonia during week 1 or 2 are a high-risk group, and that use of amphotericin B indicates a particularly poor prognosis, although we present data suggesting that earlier use of amphotericin might be beneficial. Furthermore, since achievement of CR was an important prognostic factor in all groups, WBC transfusions particularly from donors given G-CSF should be considered as a therapeutic option. Finally, since time to failure of induction therapy and time to CR were similar in high-risk patients, new chemotherapy regimens could potentially improve both the CR rate and the outcome of pneumonia.

Patients with newly diagnosed acute myelogenous leukemia (AML) with persistent leukemia after their first course (CO1) of induction chemotherapy are generally given a second similar course, although their outcome is known to be worse than CO1 responders even when a complete remission (CR) is achieved. To identify specific patients who should or should not receive a second induction course identical to the first we analyzed outcome in 370 patients with persistent AML after CO1 who received a second identical course. One hundred and forty-two (38%) achieved CR on this course; median subsequent disease-free survival (DFS) in these 142 was 29 weeks and 10% were alive in CR at 5 years. The 5-year DFS of CO2 responders was significantly lower than that of CO1 responders (10 vs 24%, P0.001). Logistic regression identified pretreatment cytogenetic abnormalities (except inv 16, t(8;21), or t(15;17)), presence of an antecedent hematologic disorder or secondary AML as each having unfavorable prognostic import similar to the case in untreated patients. Treatment with "high-dose' rather than standard-dose cytarabine increased the probability of 2nd course CR. The occurrence of pneumonia, sepsis, or major hemorrhage were prognostically unfavorable, primarily in the high-dose cytarabine group, and, once in CR, DFS was shorter in this group. Equations predicting probability of 2nd course CR were derived. If validated prospectively these could be used to assign patients to either receive a second course of initial induction therapy or to change to salvage or investigational therapy after the first course. Alternatively, they could be used to stratify patients entering a prospective randomized trial comparing these two strategies.

A 24-member kindred is described in which four cases of acute myeloid leukemia (AML), and one case of myelodysplastic syndrome (MDS) occurred over three generations. The proband was diagnosed with AML at age 47; within 6 months, her sister, age 41, was diagnosed with MDS. The proband's father, grandfather and a paternal uncle all died of AML, preceded by a pre-leukemic phase. The five cases had several clinical features in common. In the two sisters and their paternal uncle, cytogenetic analyses of bone marrow cells revealed a common abnormality characterized by loss of the long arm of chromosome 5, del(5q). No constitutional cytogenetic abnormality was detected in mitogen-stimulated peripheral blood lymphocytes from the proband. In addition, there was no history of common environmental or occupational exposure in the family. The occurrence of AML and MDS in three generations of this family most likely resulted from a single gene defect with an autosomal dominant pattern of inheritance. The association with the somatic loss of 5q material in the leukemia cells of affected members suggests that a germline mutation of a leukemia suppressor gene located on 5q might be the primary event responsible for hereditary susceptibility to leukemia in this family.

A human myeloid leukemia cell line, KBM-7, was developed from a patient in the blastic phase of chronic myeloid leukemia (CML). We characterized its morphology, immunophenotype, cytogenetics, and proliferative capacity. Developed in the absence of exogenous lymphokines, KBM-7 in vitro cloning capacity actually decreased when colony-stimulating factors were added. The cells had an aberrant immature myeloid phenotype, a doubling time of 22 h in suspension cultures and a high cloning efficiency in semisolid system (24 +/- 3)%. Early passages contained one near-haploid (predominant) and one hyperdiploid stem line. Gradually the hyperdiploid stem line became predominant, reaching an average of 49 chromosomes per cell. Cells from passage 89 had two Philadelphia chromosomes [t(9;22)(q34;q11)] and lacked normal copies of chromosomes 9 and 22. Detailed molecular characterization of the breakpoint in the t(9;22)(q34;q11) revealed that KBM-7 had the BCR 2/ABL II splice junction. The cells had high protein kinase (p210BCR-ABL) activity and carried two identified variants of an ABL-BCR message. There was no evidence that normal BCR or c-ABL messages were expressed, assessed with the reverse-transcriptase polymerase chain reaction. When KBM-7 cells were heterotransplanted into nude mice without immunosuppressive pretreatment, one of three mice injected with 1 x 10(7) cells and all mice injected with 1 x 10(8) cells developed slowly growing granulocytic sarcomas within 6-8 weeks. These tumors were locally invasive but did not metastasize. We conclude that the KBM-7 cell line will be of value for investigating molecular events underlying neoplastic transformation in CML, in particular for studying the effects of BCR-ABL and ABL-BCR on the proliferation of CML cells in the absence of normal BCR and c-ABL messages.

Although central nervous system (CNS) leukemic relapse is frequent in adult acute lymphocytic leukemia (ALL), the need for prophylaxis in different risk groups for CNS relapse, the value of high-dose systemic and intrathecal (IT) chemotherapy, and the timing of prophylaxis are not well defined. This analysis was conducted to investigate these questions and to assess the value of a risk-oriented CNS prophylaxis approach. We analyzed the incidence of CNS leukemia after initiation of therapy in patients treated on 4 consecutive trials for adult ALL including different CNS prophylactic modalities. The treatment groups included (1) the program preceeding the vincristine-Adriamycin-dexamethasone (VAD) regimen, with no CNS prophylaxis; (2) the VAD regimen with prophylaxis using high-dose systemic chemotherapy; (3) the modified VAD program with high-dose systemic chemotherapy to all patients and IT chemotherapy for high-risk patients after achieving complete remission; and (4) the hyperCVAD program with early high-dose systemic and IT chemotherapy starting during induction to all patients, with more IT injections (16IT) administered to the high-risk group for CNS relapse compared with the low-risk group (4IT). A total of 391 patients were included, 73 of whom were treated with preVAD, 112 with VAD, 114 with modified VAD, and 92 with hyperCVAD. The overall CNS relapse rates were 31%, 18%, 17%, and 3%, respectively for the 4 groups (P.001). For the high-risk group for CNS relapse, they were 42%, 26%, 20%, and 2%, respectively (P.001). The differences in CNS relapse rates in the low-risk group were not statistically significant. At 3 years, the overall CNS leukemia event-free rates were 48%, 76%, and 98%, respectively (P.001). In the high-risk group, the CNS event-free rates were 38%, 66%, 75%, and 98%, respectively (P.001); however, there was no difference in the low-risk group. We conclude that (1) high-dose systemic chemotherapy is a useful prophylactic measure; (2) early IT chemotherapy is necessary to reduce the incidence of CNS leukemia overall and in the high-risk group; and (3) a risk-oriented approach is appropriate to tailor the intensity of CNS prophylaxis.

Abnormalities of chromosome 16 in AML include del(16q), inv(16) and t(16;16). These three groups have been categorized together and have been associated with high complete remission (CR) and survival rates following Ara-C-based chemotherapy. We have reviewed the 63 AML or MDS patients with an abnormality of chromosome 16 treated at MD Anderson Cancer Center (MDACC) over the past 18 years. Marked differences in survival and remission duration (RD) were noted between the inv(16) or t(16;16) patients and those with del(16q), whose outcome was no better than other M4 AML or MDS patients treated during the same period. Other differences characterizing del(16q) included a lack of CNS relapses, lower incidences of eosinophilia and M4 FAB subtype. Half the inv(16) patients had additional karyotypic abnormalities. The overall survival and remission duration for those patients were no different from those for patients with inv(16) alone, although the probability of remaining in first CR at 2 years was higher in the inv(16) alone group. There was no difference in overall survival for the 45 patients who received HDAC vs those who did not. The incidence of CNS relapse was, however, markedly reduced for the HDAC patients. Eosinophilia did not correlate with improved survival. We conclude that del(16q) confers a different prognosis from inv(16) and t(16;16) and for the purposes of prognostication or treatment recommendations should no longer be categorized with them. Additional karyotypic changes however, which accompany inv(16) in 50% of cases do not influence the overall outcome compared to patients with inv(16) alone.

Aberrant expression of the tumor suppressor gene RB1 is associated with a variety of solid tumors and hematopoietic neoplasms. Certain cancer cell lines in which the protein encoded by RB1 (p110RB) is absent have been reported to show decreased growth rate, clonogenicity, or tumorigenicity following insertion of a transcriptionally active RB1 gene. We asked whether these RB-deficient cells could be growth inhibited by direct exposure to purified p110RB. We report a decrease in uptake of tritiated thymidine by 5637 bladder carcinoma cells (RB-negative) when purified recombinant p110RB is added to culture media. Internalization of the protein by cells and translocation to the nucleus are demonstrated by immunohistochemistry, FACS, and detection of radiolabeled protein in subcellular fractions. Next, we chose a well-described leukemia cell culture model to investigate the potential effect of recombinant p110RB in clinical disease. We observed dose-related decreases in cell number of colony formation in vitro in 8 of 20 acute myelogenous leukemia samples, 7 of which did show endogenous p110RB detectable by immunohistochemistry. Histological appearance following exposure to p110RB shows cytoplasmic vacuolization and nuclear lobulation of degenerating cells. We conclude that purified p110RB added to culture media is internalized by cells, translocated to the nucleus, and exerts a growth-inhibitory effect on certain cancer cell types.

Trisomy 21 is the second most common trisomy in patients with acute myelogenous leukemia (AML) and myelodysplastic syndromes (MDS). However, its clinical and prognostic significance is not known. We analyzed the records of 1187 consecutive patients with untreated AML or MDS. Thirty-seven (3.3%) had trisomy 21: four (0.3%) as the only cytogenetic abnormality and 33 (2.7%) with other cytogenetic abnormalities (-5 and/or -7 in 15, +8 in nine, t(15;17) in three, inv(16) in three, t(8;21) in one, and hyperdiploid with several other additional chromosomes in two). Twenty-eight patients had AML and nine MDS. No patients had megakaryocytic phenotype (M7), common in patients with constitutional trisomy 21 (Down's syndrome) and AML. Overall, 57% achieved complete remission (CR), with median CR duration of 39 weeks, and median survival of 31 weeks. When patients with additional cytogenetic abnormalities were compared to patients with similar abnormalities but no trisomy 21, their clinical features as well as their CR rate, CR duration and survival were similar, with or without trisomy 21. We conclude that trisomy 21 in AML typically presents in conjunction with other cytogenetic abnormalities, especially -5/-7 and +8 whose presence rather than the presence of +21 dictates the clinical outcome.

The long-term results in 130 patients with newly diagnosed acute myelogenous leukemia treated with continuous infusion high-dose ara-C (1.5 gm/m2/day x 4 days, CIHDAC) were compared with those in 264 patients treated in previous studies with standard dose ara-C (70-90 mg/m2/d x 7 days) plus either adriamycin (Ad-OAP), or amsacrine (AMSA-OAP). All patients have been followed at least 5 years. Patients in first CR at 5 years (FCR5) treated on protocols prior to CIHDAC had only 5% chance of relapse (median subsequent follow-up of 9 years). Therefore, we considered patients in FCR5 potentially cured. The two groups were similar with respect to known prognostic factors and CR rates. Although remission duration and survival were shorter with CIHDAC than Ad-OAP/AMSA-OAP, the percent of patients potentially cured was similar (10 vs. 15%). Marked differences between regimens were seen in inv(16) and t(15;17) patients. CIHDAC was better for patients with inv(16) with more patients in FCR5 (80 vs. 38%), longer remission duration and survival, and lower incidence of CNS relapse (0 vs. 43%). The Ad-OAP/AMSA-OAP protocols were superior in patients with t(15;17). We also measured steady-state ara-CTP concentrations (ara-CTPss) in 54 CIHDAC-treated patients presenting with high-blast count. While there was no correlation between ara-CTPss and response duration, all five patients in FCR5 in whom ara-CTPss was measured had high concentrations. These data support the concept that patients with AML should be treated differently according to cytogenetics. Inv(16) patients should be treated with high-dose ara-C while t(15;17) should rely more on anthracycline exposure.

A number of effective treatments are available for patients with hairy cell leukemia (HCL). 2-Chlorodeoxyadenosine (2-CdA) induces more than 80% complete responses, but is associated with profound suppression of CD4+ lymphocyte counts. However, the duration of each is uncertain. We have analyzed a previously reported cohort of 40 patients who had responded to 2-CdA. Eight patients (20%) have relapsed at a median of 16 months (range, 3 to 23 months). The remaining 32 patients were observed for a median of 30 months (range, 7 to 43 months). No patients have died. At 3 years, the actuarial disease-free survival rate is 77% (95% confidence interval, 70% to 84%). The median CD4+ lymphocyte count before therapy was 743/microL (range, 58 to 2,201/microL). The median CD4+ nadir after treatment was 139/microL (range, 25 to 580/microL). There was a single opportunistic infection and no second malignancies observed. Although there was evidence of some improvement in CD4+ lymphocyte counts on sequential testing, CD4+ counts remained significantly lower than baseline (P.0001) at a median of 23 months after therapy (median, 237/microL; range, 25 to 514/microL), and were also lower than baseline (P.002) in those patients with more than 1 year of follow-up (median, 27 months; range, 13 to 42 months). The median time to reach an absolute CD4+ lymphocyte count of 365/microL, the lower limit of the normal range, was 40 months. Although responses to 2-CdA are durable in the majority of patients with HCL, the uncertain long-term consequences of the observed CD4+ lymphocytopenia suggest caution in the broad application of this therapy.

The nonrandom chromosomal translocation t(8;21)(q22;q22) can be found frequently in acute myelogenous leukemia with maturation (AML-M2). The breakpoint of this translocation has been cloned and characterized, and fusion transcript AML1/ETO has been identified. Reverse transcription polymerase chain reaction (RT-PCR) can be used to amplify the breakpoint site of AML1/ETO in t(8;21)-positive AML-M2 patients. The chimeric transcript can be detected in all 16 (100%) t(8;21)-positive AML-M2 patients. In all samples, the size of the amplified DNA fragments and pattern of restriction digest were identical, indicating that the t(8;21) translocation breakpoint occurs within a single intron of the AML1 and ETO genes. Interestingly, this fusion transcript was also detected in one of 13 AML-M2 patients without the t(8;21) translocation, indicating that a masked translocation involving chromosomes 8 and 21, exists in AML. Minimal residual disease was detected by semi-nested RT-PCR in all four patients tested, who had been in complete remission for 12, 15, 34, and 52 months, respectively. These results indicate that RT-PCR amplification of the AML1/ETO fusion transcript is a powerful tool for diagnosing and monitoring minimal residual disease in AML-M2 patients.

Sequence analysis of the translocation breakpoint junctions on the der(14q-) chromosome in six patients carrying a t(10;14) chromosome translocation revealed that the breakpoint occurred 5' to the HOX11 protooncogene at the breakpoint cluster region. HOX11 coding sequence was not effected. The translocation resulted in the joining of the V-(D)-J recombination signals 5' to the T-cell receptor D delta 2 segment on chromosome 14 with chromosome 10 at a location within a heptamer-like sequence. At the breakpoint junctions, the insertion of extra nucleotides, N-nucleotides, including P-nucleotides, was evident. The mechanism involved in this process is discussed.

Molecular genetics is a powerful new tool for detection, diagnosis, evaluation of treatment, and understanding of the biology of malignant disease. The first malignant disease to be characterized by a specific cytogenetic aneuploid abnormality was chronic myelogenous leukemia (CML) which was first recognized in 1962 as an abnormal shortening of the long arm of either chromosome 21 or 22, the smallest chromosomes in the human genome. This cytogenetic abnormality was subsequently shown to represent not a loss of genetic material, but a pseudodiploid aneuploidy with a reciprocal translocation of chromosomal material from the long arms of chromosome 9 and 22. The discovery that the abl oncogene was located at the breakpoint on chromosome 9 allowed the molecular geneticists to identify the genetic material adjacent to the translocated material from chromosome 9 and because the breakpoint on chromosome 22 was variable but restricted to a small region, it was named the breakpoint cluster region (BCR). The BCR/abl gene has been found exclusively in leukemic cells from patients with CML. The somatic cells of the host are not involved and at least to date, every individual that has been found to have this gene has also had the disease CML. Approximately 15 % of CML patients lack the Philadelphia chromosome on cytogenetics. However, approximately half of these patients can be shown to have the BCR/abl neogene present as a masked translocation.

The fusion transcript AML1/ETO was detected in the bone marrow of two t(8;21)-negative acute myelogenous leukemia (AML) patients by means of reverse transcription-polymerase chain reaction. This fusion transcript is identical to the one transcribed from the t(8;21) translocation base, as deduced from (a) the size and restriction pattern of the amplified DNA fragment and (b) the DNA sequence analysis of the fusion junction. We also showed that the ETO gene is highly expressed in these patients, much as it is in the t(8;21)-positive AML. Southern blot analysis showed rearrangement of the AML1 gene in one of the patients. Together, our results demonstrate that there is a masked t(8;21) translocation in AML that is not detectable by cytogenetic analysis but is able to transcribe an AML1/ETO fusion transcript similar to that transcribed in t(8;21)-positive AML-M2 patients.

The clinical response and survival of 113 patients with at least 3-year follow-up after treatment with fludarabine as a single agent for chronic lymphocytic leukemia has been evaluated. Seventy-eight patients were previously treated and 35 were untreated. The response to therapy and survival were strongly correlated with the degree of previous therapy, the stage of disease, and whether or not the patients were refractory to alkylating agents. Other characteristics associated with survival were the age of the patient and the serum albumin level at the start of therapy. The median time to progression of responders who had not received prior therapy was 33 months and was 21 months for previously treated patients. Survival after progression of disease was also strongly correlated with the degree of prior therapy. No successful salvage regimen after initial fludarabine therapy was shown for patients refractory to alkylating agents, although fludarabine achieved further remissions in patients who had received fludarabine as their initial treatment or were not refractory to alkylating agents. The morbidity of patients in unmaintained remission on discontinuation of fludarabine was low, with less than one episode of infection per patient-year at risk. The morbidity during this time was correlated with clinical response and whether the patients had received prior therapy. Although fludarabine is a very effective cytoreductive regimen, most patients, including those who achieved true complete remissions, will have recurrent disease. Longer follow-up and comparative trials are required before the effect of fludarabine on survival is shown.

Bone marrow samples collected from acute myeloblastic leukemia (AML) patients in complete clinical and hematological remission were studied for the persistence of cytogenetic abnormalities. AML patients from the three favorable cytogenetic categories [inv 16, t(8;21) and t(15;17)] and patients from the unfavorable cytogenetic categories (+8, -5, -7 and Philadelphia-positive) were studied. Seventy-one patients had evaluable metaphase spreads in remission marrows and 20 (28%) had one or more abnormal metaphases identical to that present in the pretreatment marrow. All 20 of these patients relapsed within 78 weeks, thus there were no false positive studies. Fifty-one patients had only diploid metaphases in their complete remission marrow, 25 relapsed, and 21 remained in continuous complete remission. Thus there was a 49% false negative rate of this study. These data indicate that the failure to detect residual chromosomally abnormal cells in the bone marrow does not guarantee continuous complete remission. Cytogenetic study was most useful in the favorable cytogenetic groups and least useful in the unfavorable groups. The persistence of normal metaphases in pretreatment marrows did not affect outcome or risk of recurrence. Twenty-five of 34 evaluable patients who relapsed after remission had either the identical cytogenetic abnormality present in the pretreatment marrow or showed the identical abnormality with additional chromosomal changes. Thus study indicates that cytogenetic examinations of complete remission bone marrow samples in patients with AML provides an objective method for detecting residual leukemia, and identifies patients with a potential for prolonged disease-free survival.

The significance of the polymerase chain reaction (PCR) in the detection of minimal residual disease in Philadelphia chromosome (Ph')-positive chronic myelogenous leukemia (CML) following interferon therapy was investigated. Forty remission blood samples obtained at various remission time points from 29 patients in complete cytogenetic remission were analyzed. All 40 samples showed minimal residual Ph'-positive cells by PCR: 22 in remission for less than 12 months, 12 in remission for 12 to 24 months, four in remission for 25 to 60 months, and two in remission for more than 60 months. Of these 29 patients, seven relapsed at 4, 6, 9, 14, 17, 19, and 50 months after their first PCR-positivity during remission. One developed extramedullary myelopoiesis at 49 months after PCR-positivity. The remaining 21 patients remained in complete hematologic and cytogenetic remission with median follow-up of 13 months (range, 4 to 36 months) after PCR analysis. These findings indicate that PCR-positivity is not associated with immediate disease recurrence. Long-term follow-up is essential to determine the relevance of PCR-positivity, since late recurrence is observed in our study.

Evidence suggests that prognosis in patients with myelodysplastic syndromes (MDS) or acute myelogenous leukemia (AML) depends more on karyotype than on formal classification as either MDS or AML according to the French-American-British (FAB) system. We provide further evidence of overlap between these two entities, reporting 4 patients who presented with either inv(16) (p13q22), del(16) (q22), or t(8;21) despite an FAB diagnosis of MDS rather than the diagnosis of AML with which these abnormalities are generally associated. In 3 patients, disease was relatively long-standing (3-10 months) prior to diagnosis, suggesting that the association between MDS and these cytogenetic abnormalities may not merely reflect a transient phenomenon. Two patients with inv(16) and the MDS subtype refractory anemia with excess blasts in transformation (RAEB-t) received AML-type chemotherapy as did a patient with t(8;21) and RAEB-t. All entered CR paralleling the high CR rate seen in patients with AML and these abnormalities. Our data support the concept that MDS and AML may be different manifestations of the same disease.

The retinoic acid receptor-alpha (RAR-alpha) gene was previously localized to chromosome 17q21, a region close to the t(15;17) (q22;q21) abnormality in acute promyelocytic leukemia (APL). We used the RAR-alpha gene as a probe and found that eight of nine APL patient samples with t(15;17) (q22;q21) showed rearranged bands. A tenth APL patient was diploid and demonstrated no rearrangement. One patient who had rearrangement as an acute leukemia did not have rearrangement in remission. The results obtained from intron/exon mapping of the RAR-alpha gene demonstrated that breakpoints of seven of the eight patients occurred within intron 1. Northern blot analysis of leukemic samples indicated the expression of two RAR-alpha mRNA of 2.7 and 3.7 kb. However, two additional mRNA of 4.1 and 3.2 kb were found in an APL patient. We conclude that the RAR-alpha gene is directly involved in the t(15;17) translocation in APL and may transcribe aberrant messages.

The level of myeloperoxidase (MPO) mRNA is reduced significantly after HL-60 induced differentiation with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). We examined the chromatin structural changes of the MPO gene during TPA induction. Before TPA induction about nine DNase I hypersensitive sites (HS) were found on the 5' upstream and at various intron regions of the MPO gene. A new HS was found on intron 8 within 4 h of induction; its appearance preceded down regulation of the MPO gene. At the same time DNase I HS found in 0.3 and 1-1.5 kb upstream of the MPO CAP site, were significantly reduced or disappeared after TPA induction. These chromatin structural changes could be closely linked to the mechanism which regulates the MPO gene expression.

We have a broad spectrum of treatment techniques which can render patients with diagnosable malignancy free of disease (N.E.D. or C.R). Such patients depending on the characteristics of the original tumor are at variable risks of recurrence of disease. It is clear that patients in complete remission can benefit from adjuvant treatment given during the disease free period. The basis for such adjuvant therapy depends on the probability that the patient will develop recurrent disease. The prescription of adjuvant treatment results in offering treatment to some patients who are already cured of their disease in an effort to prolong the disease free period for patients who have residual disease. Thus, a pressing problem in the field of adjuvant therapy is the development of techniques for the objective detection of residual disease which would serve as a guide to the treating physician and would maximize the benefit/risk ratios for each individual patient.

Seven adult patients with untreated acute lymphocytic leukemia (ALL) who manifested 5% to 40% myeloperoxidase (MPO)-positive blasts by electron microscopy (EM) are reported. Six patients had an L2 morphology, and one had an L1 morphology by the French-American-British (FAB) classification. The immunophenotype was T cell in four patients. Molecular analysis showed rearrangement of the immunoglobulin JH in four patients, three of them also having rearrangement of the T-cell receptor beta or gamma. Induction chemotherapy with vincristine-doxorubicin-dexamethasone (VAD) produced a complete remission in five of six patients (83%). Our findings suggest the existence of a previously undescribed subtype of mixed-lineage leukemia, which by morphology and immunophenotype often appears as T-cell ALL but exhibits MPO-positive blasts by EM.