Melanoma-associated markers expression in blood: MUC-18 is associated with advanced stages in melanoma patients.

BACKGROUND: Multimarker reverse transcriptase-polymerase chain reaction (RT-PCR) was originally reported to reveal melanoma-associated mRNAs (MAMs) in melanoma cells but not in the peripheral blood of healthy individuals. OBJECTIVES: To evaluate the expression of MAMs in the peripheral blood of melanoma patients at different American Joint Committee on Cancer (AJCC) stages, and to correlate their presence with early and/or advanced stages of the disease. MATERIALS AND METHODS: One hundred blood samples of melanoma patients (AJCC I-IV) were analysed using multimarker RT-PCR to assess the co-expression of Tyr-OH, MART-1, MAGE-3, MUC-18/MCAM and p97. Patients were stratified into two disease categories: early and advanced stages. The former includes in situ and melanoma stages AJCC I-II, the latter AJCC III-IV. chi(2) and Fisher's exact tests were used to statistically evaluate the association between each MAM and disease categories. The recognized significant associations were subsequently resubmitted to univariate logistic regression. Furthermore, sensitivity and specificity were established. RESULTS: At least one MAM could be detected in 24% of our series. Tyr-OH was the most common marker (14%), followed by MUC-18 (12%), MART-1 (5%), MAGE-3 (4%) and p97 (3%). No significant association among Tyr-OH, MART-1, MAGE-3, p97 and disease stages were evidenced. Only MUC-18 was statistically associated (P < 0.009) with advanced stages alone or co-expressed with other MAMs. According to logistic regression univariate analysis, MUC-18 increases the probability (odds ratio: 33) being in advanced stages and the incidence of recurrences (95% CI 2.9-374). CONCLUSIONS: MUC-18 RT-PCR assay could be proposed as an adjunctive molecular method in the management of melanoma patients and is useful in the monitoring of study protocols.

Molecular evaluation of the NUP98/RAP1GDS1 gene frequency in adults with T-acute lymphoblastic leukemia.

The NUP98/RAP1GDS1 (NRG) is a new fusion gene, originating from the t(4;11)(q21;p15) translocation, that characterizes a subset of T-cell acute lymphoblastic leukemia (T-ALL). In this study we analyzed 43 T-ALL patients for the expression of this new molecular marker using a reverse transcription-polymerase chain reaction (RT-PCR) system, which is more sensitive and specific than cytogenetics alone, confirming that NRG-positive ALLs are infrequent, accounting for approximately 5% of cases.

HLA class I in acute promyelocytic leukemia (APL): possible correlation with clinical outcome.

The majority of patients with acute promyelocytic leukemia (APL) possess either a bcr1 or a bcr3 type fusion between PML and RARalpha genes. The junction sequences may possibly be a target for immune response and influence susceptibility to the disease. In this case, HLA class I allele frequencies would be different between bcr1 and bcr3 patients. To test this hypothesis, we typed 102 APL patients for HLA-A, -B and -Cw alleles. The A*1, A*30, B*51, B*41, Cw*0602, and Cw*1701 alleles showed a different distribution between bcr1 and bcr3 patients, but in no case was this statistically significant after correction for the number of comparisons or was confirmed in an independent panel. Moreover, no difference was detected between bcr1 and bcr3 when HLA alleles were grouped according to their peptide binding specificities. Comparing HLA frequencies, clinical features at diagnosis and clinical outcome of the 64 patients homogeneously treated with all-trans retinoic acid and idarubicin (AIDA protocol) we observed a statistically significant association between HLA-B*13 and risk of relapse by univariate and multivariate regression analysis. Should this finding be confirmed in larger future studies, this observation would be of outmost importance in identifying patients at high risk of relapse in which more aggressive consolidation therapies should be used.

A prospective study of residual-disease monitoring of the ALL1/AF4 transcript in patients with t(4;11) acute lymphoblastic leukemia.

Twenty-five patients (22 adults and 3 infants) with ALL1/AF4-positive acute lymphoblastic leukemia (ALL) were prospectively monitored by reverse transcriptase-polymerase chain reaction (RT-PCR) between January 1992 and July 1999. After high-dose induction and consolidation chemotherapy without bone marrow transplantation, all patients had a complete hematologic remission. Using nested RT-PCR (sensitivity 10(-4)), we observed conversion to PCR negativity in 11 (44%) of the patients. Thirteen of the 14 patients who did not have a molecular remission had a relapse at a median time of 4 months (range, 1 - 20 months). Of the 11 patients who had a conversion to PCR negativity, 5 reconverted to PCR positivity within 1 to 14 months. These 5 patients all progressed to hematologic relapse after 2, 3, 4, 4, and 7 months, respectively. Of the remaining 6 patients, 4 are in persistent hematologic and molecular remission at 12, 14, 88, and 96 months, whereas 2 are early in their follow-up. Actuarial probabilities of relapse and overall survival were 100% and 0% at 14 and 24 months and 67% and 43% at 96 and 100 months, respectively, in patients who had persistent RT-PCR positivity and in those who had a molecular remission. For both relapse and survival, the differences observed between the two groups were significant (P =.003 and P <.005, respectively). This study, which represents the first prospective analysis of residual-disease monitoring carried out in a substantial series of patients with t(4;11)-positive ALL, emphasizes the clinical relevance of RT-PCR-based methods to monitor minimal residual disease in this leukemia subset. (Blood. 2000;95:96-101)

HLA-C and HLA-DQB1 compatibility in unrelated cord blood transplants.

BACKGROUND AND OBJECTIVE: Umbilical cord blood (UCB) cells have been definitively proved to be a source of hematopoietic stem cells with repopulating capacity when transplanted into pediatric hosts with neoplastic or non-neoplastic disease. Moreover, due to the immaturity of the UCB lymphoid compartment, these transplants are usually associated with a low incidence and severity of GvHD. This clinical observation and the immaturity of the UCB lymphoid compartment justify the acceptance of UCB units which differ from their recipient by 1 or 2 HLA antigens of the six HLA A, B and DRB1 antigens conventionally typed. Whether the number and type of HLA disparities affect clinical outcome of UCB transplants has not, however, been clearly demonstrated yet. DESIGN AND METHODS: In the present study on 14 pediatric patients with high risk leukemia transplanted with UCB from unrelated donors, evaluation of HLA compatibility was extended to HLA-C and DQB1 genes and correlated to the engraftment rate and occurrence of GvHD. Conditioning regimen and GvHD prophylaxis were identical in all cases. HLA-A and B antigens were typed by serology, whereas DNA based methods were used to define HLA-C gene groups, and HLA-DRB1 and DQB1 alleles. RESULTS: Conventional HLA-A, B and DRB1 typing demonstrated that 12 recipient/donor pairs differed at one HLA locus, while 2 pairs had 2 HLA disparities. The extended HLA-typing showed that only one out of the six pairs with a different HLA-A locus had additional mismatches at HLA-C and DQB1 loci, whereas all the remaining 8 pairs, which already differed at HLA-B and/or DRB1 loci after conventional typing, had additional HLA-C and/or DQB1 mismatches (p = 0.002). By contrast, engraftment rate and occurrence of GvHD did not significantly correlate with level of HLA-mismatches even after extended HLA-typing. INTERPRETATION AND CONCLUSIONS: The present data show that additional mismatched HLA-C and/or DQB1 antigens are significantly more frequent in pairs which after conventional HLA-typing differed at HLA-B and/or DRB1 loci, than in those showing one HLA-A mismatch. This observation provides an additional criterion for selection of UCB donors with the closest HLA-match when more than one unit are available. We did not, however, observe any correlation between engraftment rate, occurrence of GvHD and degree of HLA disparities detected either by standard or extended typing. These data support the notion that certain HLA differences do not affect the clinical outcome of UCB transplants and indicate that the expensive and time consuming molecular typing of HLA-C and DQB1 loci might be avoided for UCB donor selection.

A prospective molecular study of chimaerism in patients with haematological malignancies receiving unrelated cord blood or bone marrow transplants: detection of mixed chimaerism predicts graft failure with or without early autologous reconstitution in cord blood recipients.

We prospectively studied the chimaerism status in the bone marrow (BM) and peripheral blood (PB) of 23 patients receiving umbilical cord (UCB, 14 cases) or BM (nine cases) transplants from unrelated donors by PCR amplification of four individual-specific VNTR genetic loci. Haematological engraftment, with persistent full donor pattern. was observed in 10/14 (72%) patients receiving UCB and in 9/9 (100%) patients transplanted with marrow from an unrelated donor (MUD). In contrast, the remaining four patients converted to an autologous pattern. Three out of these four patients had an early autologous haematological reconstitution reaching a neutrophil level >0.5 x 10(9)/l at days 27, 33 and 37 after transplant, respectively. In all three of these patients, chimaerism analysis demonstrated an early appearance of donor cells (i.e. within 35 d after UCB transplant) showing a transient full donor (one case) or mixed chimaerism condition (two cases). Despite the early autologous haemopoietic reconstitution, one of the three patients died of GVHD at day 60, which was explained by the demonstration of low levels of donor lymphoid cells. In the MUD group all nine patients converted to a persistent full donor pattern with haematological reconstitution, accompanied in two of them by transient mixed chimaerism lasting to days 60 and 270 after transplant. Our data show that monitoring of chimaerism may predict graft failure with or without early autologous haemopoietic reconstitution in patients receiving unrelated UCB transplants. Furthermore, chimaerism analysis may identify, in patients with autologous reconstitution, those at risk of severe GVHD in whom immunosuppressive therapy should not be discontinued.

Umbilical cord blood transplant from unrelated HLA-mismatched donors in children with high risk leukemia.

In the last 3 years, 14 children with high-risk leukemia (11 ALL, 2 AML and 1 CML) underwent cord blood transplantation from unrelated HLA-mismatched donors at a median of 99 days from the start of search. Eight patients were transplanted in second CR, one in accelerated phase, three at relapse and two patients in first CR. Conditioning regimen (fractionated TBI, etoposide, CY and anti-lymphocyte serum) and prophylaxis of GVHD (CsA and 6-methylprednisolone) were identical for all patients. Neutrophils >0.5x10(9)/l were reached at a median of 33 days from transplant, but in four cases we observed an autologous hematopoietic reconstitution (three spontaneous, one after autologous BM rescue). Acute and chronic GVHD were observed in 10/14 and 3/8 evaluable cases, respectively. Three patients died of transplant-related toxicity and three patients relapsed. The probabilities of event-free, disease-free and overall survival were 50, 53 and 64%, respectively. Cord blood transplant from HLA-mismatched unrelated donor is a valid option for the treatment of children with high-risk leukemia. With our eligibility criteria, conditioning regimen and prophylaxis of graft-versus-host disease, the main obstacles to successful transplant were represented by graft failure and fatal acute GVHD.

ALL1 gene alterations in acute leukemia: biological and clinical aspects.

BACKGROUND AND OBJECTIVE: The ALL1 gene, also referred to as MLL, HRX or Htrx1, is interrupted in the vast majority of translocations involving the chromosome band 11q23. Alterations in this gene are reported in approximately 5-10% of acute leukemias (AL) and characterize different leukemic subtypes such as infant (< 12 months of age) AL, topoisomerase II inhibitors-related (TR) AL and a small subset of de novo AML and ALL. Distinguishing features of ALL1 alterations include the striking heterogeneity of its recombinations, i.e., more than 30 chromosome partners have been described in ALL1 rearrangements, and the lack of association with a definite lineage. The objective of this article is to review the biological and structural properties of ALL1 gene and its various fusion proteins, and to discuss the clinical relevance of these lesions with special emphasis on their role in molecular diagnosis and monitoring of minimal residual disease. EVIDENCE AND INFORMATION SOURCES: The material examined in the present review includes data published by the authors in this field, articles and abstracts published in journals covered by the Science Citation Index and Medline, as well as some more recent personal unpublished observations. STATE OF THE ART: The ALL1 gene spans approximately 90 kb of DNA in length, and consists of 36 exons, ranging in size from 65 bp to 4249 bp. ALL1 codifies for a major transcript of approximately or equal to 15 kb. It encodes a protein of more than 3910 amino acids, containing three regions sharing sequence homology with the Drosophila trithorax gene. These homologies suggest that ALL1 is a transcription factor controlling development and/or differentiation of human cells. To date, twelve ALL1 partner genes have been characterized which are involved in the following translocations: t(4;11), t(9;11), t(6;11), t(11;19), t(1;11) t(10;11), t(11;16), t(11;17) and t(X;11). Since all these genes do not share relevant homologies among each other, their putative role in ALL1 activation still remains to be clarified. The analysis of ALL1 breakpoint cluster region (bcr) shows that several DNA motifs implicated in illegitimate recombination events are located within the bcr. Thus, mapping of breakpoints in the different subtypes of ALL1 +ve leukemia may help in understanding the events leading to translocations in human ALs. In this respect, data on ALL1 breakpoint localization suggest that similar pathogenetic mechanisms may underlie infant and TR AL and that these events might differ from those occurring in de novo AL. The availability of this molecular marker provides a new tool for diagnostic purposes and characterization of ALs and for monitoring of minimal residual disease. To date, the prognostic value of ALL1 rearrangements has been clearly demonstrated for infant ALs only, whereas the clinical relevance of ALL1 rearrangements in the other leukemic subtypes needs further evaluation by future prospective studies on a larger number of patients homogeneously treated. As concerning studies on minimal residual disease, data on PCR monitoring of the ALL1/AF4 fusion transcript, resulting from the t(4;11) translocation, show the clinical relevance of this molecular test in predicting outcome and, as a consequence, in designing individual post-remission therapies. PERSPECTIVES: It is expected that future studies will provide more detailed information regarding either the normal ALL1 function and/or the leukemogenic effect of ALL1 alterations, together with a better definition of the prognostic relevance of the hybrid proteins formed by this gene at diagnosis and during remission of disease.

Infant acute leukemias show the same biased distribution of ALL1 gene breaks as topoisomerase II related secondary acute leukemias.

The ALL1 gene (also called MLL, HRX, or Htrx1) at the cytogenetic band 11q23 is consistently altered by chromosome rearrangements in acute leukemias (ALs) of early infancy, in ALs developed after exposure to topoisomerase (topo) II-inhibitory drugs, and in a small subset of de novo ALs in children and adults. Because exposure to natural or medicinal substances blocking topo II during pregnancy have been proposed as etiological agents for infant leukemia, we have compared the distribution of ALL1 gene breakpoints in infant leukemias with an altered ALL1 gene configuration to those in secondary leukemia associated with prior exposure to topo II targeting drugs and in reference to the major topo consensus binding site in exon 9. ALL1 gene breakpoint distribution was determined by Southern blot hybridization and/or reverse transcription-PCR of the ALL1/AF4 fusion cDNA in 70 patients. Using restriction enzyme analysis, the 8.3-kb ALL1 breakpoint cluster region was divided in a centromeric portion of 3.5 kb (region A) and telomeric portion of a 4.8 kb (region B). ALL1 breakpoint were located in region A in 8 of 28 (28.5%) cases of infant ALs, 16 of 24 (66%) cases of de novo ALs, and 0 of 5 cases of therapy-related (TR) ALs. Conversely, ALL1 breakpoints in region B were detected in 20 of 28 (71.5%) cases of infant AL, 8 of 24 (33%) cases of de novo AL, and 5 of 5 (100%) cases of TR AL (P = 0.002). These results were confirmed by direct sequencing of the ALL1/AF4 fusion transcript in 30 cases (19 infants and 11 child and adult de novo cases). The analysis of ALL1/AF4 junction types showed that children and adults with de novo leukemia had ALL1 breakpoints in intron 6 (9 cases) or intron 7 (2 cases), whereas breakpoints in infant cases were mainly located in intron 8 (14 cases) and less frequently in intron 6 (4 cases) and intron 7 (1 case). The difference in ALL1 breakpoint location between infant and noninfant AL patients with ALL1/AF4 fusion was statistically significant (P = 0.00005). These data demonstrated that infant and TR ALs share a similar biased clustering of ALL1 gene breakpoints, which supports the possibility that topo II inhibitors may also operate in utero and play a crucial role in the etiology of infant leukemia.

Sequential molecular monitoring of chimerism in chronic myeloid leukemia patients receiving donor lymphocyte transfusion for relapse after bone marrow transplantation.

Recent observations of chimerism in patients relapsed following an allotransplant suggest the persistence of immunotolerance, thus offering a biologic rationale for the use of donor lymphocyte transfusion (DLT). In this study, we have analyzed by PCR amplification of several VNTR regions, sequential bone marrow and peripheral blood DNA samples in four patients who received DLT for CML relapse after bone marrow transplantation. Prior to DLT, all patients showed mixed chimerism in peripheral blood cells while two had mixed chimerism and two no chimerism in the BM. None of these four patients showed evidence of chimerism at the cytogenetic level (all had 100% +ve metaphases). After DLT, a complete hematologic and molecular remission (ie disappearance of the BCR/ABL fusion transcript) was obtained in the two patients who had bone marrow mixed chimerism prior to DLT. The two patients without evidence of marrow chimerism prior to DLT converted to a pattern of mixed chimerism after DLT, but both developed a severe bone marrow aplasia occurring at day 56 and 36, respectively. With regard to the sequential analysis of bone marrow chimerism after DLT we observed that: (1) the disappearance of BCR/ABL +ve cells paralleled the conversion to a pattern of full donor chimerism; and (2) the time interval to achieve CR was inversely correlated with the percentage of donor DNA in bone marrow. In conclusion, we have shown here that the assessment of bone marrow pre-DLT chimerism by PCR analysis might predict the response in patients with favorable characteristics, and also might identify patients at high risk of developing severe myelosuppression.

Clinical relevance of residual disease monitoring by polymerase chain reaction in patients with ALL-1/AF-4 positive-acute lymphoblastic leukaemia.

In this study we used reverse transcriptase-polymerase chain reaction (RT-PCR) for the longitudinal monitoring of minimal residual disease in 12 patients with All-1/AF-4 positive ALL. Of these, seven also showed at presentation a typical t(4;11) cytogenetic translocation. Seven patients were infants <18 months of age and five were adults. Eleven patients were treated with high-dose intensive induction and consolidation chemotherapy without bone marrow transplantation and one received conservative treatment due to poor performance status. Three had resistant disease, four relapsed within 12 months after achieving complete remission, and five are in continuous complete remission (CCR) at 32, 39, 52, 53 and 61 months from diagnosis, respectively. The sequential analysis of the ALL-1/AF-4 hybrid transcript showed a persistently negative RT-PCR in the five CCR long-term survivors. The PCR analysis resulted persistently positive in the remaining seven cases, including the four cases who relapsed after the achievement of clinical CR. These data emphasize the clinical relevance of PCR monitoring analysis in t(4;11) ALL patients and should be considered in order to better determine variable post-remission treatment according to risk prediction.

Analysis of the BCL-6 gene configuration in diffuse B-cell non-Hodgkin's lymphomas and Hodgkin's disease.

BCL-6 is a novel proto-oncogene that codes for a zinc-finger protein sharing homologies with many transcription factors. It has recently been shown that BCL-6 is involved in chromosome band 3q27 aberrations in non-Hodgkin's lymphomas (NHLs) and BCL-6 rearrangements have been detected in 34-45 per cent of diffuse large cell lymphomas with B immunophenotype. We have studied the BCL-6 gene configuration by Southern blot analysis in 60 cases of B-cell NHL and in 17 cases of Hodgkin's disease (HD). BCL-6 was rearranged in 15/46 (32.6 per cent) diffuse B-large cell lymphomas, mainly with centroblastic morphology, and in 2/11 (18.2 per cent) follicular (centroblastic-centrocytic) lymphomas. Conversely, all cases of HD, including four cases of lymphocyte predominant, nodular type (nodular paragranuloma), had a germline configuration. These findings confirm that BCL-6 is rearranged in a significant percentage of diffuse B-large cell lymphomas, suggesting that this proto-oncogene might have a pathogenetic role in this subset of NHLs, but our preliminary analysis suggests that BCL-6 lesions are not involved in the pathogenesis of HD. However, further investigations using more sensitive techniques are required to confirm these findings.

ALL-1 gene rearrangements in acute myeloid leukemia: association with M4-M5 French-American-British classification subtypes and young age.

We have analyzed by Southern blotting the ALL-1 (MLL, HRX, Hrtx 1) gene configuration in a series of 126 patients with acute myeloid leukemia (AML) representative of all ages and French-American-British Classification groups and correlated this genetic feature with clinical and biological features at diagnosis. ALL-1 gene rearrangements were detected in 17 of the 74 cases with M4-M5 (myelomonocytic and monocytic) AML and in 2 of the 52 cases with other leukemic subtypes (P < 0.01). Within the series of 74 M4-M5 patients, ALL-1 rearrangements were significantly associated with French-American-British Classification M5 (P = 0.009), high WBC (P = 0.002), and young age. In particular, all 5 infant (< 1.5 years) AML cases, 6 of the 19 (31%) patients between 1.5 and 18 years of age, and 6 of the 50 (12%) patients > 18 years old showed an altered ALL-1 genomic configuration (P < 0.001). Immunophenotypic characterization revealed coexpression of lymphoid and myeloid markers in 6 of 17 ALL-1 rearranged M4-M5 cases. The IgH gene configuration was studied in 77 of 126 AMLs. Five patients (6%) showed IgH clonal rearrangements and all were in the ALL-1 rearranged group (P < 0.0001). Our findings indicate that ALL-1 rearrangement is the commonest genetic alteration presently detectable in M4-M5 AML, particularly in childhood where it is found in up to one-third of all cases. The association of IgH rearrangements with ALL-1 alterations in AML, coupled to the frequent detection in this subset of lymphoid associated markers, further supports the origin of these tumors from a common multipotent precursor with bipotential lymphoid and monocytic differentiation capability.

Xenopus laevis ribosomal protein L22: full-length cDNA sequence and expression analysis.

A cDNA clone was isolated from a Xenopus laevis embryo library and sequenced. Primer extension experiments indicated the full-length nature of the insert and the encoded product was identified on a two dimensional gel as ribosomal protein (r-protein) L22. The 510-bp L22 cDNA sequence presents short untranslated regions and a 5'-end polypyrimidine tract found in all other vertebrate r-protein mRNA (rp mRNA) so far analyzed. Both the nucleotide (nt) and the deduced amino acid (aa) sequences have been compared with the homologous sequences from other species. The L22 nt sequence is about 70% similar to the mammalian L27a rp mRNA and about 60% homologous to the Drosophila, Tetrahymena and yeast corresponding mRNAs. The 148-aa sequence presents a higher conservation, being 90% similar to the mammalian sequence and more than 70% to the other species. Expression analysis showed that, both during X. laevis embryogenesis and in X. laevis cultured cells during growth-rate changes, L22 synthesis is translationally regulated. Therefore X. laevis L22 mRNA is a new example of the correlation between the polypyrimidine terminal tract and the translational regulation observed in other rp mRNAs.

Prognostic relevance of ALL-1 gene rearrangement in infant acute leukemias.

We and others have recently reported a high frequency (70-80%) of ALL-1 (MLL, HRX, HTRX) gene rearrangements in infants with acute leukemias (AL) aged less than 1 year. Preliminary observations in limited series also suggested that ALL-1 gene configuration is an important prognostic factor in this leukemic subset. We have now extended our study to a series of 45 AL patients aged between 0 and 18 months. The genomic configuration of ALL-1 in leukemic DNAs was determined by Southern blot hybridization and correlated with biological and clinical features at presentation, as well as with treatment outcome. Twenty-nine out of 45 (64%) patients showed ALL-1 rearrangements, including 4/11 (36%) infants aged between 13 and 18 months. Considering morphological types, 24/38 cases with acute lymphoblastic leukemia and 5/7 patients with acute myeloid leukemia showed ALL-1 rearrangements. The features more frequently found in association with ALL-1 rearrangements were hyperleukocytosis (P < 0.007) and CD19+/CD10- blast immunophenotype (P < 0.02). ALL-1 status was an independent prognostic marker of event-free survival (EFS) in a multivariate model including age, sex and WBC count, and maintained its statistical significance when FAB morphology was considered in the analysis by including AML patients. Considering the ALL cases the actuarial EFS was 57 and 9% for infants with germline and rearranged ALL-1 configuration, respectively (P = 0.008). A high frequency of ALL-1 gene alterations in infant AL is confirmed by this study. In addition, our results emphasize the need for extending the analysis of ALL-1 gene status to infants with AL aged > 12 months. We show that this genetic lesion is the most important variable negatively affecting prognosis in a multivariate model including other known risk factors. This latter observation should influence the choice of risk-adapted treatment strategies in this AL subset.