ALBERT

Description: We used starch gel electrophoresis to investigate levels of genetic variation between trees of interior Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco var. glauca) that were phenotypically resistant versus susceptible to defoliation by the western spruce budworm (Choristoneura occidentalis Freeman). We also investigated the association between allozyme variation and tree growth traits. Overall, the phenotypically resistant trees had a lower allelic heterozygosity (p = 0.020) compared with susceptible trees. However, this difference between resistant and susceptible trees primarily occurred at the Buena Vista, Colorado, site rather than the Deckers, Colorado, and Jacob Lake, Arizona, sites. Among 25 loci we examined, the resistant trees also had a higher frequency of the most common alleles (p = 0.057) and a higher proportion of homozygous genotypes, especially at loci FEST-1 (p = 0.004), ACO-1 (p = 0.080), and 6PGD-1 (p = 0.084). The higher allelic heterozygosity in susceptible trees was mainly due to their higher proportion of uncommon and (or) rare alleles. Compared with susceptible trees, resistant trees had higher mean radial growth rates (p = 0.047) and less temporal variability in growth rate over 25 years (p = 0.037). Mean radial growth rate and average tree heterozygosity were not related at any site (p = 0.316). Relationships between temporal variability in growth rate and tree heterozygosity were inconsistent among sites. Our results suggest that phenotypic differences in resistance of interior Douglas-fir to western spruce budworm defoliation are partly caused by genetic differences among trees.

Description: The nucleoside analogue pentostatin has clinical activity in B-Cell Chronic Lymphocytic Leukemia (CLL) and has shown significant activity and minimal toxicity when combined with cyclophosphamide for previously treated CLL. Building on this, we initiated a trial in 2002 of combined pentostatin (2mg/m2), cyclophosphamide (600 mg/m2) and rituximab (375mg/m2). Of the 33 enrolled eligible patients included in these analyses, seventeen patients were in the high Rai risk group, 25 were male, and median age for this cohort was 62 yrs (range: 40–79); 17 were non-mutated for the immunoglobulin heavy chain variable region gene, and the majority (67%) were CD38 negative. Only 5 patients had no detectable chromosomal abnormalities by FISH at baseline, 20 had a single FISH anomaly, and 8 had 2 or more FISH anomalies. Of all 28 pts with any anomaly, the following specific abnormalities were detected: 13q- (n=17), +12 (n=8), 11q- (n=7), 17p- (n=2), t(14;18) (n=1), 6q- (n=1), and MDM2 (n=1). Of the 33 patients, 22 had grade 3+ toxicity; 16 patients had non-hematologic toxicity wtih the most common symptoms being nausea (6) and vomiting (4). One patient died on study of grade 5 hypoxia as well as hypotension and this was deemed possibly related to treatment. Almost all patients (32/33; 97%) had a best response of PR or better. Including review of bone marrows done 2 months post-treatment, there were 11 CRs (complete response), 7 nPRs (nodal partial response) and 13 PRs. No differences were observed between type of response and mutation status or CD38+ status. Post-treatment FISH analyses were available on 27 patients; results for 25 patients became normal after treatment. Of the remaining 2, one patient was 13q- x1 and went from 94% to 27.5% abnormal nuclei after treatment; the other is the patient who died on study. To establish minimal residual disease (MRD) post-response, we used three color flow cytometry to detect CD5+/CD19+/CD79b− B cells. This approach found all patients had a reduction in CLL B cells with a median reduction of 91% (range: 5 – 100%). Of interest, all 3 response groups (CR vs. nPR vs. PR) had patients with significant reductions in CLL B cells (i.e., 〉90%). The nPR group was most variable in terms of MRD (median 47%; range: 5–93%) compared to the CR (median: 91%; range: 42–99.9%) and PR (median: 97%; range: 46–100%) groups. In conclusion this novel regimen of pentostatin, cyclophosphamide and rituximab has demonstrated significant clinical activity irrespective of risk stratification parameters with rapid induction of responses, achievement of minimal residual disease in some, and modest toxicity. Patients continue to be accrued to further explore correlative measures of response to treatment.

Description: Background: Over the last decade, increasing numbers of families with at least 2 cases of CLL or other B-cell lymphoproliferative disorders have been described. Methods: Families seen at Mayo Clinic with at least one individual diagnosed with CLL and one or more first or second degree relative diagnosed with CLL or a B-cell lymphoproliferative disorder(LPD) were identified. Clinical characteristics and risk stratification test results (cytogenetic abnormalities by FISH, level of CD38 expression, and IgVH gene mutation status) were extracted and reviewed. Serum, intracellular and CLL B cell secreted levels of pro- and anti-angiogenic cytokines (VEGF, BFGF, and TSP) were also compared between cases of familial and sporadic CLL. Results: Seventy-one families seen at Mayo Clinic were identified meeting criteria for familial CLL. Of the 71 index CLL families, 78.9% (n=56) had at least one other first or second-degree relative with CLL or a B cell LPD, 15.5% (n=11) had 2 other affected members, and 5.6% (n=4) had 3 or more affected members. Of affected family members with B-cell LPDs other than CLL, 64% (n=25) had non-Hodgkin lymphoma, 21% (n=8) had Hodgkin disease, 10% (n=4) had multiple myeloma or Waldenstrom macroglobulinemia, and 5% (n=2) had another lymphoid leukemia (1 ALL, 1 hairy cell leukemia). The median age was 62, with 63% males and 37% females. The majority of individuals had an early Rai stage at diagnosis (stage 0–66%, stage I-19%). Results of cytogenetic testing by FISH, CD38 status, IgVH gene mutation status appear similar in familial and sporadic CLL cases seen at Mayo clinic during the same time interval. No clear pattern of IgVH gene mutation status, IgVH gene family usage, level of CD38 expression, or cytogenetic abnormalities by FISH was seen among multiple affected members from the same family. There were also no significant differences noted in serum, intracellular, and CLL B cell secreted levels of VEGF, BFGF or TSP in the familial samples when compared to a cohort of non-familial samples. Conclusions: Expression of cytogenetics by FISH, immunophenotype, IgVH mutation status and levels of serum, intracellular and cytoplasmic pro- and anti-angiogenic factors(VEGF, BFGF, and TSP) were similar in individuals with familial and sporadic CLL.

Description: We investigated a new method using fluorescence in situ hybridization and DNA probes that span the common breakpoints of t(9;22)(q34;q11.2) and that detect double BCR/ABL fusion (D-FISH) in bone marrow cells with this translocation, one on the abnormal chromosome 9 and one on the Philadelphia chromosome (Ph chromosome). D-FISH patterns were abnormal in 30 of 30 specimens with classic, simple, complex, and masked Ph chromosomes. Based on 200 nuclei from each of 30 normal specimens, the mean percentage of false-positive cells was 0.25 ± 0.39. Thirty-seven specimens from 10 patients were studied before treatment and two or more times at 4-month intervals after treatment with interferon-α2b (IFN-α2b) with or without ara-C. Based on 200 nuclei, the results of D-FISH in these specimens correlated closely with quantitative cytogenetics and accurately quantified disease within a few percent. We studied 6,000 nuclei for each of six specimens, three normal and three from patients with chronic myeloid leukemia (CML) in cytogenetic remission. The normal cutoff for 6,000 nuclei was 0.079% and patients in cytogenetic remission had residual disease ranging from 7 (0.117%) to 53 (0.883%) Ph-positive nuclei. We conclude that D-FISH can detect the Ph chromosome and its variant translocations and accurately quantify disease in CML at diagnosis and at all times after treatment, including cytogenetic remission.

Description: We studied 25 T-cell chronic lymphocytic leukemia (T-CLL) cases collected over a 15-year period. Immunophenotypic analysis was performed in each case; 12 cases were evaluated by cytogenetics, and gene rearrangement studies were performed in 14 cases. The median age was 57 years with a male predominance (M:F, 15:10). The median presenting lymphocyte count was 36.3 x 10(9)/L (range, 3.9 to 438 x 10(9)/L). Fourteen patients (56%) had shotty adenopathy and ten (40%) had mild-to-moderate splenomegaly at presentation; four (16%) had erythematous skin lesions. The lymphocytes were predominantly small; some cases had a minor component of medium-sized cells (〈 10%). The nuclear: cytoplasmic ratios were uniformly high with round to oval nuclei; however, a wide spectrum of nuclear outlines could be found, ranging from minimally to markedly convoluted. Nucleoli were either absent or small and inconspicuous. These lymphocytes did not have the morphology of prolymphocytes and did not contain cytoplasmic granules. Bone marrow infiltration was generally in an interstitial pattern; the degree of involvement ranged from 15% to 90%. Immunophenotyping showed that the lymphocytes were mature T-cells with a predominant CD4+ immunophenotype. Three cases displayed a CD8+ immunophenotype. The patients were treated with a variety of chemotherapeutic regimens with only a minimal response observed in two of 20 patients. We conclude that T-CLL is an uncommon chronic lymphoproliferative disorder (CLPD) that can be morphologically similar to B-CLL, is distinct from T- prolymphocytic leukemia, and has an aggressive clinical course that is refractory to therapy. It may also be difficult to distinguish T-CLL from other T-CLPD, especially the leukemic phase of peripheral T-cell lymphoma and some cases of Sezary syndrome.

Description: The t(6;9)(p23;q34) translocation, which results in the formation of a chimeric fusion gene DEK/CAN on the der(6) chromosome, is a rare recurring cytogenetic aberration reported in patients (pts) with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Because the abnormality is an infrequent finding in AML with most reports describing 2 to 8 cases, the US Intergroup Cytogenetics Consortium investigated the frequency and clinical, pathologic and cytogenetic characteristics of t(6;9) leukemia among pts registered to 19 different treatment protocols. Among 6567 pts with evaluable karyotypes, 62 (0.9%) had t(6;9): 30 on pediatric trials (mean 12 yrs; 15/15 male/female) and 32 on adult trials (mean 38 yrs; 21/11 male/female), compared to the mean age of 8 yrs for pts on pediatric AML/MDS trials and 54 yrs for pts on adult AML/MDS trials. Three cases (5%) showed a complex (3- or 4-way) variant translocation and only 7 (11%) of the 62 pts showed secondary aberrations: 3 (10%) of 30 pediatric cases and 4 (13%) of 32 of the adult cases. The majority of t(6;9) cases were classified as FAB-M2 (34%), M4 (31%) or M1 (19%). Although the immunophenotyping (N=7) and morphology data (N=17) were limited, increased basophilia and Auer rods were observed and the blasts showed CD13, CD15, and CD33 expression, in agreement with a previously reported preliminary study (Am J Clin Pathol107:430–437,1997). Four pts (1 pediatric and 3 adults) had MDS. Among the remaining 58 pts, 25 (78%) adults had previously untreated AML (16 de novo, 2 secondary, and 7 unknown secondary/de novo status) while all 29 pediatric AML patients had de novo AML. For the 54 patients with previously untreated AML, complete remission rates were slightly higher, but not statistically significantly (p=.20) in children (69%), when compared to adults (52%). Disease-free survival (DFS) (combined median 8.8 mo, 95% CI, 5.1–13.7) and overall survival (OS) (combined median 11.9 mo, 95% CI, 10.0–14.3) were poor regardless of age, a finding in distinct contrast to the t(8:21) favorable risk group also commonly observed in M2/M4 AML. Kaplan-Meier estimates of 3-yr survival were 25% for pediatric cases and 9% for adults. Analysis of stem cell transplantation (SCT) was inconclusive due to the small number of transplanted patients (N=15), but suggested that allogeneic SCT might be associated with better OS than no SCT (hazard ratio [HR] 0.39 after SCT, 95% CI 0.14 – 1.11), while autologous SCT might not (HR 1.49, 95% CI 0.57–3.85). Based on this study of t(6;9), largest to date and previously published data, AML with t(6;9) leukemia is a distinct AML subgroup with distinguishing clinicopathological features including poor outcome in relatively young patients, not explained by other known poor prognostic factors that warrants novel therapeutic strategies. Similar to other recurring cytogenetic abnormality subtypes of de novo acute myeloid leukemia of the WHO classification, t(6;9) may warrant a specific leukemia disease subtype.

Description: Activating FLT3 mutations are the most common genetic aberrations in acute myeloid leukemia (AML), resulting in the constitutive activation of this receptor tyrosine kinase (RTK), but such mutations are rarely found in acute lymphoblastic leukemia (ALL). Here we describe a unique subset of de novo adult T-cell ALL (T-ALL) cases that coexpress CD117/KIT and cytoplasmic CD3 (CD117/KIT+ ALL). Activating mutations in the FLT3 RTK gene were found in each of 3 CD117/KIT+ cases that were analyzed, but not in 52 other adult T-ALL samples from the same series that lacked CD117/KIT expression. Our results indicate the need for clinical trials to test the efficacy of drugs that inhibit the FLT3 RTK in this subset of patients with T-ALL. (Blood. 2004;104:558-560)

Description: Background: Multiple myeloma (MM) has been studied by conventional cytogenetics and fluorescence in situ hybridization using DNA probes (FISH) on interphase nuclei and metaphase cells, but the relative clinical significance of proliferating (metaphase) versus non-proliferating (interphase) cells at diagnosis is poorly understood. We investigated a consecutive series of 154 patients (pts) with newly diagnosed untreated MM, and compared results for metaphase and interphase cells with survival. Methods: Bone marrow within 30 days of diagnosis was analyzed by standard cytogenetics. Leftover cells were stored at −70°C. Interphase and metaphase FISH was done using probes for IGH, FGFR3, CCND1, c-MAF, D13S319, LAMP1, p53 and D17Z1 to detect t(4;14), t(11;14), t(14;16) and chromosome 13 or 17 anomalies, respectively. We analyzed

Description: MDS are a heterogeneous group of clonal disorders characterized by the development of one or more symptomatic cytopenias. We evaluated the role of erythropoietin (EPO) with or without granulocyte colony stimulating factor (G-CSF) for the treatment of anemic patients with MDS. Patients with refractory anemia (RA n=40), RA with ringed sideroblasts( RARS n=36), and RA with excess blasts (RAEB n= 29) were initially randomized to either supportive therapy (ST) or EPO, 150 u/kg/d. Patients on the ST arm could cross over to the EPO arm if after at least a 4 month period of observation they had a ≥ 50% increase in their transfusion requirement. In patients who did not respond to or did not maintain their response to EPO( 150 u/kg) , G-CSF, 1mcg/kg/d was administered in addition to the EPO. Patients who did not respond after 4 months received an increased dose of EPO at 300u/kg/d + G-CSF. Bone marrow studies and iron stores were evaluated before each change in treatment. Response criteria were CR (complete response), GR (good erythroid response), which required a sustained increase of the pre-transfused Hgb value of 〉 2 g/dl or loss of the transfusion requirement or PR (partial response), defined as a decrease in the transfusion requirement by ≥ 50% or an increase in the Hgb by 〉 1 g/dl above the pretreatment level. A GR or PR must be sustained for at least 4 months. Transformation to acute leukemia was defined as ≥ 30% blasts in the bone marrow. 118 patients were enrolled , 109( 53 EPO, 56 ST) were eligible and 105 were evaluable for response. The median age was 73 (range 37–88 years). The response rate (CR+GR+PR) in the EPO arm was 35% vs. 9% in the ST arm (p=. 002). Of the 23 patients who crossed over from the ST arm, 30% responded to EPO (GR+PR). Six of 27 patients (22%) who received EPO 150u/kg + G-CSF responded (CR+GR+PR). Ten patients received EPO 300 u/kg + G-CSF and 6(60%) responded (CR+GR+PR). Transformation to acute leukemia occurred in 3.6% and 0% in the supportive therapy and EPO arm respectively( P=.50). A complete response (CR) was documented in 2 patients, 1 each on EPO and EPO+G-CSF. There was a survival difference for patients who had an erythroid response: median survival 53 months for responders vs 26 months for non-responders (p=.009). Neither EPO nor the addition of G-CSF was associated with an increase rate of transformation to acute leukemia. Toxicities were comparable in all the treatment arms. The pretreatment serum EPO level correlated with the response to treatment (P=.004): median EPO of 48 vs. 140 mu/ml for responders versus non-responders. Responses were observed in all subtypes. Responses were greater in RA〉RARS〉RAEB. In conclusion, EPO administration was associated with a significant increase in Hgb and a decrease in the transfusion requirement in anemic patients with MDS. Low doses of G-CSF + EPO were effective in EPO nonresponsive or refractory patients. In MDS patients EPO + low dose G-CSF was not associated with progression or transformation to acute leukemia.

Description: Background: In B-CLL, the observation of interphase cells with hemizygous D13S319 deletion at 13q14 (13q-x1) as a sole anomaly in blood is widely considered a favorable prognosis. The observation of cells with 11q-, +12 or 17p- has been associated with a relatively poor prognosis. Over the past 1.5 yrs, 16.2% (174/1,076) of patients (pts) referred for fluorescence in situ hybridization (FISH) testing for B-CLL in our clinical practice had a clone with homozygous D13S319 deletion (13q-x2), but the prognostic significance of this observation is poorly understood. Moreover, 39.3% (142/361) of pts with unfavorable FISH anomalies have 13q-x1 and/or 13q-x2 and the clinical significance of this observation is also unknown. Thus, we investigated pts with 13q- (with or without other chromosome anomalies) to establish the relative clinical significance of 13q- in B-CLL. Methods: We studied 333 pts with B-CLL sampled between 9/1999 and 6/2004 who had FISH performed on interphase nuclei from blood. The FISH probe set was designed to detect 6q-, 11q-, +12, 13q-, 17p-, and translocations involving IgH at 14q32. We classified pts into four groups: 13q-x1 only (group 1), 13q-x1 and 13q-x2 only (group 2), 13q-x2 only (group 3) and 13q-x1 and/or 13q-x2 plus other FISH anomalies (group 4). FISH groups were compared with gender, age, Rai stage, treatment status, time to treatment, CD38 and IgVH mutation. Results: Of the 333 pts, 171 (51.3%) had a 13q-: 71 were in group 1, 25 in group 2, 26 in group 3 and 49 in group 4. %CD38+ differed significantly across FISH groups; in pairwise analyses, the proportion of pts with 〉30% CD38+ was significantly greater for pts in group 4 vs. group 3 (p=0.0015) although no significant differences were observed for group 3 vs. group 1 or vs. group 2. Pts in group 3 were not significantly different from other FISH groups for Rai stage, IgVH mutation or gender. The median percentage of abnormal nuclei for pts with group 1 was 54.5% vs. 79.5% for pts in group 4 (p