Citations

Abstract Background: Pre-treatment cytogenetics is a known predictor of outcome in haematological malignancies. However, its value in adult ALL is generally limited to the presence of the Philadelphia (Ph) translocation; mostly due to the low incidence of other recurrent chromosomal abnormalities. Methods: Since 1993, cytogenetic data from patients enrolled on a joint MRC/ECOG trial have been collated and reviewed centrally in the UK and US. Karyotype data from patients registered before the Imatinib amendment were pooled using standard cytogenetic definitions and classification criteria. Results: A successful cytogenetic result was achieved in 938/1235 (76%) cases; while 297 (24%) failed with <20 normal metaphases analyzed. Patients were classified into the following mutually exclusive groups: t(9;22)(q34;q11) n=193(21%); t(4;11)(q21;q23) n=50(5%); other MLL translocations n=11(1%), t(8;14)(q24;q32) and variants n=14(1%); t(1;19)(q23;p13) n=23(2%); T cell receptor translocations n=35(4%); low hypodiploidy (30–39 chromosomes) /near triploidy n=23(2%); high hyperdiploidy n=83(9%); tetraploidy n=12(1%); complex karyotype (>=5 aberrations) n=48(5%); other abnormal n=255(27%); normal n=191(20%). MRC and ECOG patients showed similar cytogenetic distributions, except for t(9;22), which was significantly more prevalent in the ECOG cohort (27% v 18%, p<0.001). However, the incidence of t(9;22) increased with age and ECOG patients were significantly older. Overall, the 5 year event free survival (EFS) was 32% (95% CI 30%–35%) with a median follow-up time of 5.1 years and did not differ between the two cohorts. The EFS of t(9;22) patients [15% (11%–21%)] was significantly inferior compared with all other cases [36% (32%–39%)] (p<0.001) and was independent of age and white cell count (WCC). Univariate logrank analyses identified t(4;11) and high hyperdiploidy as indicators of poor and good prognosis, respectively, compared with other Ph negative cases, but neither was independent of age and WCC. Three subgroups were shown to have a significantly worse EFS compared to other Ph negative cases by logrank analyses after adjusting for age and WCC: t(8;14) 14% (2%–37%) v 36% (32%–40%) (p=0.01); low hypodiploidy 22% (8%–40%) v 36% (32%–40%) (p=0.02); complex karyotype 21% (10%–33%) v 32% (29%–35%) (p=0.005). Neither of the latter two subgroups were associated with other poor risk features such as age, WCC or failure to achieve a remission within 4 weeks. Conclusions: This is the largest cytogenetic dataset of adult ALL reported to date and demonstrates the importance of combining cohorts to increase the number of cases available for analysis. The observation that low hypodiploidy and, for the first time, karyotype complexity confer a poor risk, demonstrates that cytogenetic subgroups other than the Ph translocation can and should be used to risk stratify adults with ALL to alternative treatments.

Abstract Background: AT’s have become the standard of care in the management of patients with MM, effecting 10-year survival rates of more than 30%. Long-term sequelae are uncommon, so that even 3rd and 4th AT have been applied. MDS is a well recognized complication of cancer therapy and has been reported in the context of both standard and AT-supported high-dose therapies for lymphoma and MM. It remains unclear whether t-MDS development is caused by prolonged “low” dose chemotherapy or by pre-transplant regimens. The purpose of this study was to survey a cytogenetic (CG) data base (DB) of > 30.000 samples submitted with each bone marrow examination involving 2152 patients receiving at least 1 AT. Patients and Methods: 1886 patients were identified with all relevant information as well as pre- and post-AT CG data available receiving at least 1 AT prior to January 2004. Common criteria for MDS-CA were employed. Routine CG follow-up studies were performed at 3–6 month intervals. MDS-CA development was examined in relationship to age, duration and type of therapy prior to transplant preparative treatment, blood counts, PBSC collection, AT regimen and number, post-transplant platelet and granulocyte recovery, as well as in the context of MM parameters. Interphase FISH analysis for MDS was also performed on PBSC in 42 patients (to detect early MDS cytogenetic changes caused by standard-doses therapies preceding AT) and correlated with MDS-metaphase CA post-AT in 7 patients. Results: Eighty-five patients developed MDS-CA at a median of 33 months after AT (range, 5 to 126 mo); the 10-year incidence estimate was 7%. MDS-metaphase CA development was independently associated with older age (> 55yr), lower CD 34 yield with first collection (< 18 million/kg) and lower platelet recovery 3 mo post-AT. The 10-year probability of MDS-metaphase CA was 1% among 315 patients with 0 risk factor, 6% among 1367 patients with 1 or 2 and 13% among the 199 patients with 3 risk factors (p<.0001). Univariately, >12 mo of pre-AT standard therapy increased the cumulative incidence of MDS-metaphase CA (p=0.04). The median time to MDS-metaphase CA development was shorter (24 mo) in the 55 patients with 2 or 3 risk factors compared to the 28 patients with 0 or 1 risk factors (41 mo; p=0.01). Conclusion: This report is the largest ever on the detection of MDS by metaphase CG, obtained in the context of AT for MM. Results confirm earlier findings that MDS-metaphase CA is infrequent when PBSC are procured early after the start of chemotherapies, in those with robust stem cell collection and prompt platelet recovery post-transplantation (evidence of non-damaged hematopoiesis) and among the younger age group. Thus, t-MDS after AT-supported high-dose therapy can be traced to stem cell damage likely inflicted by and not completely repaired after prolonged pre-AT therapies and accentuated by the post-AT replicative stress, especially in older patients. MDS- FISH data of PBSC will be presented at the meeting.

Abstract BACKGROUND: The molecular etiology of idiopathic acquired sideroblastic anemia (IASA), now considered a form of myelodysplastic syndrome, is currently unknown. Romslo et al (Blood 1982) reported a patient with IASA who had moderately elevated free erythrocytic protoporphyrin (FEP); this observation is now frequently made in IASA, helping distinguish IASA (high FEP) from inherited sideroblastosis (low FEP). In addition, rare patients with erythropoietic protoporphyria (EPP)—defined by cutaneous photosensitivity, dramatically elevated FEP levels, and germline point mutations in the ferrochelatase (FECH) gene at 18q21.3—have ringed sideroblasts in their marrow. We hypothesized that patients with IASA might have acquired somatic FECH mutations. METHODS AND RESULTS: We designed a denaturing high performance liquid chromatography (DHPLC) assay to explore this possibility and to avoid problems related to mutation screening in the setting of mixed clonality. To validate the DHPLC assay, the coding region of the FECH gene from 2 molecularly undiagnosed EPP patients without liver disease was analyzed. In one patient, both a heterozygous 69delG mutation (GenBank Accession NM_000140) and heterozygosity for the FECH expression-modulating IVS3-48C/T polymorphism were detected. In the other patient, no FECH coding mutations or polymorphisms were detected, either by DHPLC or conventional dye sequencing. FEP measurements were then obtained on 2 IASA patients and were elevated (65 and 115 mcg/dL; normal 1–10 mcg/dL) with normal urine and fecal porphyrins. Genomic DNA obtained from these 2 patients as well as archival DNA from 30 other patients with IASA was amplified and tested for FECH mutations. No coding region mutations were detected. Synonymous polymorphisms in exon 7 (rs536765) and 9 (rs536560) were found in 6/32 (19%; normal heterozygosity 0.398) and 14/32 (44%; normal heterozygosity 0.402) samples, respectively. The IVS3-48C/T polymorphism (rs2272783) was found in 3/32 (9%) (prevalence in the general population is 11%, Gouya Nat Genet 2002). In addition, 3 intronic polymorphisms not in the refSNP database were detected: IVS8+34 C/T (2/32), IVS8-61delG (5/32), and IVS9-59delA (2/32). CONCLUSION: IASA is not associated with coding mutations in FECH. Elevation of FEP in ASA must instead be due to the lack of mitochondrial iron in the proper form for incorporation into the porphyrin ring; attention should instead focus on factors responsible for maintaining the appropriate redox state and compartmentalization of iron. DHPLC can detect FECH mutations and polymorphisms, but because of the high frequency of the latter and the consequent need to sequence multiple exons, it is not a practical screening tool for studying undiagnosed EPP patients.

Abstract Background: The FIP1L1-PDGFRA fusion, an imatinib-sensitive molecular target, is detected in a subset of cases with moderate-to-severe eosinophilia (eosinophil count ≥1500/μL). Using 2 FISH strategies to detect loss of the CHIC2 locus as a surrogate marker of the FIP1L1-PDGFRA fusion, we previously found 14% of cases to be CHIC2-deleted when studying bone marrow smears from 81 adult patients with moderate-to-severe, non-reactive eosinophilia at our institution (Blood 2004, 104:3038–3045). Since then, the FISH test has been made available for routine clinical use both within our institution, and to external physicians as a mail-in test, for the workup of patients with eosinophilic disorders, including systemic mast cell disease (SMCD). Study design: FISH data, subsequent to the test’s introduction into routine clinical practice (from Nov. 2003), were retrospectively analyzed for CHIC2-deletion. Data from the initial pilot study of 81 patients was not used in this analysis (Table). The imatinib treatment analysis, however, included all CHIC2-deleted patients treated at our institution, including those from the pilot study (Table). Results: A total of 741 patients were studied in the post-pilot phase. Of these, 20 cases were found to be CHIC2-deleted, with an overall prevalence of 2.7% (Table). Of the 219 patients seen at our institution, 13 (all male) were found to be CHIC2-deleted (Table) (prevalence=5.9%), and of these, 10 have been treated with imatinib (100 mg/day starting dose) to date. Nine patients are evaluable for response - all achieved a complete clinical and histological remission (7 patients were tested by FISH post-treatment - all achieved cytogenetic remission). One patient received concomitant corticosteroids for co-morbidities and could not be evaluated for response. After a median follow-up of 24 months (range 17 to 39 months), all 9 patients continue to be in remission, with a normal eosinophil count. At last follow-up, one patient had discontinued imatinib and the others were on maintenance therapy at the following dose: 100mg/wk (n=1), 100mg BIW (n=1), 100mg TIW (n=2), 50mg QOD (n=1), and 100 mg/day (n=3). Conclusions: FIP1L1-PDGFRA prevalence in non-selected patients with eosinophilia is lower than anticipated from prior studies. Patients with FIP1L1-PDGFRA exhibit durable responses to imatinib therapy. It may be feasible to maintain some patients in remission on an empirically derived, intermittent schedule of low-dose imatinib. Pilot Data Post-pilot Data (n=741) Total Mayo samples Mayo samples Mail-in samples Number of Patients 81 138 603 822 Number CHIC2-deleted 11 2 18 31 Prevalence 14% 1.4% 3.0% 3.8% Number Imatinib treated 8 2 n/a 10

Abstract Background: Multiple myeloma (MM) is a plasma cell malignancy involving complex cytogenetic dysregulation of genes and heterogeneous expression of multiple B cell and plasma cell (PC) surface antigens. 60% of MM patients have cytogenetic translocations of the IGH locus. In 16% of myeloma patients, the partner oncogene is cyclin D1 on 11q13 causing t(11;14)(q13;q32) (Fonseca et al. Blood. 2002). This translocation is more common in patients with a lymphoplasmacytic morphology and confers a more favorable prognosis (Fonseca et al. Blood. 2003). A subset of MM patients (20%) harbor a clonotypic population of CD20+ bone marrow plasma cells (BMPC). These patients have a shortened survival time (SanMiguel et al. Br J Haematol. 1991). There is a strong association with CD20+ surface expression and t(11;14)(Robillard et al. Blood. 2003) and CD20+ PC has been suggested to be clonogenic and important in the pathogenesis of MM. A small Phase II study showed only a modest response to the humanized anti-CD20 monoclonal antibody rituximab in MM patients with CD20+PC (1 PR and 5SD) (Treon et al. J. Immunother. 2002). We report a response to treatment with rituximab in a MM patient with CD20+ BMPC and t(11;14)(q13;q32) by FISH. Case: The patient is a 68 year old male with a 1.5 year history of MM. Initial diagnosis of ISS stage I disease was made after a three year history of IgA kappa monoclonal gammopathy of undetermined significance (MGUS). At diagnosis of MM, he had symptomatic anemia Hgb10.5g/dL, rising serum IgA of 3950 mg/dL, serum m-spike of 4.1 g/dL (IgA kappa), PCLI (plasma cell labeling index) of 1%, and a small monoclonal IgA kappa with kappa fragment in the urine. BM showed 44% monoclonal kappa plasma cells with 90% CD20+,clonal PC on flow cytometry. Karyotype was normal; 46, XY, but FISH showed evidence of an IGH translocation t(11;14)(q13;q32) in 60% of PC. Immunohistochemical staining was positive for cyclin D1. Given the marked elevation of CD20+ PC and early stage of disease, initial treatment was employed with four weekly cycles of rituximab (375mg/m2). IgA fell from 4750 mg/dL to a nadir of 2990 mg/dL, (m-spike; 4.5g/dL to 3.2 g/dL) five weeks after completion of treatment. The monoclonal protein (MP) in the urine disappeared. Hgb improved to 12.5g, with erythropoietin. Three months later, IgA rose to 3260mg/dL (m-spike 3.2 g/dL) and urinary MP returned. The patient remained asymptomatic. A second course of weekly rituximab was started. IgA level and serum m-spike remained fairly stable over the next 7 months. All treatment was tolerated well except for initial transient rigors, fever, and nausea. Despite bi-monthly maintenance rituximab, the patient devloped a rising IgA of 4940 mg/dL, m-spike of 4.7, falling Hgb and a rising PCLI of 1.5%, 15 months after his original diagnosis of MM. Of particular concern is the development of new, unfavorable, cytogenetic aberrations by FISH with BMPC now showing deletion 13 in 93% and mutation of p53 (17p13) in 98%.100% show fusion of CCND1 and IGH and PC now only partially express CD20. Conclusion: This report suggests that MM patients with CD20+ BMPC and t(11;14)(q13;q32) may represent a target population for anti-CD20 therapy. Our report supports the concept proposed that targeting this clonotypic subset of B cells possibly interrupts a critical oncogenic pathway that is important in the pathogenesis of the clonogenic development of MM.

Abstract Background: Cytogenetic risk categories based on conventional chromosome studies are widely used in clinical practice to make treatment decisions for AML. We evaluated the efficacy of interphase FISH to detect chromosome anomalies in the workup of young (<60 years) patients with AML. Methods: Study subjects were enrolled in E1900, a front-line Eastern Cooperative Oncology Group (ECOG) clinical trial for AML. This is an on-going Phase III clinical trial with Daunorubicin dose-intensification ± Gemtuzumab-Ozogamicin consolidation therapy prior to stem cell transplant. This trial opened December 2002; as of February 2005, 223 patients were enrolled. The protocol was designed to collect bone marrow for both cytogenetic and FISH studies at study entry (diagnosis). Cytogenetic studies were done by local laboratories with results reviewed centrally by the ECOG Cytogenetics Committee. Each case was classified as acceptable or unacceptable based on predefined ECOG cytogenetic criteria. FISH for each patient was performed in the ECOG FISH laboratory at Mayo Clinic and utilized eight probe sets to detect t(8;21), t(9;22), t(11;var), t(15;17), inv(16), +8, −5/5q, and −7/7q (Vysis, Downer Grove, IL). Results: 64 (29%) of 223 specimens had incomplete cytogenetic and/or FISH results. We analyzed the remaining 159 (71%) specimens with complete cytogenetic and FISH results. Results for each specimen were classified by probe set into one of the following categories: Normal cytogenetics and normal FISH; Abnormal cytogenetics and abnormal FISH for the anomaly the probe was designed to detect; Abnormal cytogenetics and abnormal FISH for an anomaly the probe was not primarily designed to detect; Normal cytogenetics and abnormal FISH; Abnormal cytogenetics and normal FISH; or Abnormal cytogenetics and abnormal FISH that further defined the karyotype. Figure 1: Results for 159 patients by category and FISH probe set: *t(8;21); t(9;22); t(11;var); t(15;17); inv(16); cen(8); del(5/5q); de(7/7q). Figure 1:. Results for 159 patients by category and FISH probe set: *t(8;21); t(9;22); t(11;var); t(15;17); inv(16); cen(8); del(5/5q); de(7/7q). The concordance rate between cytogenetic and FISH results ranged from 97 to 100% for all probe sets and kappa analysis for concordance had a p value of <0.0001. Of the total 159 cases, discrepancies between FISH and cytogenetic results occurred in only 4 cases; two with normal cytogenetics and abnormal FISH and two with abnormal cytogenetics and normal FISH results. Conclusions: The high level of agreement between cytogenetics and FISH demonstrates the accuracy of a panel of 8 FISH probe sets for the detection of significant abnormalities in AML. The data from this investigation support the use of FISH as an adjunct in cases of failed cytogenetic analyses to increase the yield of useful cytogenetic results in large cooperative trials. Furthermore, because of the strong correlation between cytogenetics and FISH, our results demonstrate the potential of FISH as a follow-up study of minimal residual disease in ECOG trials.

Abstract Background: Loss of p53 is detected by fluorescence in situ hybridization (FISH) in interphase nuclei in 8–10% of patients with B-CLL and has been associated with aggressive disease and poor response to therapy. Results of chromosome studies suggest loss of p53 is often due to structural anomalies of chromosome 17 rather than monosomy 17. To characterize chromosome 17 anomalies in B-CLL, we used interphase and metaphase FISH to study a series of patients with this disorder. Methods: We studied 16 patients with B-CLL who had chromosome 17 anomalies detected by conventional chromosome studies or interphase FISH. The study cohort consisted of 8 females and 8 males (age 54 – 73 years). Clinical data was available for 15 patients: Rai stages were I, II, III and IV for 1, 2, 6 and 6 patients, respectively; 14 patients received treatment and 10 have died. Metaphase and interphase FISH studies were performed using left-over fixed bone marrow or peripheral blood cell pellets from conventional cytogenetic studies. To identify recurrent chromosome 17 breakpoint cluster regions, patients were first studied with p53(17p13.1) and D17Z1(centromere17), and then with FISH probes for SMS(17p11.2) and RARA(17q21). For patients retaining the SMS probe site, we did FISH for HNPP(17p11.2–12) and D17Z1(centromere17). A total of 200 interphase nuclei and up to 20 metaphases were analyzed by each probe set. Results: The karyotype was normal for 3 patients and complex involving various abnormalities of chromosome 17 for the remaining 13. All patients showed loss of one p53 probe site with retention of both chromosome 17 centromeres. The SMS locus was absent in 13 patients indicating a breakpoint cluster region between the centromere and SMS probe site. In the remaining 3 patients, HNPP was absent, but the SMS probe site was retained indicating chromosome breakage between the SMS and HNPP probe sites. Metaphase FISH results indicate loss of p53 was due to an isochromosome 17p11.2 in 4 patients and an unbalanced translocation involving 17p11.2 in 12 patients. Conclusions: This study shows that loss of p53 in B-CLL cells is due to isochromosome formation or unbalanced translocations that involve a ~6,400 kb breakpoint cluster region adjacent to the centromere on the short arm of chromosome 17. Importantly, our findings indicate that loss of p53 is not due to loss of chromosome 17, simple deletions of p53, or chromosome breakage near the p53 locus. The region between the centromere and HNPP on chromosome 17 is known to be rich in low copy repeats and homologous DNA sequences are dispersed throughout the genome. In somatic cells, these repeats can result in pairing of the same or different chromosomes; crossing over within these homologous regions can result in translocations or isochromosomes. Undoubtedly, this genetic mechanism plays an important role in the origin of structural anomalies of chromosome 17 in B-CLL.

Abstract On intergroup phase III trial, MRC UKALLXII/ECOG2993, 25% of 613 ECOG patients were positive for Ph or its molecular equivalent, BCR/ABL; all were B-lineage ALLs (B-ALL). When compared to Ph(−) B-ALL, Ph(+) ALL had more blasts expressing CD25 (α-chain of interleukin-2 [IL-2] receptor) (p=3E-31), CD34 (p=4E-10), CD13 (p=3.9E-16) and CD33 (p=3.5E-09), and blasts with higher density of CD10 (p=9.6E-12). There was no difference in antigen expression profiles between pts with e1a2 (N=107) and b2a2/b3a2 (N=48) BCR/ABL transcripts. Complete remission (CR) rate was lower (p= 7.4E-6) and overall survival (OS) was shorter in Ph(+) compared with Ph(−) B-ALL (p=6.5E-5). There was no difference in outcome between e1a2 and b2a2/b3a2 pts (p=0.65). CD25 was the most powerful prognostic indicator in Ph(+) cases, both for CR (p=0.004) and OS (p=1.3E-05). Strikingly, CD25 negatively affected OS in a continuous fashion; 1% increase in CD25 was related to a 1% increase in the hazard of death. On the other hand, CD25 expression was not associated with OS in Ph(−) pts (p=0.66). Among 152 Ph(+) pts, 30 lacked CD25 expression, 24 with e1a2 and 6 with b2a2/b3a2 transcripts. When analyzed separately, CD25(−)Ph(+) pts had outcome indistinguishable from that of Ph(−) pts (p=0.89). Compared to CD25(+)Ph(+) pts, CD25(−) Ph(+) pts had lower WBC counts at presentation (p=4.1E-05). Both CD25(+) and CD25(−) Ph(+) blasts expressed the γ-chain but not the β-chain of the IL-2 receptor, arguing against the presence of a functional IL-2 receptor. There was no association between CD25 expression or outcome and the presence of additional cytogenetic abnormalities seen in 52% of Ph(+) pts. To further examine the prognostic impact of CD25, BCR/ABL levels were quantitated by real-time PCR in 136 Ph(+) pts at presentation. There was no correlation between presenting BCR/ABL levels and blast count (p=0.98), achievement of CR (p=0.73), or OS (p=0.67). However, higher blast counts at diagnosis were associated with shorter survival (p=0.003). BCR/ABL levels were measured in 43 pts 1 month after induction therapy. Changes in BCR/ABL after therapy were classified as undetectable, >90% reduction, >50% reduction, or no change. While CR status did not correlate with BCR/ABL reduction (p=0.97), the chance of survival ranked in the order of undetectable (p=0.01), >90% reduction (p=0.05), >50% reduction (p=0.37), or no change (p=0.87). CD25 expression at presentation correlated with that order (p=0.002). CD25(−)Ph(+) pts fell into the group with undetectable BCR/ABL levels after treatment, whereas pts with highest initial CD25 expression fell into the group with no change. In conclusion, CD25(−)Ph(+) blasts are more sensitive to non-TKI therapy than CD25(+) Ph(+) blasts. Outcome in Ph(+) ALL is thus determined by CD25 expression rather than the presence of Ph per se. RESPONSE CD25(+)Ph(+) CD25(−)Ph(+) Ph(−) B-Lin ALL n 122 30 308 % CR Rate 57% 71% 81% MedianOS (months) 12.2 30.2 27.7

Abstract Fluorescence in situ hybridization (FISH) probes and analysis methods for B-cell Chronic Lymphocytic Leukemia (CLL) vary extensively among cytogenetic laboratories. This is not unexpected, as neither national nor international standards have been established for most FISH studies. Lack of standardization is problematic when data collected at multiple institutions are used for clinical correlative studies. To circumvent such problems, the five participating laboratories in the CLL Research Consortium (CRC) designed and executed a joint CLL FISH validation study. Methods: Initially a survey was sent to assess equipment, methods and experience with FISH for CLL. In a pilot study to compare laboratory performance in scoring patient samples, slides from ten patients were prepared and sent to each participating lab to be hybridized with five probe sets (= 50 hybridizations) and analyzed according to their local protocol. In a second pilot study, slides from two patient samples and identical probe sets were sent to the participating labs where hybridization and analysis were carried out according to their local protocol. Next, technologists and directors from all participating labs attended a workshop where technologists working in pairs scored nuclei together, techniques and scoring criteria were established, and consensus reached on other concerns. In a proficiency test nine months after the workshop, slides from two patient samples (10 hybridizations) were hybridized and scored according to each lab’s protocol and results shared using a common reporting form. Results: Survey results indicated that four labs used the same commercially available CLL FISH panel, and one used a combination of probes from the same vendor plus several home-brew probes. Each lab scored between 100 and 200 nuclei per hybridization site, and each independently set normal cutoff values. The FISH panel included probes to detect 11q, 13q, and 17p deletions, trisomy 12, and IGH gene rearrangement. One lab included probes to detect 6q deletion. In the first pilot study each lab used their hybridization methods, probe sets, and scoring criteria. Differences among labs were observed due to variations in probe strategy, reporting of anomalies, and perhaps most important, scoring criteria. Probe strategy differences resulted in variable reporting of 11q- vs monosomy 11 and 12q duplication vs trisomy 12. Some participants reported 13q-x1 and 13q-x2 as subclones and some reported only 13q-. One lab reported an IGH rearrangement whereas the others scored IGH as normal. In the second pilot study each lab used the same methods and probe sets to facilitate comparison of scoring by the technologists. All labs correctly identified the abnormalities, and there were no false positive results. Minor scoring differences were attributed to variation in scoring criteria or inexperience with an unfamiliar FISH probe strategy. The proficiency test that followed the workshop demonstrated 100% concordance in identification of abnormalities. Inter-lab scoring was much improved compared to the first pilot study. The only exceptions were a 13q- range of 72–90% in one case, and a 17p- range of 38–67% in another case. Conclusion: The pilot studies identified a need to develop common scoring criteria. The subsequent workshop and proficiency test demonstrated that the collaborative effort resulted in more standardized scoring among the CRC laboratories. Our collaborative study emphasizes the need to establish rigorous standards and guidelines for FISH procedures and scoring criteria. Standardization of FISH methods among participating laboratories will enhance the confidence in FISH studies for both clinical applications and cooperative intergroup clinical research.

Abstract MRC UKALLXII/ECOG2993 accrued 739 ECOG pts over 13 years. ECOG’s reference laboratories centrally characterized 613 (91%) and 505 were B-Lin ALLs. Expression of 32 antigens (Ags), including 9 myeloid-associated Ags, was determined by multiparameter flow cytometry on gated blasts, both as percentage of Ag expressing blasts and intensity of antibody staining (equivalent of Ag density). To test for the potential therapeutic efficacy of monoclonal antibodies, such as rituximab, epratuzumab, alemtuzumab, or gemtuzumab in future trials, expression of Ags for these antibodies (CD20, CD22, CD52, or CD33, respectively) was determined. In E2993, 14% of pts typed as Pro-B (CD10 negative), 71% as Early Pre-B (CD10 positive), 13% as Pre-B (intracytoplasmic mu positive), and 2% as Mature B-ALL (surface mu chains positive). CD20 expression correlated with the stage of B-lymphoid maturation (p=5.4E-13) (see table). Median CD20 intensity of fluorescence was lowest in Pro-B and highest in Mature B-ALL. Membrane expression of CD22 was lower in Pro-B compared with the other three groups, Early Pre-B, Pre-B and Mature B (p=1.0E-5) but indistinguishable among the latter three (p=0.21). While levels of CD20 and CD22 had no effect on complete remission (CR) and overall survival (OS) univariatly, higher CD22 expression was significantly correlated with better OS (p=0.02) in a bivariate Cox model of CD20 and CD22. With respect to myeloid Ags, CD33/CD13 expression was associated with Early Pre-B (p=0.02), and CD65(s)/CD15(s) with Pro-B ALL (p<0.0001). While CD65(s) and CD15(s) did not affect outcome, the combined expression of CD33/ CD13 conferred an inferior prognosis; this effect was due to the association of CD33/ CD13 with BCR/ABL positivity (p<0.0001). BCR/ABL negative B-Lin ALL (N=350) expressed CD33 on a median of only 4% of blasts, compared with a median of 54% in BCR/ABL positive ALL (N=152) (p=3.5E-09). However, intensity of CD33 staining was significantly lower in both BCR/ABL negative and positive blasts (median 3.1) compared with leukemic myeloblasts (median 56) (p=<0.001). Median CD52 expression was higher in BCR/ABL positive versus negative B-Lin ALL (p=3.5E-06); in neither group was CD52 associated with outcome. We conclude that antibodies to CD52 are better suited for BCR/ ABL positive than negative B-Lin ALL. Since expression of CD33 in ALL is weak, CD33- independent effects of gemtuzumab must be investigated. Low CD20 combined with high CD22 expression confers superior OS. The expression of these Ags in most B-Lin ALL pts justifies the incorporation of antibodies into frontline chemotherapy regimens. B-Lin ALL Stage CD20 Percentage Median Intensity CD22 Percentage Median Intensity ≥20% ≥50% ≥75% ≥20% ≥50% ≥75% Pro-B 21 10 3 2 88 72 55 12 Early PreB 66 48 37 21 96 90 84 28 Pre-B 61 46 37 21 100 90 81 37 Mature B 87 80 64 106 92 84 80 9