High frequency of immunophenotype changes in acute myeloid leukemia at relapse: implications for residual disease detection (Cancer and Leukemia Group B Study 8361)

Multiparameter flow cytometry (MFC) has the potential to allow for sensitive and specific monitoring of residual disease (RD) in acute myeloid leukemia (AML). The use of MFC for RD monitoring assumes that AML cells identified by their immunophenotype at diagnosis can be detected during remission and at relapse. AML cells from 136 patients were immunophenotyped by MFC at diagnosis and at first relapse using 9 panels of 3 monoclonal antibodies. Immunophenotype changes occurred in 124 patients (91%); they consisted of gains or losses of discrete leukemia cell populations resolved by MFC (42 patients) and gains or losses of antigens on leukemia cell populations present at both time points (108 patients). Antigen expression defining unusual phenotypes changed frequently: CD13, CD33, and CD34, absent at diagnosis in 3, 33, and 47 cases, respectively, were gained at relapse in 2 (67%), 15 (45%), and 17 (36%); CD56, CD19, and CD14, present at diagnosis in 5, 16, and 20 cases, were lost at relapse in 2 (40%), 6 (38%), and 8 (40%). Leukemia cell gates created in pretreatment samples using each 3-antibody panel allowed identification of relapse AML cells in only 68% to 91% of cases, but use of 8 3-antibody panels, which included antibodies to a total of 16 antigens, allowed identification of relapse AML cells in all cases. Thus, the immunophenotype of AML cells is markedly unstable; nevertheless, despite this instability, MFC has the potential to identify RD in AML if multiple antibody panels are used at all time points. (Blood. 2001;97:3574-3580)     

Significance of trilineage myelodysplasia in de novo acute myeloid leukaemia during remission rather than at diagnosis

Patients with trilineage myelodysplasia (TMDS) in de novo acute myeloid leukaemia (AML) at diagnosis and remission were clinically evaluated between 1983 and 1996. AML with TMDS (AML/TMDS) was observed in 20 (12%) of 162 patients with de novo AML at diagnosis. Complete remission (CR) was achieved with combination chemotherapy in 12 (67%) of 18 AML/TMDS cases. This CR rate was relatively worse than the rate of 78% (106/136 cases) of AML without TMDS, but this difference was not significant. Disease-free survival curves also showed no difference between AML/TMDS and AML without TMDS. During remission, eight (67%) of 12 AML/TMDS cases had myelodysplastic remission marrow (AML/MRM). AML/MRM was also seen in seven (7%) of 106 AML cases without TMDS. The actuarial disease-free survival was significantly lower in AML/MRM than in AML without MRM ( P  = 0.0003). All of the AML/MRM cases exhibited early leukaemic relapse; median remission duration was only 9 months. Clonal changes occurred in two cases of AML/TMDS and five cases of AML/MRM at the time of relapse. These findings suggest that TMDS during remission predicts a poorer prognosis and early leukaemic relapse when compared with the absence of TMDS.     

Shifts in expression of immunological cell markers in relapsed acute leukemia

The immunophenotypic features of leukemia blast cells were analyzed in a group of 156 patients with different immunological subtypes of acute leukemia, both lymphoblastic and myeloblastic. Of the 58 patients for whom immunologic studies were performed at relapse, 42 (72%) showed changes in the expression of immunologic markers. The minor shifts in B-ALL were observed most frequently and concerned of the loss of CD34 antigen in 17 cases and the loss of cALLA (CD10) in 7 cases of B-ALL at the first relapse. The acquisition of cell markers was not frequently observed, only in four cases could be seen. HLA-DR molecules remained relatively constant from diagnosis to relapse. In 2 from 3 T-ALL cases the loss of CD1 and CD2 markers, respectively, was noticed at relapse. CD5 and CD7 markers were relatively stable. In AML cases at relapse the acquisition of CD13 marker (in 4 from 7 cases) was often observed. It was interesting that comparing to the B-ALL cases, the loss of CD34 marker in AML cases was stray. In one case the acquisition of this antigen at relapse was actually observed. The major interlineage shift was detected in one case of B-ALL, that was newly diagnosed at relapse as AML M4 and presented different cytogenetic features. This case provides strong connection with the treatment, as more recently epipodophyllotoxins (vumon in our patient) have been linked to the development of secondary AML associated with a shorter latency period. The immunophenotypic changes frequently occur at relapse in all acute leukemia types. The shifts (loss or acquisition) in expression of individual markers at relapse are bound with the first diagnosis and may have a relationship to the treatment and are important for correct assessment of minimal residual disease.     

Testicular infiltration in acute myeloid leukemia with complex karyotype including t(8;21)

Testicular infiltration is a well-known complication in acute lymphoblastic leukemia. In acute myeloid leukemia (AML), it has rarely been described and preferably occurred in cases with myelomonocytic or monoblastic differentiation. We report on a patient with AML with complex karyotype including translocation t(8;21) who presented with testicular infiltration at the time of his third bone marrow relapse. Cytological analysis of the specimen showed infiltration with blasts displaying the typical morphology of AML with translocation t(8;21) and comparable to those detected in the bone marrow. Fine needle aspiration cytology might suffice in these cases and should be performed if testicular involvement is suspected.     

The role of prophylactic bilateral oophorectomy at the time of initial diagnosis of a unilateral ovarian metastasis in cases with colorectal adenocarcinoma

BACKGROUND/AIMS: In cases with a macroscopic unilateral ovarian metastasis of colorectal adenocarcinoma, a clear therapeutic policy regarding a prophylactic bilateral oophorectomy is lacking. METHODOLOGY: Four cases of ovarian metastases of colorectal adenocarcinoma are presented. RESULTS: Case 1 is a 63-year-old with ascending colon carcinoma, Dukes' C, and right oophorectomy due to metachronous ovarian metastasis. Case 2 is a 28-year-old with transverse colon carcinoma, Dukes' D, and right oophorectomy due to synchronous ovarian metastasis. Case 3 is a 40-year-old with rectal carcinoma, Dukes' B, and right oophorectomy due to metachronous ovarian metastasis. Case 4 is a 32-year-old with rectal carcinoma, Dukes' D, and bilateral oophorectomy due to synchronous and metachronous ovarian metastases. Later, in cases 1 and 3, in which a unilateral ovary was preserved, ovarian metastases to the preserved ovary were found and caused severe clinical symptoms. However, by that time their general condition did not permit any additional laparotomy. CONCLUSIONS: Without other extensive metastases, if demonstrable diseases are found in a unilateral ovary, a prophylactic bilateral oophorectomy is recommended.     

Surface marker expression in acute myeloid leukaemia at first relapse

Surface markers were studied at first relapse in 66 cases of acute myeloid leukaemia (AML), using a panel of five monoclonal antibodies directed to CD13, CD14, CD15, CD33 and CD34 antigens. At time of relapse, there was increased expression of CD33 (P = 0.002) and CD34 (P = 0.0001), and decreased expression of CD13 (P = 0.004) and CD15 (P = 0.0001) antigens by comparison to initial diagnosis. There was no strict correlation with the FAB classification. However, CD13 and CD33 expression changes preferentially affected granulocytic leukaemias. At relapse, CD14 and CD34 were significantly more expressed in monocytic than in granulocytic AML (P = 0.01 and 0.003 respectively). In a multivariate analysis, CD34 expression was associated with a low CR rate (P = 0.001) and short survival (P = 0.05), whereas CD15 expression was associated with long survival (P = 0.0004). These results suggest that AML tends to relapse with a less differentiated phenotype than observed at diagnosis and that AML with less differentiated phenotype is of poor prognosis after first relapse, as also observed at diagnosis.     

Translocations involving 6p22 in acute myeloid leukaemia at relapse: breakpoint characterization using microarray-based comparative genomic hybridization

The detection of chromosomal aberrations is essential for the diagnosis and therapy of acute myeloid leukaemia (AML). We report two cases of de novo AML with translocations involving the breakpoint 6p22 first detected at relapse. Chromosomes were identified by conventional and molecular cytogenetics. At diagnosis, one patient presented a normal karyotype and the other one a trisomy 11 and a del(7)(q31q36). In the first case, cytogenetic analyses at relapse revealed a t(3;6)(q21;p22). The second patient showed a t(1;6)(q21;p22) at relapse. Detailed characterization of the breakpoints on the short arm of chromosome 6 was performed using array comparative genomic hybridization (CGH) on a platform specific for chromosome 6. In both cases, array CGH showed a terminal deletion and a small internal duplication of the short arm of chromosome 6. The region 6p22 is involved in several aberrations in tumours. Translocation partners are distributed throughout the human genome. We identified 3q21, a recurrent breakpoint in AML, for the first time as a translocation partner. The fragile site FRA6C, located in 6p22.2, and possibly the genes that reside within it, may play a role in tumorigenesis. The occurrence of translocations involving 6p22 after chemotherapy or radiation therapy suggests that one or more therapeutic agents might play a role in their origin.     

Myelodysplastic relapse of de novo acute myeloid leukaemia with trilineage myelodysplasia: a previously unrecognized correlation

We describe the occurrence of an unusual mode of relapse in six of 24 patients who presented with de novo acute myeloid leukaemia (AML) associated with trilineage myelodysplasia (TMDS). After the induction of complete remission (CR) by intensive chemotherapy in five patients and following bone marrow transplantation (BMT) in one, the myelodysplastic state, but not overt AML, recurred. Relapse of myelodysplasia occurred at a median of 147 weeks (50-520) from presentation and in two instances was followed a year later by AML. In five cases, myelodysplastic relapse was treated with low-dose cytosine arabinoside given alone or with other chemotherapeutic agents. Three patients remain in CR after 1, 2 and 5 years. The reappearance of myelodysplastic features in these six patients was strongly correlated with the presence of TMDS at presentation of the AML. It was not observed once in the 136 AML patients, treated similarly, who did not have associated TMDS at presentation (P less than 0.001). Thus, relapse with myelodysplasia is not an effect of chemotherapy as has been previously postulated.     

Late relapses evolve from slow-responding subclones in t(12;21)-positive acute lymphoblastic leukemia: evidence for the persistence of a preleukemic clone

TEL/AML1-positive childhood acute lymphoblastic leukemias (ALLs) generally have low-risk features, but still about 20% of patients relapse. Our initial molecular genetic analyses in 2 off-treatment relapses suggested that the initial and relapse clones represent different subclones that evolved from a common TEL/AML1-positive, treatment-resistant precursor. In order to further elaborate on this hypothesis, we studied 2 patients with late systemic relapses of their TEL/AML1-positive ALL (41 months and 49 months after initial diagnosis, respectively) who had distinct clonal antigen receptor gene rearrangements at diagnosis and relapse. These clone-specific markers enabled us to determine the responsiveness of the individual clones to treatment. The matching genomic TEL/AML1 breakpoints of the initial and the relapse clones in these patients confirmed their origin from a common progenitor cell. This proof was especially important in one of these 2 leukemias without a common antigen receptor gene rearrangement. Our retrospective analysis revealed that in both cases the relapse clone was already present at diagnosis. Despite their small sizes (5 x 10(-3) and 1 x 10(-4), respectively), we were able to detect their much slower responses to therapy compared with the dominant leukemic clone. Moreover, in all instances, these initially slow-responding clones, after they had developed into the relapse leukemia, were rapidly eradicated by the relapse treatment, underlining their different biology at the 2 time points of leukemia manifestation. We thus hypothesize that the minor clone was not fully malignant at initial diagnosis but acquired further mutations that may be necessary for the manifestation of relapse.     

The (4;11) translocation in acute leukaemia of childhood: the importance of additional chromosomal aberrations

Case reports of four girls and one boy with acute lymphoblastic leukaemia (ALL) or acute myeloid leukaemia (AML) and t(4;11) are presented. The incidence of t(4;11) ascertained at diagnosis in ALL was 2.6% and in AML 5.3%. Four of the children were under 2 years and one was 11 years at diagnosis. Leucocyte counts above 71 X 10(9)/l and liver, spleen and node enlargement were found in all cases. Blasts of the four cases tested at diagnosis were negative to the c-ALL antigen and either TdT+ (ALL) or TdT- (AML M1). Maximum survival was less than 8 months. Additional chromosomal change was found at diagnosis in two cases and in relapse in a third. In the case of AML t(4;11) (q21;p15) was present as a second translocation. Additional numerical changes, in these and other reported cases, included + 6, commonly found in ALL, +8, +19, more often reported in AML. It is suggested that additional chromosomal changes in these cases support cytochemical and surface marker evidence that t(4;11) has a pluripotent target cell, similar to that of the Philadelphia translocation.     

Outcome of allogeneic hematopoietic stem cell transplantation in adult patients with acute myeloid leukemia harboring trisomy 8

Trisomy 8 (+8) is one of the most common cytogenetic abnormalities in adult patients with acute myeloid leukemia (AML). However, the outcome of allogeneic hematopoietic stem cell transplantation (HSCT) in adult patients with AML harboring +8 remains unclear. To evaluate, the outcome and prognostic factors in patients with AML harboring +8 as the only chromosomal abnormality or in association with other abnormalities, we retrospectively analyzed the Japanese registration data of 631 adult patients with AML harboring +8 treated with allogeneic HSCT between 1990 and 2013. In total, 388 (61%) patients were not in remission at the time of HSCT. With a median follow-up of 38.5 months, the probability of overall survival and the cumulative incidence of relapse at 3 years were 40 and 34%, respectively. In the multivariate analysis, two or more additional cytogenetic abnormalities and not being in remission at the time of HSCT were significantly associated with a higher overall mortality and relapse. Nevertheless, no significant impact on the outcome was observed in cases with one cytogenetic abnormality in addition to +8. Although more than 60% of the patients received HSCT when not in remission, allogeneic HSCT offered a curative option for adult patients with AML harboring +8.     

Granulosa-stromal tumor of the ovary: a case of mixed germ cell-cord stromal tumor of the ovary with endocrinological considerations

Granulosa-stromal tumors comprise 5 to 8% of all primary ovarian neoplasms. The first clinical manifestation is precocious puberty in most prepuberal patients. We report a case of mixed germ cell-cord stromal tumor of ovary in a 7.2 years old girl, who presented with isosexual pseudo-precocious puberty of progressive outcome. Serum testosterone, estradiol and 17alphaOH-progesterone levels were increased. Abdominal-pelvic ultrasound revealed a right ovarian mass. Unilateral salpingo-oophorectomy was performed with complete resection of the tumor. The patient is well 7 years after surgery with normal pubertal and growth development and no signs of tumor relapse. We review the clinical manifestations of ovarian tumors, classification and staging of sex cord-stromal tumors, follow-up, tumor markers, treatment and prognosis.     

Segmental uniparental disomy is a commonly acquired genetic event in relapsed acute myeloid leukemia

Despite advances in the curative treatment of acute myeloid leukemia (AML), recurrence will occur in the majority of cases. At diagnosis, acquisition of segmental uniparental disomy (UPD) by mitotic recombination has been reported in 15% to 20% of AML cases, associated with homozygous mutations in the region of loss of heterozygosity. This study aimed to discover if clonal evolution from heterozygous to homozygous mutations by mitotic recombination provides a mechanism for relapse. DNA from 27 paired diagnostic and relapsed AML samples were analyzed using genotyping arrays. Newly acquired segmental UPDs were observed at relapse in 11 AML samples (40%). Six were segmental UPDs of chromosome 13q, which were shown to lead to a change from heterozygosity to homozygosity for internal tandem duplication mutation of FLT3 (FLT3 ITD). Three further AML samples had evidence of acquired segmental UPD of 13q in a subclone of the relapsed leukemia. One patient acquired segmental UPD of 19q that led to homozygosity for a CEBPA mutation 207C>T. Finally, a single patient with AML acquired segmental UPD of chromosome 4q, for which the candidate gene is unknown. We conclude that acquisition of segmental UPD and the resulting homozygous mutation is a common event associated with relapse of AML.     

FLT3 length mutations as marker for follow-up studies in acute myeloid leukaemia

Length mutations within the FLT3 gene (FLT3-LM) can be found in 23% of acute myeloid leukaemia (AML) and thus are the most frequent mutations in AML. FLT3-LM are highly correlated with AML with normal karyotype and other cytogenetic aberrations of the prognostically intermediate group. This group is supposed to be a mixed group of AML with differences in the underlying pathogenesis. For more individualized treatment options it would be helpful to better characterize this large AML group not only by molecular mutations but also use these markers for the definition of minimal residual disease (MRD). However, so far the cytogenetically intermediate AML has been lacking suitable markers for PCR-based MRD detection like the fusion genes in the prognostically favourable subgroups. The suitability of the FLT3-LM as a target for PCR-based MRD was discussed controversially as it seemed to be a rather unstable marker. Thus, we aimed at the evaluation of FLT3-LM as a marker for residual disease in a large cohort of AML. Paired samples of 97 patients with AML at diagnosis and at relapse were analyzed. It could be shown that in only four cases a loss of the length mutation was detected. This is in the range of other well-characterized AML relapsing with a different geno- and/or phenotype. In contrast, a change in the ratio of the mutated allele in comparison to the wild-type allele was frequently observed. In detail, the FLT3-LM showed a tendency to accumulate during disease progression and was found more frequently at relapse than at diagnosis. In addition, 45 patients were analyzed at different time points during and after therapy. Using conventional PCR it clearly could be shown that for most of the patients positive at presentation FLT3-LM is a reliable PCR marker for monitoring treatment response. Even an early detection of relapse was possible in some cases.     

The impact of cell heterogeneity and immunophenotypic changes on monitoring minimal residual disease in acute myeloid leukemia

Monitoring of minimal residual disease (MRD) becomes increasingly important for the more accurate stratification of the therapy in acute leukemia. The purpose of this study was to characterize in detail the phenotypes of heterogeneous population in various AML subtypes and to identify the leukemia associated aberrant phenotype (LAP) in individual patients with AML for precise investigation of MRD. The impact of heterogeneity of pathological populations, the effectiveness of location AML blasts on CD45/SSC dot plots in AML patients during follow-up and phenotype changes on MRD monitoring were evaluated in the second step. Bone marrow samples from 63 patients with AML were analyzed at diagnosis, 33 were selected for monitoring of MRD during follow-up and 13 analyzed at relapse using a wide antibody panel in quadruple combinations by multiparameter flow cytometry. In 88% of AML patients at least one LAP was defined at diagnosis, two or more aberrancies coexisted in 60% of them. The total number of LAPs identified by application of various combinations of antibodies was 112 (mean = 2.04 LAP/patient) and the median percentage of blasts carrying the LAP was 53.57%. In half of the patients, we were able to detect the presence of at least two subpopulations, which not always shared the same aberrancy. Although AML cells often have light scattering properties similar to those of normal (myeloid and B-lymphoid precursors, basophiles etc.) in a fraction of cases we found also very useful the location on CD45/SSC dot plots for MRD discrimination. In 13 patients relapse occurred and although we found in 69% changes of phenotype when comparing diagnosis and the first relapse, at least one LAP was constant in 92%. According to our observations, in majority of patients with AML monitoring of MRD by multiparameter flow cytometry is feasible although in some cases could present some specific difficulties owing to their immunophenotypic heterogeneity, similarity with other cell subpopulations or shifts at relapse. In conclusion, investigation of MRD should be based on the phenotypic characteristics of each subpopulation even if it is present in low frequencies.     

The relationship between bcl-2 expression and response to chemotherapy in acute leukaemia

Summary. Immunocytochemistry was used to assess bcl-2 expression in blasts obtained from the bone marrow of 28 patients with acute lymphoblastic leukaemia (ALL) (16 children and six adults at presentation and three children and three adults on relapse) and 20 with acute myeloid leukaemia (AML) (19 adults and one child, 13 with de novo AML, 11 at presentation and two on relapse, and seven secondary to myelodysplasia or chronic myeloid leukaemia). Slides were examined both for the percentage of positive cells and for the intensity of staining using a five-point scale. There was a statistically significant increase in both the percentage of positive cells seen (P < 0.002) and the intensity of staining (P < 0.01) between samples obtained at relapse and those at presentation in ALL. There was a significantly greater intensity of staining in cells from patients with ALL (P < 0.05) and AML (P < 0.05) who failed to achieve remission after chemotherapy than in those who responded. The intensity of staining in cases of secondary AML was lower than that in de novo disease (P < 0.01). These results suggest that expression of bcl-2 may be an important prognostic feature in both de novo AML and in ALL, but not in secondary AML.     

Three AML patients with existing or pre-existing intracerebral granulocytic sarcomas who were successfully treated with allogeneic bone marrow transplantations

We report three acute myelogenous leukemia (AML) patients who developed intracerebral granulocytic sarcomas (GS) and were successfully treated with allogeneic BMT (allo-BMT). The diagnosis of one patient was AML M2 with myelofibrosis, and the other two patients were AML M4 with eosinophilia (AML M4 Eo), according to the FAB classification. Two patients first experienced a relapse in the brain that resulted in the formation of GS, followed by a relapse in the bone marrow. The remaining patient developed an optic nerve GS after suffering a bone marrow relapse. All three patients received irradiation for the GS and systemic chemotherapy before the allo-BMT. TBI was used for conditioning, and GVHD prophylaxis was with cyclosporine (CsA) and short-term MTX in all three cases. These patients are currently 9 to 37 months post-BMT without relapse. Thus, our experience suggests that allo-BMT is an effective treatment for AML patients with existing or pre-existing intracerebral GS.