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Leukemia Research 26 (2002) 909–918 Bone marrow biopsy in adult acute lymphoblastic leukemia: morphological characteristics and contribution to the study of prognostic factors Xavier Thomas a,∗, Quoc-Hung Le a, Catalin Danaı¨la a, Véronique Lhéritier a, Martine Ffrench b a Service d’Hématologie Clinique, Hoˆpital Edouard Herriot, 69437 Lyon Cedex 03, France b Laboratoire d’Histologie et de Cytologie Analytique, Hoˆpital Edouard Herriot, 69437 Lyon Cedex 03, France Received 22 October 2001; accepted 4 February 2002 Abstract Bone marrow (BM) sections were examined in 128 untreated adult patients with newly diagnosed acute lymphoblastic leukemia (ALL), seen in our institution over a 19-year period. BM biopsy was performed in order to assess the incidence, degree and prognostic significance of histological data of the disease. BM features studied were reticular fibrosis, total cellularity, residual hematopoiesis, mitotic activity, and blastic infiltration. T-cell lineage ALL were diagnosed in 23% of the cases, while B-cell lineage ALL represented 70% of the cases. There were 7% of non-T-non-B-cell lineage ALL. The percentage of BM leukemic cells was related to cellularity (P = 0.02), while it was related to the disappearance of normal cell lines (P < 0.0001). BM cellularity was related to the percentage of circulating leukemic cells at diagnosis (P = 0.006). Residual hematopoiesis was related to a higher initial granulocyte count (P = 0.04) and lower percentage of circulating blasts (P = 0.04). The degree of fibrosis was inversely related to that of BM cellularity (P = 0.04). All patients, but four, received standard ALL induction chemotherapy according to different successive protocols. In this whole cohort of patients, complete remission (CR) rate was 78%. Median disease-free survival (DFS) and median overall survival (OS) were 13.7 months and 20.2 months, respectively. In univariate analysis, CR rate was positively affected by mitotic activity (P = 0.01) and residual hematopoiesis (P = 0.008). OS was positively influenced by a higher leukemic cell mitotic activity (P = 0.03) and the persistence of more than two residual normal cell lines in BM (P = 0.04). Patients presenting with both of those characteristics had better outcome than patients who did not, as well as, in terms of CR (P = 0.03), or DFS (P = 0.002), or OS (P = 0.003). T-cell lineage ALL and L3 ALL did not significantly influence those results. Our findings did not confirm that among marrow features, reticular fibrosis has any prognostic value. A multivariate analysis of both clinical and histological data was performed to test their prognostic relevance. In a model including age, immunophenotype, Philadelphia chromosome status, mitotic index, and level of normal residual hematopoiesis, the only significant predictor of CR achievement were the persistence of normal residual hematopoietic cell lines (P = 0.01) and the mitotic activity of leukemic cells (P = 0.002). Philadelphia chromosome status (P = 0.03) and age (P < 0.0001) were of prognostic value, respectively for DFS and OS. We conclude that some characteristics of BM biopsy afford not only descriptive but also prognostic information for predicting the outcome. The persistence of normal residual hematopoiesis and intense leukemic cells mitotic activity were both factors of favorable outcome, while BM fibrosis did not display any prognostic value. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Bone marrow biopsy; Fibrosis; Acute lymphoblastic leukemia; Prognosis 1. Introduction The prognosis of acute lymphoblastic leukemia (ALL) has improved over the last 20 years, mainly because of more intensive chemotherapy, improvements in supportive ther- apy and the introduction of new drugs, and also because of risk-stratified approach for treatment according to prog- ∗ Corresponding author. Tel.: +33-4-72117395; fax: +33-4-72117404. E-mail address: xavier.thomas@chu-lyon.fr (X. Thomas). nostic factors. Parameters measured at diagnosis such as age, immunophenotype, laboratory values or clinical man- ifestations can be predictive for either complete remission (CR) achievement or for remission duration [1,2]. These features have been utilized to identify patients with low- or high-risk of relapse, in order to allocate them to regimen with risk-adapted treatment intensity. Several prognostic models have been proposed [1,3,4]. High-risk-groups represent the majority of adult ALL cases. Hoelzer et al. identified time to CR as greater than 4 weeks, age older than 35 years, 0145-2126/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S0 1 4 5 -2126 (02 )00034 -6 910 X. Thomas et al. / Leukemia Research 26 (2002) 909–918 leukocyte count above 30 × 109/l, and null ALL pheno- type as poor prognostic features [1]. Based on these criteria, we recently introduced a new risk-oriented post-remission treatment policy (collaborative LALA-94 trial) identifying standard- and high-risk ALL, Philadelphia-positive (Ph+) ALL, central nervous system-positive (CNS+) ALL. The diagnosis and classification of ALL are typically based on morphological and immunophenotypic studies of peripheral blood (PB) and aspired bone marrow (BM) leukemic cells. The histological examination of a BM core biopsy is usually performed only in cases with a dry tap aspiration or in those cases with inadequate cell numbers on the smears. However, BM biopsy has also proven to be of value in predicting the outcome of patients with several hematological disorders [5–9]. The prognostic value of BM histologic pattern in ALL has been until now poorly investigated. However, a progressive increase in marrow reticulin has been associated with impending relapse [10]. In this setting, ALL presenting with fibrosis features were until now systematically entered in high-risk-group, and thus received intensified therapy. In order to really determine the incidence and the prog- nostic and therapeutic significance of BM histopathological parameters seen in adult ALL, we retrospectively reviewed BM biopsies performed on 128 newly diagnosed patients seen in our institution over a 18-year period. 2. Materials and methods 2.1. Patients Between February 1980 and October 1998, a total of 330 consecutive adult patients aged 15 years or more with newly diagnosed ALL were referred to our institution. During that time period, BM biopsy was routinely planned at the time Fig. 1. Percentage of patients with de novo ALL undergoing BM biopsy according to the year of admission. BM biopsy was systematically planned at diagnosis beginning in February 1980. However, BM biopsy was effectively performed in only about 50% of cases between 1980 and 1991. After 1991, BM biopsy was performed less and less. Its use was definitively stopped by October 1998 because of its difficult systematic performance. of diagnosis. Actually, BM biopsy was not systematically performed (Fig. 1). Overall, BM biopsy was available for evaluation in 128 patients (39%). Diagnosis of ALL was based on May-Grünwald-Giemsa (MGG) smears of the BM aspirates. Leukemic cells were classified according to the French–American–British (FAB) morphological and cyto- chemical criteria [11]. Immunophenotyping of leukemic cells was attempted in patients to define early-pre-B, pre-B, B-cell, and T-cell subtypes of ALL. The percentage of positive cells was calculated by counting the number of positive cells comparatively to the controls. The criteria for antigen positivity was an expression of surface antigens by at least 20%, and of intracytoplasmic antigens by at least 10% of the leukemic cells, as previously described [12]. Based on the pattern of reactivity, lymphoblasts were classified as T (CD7+, CD5+ or CD2+), early-pre-B (CD19+, HLA-DR+, CD10±, CD22±, CD7-, cIg-, sIg-), pre-B (cIg+), or B (sIg+) [2]. The level of differentiation into early pre-B stage was evaluated by the presence of the CD10 and CD20 antigens. Myeloid-antigen-positive (My+) ALL was defined as co-expression of lymphoid markers and at least two myeloid-lineage-associated antigen (CD13/CD14/CD15/CD33) on ≥ 20% of the lymphoblasts. Cytogenetic analysis was performed on BM and/or on PB cells before initiation of therapy, using short unstimulated cultures and RHG banding. Karyotypes were analyzed according to the International System for Human Cytoge- netic Nomenclature [13]. Two different classifications were used. In the first classification, patients were grouped ac- cording to the presence or absence of normal metaphases (NN/AN/AA). Twenty normal metaphases were required to classify a karyotype in the normal group. In the second chro- mosome grouping, karyotypes were classified according to ploidy groups or structural (recurrent) abnormalities. Mole- cular characterization of BCR/ABL, E2A-PBX1, HRX-AF4 rearrangements at diagnosis were routinely performed since X. Thomas et al. / Leukemia Research 26 (2002) 909–918 911 1992 using a reverse transcription-polymerase chain reac- tion (RT-PCR) technique. Chromosomal fluorescence in situ hybridization (FISH) was occasionally used since 1994 with labeled probes for mBCR/ or MBCR/ABL translocation. Central nervous system (CNS) leukemia was diagnosed on the basis of cranial nerve palsies with or without leukemic blasts in the cerebrospinal fluid, or when at least five mononuclear cells per microlitre of cerebrospinal fluid with blasts were present in a cytospin preparation. All non-T-cell lineage ALL patients were classified as being at “standard- or higher-risk”of relapse, based on Hoelzer’s criteria [14] and defined as having at least one of the fol- lowing factors: Age > 35 years, WBC counts >30 × 109/l, and Ph+ or 11q23 chromosomal abnormality. 2.2. Treatment Four of the 128 patients who underwent BM biopsy did not receive standard ALL chemotherapy. One patient died early before any chemotherapy could be given. One patient was treated only with corticosteroids and vincristine because of advanced age and poor medical condition. Two patients were misdiagnosed initially with acute non-lymphoblastic leukemia (ANLL) and treated on an ANLL protocol. In all 124 other patients, induction chemotherapy con- sisted of a corticosteroid, vincristine, ±cyclophosphamide, ±anthracycline, ±bleomycin combination. One hundred and four of those 124 patients were treated according to seven successive treatments based on therapeutic schedules of LALA protocols [15–21]. Four patients received L10 schedule [22], eight were treated according to EORTC pro- tocol 58741 [23], seven with ACVBP schedule [24], and one according to L3 specific trial [25]. Marrow response status was determined by BM aspirates at about day 28 of induction chemotherapy. Patients who did not achieve CR in one course of chemotherapy, as evaluated by the persistence of blast in day 28 BM aspirate, received salvage therapy ac- cording to the protocol design in which they were included. Patients with CR after induction or salvage were given con- solidation chemotherapy according to the protocol design in which they were included. As post-induction therapy, 71 cases followed a chemotherapy program. Adjustments were made to the dose or timing of consolidation therapy only under exceptional circumstances. Doses and timing of maintenance cycles were adjusted frequently to prevent unacceptable neutropenia or thrombopenia. Twenty-nine patients, aged less than 50 years, received early BM or PB stem cell transplantation (SCT). Twenty-four underwent allogeneic bone marrow transplant (BMT) from a HLA sibling donor, while five patients received autologous SCT. 2.3. Supportive care During induction therapy, transfusional support was gen- erally provided to maintain the hemoglobin level at >80 g/l and the platelet count at >20 × 109/l. Febrile neutropenic patients received broad-spectrum antibiotic coverage until culture and sensitivity results were available. Prophylactic antibiotics were not administered. 2.4. Evaluation of therapy CR was defined according to the CALGB criteria as less than 5% blasts in BM aspirates with evidence of mat- uration of cell lines and restoration of PB counts [26]. Patients failing induction therapy were categorized as fail- ures and divided into: (i) death during induction (death occurred while the patient was receiving induction ther- apy), and (ii) resistant (the patient survived induction but resistant leukemia redeveloped). Overall survival (OS) was defined as the time from diagnosis to death or the last follow-up. Disease-free survival (DFS) was calculated in patients who achieved CR from CR to the time of relapse, death from any cause, or last follow-up. Hematological relapse was considered when more than 5% blasts were seen in two BM aspirates obtained at a 15-day interval [26]. 2.5. Bone marrow biopsy Biopsies were obtained from posterior iliac crest with a Jamshidi or an Islam needle. Samples were then pro- cessed into plastic according to the methods previously described [27]. Briefly, undecalcified bone specimens were fixed in PBS-buffered formalin, dehydrated in acetone, em- bedded in a Kushida’s low-viscosity glycol-methacrylate Immuno-BedTM resin (Polysciences Europe GmbH, Eppel- heim, Germany) [28]. Sections were cut at a thickness of 2�m using a glass knife in a Sorval JB4 microtome. Sec- tions were stained with usual histological procedures using Giemsa and eosin panoptic staining for cytological detail, and by Gomori’s silver impregnation to show reticulin fibres. Characteristics recorded from each biopsy sample in- cluded: total cellularity, mitotic activity, blastic infiltration, residual hematopoiesis, and reticular fibrosis. The crite- ria for evaluation and grading of these characteristics are shown in Table 1. The total cellularity of each BM section was assessed visually under low-power microscopy and expressed as a percentage of the total marrow volume. The leukemic infiltrate was expressed as a percentage observed among total cells. The stromal fibre content was also graded. Persisting normal hematopoiesis was evaluated as the num- ber of persisting normal cell line activity. Persistence of megakaryocytic, erythroid or granulocytic lineage from precursors to differentiated cells defined persistent normal hemopoiesis regarding this specific cell line. Mitotic activ- ity was expressed as a percentage of mitotic cells observed among blastic infiltration. All samples were examined at the time of initial diagnosis at least on five different fields (50× magnification), and reviewed by the same observer at time of study. Discordant data were excluded from later analysis. 912 X. Thomas et al. / Leukemia Research 26 (2002) 909–918 Table 1 Histological data analyzed on bone marrow biopsy Histological data Grade Characteristic Reticular fibrosis 0 No reticulin increase 1 Focal, minimal fibrosis 2 Multifocal or diffuse non-confluent fibrosis 3 Marked, diffuse fibrosis Total cellularity 1 ≤50% 2 Between 50 and 90% 3 ≥90% Residual hematopoiesis 0 No persistent cell line 1 Persistence of one-cell line 2 Persistence of two-cell lines 3 Persistence of three-cell lines Mitotic activity 1 ≤1/400 2 Between 1/400 and 1/100 3 ≥1/100 Blastic infiltration 1 ≤50% 2 Between 50 and 90% 3 ≥90% 2.6. Statistical methods Univariate analysis of associations between categorical factors was performed using the χ2 statistics. For contin- uously distributed variables other than event times, differ- ences between groups were also tested using the Wilcoxon rank-sum test. The end points used to assess the prognostic importance of the different variables were the outcome of induction chemotherapy, the DFS for patients who achieved a CR, and the OS duration. The 95% confidence intervals (CIs) on proportions of CR patients were calculated using the exact binomial formula. Survival and DFS curves were estimated by the Kaplan–Meier method, and their 95% symmetrical CI was calculated according to Greenwood’s method. For analysis of OS and DFS, patients receiving al- logeneic or autologous BM transplantation in first CR were censored at the time of transplantation. Survival curves were compared using the log-rank test [29]. Only probability val- ues less than 0.05 were considered statistically significant. Multivariate analysis was made by a multiple logistic regres- sion for the CR rate and by the Cox’s proportional hazard model for DFS and OS [30]. The BM biopsy data selected for possible inclusion in the regression method were those for which there was some indication of a significant associ- ation with survival in univariate analysis. In order to analyze whether the BM biopsy characteristics have independent prognostic value, we also included in the multivariate analy- sis: age, immunophenotype, and karyotype. Those variables were included because they had proved to be the best combi- nation of patient’s and disease characteristics for predicting survival in previous studies [1,17]. Goodness of fit of the models was tested using the likelihood ratio statistics. Com- putations were performed using BMDP PC-90 statistical program (BMDP Statistical Software, Los Angeles, CA, USA). In order to assess the prognostic value of BM histological data, treatment outcome was studied after patients were sep- arated onto different categories. For the purpose of the sta- tistical analysis, total cellularity was separated into “poor” (grade 1 or grade 2) and “rich” (grade 3), mitotic activity into “moderate” (grade 1 or grade 2) and “intense” (grade 3), residual hematopoiesis into “null or low” (grade 0 or grade 1) and “persistent” (grade 2 or grade 3), and reticular fi- brosis into “null or fine” (grade 0 or grade 1) and “coarse” (grade 2 or grade 3). 3. Results 3.1. Characteristics of patients Clinical and biological characteristics at diagnosis of the 128 patients (77 males and 51 females) are summarized in Table 2. A hundred and nine patients were less than 60 years of age (85%). Six patients (5%) presented with a previous history of malignant disease. Median leukocyte count of the entire cohort was 9.5×109/l (range, 0.8–345×109/l) on ad- mission. About 43% of patients had fever or infection, 20% had weight loss >5%, 31% had hemorrhage, and 3% pre- sented with WHO performance status >2. Tumoral syndrome Table 2 Main clinical and biological characteristics of the 128 adult ALL patients Characteristics At diagnosis Clinical characteristics Age (years) 38 (14–77)a Sex (males/females) 77/51b Tumoral syndrome (yes/no/NDc) 81/46/1 CNS involvement (yes/no/ND) 6/120/2 Biological characteristics Hemoglobin (g/l) 93 (36–176) WBC count (×109/l) 9.5 (0.8–345) Platelet count (×109/l) 51 (4–363) Circulating leukemic cells (%) 47 ( 0–100) Bone marrow leukemic cells (%) 90 (45–100) FAB classification (L1/L2/L3/unclassified/ND) 66/34/11/13/4 Immunophenotyped (B/T/non-B-non-T lineage ALL) 87/28/9 Karyotypee (Ph+/11q23/t(8;14)/ hyperdiploidy ≥50) 14/4/8/8 Risk-groupf (standard-risk/high-risk) 26/74 LDH (UI/l) 775 (176–9980) Uric acid (�mol/l) 346 (115–905) Creatininemia (�mol/l) 85 (34–523) a Median (range). b Number of patients. c Not determined. d Determined in 124 patients. e Determined in 98 patients. f According to Hoelzer’s criteria. X. Thomas et al. / Leukemia Research 26 (2002) 909–918 913 was seen only in 81 out of the 127 cases with available data (64%), of which 6 cases presented with CNS involvement (5%). Lymph nodes were palpable in 52%. Liver and spleen were more than 3 cm from the costal margin in 27 and 37%, respectively. Mediastinal enlargement was seen only in 12 patients. Among the 124 patients classified in FAB, 66 (53%) showed L1, 34 (27%) showed L2, and 11 (9%) showed L3 morphology. Thirteen patients (11%) showed undifferenti- ated leukemia. The immunophenotypic features were deter- mined in 124 cases. T lymphoid markers were detected in 28 cases (23%), while 87 patients (7