Relapse of acute promyelocytic leukemia with PML-RARalpha mutant subclones independent of proximate all-trans retinoic acid selection pressure.

Relapse of acute promyelocytic leukemia (APL) following all-trans retinoic acid (ATRA) therapy has been associated with the acquisition of mutations in the high-affinity ATRA binding site in PML-RARalpha, but little information is available about the selection dynamics of the mutation-harboring subclones. In this study, 6/18 patients treated with sequential ATRA and chemotherapy on protocol INT0129 relapsed with complete replacement of the nonmutant pretreatment APL cell population by a PML-RARalpha mutant subclone. Two patients relapsed in proximity of ATRA treatment; however, in four patients there was a 6-48 month hiatus between the last ATRA treatment and relapse. The mutant subclones were not detectable in samples tested > or = 3 months before relapse at > or = 1 in 10(2) (10(-2)) sensitivity. In one patient, a functionally weak mutation was detected at 10(-4) sensitivity before therapy but only limited pre-relapse enrichment of the mutant subclone was observed on subsequent ATRA therapy. These results indicate that proximate ATRA selection pressure is frequently not the main determinant for the emergence of strongly dominant PML-RARalpha mutant subclones and suggest that APL subclones harboring PML-RARalpha mutations are predisposed to the acquisition of secondary genetic/epigenetic alterations that result in a growth/survival advantage.

Acute promyelocytic leukemia cell line AP-1060 established as a cytokine-dependent culture from a patient clinically resistant to all-trans retinoic acid and arsenic trioxide.

AP-1060 is a newly established acute promyelocytic leukemia (APL) cell line from a multiple-relapse patient clinically resistant to both all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). The line was initially derived as a granulocyte colony-stimulating factor-dependent strain that underwent replicative senescence and, following ethylnitrosourea treatment, as a phenotypically similar immortalized line. Immortalization was associated with broadened cytokine sensitivity but not growth autonomy, in contrast to three previously derived APL lines. Both the AP-1060 strain and line had shortened telomeres and low telomerase activity, while the line had higher expression of many genes associated with macromolecular synthesis. The karyotype was 46,XY,t(3;14)(p21.1;q11.2),t(15;17)(q22;q11)[100%]; the unique t(3;14) was observed in 4/9 t(15;17)-positive metaphase cells at previous relapse on ATRA therapy. The PML-RARalpha mRNA harbored a missense mutation in the RARalpha-region ligand-binding domain (Pro900Ser). This was associated with a right-shift and sharpening of the ATRA-induced maturation response compared to ATRA-sensitive NB4 cells, which corresponded to the transcriptional activation by PML-RARalphaPro900Ser of a cotransfected ATRA-targeted reporter vector in COS-1 cells. AP-1060 also manifested relative resistance to ATO-induced apoptosis at >/=1 microM, while 0.25 microM ATO stimulated limited atypical maturation. These findings suggest that AP-1060 will be useful for further assessing molecular elements involved in APL progression and drug response/resistance.

Hodgkin's lymphoma in Chinese migrants to British Columbia: a 25-year survey.

BACKGROUND: Compared with the West, Hodgkin's lymphoma in Oriental countries is characterized by a lower incidence rate and a higher proportion of mixed cellularity histology. Both environmental and genetic factors may be involved. PATIENTS AND METHODS: The incidence and pattern of pathology of Hodgkin's lymphoma in the migrant Chinese population (0.4 million) in British Columbia (population 3.2 million) were studied. From a computerized database, all Hodgkin's lymphoma cases diagnosed in British Columbia from 1970 to 1997 were identified. Chinese descent was determined using patient surname by standard methodology and verified from the treatment record or by patient interview. The corresponding figures from the Chinese population in Hong Kong were used for comparison. For incidence rates, the age-specific incidence of Hodgkin's lymphoma in Hong Kong was obtained from the government cancer registry. For comparison of histology subtypes, 200 Hodgkin's lymphoma records from a Hong Kong regional referral center for the same time period were reviewed. Crude and age-standardized incidence rates were calculated by 5-year intervals in terms of age and calendar year, and relative rates were compared between the three populations. RESULTS: From 1970 to 1997, Hodgkin's lymphoma was diagnosed in 34 Chinese patients in BC, with 24 cases diagnosed from 1970 to 1994. Thus, the crude and age-adjusted incidence rates from 1970 to 1994 were 0.91 and 1.14 per 100,000 per year in the British Columbia Chinese migrant population. Within the same period, 1862 cases of Hodgkin's lymphoma were diagnosed in British Columbia, giving a provincial background crude and age-adjusted incidence rates of 5.2 and 4.87 per 100,000 per year. The number of cases in the Hong Kong Chinese population (1970-1994) was 404, giving crude and age-adjusted incidence rates of 0.32 and 0.31 per 100,000 per year, respectively. Corrected for age and calendar year trends, the observed 25-year incidence of Hodgkin's lymphoma in British Columbia Chinese was significantly lower than expected from the British Columbia background population [24 observed versus 71 expected cases; standardized incidence ratio (SIR) = 0.34; 90% confidence interval (CI) 0.24-0.48; P <0.0001]. On the other hand, it is higher than that expected by extrapolating from the Hong Kong Chinese population (24 observed versus 8.5 expected cases; SIR = 2.81; 90% CI 1.94-3.95; P <0.0001). The difference is mainly accounted for by young patients with nodular sclerosis type disease in the migrant population. CONCLUSIONS: Although any conclusion about the impact of migration on Hodgkin's lymphoma incidence and types in the Chinese population must be considered tentative due to the small number of observed cases and confounding variables such as age, changing diagnostic standards and secular trends in Hodgkin's lymphoma rates, our data demonstrate a tendency for the Chinese population of British Columbia to take on a Western pattern of Hodgkin's lymphoma. This observation provides additional evidence that both genetic and environmental influences play a role in the pathogenesis of this lymphoma, and that environmental factors can exert their influence over a relatively short period of time.

Retinoic acid resistance in acute promyelocytic leukemia.

Primary resistance of PML-RARalpha-positive acute promyelocytic leukemia (APL) to the induction of clinical remission (CR) by all-trans retinoic acid (ATRA) is rare but markedly increases in frequency after > or =2 relapses from chemotherapy-induced CRs. Nevertheless, even in de novo cases, the primary response of ATRA-naive cases is variable by several measures, suggesting involvement of heterogeneous molecular elements. Secondary, acquired ATRA resistance occurs in most patients treated with ATRA alone and in many patients who relapse from combination ATRA chemotherapy regimens despite limited ATRA exposure. Although early studies suggested that an adaptive hypercatabolic response to pharmacological ATRA levels is the principal mechanism of ATRA resistance, recent studies suggest that molecular disturbances in APL cells have a predominant role, particularly if disease relapse occurs a few months after discontinuing ATRA therapy. This review summarizes the systemic and APL cellular elements that have been linked to clinical ATRA resistance with emphasis on identifying areas of deficient information and important topics for further investigation. Overall, the subject review strongly supports the hypothesis that, although APL is an infrequent and nearly cured disease, much can be gained by understanding the complex relationship of ATRA resistance to the progression and relapse of APL, which has important implications for other leukemias and malignancies.

Pre-clinical validation of a novel, highly sensitive assay to detect PML-RARalpha mRNA using real-time reverse-transcription polymerase chain reaction.

We have developed a sensitive and quantitative reverse-transcription polymerase chain reaction (RT-PCR) assay for detection of PML-RARalpha, the fusion oncogene present as a specific marker in >99% of cases of acute promyelocytic leukemia (APL). The assay is linear over at least 5 orders of magnitude of input DNA or RNA, and detects as few as 4 copies of PML-RARalpha plasmid DNA. PML-RARalpha transcripts could be detected in mixtures containing 2 to 5 pg of RNA from fusion-containing cells in a background of 1 microg of RNA from PML-RARalpha-negative cells. Using 1.0 to 2.5 microg of input RNA, the sensitivity of the assay was between 10(-5) and 10(-6). Furthermore, determination of GAPDH copy number in each reaction allowed an accurate assessment of sample-to-sample variation in RNA quality and reaction efficiency, with consequent definition of a detection limit for each sample assayed. Using an internal calibrator, assay precision was high, with coefficients of variation between 10 and 20%. An interlaboratory study using coded samples demonstrated excellent reproducibility and high concordance between laboratories. This assay will be used to test the hypothesis that sensitive and quantitative measurement of leukemic burden, during or after therapy of APL, can stratify patients into discrete risk groups, and thereby serve as a basis for risk-adapted therapy in APL.

Altered ligand binding and transcriptional regulation by mutations in the PML/RARalpha ligand-binding domain arising in retinoic acid-resistant patients with acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is characterized by a specific translocation, t(15;17), that fuses the promyelocytic leukemia (PML) gene with the RA receptor RARalpha. Pharmacologic doses of retinoic acid (RA) induce differentiation in human APL cells and complete clinical remissions. Unfortunately, APL cells develop resistance to RA in vitro and in vivo. Recently, mutations in PML/RARalpha have been described in APL cells from patients clinically resistant to RA therapy. The mutations cluster in 2 regions that are involved in forming the binding pocket for RA. These mutant PML/RARalpha proteins have been expressed in vitro, which shows that they cause a diversity of alterations in binding to ligand and to nuclear coregulators of transcription, leading to varying degrees of inhibition of retinoid-induced transcription. This contrasts with the nearly complete dominant negative activity of mutations in PML/RARalpha previously characterized in cell lines developing RA resistance in vitro. Current data from this study provide additional insight into the molecular mechanisms of resistance to RA and suggest that alterations in the ability of mutants to interact with coregulators can be determinant in the molecular mechanism of resistance to RA. In particular, ligand-induced binding to the coactivator ACTR correlated better with transcriptional activation of RA response elements than the ligand-induced release of the corepressor SMRT. The diversity of effects that are seen in patient-derived mutations may help explain the partial success to date of attempts to overcome this mechanism of resistance in patients by the clinical use of histone deacetylase inhibitors.

Using evaluation methods to guide the development of a tobacco-use prevention curriculum for youth: a case study.

BACKGROUND: Fundamental to the development of educational programs and curricula is the evaluation of processes and outcomes. Unfortunately, many otherwise well-designed programs do not incorporate stringent evaluation methods and are limited in measuring program development and effectiveness. Using an advertising lesson in a school-based tobacco-use prevention curriculum as a case study, the authors examine the role of evaluation in the development, implementation, and enhancement of the curricular lesson. METHODS: A four-phase formative and summative evaluation design was developed to divide the program-evaluation continuum into a structured process that would aid in the management of the evaluation, as well as assess curricular components. RESULTS AND CONCLUSIONS: Formative and summative evaluation can provide important guidance in the development, implementation, and enhancement of educational curricula. Evaluation strategies identified unexpected barriers and allowed the project team to make necessary "time-relevant" curricular adjustments during each stage of the process.

Molecular analysis and clinical outcome of adult APL patients with the type V PML-RARalpha isoform: results from intergroup protocol 0129.

The type V (for variable) promyelocytic leukemia retinoic acid receptor (PML-RAR)alpha transcript, found in approximately 8% of adult patients with acute promyelocytic leukemia (APL), is defined molecularly by truncation of PML exon 6 and frequent insertion of genetic material from RARalpha intron 2. To more fully characterize the molecular features of PML-RARalpha V-type transcripts and to determine whether V-form APL patients have a distinct clinical presentation or prognosis, we analyzed 18 adult V-form APL patients enrolled on Intergroup protocol 0129 (INT-0129). Truncations in PML exon 6 ranged from 8 to 146 nucleotides, and 3 to 127 extra nucleotides (1 to 42 extra amino acids) were inserted at the PML exon 6/RARalpha exon 3 junction in 13 cases. No distinguishing morphologic, cytogenetic, or immunophenotypic features of V-form blasts were identified. A total of 5 of 7 patients induced with ATRA and 8 of 11 patients who received chemotherapy for induction achieved complete remission (CR). Six patients have relapsed, 4 after chemotherapy induction and 2 after ATRA. Nine patients (50%) are alive, 6 in continuous CR, 2 after salvage therapy for relapsed or refractory disease, and 1 after alternative treatment following early removal from protocol. Although the failure rate for V-form APL patients was high (61%), the low power of the current study to detect clinically significant differences precludes a meaningful comparison of clinical outcomes between the 18 V-form cases and non-V-form adult APL patients enrolled on INT-0129. (Blood. 2000;95:398-403)

Arsenic trioxide induces apoptosis of myeloid leukemia cells by activation of caspases.

The primary objective of this study was to determine whether caspases are involved in arsenic trioxide(ATO)-induced apoptosis of human myeloid leukemia cells. A secondary objective was to determine whether apoptosis induced by ATO compared with VP-16 is differentially affected by an activator of protein kinase C (PKC), phorbol 12-myristate 13-acetate (PMA), which has been reported to inhibit apoptosis induced by some chemotherapeutic agents. NB4 and HL60 cells were incubated with ATO in the presence and absence of the caspase protease inhibitors Z-VAD.fmk or Y-VAD.cho. Apoptosis was assessed by morphology, DNA laddering and flow cytometry. Poly (ADP-ribose) polymerase (PARP) cleavage was used as a marker for the activation of caspases. PARP cleavage occurred during ATO-induced apoptosis in both NB4 and HL60 cells. Z-VAD.fmk, a broad-spectrum inhibitor, could block ATO-induced apoptosis and PARP cleavage, whilst Y-VAD.cho, a selective inhibitor of caspase 1, had no such effect. PMA pre-incubation for up to 8 hours under conditions known to activate PKC had no effect on either ATO- or VP-16-induced apoptosis. We conclude that in cultured myeloid leukemia cells ATO-induced apoptosis is executed by caspases from the distal, PARP-cleaving part of the activation cascade and that PKC activation has no effect on apoptosis induced by either ATO or VP-16 in these cells.

The molecular biology of acute promyelocytic leukemia.

The preceding two years have witnessed an explosion in the accumulation of knowledge relating to the molecular pathogenesis of APL. Critical advances include: The molecular delineation of atypical APL cases with alternative RAR alpha fusion partners, and the demonstration that cells from 2 of the 3 types of 'atypical' APL retain sensitivity to ATRA. Perhaps the key question is why such cases are so rare. However, at a minimum, the presence of such cases argues persuasively that disruption of the retinoid signaling pathway is a (perhaps the) key pathogenetic feature of APL. Although certainly not 'passive' partners, it is likely that PML, PLZF, NPM, and NuMA serve similar functions in the pathogenesis of APL. The demonstration, in transgenic mice, that PML-RAR alpha (and PLZF-RAR alpha) can disrupt normal hematopoiesis and, given sufficient time, cause an APL-like syndrome. the variation in phenotype of the mice, which appears to be a consequence of the specific expression vector used, emphasizes the cell-type-specific nature of PML-RAR alpha function. Continuing functional analysis of PML, PLZF, and RAR alpha. In particular, the demonstration that PML and PLZF can form heterodimers provides a critical functional link between these proteins and offers a tantalizing glimpse at how both, when linked with RAR alpha, can cause APL. The demonstration that PML-RAR alpha is degraded, perhaps via a ubiquitin-dependent pathway, in response to ATRA. This result offers a unifying, if not yet proven, hypothesis to explain the sensitivity of leukemic promyelocytes to ATRA. Unfortunately, it is not known if ATRA can also cause degradation of NPM-RAR alpha or NuMA-RAR alpha (atypical cytogenetic APL variants that retain ATRA responsiveness). Whether PML-RAR alpha degradation is a cause, or consequence, of promyelocytic maturation remains unclear. Continuing insight into retinoid resistance, including the first demonstration of mutations in the PML-RAR alpha molecule from ATRA-resistant patients. The definitive demonstration that the two major PML-RAR alpha isoforms, while having subtle differences in biological activity and producing slightly different APL phenotypes, nevertheless do not, in and of themselves, have prognostic significance in patients treated with ATRA/chemotherapy combinations. Further functional analysis of PML-RAR alpha in vitro. The fascinating finding that PML-RAR alpha is cytotoxic to most cell types suggests that it must function as an oncogene in a very specialized milieu. In addition, the demonstration that both the DBD (from RAR alpha) and dimerization interface (from PML) are required for full in vitro functional activity, coupled with the finding that PML itself is a strong transcriptional suppressor, suggests that PML-RAR alpha may directly repress transcription of RA target genes. The challenge in APL research now is to integrate the above findings into a cohesive, unifying model that explains the biology of APL at a molecular level. The creation and validation of such a model will clarity whether APL is a fortunate medical curiosity or whether it will serve as a paradigm for the development of effective differentiation therapies in other types of human cancers.

Constitutive expression of cellular retinoic acid binding protein II and lack of correlation with sensitivity to all-trans retinoic acid in acute promyelocytic leukemia cells.

The up-regulation of cellular retinoic acid binding protein-II (CRABP-II) has been invoked as an important mechanism of clinically acquired resistance to all-trans retinoic acid (RA) therapy in acute promyelocytic leukemia (APL). To test this hypothesis, we used quantitative reverse transcription-PCR and fast performance liquid chromatography procedures to examine the levels of CRABP-II mRNA and RA binding activity in APL patient samples. We found that CRABP-II mRNA in APL cells from pretreatment patients (n = 36) was constitutively expressed at relatively high levels (median, 0.92; range, 0.16-4.13) relative to the level in CRABP-H protein-expressing NB4 cells (arbitrarily set at 1.0 unit). Consistent with this finding, the RA binding activity of CRABP in APL cells from three pretreatment cases (range, 27.2-53.2 fmol/mg protein) was similar to that of NB4 cells (22.6 +/- 5.4 fmol/mg protein). Furthermore, in the pretreatment samples, there was no association between CRABP-H mRNA expression level and APL cellular sensitivity to RA-induced differentiation in vitro. After 45 days of remission induction therapy on Eastern Cooperative Oncology Group protocol E2491, CRABP-II mRNA was modestly increased from day 0 values in patients treated with either RA (median increase, 0.41) or chemotherapy (median increase, 0.56), and there was no significant difference between the two treatment groups (P = 0.91). In patients studied after relapse from RA therapy (n = 7), there was a significant decline in APL cell sensitivity to RA-induced differentiation in vitro compared with patients after relapse from chemotherapy (n = 5; P = 0.015-0.055 at three RA concentrations tested), but in the RA relapse cases, there was no change from pretreatment levels of CRABP-II mRNA (median, 0.98) or, in three relapse cases studied, of RA protein binding activity (range, 22.1-70.7 fmol/mg protein). Taken together, our data strongly imply that variations in CRABP-II expression and RA binding activity are not causally related to the development of clinically acquired APL cellular RA resistance, but rather, they suggest that constitutive expression of CRABP-II could have a facilitative role in the response of APL cells to RA.

Leukemic cellular retinoic acid resistance and missense mutations in the PML-RARalpha fusion gene after relapse of acute promyelocytic leukemia from treatment with all-trans retinoic acid and intensive chemotherapy.

This study evaluated whether relapse of acute promyelocytic leukemia (APL) patients from clinical remissions achieved and/or maintained with all-trans retinoic acid (RA) in combination with intensive chemotherapy is associated with leukemic cellular resistance to RA and with alterations in the PML-RARalpha fusion gene. We studied matched pretreatment and relapse specimens from 12 patients who received variable amounts of RA, primarily in nonconcurrent combination with daunorubicin and cytarabine (DA) on Eastern Cooperative Oncology Group (ECOG) protocol E2491, and from 8 patients who received DA only on protocol E2491. Of 10 RA-treated patients evaluable for a change in APL cell sensitivity to RA-induced differentiation in vitro, 8 showed diminished sensitivity at relapse, whereas, of 6 evaluable patients treated with DA alone, only 1 had marginally reduced sensitivity. From analysis of sequences encoding the principal functional domains of the PML and RARalpha portions of PML-RARalpha, we found missense mutations in relapse specimens from 3 of 12 RA-treated patients and 0 of 8 DA-treated patients. All 3 mutations were located in the ligand binding domain (LBD) of the RARalpha region of PML-RARalpha. Relative to normal RARalpha1, the mutations were Leu290Val, Arg394Trp, and Met413Thr. All pretreatment analyses were normal except for a C to T base change in the 3'-untranslated (UT) region of 1 patient that was also present after relapse from DA therapy. No mutations were detected in the corresponding sequences of the normal RARalpha or PML (partial) alleles. Minor additional PML-RARalpha isoforms encoding truncated PML proteins were detected in 2 cases. We conclude that APL cellular resistance occurs with high incidence after relapse from RA + DA therapy administered in a nonconcurrent manner and that mutations in the RARalpha region of the PML-RARalpha gene are present in and likely mechanistically involved in RA resistance in a subset of these cases.

Prospective analysis of prostate-specific markers in pelvic lymph nodes of patients with high-risk prostate cancer.

BACKGROUND: Pathologic evidence of pelvic lymph node involvement is obtained in 12%-20% of patients with localized prostate cancer that exhibits high-risk features (defined on the basis of tumor size, serum prostate-specific antigen [PSA] level, or Gleason score). The rate of systemic failure (i.e., relapse) in patients with this type of prostate cancer and no pathologic evidence of regional lymph node involvement is 55%-92% within 5 years of definitive local therapy. Since reverse transcription-polymerase chain reaction (RT-PCR) methods are likely to be more sensitive than routine pathologic examination in detecting metastatic tumor cells, we compared the ability of the two approaches to detect prostate cells in the pelvic lymph nodes of patients with localized, high-risk disease. METHODS: Fifty-eight lymph node specimens isolated from 33 patients before definitive local therapy were examined. Expression of PSA and prostate-specific membrane antigen (PSM) messenger RNAs in the specimens was assessed by means of nested RT-PCR. RESULTS: Pathologic examination identified tumor cells in the lymph nodes of four (12%) of the 33 patients, and PSA and/ or PSM expression was positive in specimens from 27 (82%) of the patients (two-sided P<.0001). The four patients with positive pathologic findings also had positive RT-PCR results. Among the 29 patients with no pathologic evidence of lymph node involvement, 23 (79%) tested positive by means of RT-PCR. In these 23 patients, PSM expression was detected more frequently than PSA expression; however, in two patients, only PSA expression was detected. CONCLUSIONS: Expression of prostate-specific markers in the pelvic lymph nodes of patients with localized, high-risk prostate cancer may indicate the presence of metastatic tumor cells. Such cells may be responsible for the high rate of systemic failure seen in these patients. Additional studies are required to determine the prognostic relevance of our findings.

Association of PML-RAR alpha fusion mRNA type with pretreatment hematologic characteristics but not treatment outcome in acute promyelocytic leukemia: an intergroup molecular study.

In each case of acute promyelocytic leukemia (APL) one of three PML-RAR alpha mRNA types is produced, depending on the break/fusion site in the PML gene that is linked to a common RAR alpha gene segment: a short (S)-form type, PML exon 3 RAR alpha exon 3; a long (L)-form type, PML exon 6 RAR alpha exon 3; or a variable (V)-form type, variably deleted PML exon 6 RAR alpha exon 3. We evaluated whether PML-RAR alpha mRNA type is associated with distinct pretreatment clinical characteristics and therapeutic outcome in previously untreated adult APL patients registered to protocol INT 0129 by the Eastern Cooperative Oncology Group, the Southwest Oncology Group, and the Cancer and Leukemia Group B. Of 279 clinically eligible cases, 230 were molecularly evaluable, and of these, 111 were randomized to receive remission induction therapy with all-trans retinoic acid (ATRA) and 119 with conventional chemotherapy. Nine cases not excluded by central pathology review were PML-RAR alpha negative, and notably, none of five of these cases treated with ATRA achieved complete remission (CR). Among 221 PML-RAR alpha-positive cases, there were 82 S-form cases (37%), 121 L-form cases (55%), and 18 V-form cases (8%). Before any antileukemic therapy, the S-form type, compared with the L-form type, was associated with higher values for the white blood cell (WBC) count (median 2,500/microL v 1,600/microL; P = .009), the percentage of blood blasts plus promyelocytes (median 29% v 8.5%; P = .03), and the absolute blood blasts plus promyelocytes (884/microL v 126/microL; P = .019). Also, an increased percentage of S-form versus L-form cases had the M3 variant phenotype, 24% v 12% (P = .036). There were no differences between S-form and L-form cases in either CR rate (79% v 69%; P = .14) or disease free survival distribution (multivariate analysis adjusting for the association of S-form type and higher WBC count; P = .40). We conclude that the S-form type is associated with previously-identified adverse risk WBC parameters but that the identification of the S-form or L-form type of PML-RAR alpha mRNA, per se, does not predict clinical outcome or add to the value of an increased WBC count as a negative prognostic indicator in APL patients.

RAR alpha1/RAR alpha2-PML mRNA expression in acute promyelocytic leukemia cells: a molecular and laboratory-clinical correlative study.

In addition to the major fusion gene PML-RAR alpha, the t(15; 17) in acute promyelocytic leukemia (APL) produces the reciprocal fusion gene RAR alpha-PML. To determine the scope of RAR alpha-containing mRNA expression in APL cells, we tested PML-RAR alpha-positive APL cells for the presence of mRNAs initiated from two distinct RAR alpha gene promoters, alpha1 and alpha2. From the normal allele, both RAR alpha1 and RAR alpha2 mRNAs were expressed in all APL cases (N = 24). From the translocated allele, RAR alpha1-PML mRNA was expressed in 77% and RAR alpha2-PML mRNA in 28% of cases (N = 98). RAR alpha2-PML mRNA was not observed in the absence of RAR alpha1-PML mRNA. There was no association between RAR alpha1-PML or RAR alpha2-PML mRNA expression and the type of PML-RAR alpha mRNA formed by either 5' or 3' breaksites in the PML gene. RAR alpha1-PML mRNAs and RAR alpha2-PML mRNAs from 5' PML breaksite cases coded for full-length RAR alpha-PML proteins but RAR alpha2-PML mRNAs from 3' PML breaksite cases encoded a truncated RAR alpha2 peptide. RAR alpha1/alpha2-PML mRNA expression was not associated with differences in APL cell sensitivity to all-trans retinoic acid(tRA)-induced differentiation in vitro or in clinical outcome after tRA or chemotherapy induction therapy (protocol E2491). Our analysis indicated that RAR alpha1/alpha2-PML mRNA expression markedly differs from normal RAR alpha1/alpha2 mRNA expression, that the difference in RAR alpha1-PML and RAR alpha2-PML mRNA expression frequency is primarily related to the genomic separation of the RAR alpha1 and RAR alpha2 coding exons, and that variations in RAR alpha1/alpha2-PML mRNA expression likely have no clinically relevant function in APL cells.

The importance of word-initial phonology: error patterns in prolonged naming efforts by aphasic patients.

Whether multiple conscious efforts at word search bring a subject closer to an elusive word and to eventual successful retrieval remains a subject of debate. Previous work with normal participants has shown that multiple attempts eventuating in correct retrieval are not usually associated with a systematic progression toward target word phonology in the intervening attempts. In this study we analyzed the naming errors produced by 30 aphasic patients who had received the Boston Naming Test. The analyses were designed to elucidate the characteristics of responses that led to eventual success. Our data showed that among aphasics, as with normal subjects, the presence of target-initial phonology in the subject's first response was the most important predictor of correct retrieval. Moreover, progression towards target phonology in the course of multiple attempts was unrelated to eventual correct retrieval.

Inhibition of all-trans-retinoic acid metabolism by fluconazole in vitro and in patients with acute promyelocytic leukemia.

All-trans-retinoic acid induces acute promyelocytic leukemia cell differentiation in vitro, and it produces greater than 90% complete remissions in patients with acute promyelocytic leukemia. Despite the high response rate, the majority of patients relapse with continued trans-retinoic acid therapy, and disease progression has been observed to be accompanied by an increase in the metabolism of trans-retinoic acid in the patients. In this study, the pharmacokinetic disposition of trans-retinoic acid was determined by HPLC in patients with acute promyelocytic leukemia before and after concurrent therapy with the triazole antimycotic agent fluconazole. Treatment with trans-retinoic acid for 1 week reduced the area under the plasma trans-retinoic acid concentration vs time curve in one patient by 67%, from 277 to 91 ng/mL/hr. Trans-retinoic acid pharmacokinetics were repeated after the second dose of fluconazole, administered 1 hour prior to the retinoid, and the AUC was found to be 401 ng/mL/hr, a greater than 4-fold increase from the pre-fluconazole level. A similar, though more modest, effect of fluconazole was seen in a second acute promyelocytic leukemia patient. The effect of fluconazole on trans-retinoic acid metabolism was examined in vitro using isolated human hepatic microsomes. Fluconazole inhibited the NADPH-dependent cytochrome P450-mediated catabolism of trans-retinoic acid in a concentration-dependent manner. Although fluconazole was approximately one-half as potent an inhibitor when compared with ketoconazole, a related antifungal drug, 60-90% inhibition was observed at the concentrations of fluconazole measured in the acute promyelocytic leukemia patients. Neither fluconazole nor ketoconazole inhibited lipid hydroperoxide-mediated metabolism of trans-retinoic acid. Since fluconazole is a well-tolerated agent frequently administered to leukemia patients, its use in combination with trans-retinoic acid merits further consideration.

Characterization of acute promyelocytic leukemia cases with PML-RAR alpha break/fusion sites in PML exon 6: identification of a subgroup with decreased in vitro responsiveness to all-trans retinoic acid.

Of 113 acute promyelocytic leukemia cases documented to have diagnostic PML-RAR alpha hybrid mRNA, 10 cases (8.8%) had fusion sites in PML gene exon 6 (V-forms) rather than in the two common hybrid mRNA configurations resulting from breaksites in either PML gene intron 6 (L-forms) or intron 3 (S-forms). In 4 V-form cases, a common break/fusion site was discovered at PML gene nucleotide (nt) 1685, abutting a 3' cryptic splice donor sequence. The fusion site was proximal to the common site in 1 case and more distal in 5 cases. The open reading frame encoding a PML-RAR alpha gene was consistently preserved, either by an in-frame fusion site or by the insertion of 3 to 127 unidentified nts. In 2 V-form cases, hybridization analysis of the reverse transcriptase-polymerase chain reaction products with a PML-RAR alpha juction probe was required for discrimination from L-form cases. Two V-form subgroups were defined by in vitro sensitivity to all-trans retinoic acid (tRA)-induced differentiation: 4 of 4 cases tested with fusion sites at or 5' to nt 1685 (subgroup E6S) had reduced sensitivity (EC50 > or = 10(-7) mol/L), whereas 4 of 4 cases with fusion sites at or 3' to nt 1709 (subgroup E6L) had high sensitivity (EC50 < 10(-8) mol/L) indistinguishable from that of L-form and S-form cases. These results provide the first link between PML-RAR alpha configuration and tRA sensitivity in vitro and support the importance of subclassifying APL cases according to PML-RAR alpha transcript type.