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Originally published as JCO Early Release 10.1200/JCO.2005.03.1021 on December 12 2005 © 2006 American Society of Clinical Oncology. Select High-Risk Genetic Features Predict Earlier Progression Following Chemoimmunotherapy With Fludarabine and Rituximab in Chronic Lymphocytic Leukemia: Justification for Risk-Adapted TherapyFrom the Division of Hematology-Oncology, Departments of Internal Medicine and Pathology, The Ohio State University, Columbus, OH; Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA; CALGB Statistical Center, Durham, NC; Department of Medicine, The University of Chicago, Chicago, IL Address reprint requests to John C. Byrd, MD, Division of Hematology-Oncology, Starling Loving Hall, Room 302, The Ohio State University, Columbus, OH 43210; john.byrd{at}osumc.edu
Purpose: Several new prognostic factors predicting rapid disease progression in chronic lymphocytic leukemia (CLL) have been identified, including unmutated Ig VH mutational status, del(11)(q23), del(17)(p13.1), and p53 mutations. To date, the impact of these same prognostic factors have not been examined relative to treatment outcome with chemoimmunotherapy. Methods: We examined the impact of these new prognostic factors on predicting treatment outcome in symptomatic, untreated CLL patients who received chemoimmunotherapy with fludarabine and rituximab as part of a completed, randomized phase II study, Cancer and Leukemia Group B (CALGB) 9712. Results: Eighty-eight patients treated as part of CALGB 9712 had detailed prognostic factor assessment performed. Using Ig VH mutational status to classify risk, there was no association between complete response rate with either unmutated Ig VH mutational status or high-risk interphase cytogenetics. However, the median progression-free survival (PFS; P = .048) and overall survival (OS; P = .01) were shorter among the Ig VH unmutated patients as compared with the Ig VH mutated patients. Using the hierarchical classification of Döhner, PFS (P = .005) and OS (P = .004) were significantly longer as the classification moved from high risk [del (11)(q22.3) or del (17)(p13.1)] to low risk.
Conclusion: These data demonstrate that high-risk CLL patients characterized by Ig VH unmutated (
Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the Western hemisphere, with a varied natural history ranging from months to decades.1 CLL is generally treated at the onset of symptomatic disease, and accepted initial treatment includes alkylator therapy (chlorambucil or cyclophosphamide) or fludarabine.2 Data from three randomized studies3-5 have demonstrated a significantly higher overall response rate (ORR), higher complete response (CR) rate, and longer progression-free survival (PFS) with fludarabine-based therapy as compared with alkylator-based therapy. Subsequent phase III studies have pursued the addition of cyclophosphamide to fludarabine and have preliminarily demonstrated superiority in terms of ORR, CR, and PFS.6 Concurrent phase II efforts adding the anti-CD20 antibody rituximab to either fludarabine3,7,8 or fludarabine and cyclophosphamide9 in previously untreated CLL patients have demonstrated high CR rates and extended PFS and overall survival (OS) as compared with historic controls. Overall, these studies have generated significant interest in phase III combination strategies for CLL. Concurrent with recent advances in therapeutic combinations for CLL, several high-risk genetic features corresponding to Ig VH mutational status,10,11 select interphase cytogenetic abnormalities [del(17)(p13.1) and del(11)(q22.3)],12 and the presence of nonsilent p53 mutations13 have been linked to early CLL progression and inferior survival in CLL. Limited retrospective investigation has suggested that del(17)(p13.1) and/or p53 mutation may predict diminished response to nucleoside analog (ie, fludarabine or pentostatin)14 and rituximab monotherapy.15 Similar small studies have suggested a shorter PFS in patients with Ig VH unmutated status who were treated with cladribine16 or autologous stem-cell transplant.17 To date, the impact of the high-risk genetic features including Ig VH mutational status, del(17)(p13.1), del(11)(q22.3), and nonsilent p53 mutations have not been examined relative to impact on response or PFS following receipt of chemoimmunotherapy for symptomatic CLL. As chemoimmunotherapy represents the most active therapy applied to CLL, understanding the impact of high-risk genetic features is of great interest. Cancer and Leukemia Group B (CALGB) 9712 was a randomized phase II study of two different schedules of rituximab combined with fludarabine, for which clinical results have been reported.3,7 In an effort to understand the impact of high-risk genetic features on outcome with chemoimmunotherapy, we studied the influence of these on PFS and OS. Here we provide evidence that more aggressive therapies such as nonmyeloablative stem-cell transplant or other new treatment approaches should be considered for symptomatic CLL patients with high-risk genetic disease.
Subjects Patients were enrolled on CALGB 97123,7 and the corresponding tissue bank study (CALGB 9665) following written informed consent. The eligibility criteria for CALGB 9712 included symptomatic, but untreated, histologically and immunophenotypically documented CLL as defined by the National Cancer Institute 1996 guidelines.18 CALGB 9712 examined administration of rituximab either concurrently (n = 51) or sequentially (n = 53) with fludarabine therapy. A total of 88 of these patients (85%) had one or more available cryopreserved vial(s) as part of the tissue bank study CALGB 9665 available for analysis.
Cell Isolation
Fluorescence In Situ Hybridization The slides were viewed using a Zeiss Axioskop fluorescence microscope equipped with the appropriate filters and imaging software (Carl Zeiss, Munich, Germany). The number of signals was evaluated in 200 cells for each probe. Standard quality control procedures were used as previously published by our group. A control sample was run concurrently with each test run. When several cytogenetic abnormalities were present in a given patient, data were categorized using the hierarchical classification described by Döhner with modifications. Specifically, abnormalities were categorized in the following order: del(17)(p13.1) and/or p53 mutation > del(11)(q22.3) > +12 > del(6)(q21) > del(13)(q14). Using this classification, a patient having both a del(17)(p13.1) and del(13)(q14) would be categorized to the del(17)(p13.1) group.
p53 Mutational Analysis
Ig VH Mutational Analysis
Statistical Analysis
The demographics of the 88 patients on CALGB 9712 who were enrolled on CALGB 9665 and had available cryopreserved cells are presented in Table 1. The median age of these patients was 63 years (range, 36 to 83 years), and the median WBC was 81.1 x 109/L (range 8.8 to 436 x 109/L). Splenomegaly was observed in 59% of the patients, and 36% of the patients had high-risk disease (Rai stage III or IV).
For this group of 88 patients, the ORR in the concurrent arm was high (n = 45; 93% ORR and 51% CR) with similar high ORRs but slightly decreased CR rates in the sequential arm (n = 43; 91% ORR and 35% CR). Neither CR nor ORR was associated with Ig VH status, p53 mutation, or the prioritized cytogenetic classification.3,7 The PFS and OS of the concurrent and sequential arms were found to be similar (data not shown). Because the association of PFS and OS with the genetic markers was similar within the two arms, all 88 patients were combined into one group for assessment of these associations. The only association of preclinical features with Ig VH mutational status, interphase cytogenetic abnormalities, or nonsilent p53 mutations was del(17)(p13.1) where all subjects were female. At the time of this analysis, 24 of the 88 patients have died, and 58 have progressed (median follow-up of the 30 censored patients is 57 months). A total of 43 of 75 patients (57%) were Ig VH unmutated CLL, utilizing a 98% germline or greater cutoff for Ig VH mutational status (on the basis of the ideal cutoff identified previously). The CR rates of the mutated and unmutated patients were 50% and 44%, respectively (P = .62). The median PFS among the Ig VH-mutated patients was 46 months (CI, 34 to NA), whereas for unmutated patients it was 31 months (CI, 22 to 42; P = .048) as shown in Figure 1A. There was also a statistically significant difference in OS between the Ig VH mutated and unmutated patients (P = .01) as shown in Figure 1B. Only two of 32 of the mutated patients (9%) have died as compared with 16 of 46 of the unmutated patients (37%). The percentage of patients with either high-risk del(11)(q22.3) or del(17)(p13.1) was 13% in the Ig VH-mutated versus 26% in the Ig VH-unmutated subgroup of CLL (P = .24). In contrast, del(13)(q14) as a sole abnormality was noted in 38% of the patients with Ig VH-mutated CLL as compared with 28% in the Ig VH-unmutated CLL (P = .46).
Of the 88 patients examined for p53 mutations, 11 patients had initial aberrant bands suggestive of mutation on denaturing gel electrophoresis screen. On confirmatory sequencing, seven of these mutations represented polymorphisms at exon 6 (n = 3) and exon 9 (n = 4) that have been well-characterized; four patients (5%) exhibited nonsilent mutations in exons 7 and 8 of the p53 gene that are known mutation hot spots. Three of these latter four patients had a del(17)(p13.1) as well. Analysis of the association of nonsilent p53 mutations with clinical outcomes was not performed because of the small number of subjects with mutations. Interphase cytogenetic analysis was performed on 88 patients. The interphase abnormalities noted in these patients are summarized in Table 2. Patients with two or more abnormalities versus the remaining group with a sole genetic abnormality or no detectable abnormality had a similar PFS and OS. Using the Dohner hierarchical classification,12 the CR rate and the median PFS for each prioritized group are summarized in Table 3. There was no significant association of the classification scheme with CR rate (P = .15). However, as the classification moved from high risk to low risk, PFS significantly improved (P = .005) as shown in Figure 2A. A comparison of the PFS of the two highest risk categories combined versus all other categories was also statistically significant, with PFS medians of 20 months (95% CI, 16 to 34 months) v 44 months (95% CI, 32 to 65 months; P = .009), respectively, as shown in Figure 2B. While only 24 of the 88 patients used in this analysis have died, still OS also significantly improved as the classification moved from the high-risk to the low-risk categories (P = .004) as shown in Figure 3A. Furthermore, the OS of the two highest categories was significantly worse than that of the other categories combined (P = .01) as shown in Figure 3B. This corroborates previous studies that have documented that patients with either del(17)(p13.1) or del(11)(q22.3) have worse OS than patients who are negative on both these markers.
Herein, we have described a cohort of CLL patients treated with chemoimmunotherapy, including fludarabine and rituximab, for which we have comprehensively assessed the newly described genetic prognostic factors, including Ig VH mutational status, interphase cytogenetics, and p53 mutational status. Previous studies with each of these parameters have demonstrated that each influence time from initial CLL diagnosis to treatment initiation, OS, and autologous stem-cell transplant.17 To date, no study in CLL has examined the impact of these abnormalities together or their clinical impact following receipt of highly effective chemoimmunotherapy. Our findings therefore, add to our understanding of appropriate treatment risk stratification in CLL. We demonstrate with borderline statistical significance that Ig VH unmutated status may contribute to short PFS and OS. Additionally, we show that high-risk interphase cytogenetics, including either del(17)(p13.1) or del(11)(q22.3), also predict for statistically inferior PFS and OS. If one or more of these findings are confirmed in large prospective studies, they likely will lead to a risk-stratified treatment approach similar to that used in acute leukemia. The frequency of each of the interphase cytogenetic abnormalities commonly observed at the time of treatment is similar to other published series.21 Patients with high risk del(11)(q22.3) or del(17)(p13.1) represent only one-fifth of the patients treated. It is of interest that all of the patients with del(17)(p13.1) responded to fludarabine and rituximab therapy, although all were partial responders and of short duration relative to all of the other interphase cytogenetic groups. Of similar interest is the high CR (53%), using the National Cancer Institute’s 1996 criteria, observed in del(11)(q22.3) patients with fludarabine and rituximab therapy. These patients nonetheless had a relatively short remission relative to the other interphase abnormalities. This may be reflective of varying amounts of disease present in the del(11)(q22.3) group versus those with other abnormalities that cannot be discerned by standard response criteria. While assessment of minimal residual disease by flow cytometry might discern such differences, it was not performed in this study. The remaining prioritized interphase cytogenetic groups, including trisomy 12, del(13)(q14), and no detectable abnormalities had a similar good outcome relative to the unfavorable groups. This report documents for the first time the poor outcome of the del(11)(q22.3) prioritized group to chemoimmunotherapy. It also suggests that for the del(11)(q22.3) abnormality, PFS is a more appropriate measurement to assess the effectiveness of alterative combination therapies. One question that arises with the overlap of Ig VH mutational status is the relative independence of each abnormality in predicting outcome. Subset analysis of this data set renders sample sizes too small to discern differences between patients who have zero, one, or more adverse prognostic factors. However, it is of interest that outcome of the subset of patients with only Ig VH unmutated status (n = 32) or high-risk cytogenetics (n = 4) have a PFS of 32 and 34 months, respectively, while those patients with both Ig VH unmutated status and high-risk cytogenetics (n = 12) have only a 22-month PFS. Future prospective studies with larger numbers of patients in each group will be required to determine the relative contribution of these two prognostic factors. All of the patients included in this article are derived from a completed, randomized phase II study of fludarabine and rituximab that yielded positive results in terms of CR rate, ORR, PFS, and OS as compared with a previously completed United States intergroup trial with similar eligibility in which patients received fludarabine alone. Given these positive results, this treatment approach of fludarabine and rituximab will likely be tested in future phase III studies. Therefore, the findings of our study are quite relevant as they suggest that there is both a favorable genetic group that clearly benefits significantly from fludarabine and rituximab, and a less fortunate group who likely would benefit from additional therapy. Given the limitations of our analysis that include a small sample size, different induction regimen, and retrospective assessment of laboratory data, an appropriate step would be to prospectively validate these findings in a genetically well-characterized group of patients. Ideally, this study would be large enough to assess the independent role of Ig VH mutational status and interphase cytogenetics on PFS and OS. If such validation occurred, risk-adapted therapy similar to that currently performed in patients with acute leukemia could ensue in which well-tolerated therapies such as fludarabine and rituximab were used in low-risk patients, while more intensive treatments could be considered for patients with high-risk genetic features. These more aggressive therapies could include alemtuzumab, which has been demonstrated to be effective for patients with p53 dysfunction and Ig VH unmutated status, or other new therapeutic agents such as flavopiridol, which also has significant activity in refractory patients. In addition, it would be important to examine other recently described prognostic factors, including ZAP-70 expression,22,23 CD38 expression,24 mcl-1 promoter insertion,25 and other mechanisms of p53 dysfunction26 that recent studies have suggested may impact PFS following receipt of treatment for symptomatic CLL. Furthermore, the proportion of CLL cells positive for specific genetic abnormalities should also be considered on the basis of a recent analysis by the British CLL-4 LRF trial.27 This validation is currently ongoing as part of the next CALGB trial of chemoimmunotherapy in the treatment of symptomatic untreated CLL.
Although all authors completed the disclosure declaration, the following author or immediate family members indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. For a detailed discription of the disclosure categories, or for more information about ASCO’s conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors.
Dollar Amount Codes (A) < $10,000 (B) $10,000–99,000 (C)
Supported in part by grants from the National Cancer Institute (CA31946) to the Cancer and Leukemia Group B (Richard L. Schilsky, MD, Chairman), R21CA101332, and UO1CA101140. Support from the Leukemia and Lymphoma Society of America (J.C.B.), and D. Warren Brown Foundation (J.C.B.). J.C.B. is a clinical scholar of the Leukemia and Lymphoma Society of America. The content of this manuscript is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute. Authors’ disclosures of potential conflicts of interest and author contributions are found at the end of this article.
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Eichhorst BF BR, Hopfinger G, Pasold R, et al: Fludarabine plus cyclophosphamide (FC) induces higher remission rates and longer progression free survival (PFS) than fludarabine (F) alone in first line therapy of advanced chronic lymphocytic leukemia (CLL): Results of a phase III study (CLL4 protocol) of the German CLL Study Group (GCLLSG). Blood 102:72a, 2003 (abstr 243) 7. Byrd JC, Peterson BL, Morrison VA, et al: Randomized phase 2 study of fludarabine with concurrent versus sequential treatment with rituximab in symptomatic, untreated patients with B-cell chronic lymphocytic leukemia: Results from Cancer and Leukemia Group B 9712 (CALGB 9712). Blood 101:6-14, 2003 8. Schulz H, Klein SK, Rehwald U, et al: Phase 2 study of a combined immunochemotherapy using rituximab and fludarabine in patients with chronic lymphocytic leukemia. Blood 100:3115-3120, 2002 9. Keating MJ MT, O’Brien S, Wierda W, et al: A high proportion of true complete remission can be obtained with a fludarabine, cyclophosphamide, rituximab combination (FCR) in chronic lymphocytic leukemia. Proc Am Soc Clin Oncol 22:569, 2003 (abstr 2289) 10. Damle RN, Wasil T, Fais F, et al: Ig V gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia. Blood 94:1840-1847, 1999 11. Hamblin TJ, Davis Z, Gardiner A, et al: Unmutated Ig V(H) genes are associated with a more aggressive form of chronic lymphocytic leukemia. Blood 94:1848-1854, 1999 12. Dohner H, Stilgenbauer S, Benner A, et al: Genomic aberrations and survival in chronic lymphocytic leukemia. N Engl J Med 343:1910-1916, 2000 13. Cordone I, Masi S, Mauro FR, et al: p53 expression in B-cell chronic lymphocytic leukemia: A marker of disease progression and poor prognosis. Blood 91:4342-4349, 1998 14. Dohner H, Fischer K, Bentz M, et al: p53 gene deletion predicts for poor survival and non-response to therapy with purine analogs in chronic B-cell leukemias. Blood 85:1580-1589, 1995 15. Byrd JC, Smith L, Hackbarth ML, et al: Interphase cytogenetic abnormalities in chronic lymphocytic leukemia may predict response to rituximab. Cancer Res 63:36-38, 2003 16. Jelinek DF, Tschumper RC, Geyer SM, et al: Analysis of clonal B-cell CD38 and immunoglobulin variable region sequence status in relation to clinical outcome for B-chronic lymphocytic leukaemia. Br J Haematol 115:854-861, 2001[CrossRef][Medline] 17. Ritgen M, Lange A, Stilgenbauer S, et al: Unmutated immunoglobulin variable heavy-chain gene status remains an adverse prognostic factor after autologous stem cell transplantation for chronic lymphocytic leukemia. Blood 101:2049-2053, 2003 18. Cheson BD, Bennett JM, Grever M, et al: National Cancer Institute-sponsored Working Group guidelines for chronic lymphocytic leukemia: Revised guidelines for diagnosis and treatment. Blood 87:4990-4997, 1996 19. Rines RD, van Orsouw NJ, Sigalas I, et al: Comprehensive mutational scanning of the p53 coding region by two-dimensional gene scanning. Carcinogenesis 19:979-984, 1998 20. Kaplan EL, Meier P: Nonparametric Estimation from Incomplete Observations. J Am Statist Assoc 53:457-481, 1958[CrossRef] 21. Krober A, Seiler T, Benner A, et al: V(H) mutation status, CD38 expression level, genomic aberrations, and survival in chronic lymphocytic leukemia. Blood 100:1110-1111, 2002 22. Crespo M, Bosch F, Villamor N, et al: ZAP-70 expression as a surrogate for immunoglobulin-variable-region mutations in chronic lymphocytic leukemia. N Engl J Med 348:1764-1775, 2003 23. Rassenti LZ, Toy TL, Chen L, et al: ZAP-70 compared with immunoglobulin heavy-chain gene mutation status as a predictor of disease progression in chronic lymphocytic leukemia. N Engl J Med 351:893-901, 2004 24. Hamblin TJ, Orchard JA, Ibbotson RE, et al: CD38 expression and immunoglobulin variable region mutations are independent prognostic variables in chronic lymphocytic leukemia, but CD38 expression may vary during the course of the disease. Blood 99:1023-1029, 2002 25. Moshynska O, Sankaran K, Pahwa P, et al: Prognostic significance of a short sequence insertion in the MCL-1 promoter in chronic lymphocytic leukemia. J Natl Cancer Inst 96:673-682, 2004 26. Lin K, Manocha S, Harris RJ, et al: High frequency of p53 dysfunction and low level of VH mutation in chronic lymphocytic leukemia patients using the VH3-21 gene segment. Blood 102:1145-1146, 2003 27. Catovsky D, Richards S, Matutes E, et al: Response to therapy and survival in CLL is influenced by genetic markers: Preliminary analysis from the LRF CLL4 trial. Blood 104:, 2004 (abstr 13) Submitted June 14, 2005; accepted October 31, 2005. This article has been cited by other articles:
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ABSTRACT
INTRODUCTION
98%) or high-risk interphase cytogenetics, including either del(17p) or del(11q), appear to have a shorter PFS and OS with chemoimmunotherapy. Larger prospective studies will be required to determine the independent influence of Ig VH mutational status and interphase cytogenetics on treatment outcome. 
