Bone Marrow Findings in Patients With Acute Promyelocytic Leukemia Treated With Arsenic Trioxide

Abstract

Objectives

Increasingly, acute promyelocytic leukemia (APL) is treated with a combination of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). This study characterizes bone marrow findings after ATRA/ATO therapy.

Methods

Bone marrow biopsies from 16 patients treated with ATRA/ATO and seven patients treated with ATRA/chemotherapy (CTX) for APL were evaluated.

Results

In ATRA/ATO cases, the marrow was likely to be hypercellular (79%) with a decreased myeloid:erythroid (M:E) ratio (88%), megaloblastoid maturation of erythroid precursors (100%), erythroid atypia (75%), and increased (88%) and atypical (75%) megakaryocytes. Significant myeloid atypia was only seen in extensive residual disease. The ATRA/CTX cases were less likely to be hypercellular (38%), have a M:E ratio of 1:1 or less (0%), exhibit significant erythroid atypia (0%), or have increased (0%) or atypical (38%) megakaryocytes.

Conclusions

Bone marrow biopsies from patients treated with ATO have unusual but characteristic features. Despite variability in marrow findings, clinical outcomes were uniformly favorable.

Acute promyelocytic leukemia (APL) is characterized by a proliferation of neoplastic promyelocytes and is associated with abnormalities in coagulation and fibrinolysis that may give rise to life-threatening bleeding and thrombosis.¹ Previously considered a highly fatal diagnosis, APL cure rates vastly improved with the advent of treatment regimens that included the vitamin A derivative, all-trans retinoic acid (ATRA), and anthracycline-based chemotherapy.² Recently, however, the treatment of low-to-intermediate risk APL, classified by a WBC count at diagnosis of less than 10 × 10⁹/L, has shifted from a combination of ATRA and anthracycline-based chemotherapy to ATRA and arsenic trioxide (ATO), with further improvements in outcomes.^3-7

ATRA and ATO work synergistically in the treatment of APL, targeting the hallmark fusion protein of the disease. The defining underlying chromosomal translocation of APL by the promyelocytic leukemia (PML) gene on chromosome 15 with the retinoic acid receptor-α (RARA) gene on chromosome 17 produces the PML-RARA fusion protein. This fusion protein inhibits the terminal differentiation of the leukemic cells and allows for their sustained proliferation.⁸ ATRA has long been viewed as an agent of terminal differentiation in APL through its action as a ligand for RARA,⁹ whereas ATO has been demonstrated to target the PML moiety of the fusion protein.¹⁰ Although the synergistic mechanism between ATRA and ATO is complex, together these agents lead to the degradation of the PML-RARA fusion protein, induction of terminal differentiation, and apoptosis of the leukemic cells.^2,8,11,12

Increasingly, patients are receiving combination therapy with ATRA and ATO for low-to-intermediate risk APL. The first cycle of this therapy is continued until morphologic remission in a bone marrow sample is observed at the time of count recovery on day 21 or beyond.³ In this setting, pathologists are frequently asked to evaluate posttreatment bone marrow biopsies as a component of disease observation and follow-up.

Much of our current knowledge of arsenic’s effect on the bone marrow derives from case reports and small case series following either treatment with ATO or, found more prominently in the literature, arsenic intoxication. One other group has described bone marrow findings following ATO treatment. In 2000, Zhang et al¹³ reviewed bone marrow findings after ATO treatment for APL in five patients with relapsed or refractory APL. They noted that after one cycle of ATO, all five patients showed some degree of morphologic response with a normo- to hypercellular marrow and myeloid predominance.

In the setting of arsenic intoxication, the following bone marrow abnormalities have been variably described: generally an erythroid hyperplasia with one report of an increased myeloid to erythroid (M:E) ratio; marrow hypercellularity; dyserythropoiesis with megaloblastoid features, nuclear irregularities, Howell-Jolly bodies, basophilic stippling, and pronounced karyorrhexis; dysgranulopoiesis including giant metamyelocytes and hypersegmented neutrophils; and abnormal megakaryocytes.^14-18

As bone marrow findings have been inconsistently reported in the setting of ATO treatment and arsenic exposure, and have yet to be defined in the setting of therapeutic doses of ATO for the treatment of primary (nonrelapsed or refractory) APL, our objective was to characterize these findings to aid in delineating effects of therapy vs true pathologic findings. Additionally, we aimed to evaluate any association between morphologic findings in the bone marrow biopsies and clinical outcomes.

Materials and Methods

Case Selection and Review

This study was approved by the Johns Hopkins Medicine Institutional Review Board. The Pathology Database System at Johns Hopkins Hospital was searched for bone marrow biopsies with new diagnoses of APL between June 2012 and August 2017. Twenty-four patient samples were identified with new diagnoses of low-to-intermediate risk APL, defined as a WBC count at diagnosis of less than 10 × 10⁹/L. Reflecting the shift in therapy for APL, 17 of these patients underwent induction therapy with ATRA/ATO between July 2013 and August 2017, and nine patients (seven low risk, two high risk) underwent induction therapy with ATRA/anthracycline-based chemotherapy (CTX) between June 2012 and April 2013.

Clinical data, including patient demographics, treatment regimens, and CBC results, were extracted from the electronic medical record. Patient bone marrow biopsies and aspirates collected after initiation of ATRA/ATO or ATRA/CTX induction were evaluated by a practicing hematopathologist (A.S.D, C.D.G., M.J.B., or K.H.B), and re-reviewed by one of the authors (A.S.D.) for the following parameters: cellularity, M:E ratio, and atypia within the erythroid, myeloid, and megakaryocytic lineages. Flow cytometry results and molecular studies (fluorescence in situ hybridization [FISH] and polymerase chain reaction [PCR]) were additionally reviewed on these samples, when available.

Histology

H&E slides from formalin-fixed and paraffin-embedded bone marrow core biopsies were reviewed along with Wright-Giemsa–stained aspirate smears. Any associated immunohistochemical stains performed at the time of the original diagnosis were also reviewed, but no additional stains were done solely for the purposes of this study. All cases were originally reviewed by a practicing hematopathologist (A.S.D., C.D.G., M.J.B., or K.H.B) and re-reviewed for the purposes of this study (A.S.D).

Flow Cytometry

Flow cytometric immunophenotyping was performed on fresh bone marrow aspirates. The material was collected in EDTA or heparin anticoagulant and processed routinely using an RBC lysis method. Cell suspensions were incubated with combinations of monoclonal antibodies (Becton Dickinson) that were used at concentrations titrated for optimal staining. In some cases prior to 2016, the panel included antibodies specific for CD2, CD3, CD5, CD7, CD10, CD11b, CD13, CD14, CD15, CD16, CD19, CD22, CD33, CD34, CD38, CD45, CD56, CD61, CD64, CD71, CD117, and HLA-DR. Specimens after 2016 were subjected to a relatively limited myeloid leukemia panel that included CD7, CD10, CD11b, CD13, CD14, CD15, CD16, CD33, CD34, CD38, CD45, CD56, CD64, CD71, CD117, CD123, and HLA-DR. Specimens collected prior to 2014 were analyzed on a BD FACSCalibur flow cytometry system (BD Biosciences). Specimens after 2014 were analyzed on BD FACSCanto 10-color system (BD Biosciences).

List mode data files were acquired and analyzed for each specimen using CellQuest and Paint-a-Gate software programs (Becton Dickinson), respectively, prior to 2014. After 2014, BD FACSDiva (BD Biosciences) and Infinicyt (Cytognos) were used for data acquisition and analysis, respectively. An antigen was considered positive if the cells of interest either showed a homogeneous distribution with the median intensity at least 20 log channels above that seen in the control or if there was a heterogeneous distribution of antigen expression, such that a subpopulation of cells was above that seen in the control. All cases were originally reviewed by a practicing hematopathologist (A.S.D, C.D.G., M.J.B., or K.H.B) and re-reviewed for the purposes of this study (A.S.D).

Cytogenetics

Cells from the bone marrow biopsies were cultured without mitogens using current techniques and subsequently harvested. Slides were prepared and G-banded using standard techniques. At least 20 metaphase cells were analyzed per specimen.

Fluorescence In Situ Hybridization Analysis

Two hundred interphase nuclei were scored following FISH with a dual-fusion FISH probe specific for fusion of the PML gene on chromosome 15q22-24 and the RARA gene on chromosome 17q21 (Abbott Molecular). For 200 nuclei, the normal cutoff is less than 1.5% of nuclei with fusion.

Molecular Analysis

Detection of the PML/RARA translocation by an mRNA-based RT-PCR assay was performed as described.¹⁹ In brief, RNA was extracted from samples using the Qiagen QIAamp RNA Blood Mini Kit according to manufacturer’s protocol. cDNA was prepared using an MMLV reverse transcription kit (Signature RT Reagents), and the assay was performed using the Asuragen Signature LTX multiplexed AML translocation reagents, with signal detection on the Luminex platform. Although a formal limit of detection was not performed, this assay provides a positive signal for the PML/RARA translocation at dilution levels of 0.1% to 1% leukemia cell line in normal cells.²⁰

Results

Patient Demographics and Clinical Findings

In the ATRA/ATO group, the median age was 53 years, and patients ranged in age from 15 to 75 years Table 1. There were seven women and nine men. All had an APL/RARA fusion, although two patients had complex variants and one had a cryptic translocation Table 2. All 16 patients were induced according to the Lo-Coco et al³ regimen in which ATRA (45 mg/m²/d) plus ATO (0.15 mg/kg/d) was continued (up to 60 days) until hematologic complete remission. None of the patients received growth factors during or after treatment. Reported side effects at the time of ATRA/ATO treatment included headache, neuropathy, and mental “fogginess.” Long-term follow-up was available for 14 patients treated at our institution, ranging from 14 to 61 months postdiagnosis. Two patients were followed at outside institutions, and follow-up was only available for these patients at 4 and 8 months. According to the most recent clinical information available for each patient, all patients in this group were alive and in remission.

Table 1.

Demographics

Characteristic	ATRA/ATO, n = 16	ATRA/CTX, n = 9
Age, y, median (range)	53 (15-75)	56 (25-71)
Men, No.	9	4
Women, No.	7	5
CBC data at diagnosis, median (range)
Hemoglobin, g/dL	8.8 (4.8-11.7)	9.8 (3.7-11.1)
Hematocrit, %	24.8 (14.3-34.1)	28.6 (10.7-32)
Platelets, × 103/μL	33 (11-166)	21 (3-103)
WBC, × 103/µL	2.6 (0.67-8.29)	4.3 (0.72-41.7)

ATO, arsenic trioxide; ATRA, all-trans retinoic acid; CTX, chemotherapy.

Table 2.

Karyotype and FISH Findings at Diagnosis

Patient	Karyotype, Bone Marrow	FISH, PML/RARA, %
ATRA/ATO
1a	46,XY,t(9;17;15)(q34;q21;q24)[16]/46,XY[4]	86b
2	47,XX,+8[11]/46,XX[9]c	86c
3	46,XY,inv(9)(p12q13),t(15;17)(q24;q21)[5]/46,XY,inv(9)(p12q13)[15]	17.5
4	46,XY,t(2;17;15)(q37;q21,q24)[21]/46,XY[1]	88
5	46,XY,t(15;17)(q24;q21)[11]/46,XY,der(15)t(15;17)(q24;q21),ider(17)(q10)t(15;17)[9]	97d
6	46,XX,t(15;17)(q24;q11.2)[20]	95.5
7	47,XY,+8,t(15;17)(q24;q21)[20]	95
8	46,XX,t(15;17)(q24;q21)[20]	ND
9	46,XX,t(15;17)(q24;q21)[15]/46,XX[5]	72.5
10	46,XY,der(15)t(15;17)(q24;q21),ider(17)(q10)t(15;17)(q24;q21)[19]/46,XY[1]	94.5
11	46,XX,t(15;17)(q24;q21)[19]/46,XX[1]	95
12	46,XX,t(15;17)(q24;q21)[20]	95.5
13	ND	Presente
14	46,XX,t(15;17)(q24;q21)[10]	96.5f
15a	46,XY,t(15;17)(q24;q21)[14]f	81.5f
16	ND	92.5
ATRA/CTX
1	46,XX,t(15;17)(q24;q21)[20]f	89.5b,f
2	46,XY,t(15;17)(q24;q21)[19]	97
3	46,XX,t(15;17)(q24;q21)[7]/46,XX[13]	79.5
4	46,XY,t(15;17)(q24;q21)[21]f	91
5	47,XX,+8,t(15;17)(q24;q21)[19]/46,XX[1]	60
6	46,XY,t(15;17)(q24;q21)[19]/45,X,-Y[1]	96
7	45,X,-Y,t(15;17)(q24;q21)[19]/46,XY[3]	98.5f
8	46,XX,der(6)t(6;8)(q25;q22),t(15;17)(q24;q21)[18]/46,XX[2]	96.5
9	46,XX,t(15;17)(q24;q21)[20]	95

ATRA, all-trans retinoic acid; CTX, chemotherapy; FISH, fluorescence in situ hybridization; ND, not done; RT-PCR, reverse transcription polymerase chain reaction.

^aPatients with significant residual disease in the biopsy.

^bWith a pattern suggesting a more complex rearrangement.

^cFISH studies show a submicroscopic insertion of the chromosome 15 probe onto chromosome 17 rather than a translocation. There is a PML/RARA fusion; however, the insertion is below the level of resolution of karyotype and is not visible due to its small size.

^d17% of abnormal cells had an additional copy of the fusion signal suggesting a more complex rearrangement.

^eOutside report indicated that FISH is positive for PML/RARA fusion; mRNA-based RT-PCR studies performed in house were positive for PML/RARA.

^fTesting performed on peripheral blood sample.

In the ATRA/CTX group, the median age was 51 years, and the range was 25 to 71 years (Table 1). There were five women and four men. All had a t(15;17) (Table 2). Follow-up was available for all patients, ranging from 38 to 72 months postdiagnosis. According to the most recent clinical information available for each patient, eight patients were alive and in remission. One patient died of relapsed APL approximately 2 years after the initial diagnosis.

Bone Marrow Findings

Timing of Biopsy

A day 28 follow-up biopsy was initially recommended by Lo-Coco et al³ for patients treated with ATRA/ATO. All 16 patients treated with ATRA/ATO in this study had an initial follow-up biopsy performed within 44 days of initiating therapy with ATRA (median, 31 days; range, 26-44 days). All biopsies were obtained during active treatment with ATO.

All patients in the ATRA/CTX group were treated with the chemotherapeutic agent daunorubicin, except for one patient who was treated with idarubicin. In the ATRA/CTX group, eight of the nine patients had a follow-up marrow biopsy within 69 days of initiating therapy with ATRA (median, 62 days; range, 37-69 days), and one patient did not have a follow-up biopsy. All eight evaluated cases showed no morphologic or immunophenotypic evidence of APL.

Morphology

In the ATRA/ATO group, the posttherapy marrow was hypercellular for age in 79% (11/14) of evaluable cases and was only rarely normocellular (14%) or hypocellular (7%) Table 3 and Image 1A. The hypocellular specimen was a biopsy performed at day 13 that showed significant residual disease. Cellularity could not be adequately assessed in two of the cases due to limited core biopsy specimens. In contrast, in the ATRA/CTX group, the marrow was more commonly normocellular for age (5/8 cases, 63%), though it was hypercellular for age in the remaining three cases.

Table 3.

Posttherapy Bone Marrow Cellularity^a

Treatment	Hypercellular	Normocellular	Hypocellular
ATRA/ATO (%)	11/14 (79)b	2/14 (14)	1/14 (7)c
ATRA/CTX (%)	3/8 (38)	5/8 (63)	0/8 (0)

ATRA, all-trans retinoic acid; ATO, arsenic trioxide; CTX, chemotherapy; FISH, fluorescence in situ hybridization.

^aHypercellular is defined as >70% young adult age 18-25 years, >60% adult age 25-75 years, and >40% elderly age >75 years; normocellular is defined as 50%-70% young adult, 40%-60% adult, and 25%-40% elderly; and hypocellular is defined as <50% young adult, <40% adult, and <20% elderly.

^bIncludes patient 16, who had active central nervous system disease at the time of the biopsy and was found to have residual disease by molecular studies on a peripheral blood specimen 2 weeks later.

^cAt 13 days after ATO; 78% PML/RARA detected by FISH.

Image 1.

Bone marrow biopsy from a 50-year-old man, 25 days after initiation of all-trans retinoic acid/arsenic trioxide therapy. A and B, Hypercellular marrow with increased morphologically abnormal megakaryocytes (A, ×40; B, ×400). C, Increased myeloid:erythroid ratio with an E-cadherin immunostain (D) highlighting erythroid precursors (H&E, ×200).

There was an erythroid hyperplasia with an M:E ratio 1:1 or less in 14 of 16 (88%) ATRA/ATO cases, and with a median M:E ratio of 1:2 (range, 10:1 to 1:6; Image 1B, Image 1C, and Image 1D).The two cases with normal or elevated M:E ratios (3:1 and 10:1) had evidence of extensive residual disease. By contrast, the M:E ratio was normal (median, 2.5:1; range, 2:1 to 3.5:1) in all seven evaluable ATRA/CTX cases. The remaining ATRA/CTX case had a limited biopsy and aspirate but appeared to have a somewhat increased M:E ratio, which was difficult to adequately quantify.

Megakaryocytes were increased in 14 of 16 (88%) ATRA/ATO specimens, and 12 (75%) of these cases showed atypia in 10% or more of the megakaryocytes. This atypia was similar to that seen in dysplasia, including small forms with hyperchromatic and hypolobated nuclei, as well as megakaryocytes with widely separated nuclear lobes Image 1B. In contrast, megakaryocytes were normal in number in all ATRA/CTX cases. There was mild atypia seen in the megakaryocytic lineage in 38% (3/8) of ATRA/CTX cases. In all three of these cases, the atypical megakaryocytes comprised approximately 10% of the overall megakaryocytes, and the changes included abnormally lobated and/or hyperchromatic nuclei.

All ATRA/ATO cases showed megaloblastoid maturation of erythroid precursors. Twelve of 16 (75%) cases demonstrated morphologic atypia of the erythroid lineages similar to erythroid dysplasia, including nuclear membrane irregularities, basophilic stippling, and/or cytoplasmic vacuoles in 10% or more of erythroid precursors (average, 22%; range, 1%-50%) Image 2A, Image 2B, and Image 2C. All ATRA/CTX cases also showed megaloblastoid maturation in the erythroid lineage, and nuclear membrane irregularities were also seen in the erythroid precursors in most cases (6/8, 75%). However, the atypical cells were far less frequent in the ATRA/CTX cases, comprising less than 10% of the cellularity in four cases and approximately 10% of the cellularity in the remaining two cases. In the latter two cases, the morphologic atypia was relatively subtle. Additionally, basophilic stippling and cytoplasmic vacuoles were not identified in the ATRA/CTX cases.

Image 2.

Representative bone marrow aspirates, all 30 days after initiation of all-trans retinoic acid/arsenic trioxide therapy. Dysplastic changes in erythroid precursors were demonstrated in 71% of cases, including cytoplasmic vacuoles in early precursors (A), nuclear membrane irregularities (B), and basophilic stippling (C). (All images, ×1,000). Flow cytometric analysis showed variable expression of CD71 in a subset of cases (D). APC-H7, allophycocyanin H7.

The myeloid lineage did not show significant morphologic atypia in the ATRA/ATO cases, with the exception of the two biopsies with significant residual disease. There was focal myeloid morphologic atypia in six of the remaining 14 cases, including frequent or chunky eosinophilic granules, eosinophil precursors with basophilic granules, and pelgeroid forms. These atypical forms comprised less than 10% of cells in all six of these cases Image 3A, Image 3B, Image 3C, and Image 3D. In contrast, no significant morphologic atypia was seen in the myeloid lineage in any of the ATRA/CTX cases.

Image 3.

Bone marrow aspirates showed focal (less than 10%) myeloid morphologic atypia in a minor subset of cases, including abundant or large eosinophilic granules (A and C), eosinophil precursors with basophilic granules (B), and pelgeroid forms (D). (All images, ×1,000). Flow cytometric analysis showed a normal pattern of myeloid maturation in all cases that do not show significant residual disease (E). Images were taken 30 days (C and E), 31 days (B), 35 days (A), and 44 days (D) after initiation of all-trans retinoic acid/arsenic trioxide therapy. APC-H7, allophycocyanin H7; PE, phycoerythrin.

Two ATRA/ATO patients (numbers 1 and 16 in Table 2) showed evidence of active clinical disease and/or morphologic evidence of significant residual disease at the time of the follow-up biopsies. Biopsies from these two patients exhibited a myeloid predominance with morphologic atypia in the myeloid lineage. The myeloid predominance and atypia likely reflect the fact that the marrow contains residual but differentiating neoplastic promyelocytes, as both ATRA and ATO are differentiating agents. One of the patients (number 1) was a 23-year-old man who had the follow-up bone marrow biopsy performed early (day 13) after initiation of therapy. FISH studies demonstrated 78% PML/RARA in the marrow aspirate, and molecular studies on the marrow were positive as well. The early timing of the biopsy likely accounts for the significant residual disease. He had hypocellular marrow (35%) with a M:E ratio of 3:1 and a normal number of megakaryocytes. In addition to the myeloid atypia, approximately 10% of the megakaryocytes and 5% of the erythroid precursors exhibited morphologic atypia, but he went on to have an uneventful clinical course and remains free of disease 61 months after diagnosis. The other patient was a 60-year-old man (number 16) who had a biopsy performed on day 30. He had active central nervous system disease at this time, which was documented by flow cytometric analysis. The marrow had a M:E ratio of 10:1 with cytoplasmic vacuoles and abnormal patterns of nuclear lobation in the myeloid cells Image 4A and Image 4B. Like the other biopsies that were performed at the end of induction, his marrow was hypercellular with increased megakaryocytes. While less than 10% of the megakaryocytes showed morphologic atypia, approximately 30% of the erythroid precursors exhibited morphologic atypia. Neither cytogenetic nor molecular studies were done on the biopsy itself, but molecular studies performed on the peripheral blood 2 weeks after the biopsy (prior to consolidation) showed residual disease. While the disease burden in the peripheral blood in patient 16 was not quantified, PCR was negative for PML/RARA on the peripheral blood of other patients in this series (numbers 13 and 15 in Table 2) who were tested preconsolidation. Despite the morphologic and molecular findings at the end of induction, patient 16 ultimately cleared both his marrow and cerebrospinal fluid. These findings indicate that residual disease at day 30 (or before) may not portend treatment failure with the ATRA/ATO regimen.

Image 4.

Significant residual disease in a 60-year-old man (patient number 16, Table 2), 30 days postinitiation of all-trans retinoic acid/arsenic trioxide therapy. The patient had active central nervous system disease at the time of the biopsy, and molecular studies on a peripheral blood specimen performed 2 weeks after the biopsy showed residual disease. A, Increased myeloid:erythroid ratio (×400). B, Atypical myeloid cells with cytoplasmic vacuoles and abnormal patterns of nuclear lobation (×1,000). C, Myeloid maturation shows an abnormal pattern of CD13 and CD16 acquisition on flow cytometric analysis. APC-H7, allophycocyanin H7; PE, phycoerythrin.

Flow Cytometry

Flow cytometric evaluation was performed on 14 ATRA/ATO cases at the time of the posttherapy biopsy. The erythroid precursors were notable for variable expression of CD71 in eight of 14 (57%) evaluated cases Image 2D. Myeloid maturation showed a relatively normal pattern of myeloid antigen acquisition (CD10, CD13, and CD16) in 12 of 14 (86%) evaluated cases Image 3E. The two markedly abnormal cases were the specimens that had a normal or elevated M:E ratio with significant residual disease Image 4C. Three specimens showed left-shifted myelopoiesis (<25% mature granulocytes), and four showed a somewhat discontinuous pattern of myeloid maturation. All specimens demonstrated no increase in blasts, as defined by moderate-intensity CD45 expression and low right-angle side scatter (median, 1.3%; range, 0%-4.2%).

In the ATRA/CTX group, flow cytometric evaluation showed no increase in blasts, no pronounced variability in the expression of CD71, and no abnormalities in myeloid maturation in any of the cases.

Cytogenetic and Molecular Studies

Cytogenetic and/or molecular studies were performed on a subset of the ATRA/ATO cases after initiation of treatment Table 4. All 10 cases that were karyotyped at the time of the posttherapy biopsy were negative for the t(15;17) seen at diagnosis. FISH studies on 13 cases showed nine negative cases, three cases with limited disease (6%, 2%, and 1.5% nuclei with PML/RARA fusion signals), and one case with extensive disease (78% PML/RARA). The case with 78% PML/RARA detected by FISH occurred in a 23-year old-man (patient 1) on a day 13 posttherapy marrow biopsy; this biopsy also showed morphologic evidence of residual disease. RT-PCR studies were positive in nine of 11 cases done at the time of biopsy. One marrow specimen had neither cytogenetic nor molecular studies performed. This marrow represented the specimen from the 60-year-old man (patient 16) who had active CNS disease and extensive morphologic evidence of residual disease on his day 30 biopsy.

Table 4.

Molecular and Cytogenetic Findings in the Bone Marrow, ATRA/ATO Group

Test	Findings
No. of cases	1	2	5	2	1	1	2a	2
Days after ATO, median (range)	13	29 (26-31)	36 (29-44)	36 (35-36)	26	37	28 (25-30)	29 (27-30)
Karyotypeb	NI	–	–	–	ND	ND	ND	ND
FISH (% PML/RARA)	+ (78)	+ (6, 1.5)	–	–	–	+ (2)	ND	ND
PCR	+	ND	+	–	+	+	+	ND

ATRA, all-trans retinoic acid; ATO, arsenic trioxide; FISH, fluorescence in situ hybridization; ND, not determined; NI, not informative; PCR, polymerase chain reaction.

^aIncludes patient 16 who had active central nervous system disease at the time of the biopsy and was found to have residual disease by molecular studies on a peripheral blood specimen 2 weeks later.

^bKaryotype negative indicates no evidence of fusion.

Cytogenetic and molecular studies were performed on a subset of posttherapy biopsies from the ATRA/CTX cases as well Table 5. All seven cases that were karyotyped had a normal complement of chromosomes with no evidence of t(15;17). FISH studies were negative in the seven evaluated cases. Molecular minimal residual disease studies were also performed on seven cases; six were negative, and one was positive, though it was noted to be very low (at the limit of detection).

Table 5.

Molecular and Cytogenetic Findings in the Bone Marrow, ATRA/CTX Group

Test	Findings
No. of cases	5	1	1	1
Days after treatment, median	61	69	64	41
Karyotypea	–	–	ND	–
FISH	–	ND	–	–
PCR	–	–	ND	+

ATRA, all-trans retinoic acid; CTX, chemotherapy; FISH, fluorescence in situ hybridization; ND, not determined; PCR, polymerase chain reaction.

^aKaryotype negative indicates no evidence of fusion.

Discussion

In this study, we found that bone marrow biopsies from patients treated with ATRA/ATO for APL had unusual but characteristic features. The bone marrow of these patients was frequently hypercellular for age with an erythroid hyperplasia, increased megakaryocytes, and morphologic atypia in the erythroid and megakaryocytic lineages. In keeping with the morphologic abnormalities noted in the erythroid lineage, flow cytometric studies showed variable expression of CD71 on erythroid precursors in the majority of cases. Though we noted focal myeloid atypia in a minority of cases, significant morphologic atypia or abnormal myeloid maturation seen via flow cytometry were only identified in cases with residual APL.

Previously, our awareness of arsenic’s effect on bone marrow stemmed from small case series and reports following either treatment with ATO or, more commonly, as a result of arsenic intoxication and poisoning. One other group, Zhang et al,¹³ has reviewed the effect of ATO on the bone marrow. In contrast to Zhang et al,¹³ who noted a normo- to hypercellular marrow with a myeloid predominance in five patients treated with ATO, we found that our patients displayed hypercellular marrow with an erythroid predominance. Zhang and coworkers¹³ did not note significant dyserythropoiesis, which was a common finding both amongst our patients and those suffering arsenic intoxication. Further, in the myeloid lineage, Zhang et al¹³ found an altered appearance of the regenerating myeloid cells, whereas in our series, significant myeloid atypia tended to be associated with residual APL. Three out of the five patients treated with ATO in the Zhang et al¹³ series had poor clinical outcomes, whereas our ATRA/ATO patients have had uniformly positive outcomes to date. The differences between our findings may stem from differences in patient population. Zhang and coworkers¹³ reviewed biopsies from patients with APL who were relapsed after and/or refractory to ATRA and chemotherapy and subsequently treated with ATO monotherapy, whereas we reviewed patients treated with ATRA/ATO at disease presentation using the Lo-Coco et al³ regimen.

While several authors report bone marrow findings in arsenic intoxication similar to ours, there are others who present conflicting results. In agreement with our data, both Feussner et al¹⁴ and Westhof et al¹⁷ noted hypercellularity in their case reports of patients who presented with arsenic intoxication. However, in a series of four patients who underwent bone marrow evaluation, Kyle and Pease¹⁶ found that two were hypercellular, one normocellular, and one hypocellular. Additionally, Lerman et al¹⁵ and Rezuke et al¹⁸ noted normocellular marrows in their case reports of patients exposed to arsenic. Similar to our finding of a decreased M:E ratio, Kyle and Pease¹⁶ found a low M:E ratio in three of four patients in their series, and Feussner et al¹⁴ and Westhof et al¹⁷ also noted this finding. Interestingly, Lerman et al¹⁵ reported an M:E ratio of 5:1 in a case report.

Parallel to our study results, all authors report some degree of dyserythropoiesis, including megaloblastic maturation, basophilic stippling, nuclear membrane irregularities, binucleate forms, and karyorrhexis.^14-18 Only Rezuke et al¹⁸ reports finding dysplastic appearing megakaryocytes, which we noted frequently among our patients.¹⁸ In contrast to our study, three authors mention changes in the myeloid lineage, including megaloblastic features and dysplasia such as giant bands and metamyelocytes, hypersegmented neutrophils, and bizarre granulocyte segmentation.^14,17,18 While we found focal myeloid atypia in patients treated with ATRA/ATO, we did not identify significant myeloid dysplasia at therapeutic concentrations. The myeloid atypia in the two cases of patients with extensive residual disease is more likely to represent differentiation of the abnormal promyelocytes of APL rather than morphologic atypia induced in regenerative marrow by ATRA/ATO.

This study is limited by the timing of the biopsies, as the ATRA/ATO specimens were typically evaluated approximately 30 days after the initiation of treatment, as recommended in the Lo-Coco et al³ regimen. The biopsies from the ATRA/CTX group were, however, all performed at a later time point. It should be noted that the earliest biopsies (37 and 41 days posttherapy) in the ATRA/CTX group were both normocellular with a normal M:E ratio (both 2.5:1), normal numbers of megakaryocytes, and dysplasia in 10% or less of megakaryocytes and less than 10% of erythroid precursors. Thus, it is unlikely that the differences between the two groups can be explained simply by the difference in timing of the follow-up marrows.

While we identified several trends seen in the marrow biopsies of patients treated with therapeutic ATO, there was some variability, including patients who had morphologically identifiable residual disease. Additionally, numerous patients had evidence of residual disease on molecular or FISH studies. Interestingly, all ATRA/ATO patients have had uniformly favorable outcomes to date, suggesting that a bone marrow biopsy performed on approximately day 28 may not have significant predictive information with regard to disease progression or survival. Current National Comprehensive Cancer Network guidelines do not advocate molecular studies after induction due to high rates of positive results, which we encountered in our series.²¹ Some authors recommend molecular monitoring via PCR of the peripheral blood after consolidation as an adequate and less invasive surrogate for marrow evaluation.²²

This study represents the largest series to date that describes in detail the bone marrow findings in the setting of therapeutic doses of ATO for APL. Patients treated with ATRA/ATO have characteristic bone marrow findings that include hypercellularity, erythroid hyperplasia, increased megakaryocytes, and morphologic atypia mimicking dysplasia in the erythroid and megakaryocytic lineages. These findings represent treatment effect and should not be interpreted as an evolving or underlying myelodysplastic syndrome. In contrast, the finding of a myeloid predominance, significant myeloid atypia, or abnormal myeloid maturation on flow cytometric analysis may serve as an indicator of significant residual APL, which may help guide clinical management.