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Hematopathology / Pseudo–Pelger-Huët Anomaly Induced by Medications Pseudo–Pelger-Huët Anomaly Induced by Medications A Clinicopathologic Study in Comparison With Myelodysplastic Syndrome–Related Pseudo–Pelger-Huët Anomaly Endi Wang, MD, PhD,1 Elizabeth Boswell, MD,1 Imran Siddiqi, MD, PhD,2 Chuanyi Mark Lu, MD,3 Siby Sebastian, PhD,1 Catherine Rehder, PhD,1 and Qin Huang, MD, PhD4 Key Words: Pseudo–Pelger-Huët anomaly; Medications; Iatrogenic; Myelodysplastic syndrome; Neutrophilic dysplasia Upon completion of this activity you will be able to: • define the characteristics of iatrogenic pseudo–Pelger-Huët anomaly (PPHA). • distinguish iatrogenic PPHA from myeloid neoplasm–associated PPHA based on differences in associated clinicopathologic features. • list the common medications that have been reported to induce PPHA. Abstract Pseudo–Pelger-Huët anomaly (PPHA) has been documented in association with transplant medications and other drugs. This iatrogenic neutrophilic dysplasia is reversible with cessation or adjustment of medications but is frequently confused with myelodysplastic syndrome (MDS) based on the conventional concept that PPHA is a marker for dysplasia. We investigated the clinicopathologic features in iatrogenic PPHA and compared them with MDS-related PPHA. The 13 cases studied included 5 bone marrow/stem cell transplantations, 3 solid organ transplantations, 1 autoimmune disease, 3 chronic lymphocytic leukemias, and 1 breast carcinoma. For 12 cases, there was follow-up evaluation, and all demonstrated at least transient normalization of neutrophilic segmentation. All 9 cases of MDS demonstrated at least 2 of the following pathologic abnormalities on bone marrow biopsy: hypercellularity (8/9), morphologic dysplasia (8/9), clonal cytogenetic abnormality (7/9), and increased blasts (3/9), whereas these abnormalities were typically absent in iatrogenic PPHA. Iatrogenic PPHA displayed a higher proportion of circulating PPHA cells than in MDS (mean, 47.4%; SD, 31.6% vs mean, 12.3%; SD, 9.8; P < .01). A diagnostic algorithm is proposed in which isolated PPHA is indicative of transient or benign PPHA unless proven otherwise. The ASCP is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The ASCP designates this educational activity for a maximum of 1 AMA PRA Category 1 Credit ™ per article. This activity qualifies as an American Board of Pathology Maintenance of Certification Part II Self-Assessment Module. The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Questions appear on p 312. Exam is located at www.ascp.org/ajcpcme. Pelger-Huët anomaly (PHA) was first described by Karl Pelger in 19281 and was further defined as a benign trait with autosomal dominant inheritance by G.J. Huët in 1931.2 This hereditary anomaly is characterized by round, oval, peanutshaped, coffee bean–shaped, or symmetric bilobed nuclei with abnormally clumped chromatin in granulocytes. Despite these morphologic abnormalities, granulocyte function, including neutrophilic chemotaxis, phagocytosis, and cytolytic activity, remains normal, and people with hereditary PHA do not have increased susceptibility to bacterial infections.3 In contrast, pseudo–Pelger-Huët anomaly (PPHA) is an acquired alteration of neutrophils closely resembling hereditary PHA in morphologic features. While its presence in some myeloid neoplasms, such as myelodysplastic syndrome (MDS), is well-established and has important diagnostic implications,4-6 PPHA can also be induced by a variety of nonneoplastic etiologies as a transient or reversible phenomenon.7-20 Among the multiple other causes, increased use of transplant medications7-10 and other drugs11-17 has become a major etiology for this morphologic deviation. Because of the conventional concept that PPHA is a morphologic marker for myelodysplasia,4-6 the presence of iatrogenically induced PPHA in the peripheral blood and/or bone marrow may cause diagnostic confusion, particularly in patients with a history of treatment for malignant neoplasms and subsequent chronic cytopenias. As the diagnostic implications of iatrogenic PPHA vs MDS-associated PPHA are markedly divergent, accurate distinction of these processes is important. We compared peripheral blood and bone marrow findings in iatrogenic PPHA with MDS-associated PPHA to characterize and define features that can aid in distinguishing these entities. © American Society for Clinical Pathology Am J Clin Pathol 2011;135:291-303 291 DOI: 10.1309/AJCPVFY95MAOBKRS 291 291 CME/SAM DOI: 10.1309/AJCPVFY95MAOBKRS Wang et al / Pseudo–Pelger-Huët Anomaly Induced by Medications Materials and Methods Case Selection For the study, 13 cases of iatrogenic PPHA and 9 cases of MDS with PPHA were identified from our bone marrow biopsy databases using the search phrase “Pelger-Huët anomaly.” These included 14 cases (7 iatrogenic PPHAs and 7 MDS with PPHA) from Duke University Medical Center, Durham, NC; 3 cases (iatrogenic PPHAs) from University of California at San Francisco; 2 cases (MDSs with PPHA) from San Francisco Veteran Affairs Medical Center, San Francisco, CA: 2 cases (iatrogenic PPHAs) from City of Hope National Medical Center, Duarte, CA; and 1 case (iatrogenic PPHA) from USC Medical Center, Los Angeles, CA. The diagnoses of iatrogenic PPHA were confirmed by clinical history, laboratory tests, and/ or, ultimately, by resumption of normal segmentation in neutrophils or a significant change in proportion of PPHA cells occurring spontaneously or in correlation with dose adjustment of relevant medications (see later text). The diagnoses in 9 cases of MDS with PPHA were confirmed according to the 2008 World Health Organization classification.21 Cytomorphologic and Histologic Evaluation Peripheral blood smears were stained with Wright stain, bone marrow aspirate smears and biopsy touch imprints were stained with Wright-Giemsa, and bone marrow core biopsies and clot sections were stained with H&E. The cases were reviewed independently by 4 hematopathologists (E.W., I.S., C.M.L., and Q.H.). PPHA neutrophils were identified by their unilobed or symmetric bilobed nuclei, abnormally clumped chromatin, and relatively abundant cytoplasm with pink or yellowish granules. We examined 200 neutrophils in peripheral blood smears, and proportions of PPHA cells were calculated as percentage of total neutrophils. In addition, other morphologic dysplasia, bone marrow cellularity, and blast counts were also evaluated. The presence of other dysplastic changes in erythroid, granulocytic, and megakaryocytic lineages was determined according to the description in the 2008 World Health Organization Classification.21 Bone marrow cellularity was defined as hypercellular when it was higher than 1 SD above the age-adjusted mean, as hypocellular when lower than 1 SD, and as normocellular when within 1 SD.22 Conventional Cytogenetic Studies Cytogenetic analysis was performed on 2 to 4 mL of bone marrow aspirate from each case. Two cultures were initiated from each fresh, anticoagulated specimen in 292 292 Am J Clin Pathol 2011;135:291-303 DOI: 10.1309/AJCPVFY95MAOBKRS complete tissue culture medium. Cells were incubated for 24 and 48 hours without mitogen stimulation, except for the 2 CLL samples, in which both B cell–stimulated and B cell–unstimulated cultures were initiated and examined. Chromosome preparations including harvesting and GTWbanding were made using standard methods. Cytogenetic abnormalities were classified according to the International System for Human Cytogenetic Nomenclature. Bone Marrow Engraftment Analysis Highly purified DNA was extracted from the pretransplantation, donor, and posttransplantation samples following routine laboratory methods. For positive selection of lymphocytes or granulocytes from posttransplantation samples, magnetically labeled antihuman CD3 or CD15 antibodies (isotype, mouse IgG1 and IgM κ, respectively) and the RoboSep automated cell separator (StemCell Technologies, Vancouver, Canada) were used. The extracted sample genomic DNA was subjected to multiplexed polymerase chain reaction (PCR)-mediated amplification reaction targeting a total of 15 autosomal short tandem repeat (STRs) markers and 1 STR marker on the pseudoautosomal region of the X and Y chromosomes (PowerPlex 16 System, Promega, Madison, WI). Following PCR amplification, the fluorescently labeled PCR products were resolved by capillary electrophoresis on the ABI 3130xl Genetic Analyzer and analyzed by GeneMapper software (Applied Biosystems, Foster City, CA) to resolve the number of repeats and relative abundance of each repeat for each STR locus. These data were then used to calculate the percentage of donor and/or recipient cells in the posttransplantation sample using the donor and pretransplantation recipient STR profiles. Assessment of Resolution of PPHA Cells To assess for resolution of PPHA neutrophils or normalization of neutrophilic segmentation, manual differentials of WBC on peripheral blood smears were evaluated following the index bone marrow biopsy for each case. Resolution of PPHA cells was defined by the absence of PPHA cells on follow-up peripheral blood smears performed after the bone marrow biopsy. In cases in which PPHA cells were absent in multiple peripheral blood samples, the earlier time of resolution was recorded as the “time of resolution.” Statistical Analysis The statistical analyses were performed with SAS version 9 (SAS Institute, Cary, NC). The Student t test and Wilcoxon-Mann-Whitney test were used to test the statistical significance of differences in circulating PPHA cells between the groups. © American Society for Clinical Pathology Hematopathology / Original Article Results Clinical Information Of the 13 cases of iatrogenic PPHA, 5 were seen following allogeneic bone marrow or stem cell transplantation for acute myeloid leukemia (AML), including 2 de novo AMLs and 3 AMLs arising from preexisting MDS. Three cases occurred in the setting of chronic lymphocytic leukemia (CLL), 2 in liver transplant recipients (history of hepatocellular carcinoma and autoimmune hepatitis, respectively), 1 in a heart transplant recipient (history of cardiomyopathy), 1 in a case of autologous stem cell transplantation with treatment for breast carcinoma, and 1 in autoimmune disease treated with mycophenolate mofetil. Among these 13 cases, 9 were men and 4 were women. Ages ranged from 20 to 75 years, with a median of 57 years ❚Table 1❚. The 9 MDS cases with PPHA components included refractory anemia with excess blasts (3 cases), refractory anemia (2 cases), therapy-related MDS (2 cases), refractory cytopenia with multilineage dysplasia (1 case), and refractory anemia with ringed sideroblasts (1 case). Among the MDS cases, 6 were men and 3 were women. Ages ranged from 53 to 82 years with a median of 67 years ❚Table 2❚. Similar to the MDS cases, all patients with iatrogenic PPHA had anemia, neutropenia, and/or thrombocytopenia at the time of bone marrow biopsy, with the exception of 1 case (case 13; Table 1). In the iatrogenic PPHA cases, bone marrow biopsies were performed to rule out relapsed acute myeloid leukemia or high-risk MDS in 5 cases following bone marrow transplantation (cases 1-5), to rule out therapy-related myeloid neoplasms in 4 cases (cases 6-9), to assess disease status of CLL in 3 cases (cases 10-12), and to rule out myelodysplastic/myeloproliferative neoplasm in 1 case (case 13). Cytomorphologic and Histologic Evaluation Circulating PPHA cells in the iatrogenic group typically displayed hypolobated nuclei with clumped chromatin ❚Image 1A❚, ❚Image 1B❚, ❚Image 1C❚, ❚Image 1D❚, ❚Image 1E❚, ❚Image 2A❚, ❚Image 2B❚, ❚Image 2C❚, and ❚Image 2D❚. The majority of the cells had nuclear contours that were round (Images 1A and 2A) or oval (Images 1B and 2B), but coffee bean–shaped (Images 1C and 2C), peanut-shaped (Images 1D and 2D), bilobed (Image 1E), and occasional other forms were also noted. Some PPHA cells also contained detached round nuclear fragments in the cytoplasm, so called Howell-Jolly–like inclusions (data not shown). Eosinophils were unaffected, retaining their lobated morphologic features ❚Image 1F❚. The median proportion of circulating PPHA cells was 33% of neutrophils (range, 11%-94%; mean, 47.4%; SD, 31.6%) in the iatrogenic PPHA group, while in the MDS group, it was 9% (range, 2%-28%; mean, 12.3%; SD, 9.8%). The difference was statistically significant between the 2 groups (P < .01). In addition, PPHA cell nuclei in the iatrogenic group were more uniformly unilobed and contained homogeneously clumped chromatin; in comparison, those in the MDS group contained irregularly hypolobated nuclei, although their nuclear chromatin was clumped in a similar manner in some cases ❚Image 3A❚ and ❚Image 3B❚. On bone marrow examination, 11 of 13 iatrogenic cases displayed normal or decreased bone marrow cellularity; the remaining 2 were CLL cases that showed increased bone marrow cellularity due to infiltration by CLL-type leukemic cells ❚Image 2F❚. Although PPHA cells or their immediate precursors were noted ❚Image 1G❚ and ❚Image 2E❚, no significant morphologic dysplasia other than PPHA was appreciable, and blasts were not increased in the iatrogenic group. In contrast, among the 9 MDS cases, 8 (89%) showed mild to marked dysplasia other than PPHA ❚Image 3C❚, ❚Image 3D❚, and ❚Image 3E❚, 3 (33%) had increased blasts (Image 3E), and 8 (89%) demonstrated significantly increased bone marrow cellularity ❚Image 3F❚. Cytogenetic Studies Cytogenetic analyses were performed on bone marrow specimens from 10 iatrogenic PPHA cases. Of these, 9 showed a normal karyotype, while 1 CLL case demonstrated a complex cytogenetic abnormality in a mitogenstimulated culture, consistent with a B-cell clone from the underlying CLL. All 9 cases of MDS had conventional cytogenetic analysis performed, and 7 of 9 showed clonal cytogenetic abnormalities ❚Image 4❚ (case 7; Table 2), while the remaining 2 cases had a normal karyotype. Bone Marrow Engraftment Analysis Among the iatrogenic PPHA cases, 3 of 5 bone marrow or stem cell transplant recipients had concurrent engraftment studies performed, with 2 showing donor cell engraftment, confirming the donor origin of PPHA cells ❚Figure 1❚ (case 1; Table 1), and 1 demonstrating recipient origin, the latter representing an engraftment failure. Donor origin of bone marrow hematopoietic elements was also seen in 2 of 4 bone marrow or stem cell transplant cases by cytogenetic studies (as determined by discordance of sex chromosome in cases 2 and 5; Table 1). Clinical Follow-up and Resolution of PPHA in Peripheral Blood All but 1 case of iatrogenic PPHA had follow-up evaluation of peripheral blood smears. All cases evaluated showed evidence of normalized neutrophilic segmentation based on manual WBC differential or examination © American Society for Clinical Pathology Am J Clin Pathol 2011;135:291-303 293 DOI: 10.1309/AJCPVFY95MAOBKRS 293 293 Wang et al / Pseudo–Pelger-Huët Anomaly Induced by Medications ❚Table 1❚ Evaluation of PPH Anomaly Associated With Medications* CBC Case No./ Sex/Age (y) History Transplant Medications WBC Count Neutrophils Hemoglobin Platelets Blasts PPH Cells (× 109/L) (× 109/L) (g/L) (× 109/L) (%) (%) 1/M/67 AML/MDS Allo BM 1.4 0.74 129 112 0 94 2/M/20 MDS Allo BM 3.5 1.8 102 31 0 21 3/M/52 4/M/68 5/F/50 6/F/68 7/M/56 8/M/24 9/M/57 10/M/71 AML/MDS AML AML AI HCC Cirrhosis Cardiomyopathy CLL Allo BM Allo BM Allo BM — Liver Liver Heart — 2.0 4.6 10.6 0.5 3.5 1.4 3.3 2.3 0.81 3.13 9.33 0.25 2.37 0.77 1.2 0.92 108 111 91 112 89 93 92 118 22 62 105 0.9 170 93 320 93 0 0 0 0 0 0 0 0 33 29 17 11 90 64 19 85 11/M/75 12/M/72 13/F/41 CLL CLL Metastatic breast cancer — — Auto BM 13.9 0.9 22.2 4.17 ND 20.2 104 131 119 87 119 257 0 0 0 57 79 17 MMF; tacrolimus; acyclovir; posaconazole; others MMF; tacrolimus; ganciclovir; cotrimoxazole; itraconazole; others MMF; tacrolimus; G-CSF; others MMF; tacrolimus; others MMF; tacrolimus; others MMF; prednisone; others MMF; tacrolimus; others MMF; tacrolimus; ganciclovir; others MMF; tacrolimus; others Co-trimoxazole; ciprofloxacin; bendamustine; acyclovir; others Fludarabine; rituximab; others Fludarabine; rituximab; others GCSF; letrozole; citalopram; lorazepam; others AI, autoimmune disease; allo BM, allogeneic BM/hematopoietic stem cell; AML, acute myeloid leukemia; Auto, autologous hematopoietic stem cell; BM, bone marrow; CLL, chronic lymphocytic leukemia; GCSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; HCC, hepatocellular carcinoma; Hyper, hypercellular; Hypo, hypocellular; MDS, myelodysplastic syndrome; MMF, mycophenolate mofetil; NA, not applicable; ND, not done; Norm, normocellular; PPH, pseudo–Pelger-Huët (neutrophils). * Values for WBCs, neutrophils, hemoglobin, and platelets are given in Système International units; conversions to conventional units are as follows: WBCs and neutrophils (/μL), divide by 0.001; hemoglobin (g/dL), divide by 10.0; and platelets (× 103/μL), divide by 1.0. † Morphologic dysplasia other than PPH anomaly. ‡ In case 8, PPH anomaly cells decreased transiently at 13 weeks, then recurred at high levels during an episode of renal failure. § In case 11, PPH anomaly cells persisted in the peripheral blood smear 1 week after the index BM biopsy, and the patient was then lost to follow-up. of follow-up peripheral blood smears, including the case with bone marrow engraftment failure (case 3; Table 1). In 1 patient (case 8; Table 1), the decrease in PPHA cells was transient, decreasing from 64% at the time of bone marrow biopsy to 8% 13 weeks later, with subsequent return of high numbers of PPHA cells. The return of PPHA neutrophils in this patient was noted during an episode of acute renal failure (slow increase in PPHA cells with rising serum creatinine level). In the remaining iatrogenic PPHA cases evaluated, neutrophilic segmentation completely normalized ❚Image 1H❚. The time to resolution ranged from 3 to 29 weeks after the index bone marrow biopsy with a median of 8.5 weeks. Discussion PPHA is an acquired alteration of neutrophils with morphologic features resembling hereditary PHA. As in the hereditary form, PPHA is characterized by neutrophils with abnormally condensed chromatin and hypolobated nuclei, which can be round, oval, peanut-shaped, coffee bean–shaped, or symmetrically bilobed. PPHA can be seen in 2 major categories of acquired granulocytic changes. 294 294 Am J Clin Pathol 2011;135:291-303 DOI: 10.1309/AJCPVFY95MAOBKRS First, it can occur in myeloid neoplasms such as MDS. In these cases, PPHA is considered a component of the malignancy,4-6 and the change persists or progresses without treatment of the underlying myeloid neoplasm. The second category of PPHA occurs in association with various infections18-20 or can be induced by certain medications.7-17 The changes in the latter category are reversible following recovery from the underlying conditions or after adjustment or discontinuation of culpable medications. Because of the conventional acceptance that PPHA is a marker for myelodysplasia,4-6 the presence of iatrogenic PPHA in the peripheral blood or bone marrow, particularly in patients with anemia, neutropenia, and/or thrombocytopenia, often causes confusion because it suggests MDS or a related myeloid neoplasm. This diagnostic confusion becomes even more critical in patients after bone marrow or hematopoietic stem cell transplantation for MDS or AML because the presence of PPHA may suggest relapsed disease or a therapy-related myeloid neoplasm. In this case series, all patients with medication-related (iatrogenic) PPHA underwent bone marrow biopsies owing to anemia, neutropenia and/or thrombocytopenia, or pancytopenia. In all of these cases, except for the 3 CLL cases, MDS or a related myeloid neoplasm was initially considered © American Society for Clinical Pathology Hematopathology / Original Article BM Blasts CytoCellularity (%) Dysplasia† genetics Resolution/ Time Engraftment to Resolution (wk) Hypo <5 No 46,XY Donor Yes/29 Hypo <5 No 46,XX Donor Yes/4 Hypo Hypo Norm Norm Norm Norm Hypo Hyper <5 <5 <5 <5 <5 <5 <5 <5 No No No No No No No No ND 46,XY 46,XY ND 46,XY 46,XY 47,XX ND Recipient ND Donor NA NA NA NA NA Yes/6 Yes/10.5 Yes/10 Yes/3 Yes/22 Yes/13‡ Yes/9 Yes/9 Hyper Norm Norm <5 <5 <5 No No No Complex NA 46,XY NA 46,XX NA A C ND§ Yes/4.5 Yes/8 in the differential diagnosis for the cytopenias. Furthermore, a diagnosis of MDS or related myeloid neoplasm was made or suggested in the diagnostic comment when PPHA was identified in peripheral blood and bone marrow aspirate smears. A typical diagnostic error is exemplified by case 7 in the series (Table 1). Briefly, this 56-year-old man developed chronic anemia 2 years after liver transplantation for hepatocellular carcinoma. Routine peripheral blood smear review revealed numerous PPHA cells, which were also noted in the subsequent bone marrow biopsy. Considering the concomitant peripheral monocytosis and clinical history, a diagnosis of “chronic myelomonocytic leukemia, probably therapy-related” was suggested in the pathology report, even though neither additional dysplastic changes nor increased blasts were identified and conventional cytogenetic analysis showed a normal male karyotype. Fortunately, the treatment was held owing to other medical issues. A follow-up peripheral blood smear demonstrated B ❚Image 1❚ (Case 1, Table 1) Reversible pseudo–Pelger-Huët anomaly (PPHA) in a hematopoietic stem cell transplant recipient. Note the hypolobated neutrophils with abnormally clumped chromatin and round (A), oval (B), and coffee bean–like (C). © American Society for Clinical Pathology Am J Clin Pathol 2011;135:291-303 295 DOI: 10.1309/AJCPVFY95MAOBKRS 295 295 Wang et al / Pseudo–Pelger-Huët Anomaly Induced by Medications D E F G H ❚Image 1❚ (cont) Peanut-like (D), and bilobed (E) nuclear contours in peripheral blood; an eosinophil with normal segmentation in peripheral blood (F); PPHA cell precursors (G, right field); and a few normal-appearing erythroid normoblasts (G, left field) on the bone marrow touch imprint and eventually normalized neutrophilic segmentations in peripheral blood (H) (A-F and H, Wright, ×1,000; G, WrightGiemsa, ×1,000). 296 296 Am J Clin Pathol 2011;135:291-303 DOI: 10.1309/AJCPVFY95MAOBKRS © American Society for Clinical Pathology Hematopathology / Original Article A B C D E F ❚Image 2❚ (Case 10, Table 1) Reversible pseudo–Pelger-Huët anomaly (PPHA) in a patient with chronic lymphocytic leukemia (CLL). Note the hypolobated neutrophils with abnormally clumped chromatin and unilobed nuclear contours in peripheral blood (A, B, C, and D, round, oval, coffee bean–like, and peanut-like nuclear shapes, respectively); 1 unilobed neutrophil or PPHA cell precursor near the center of the image in a background of CLL cells on marrow aspirate smear (E); and CLL cell infiltrate (lower field) in bone marrow on bone marrow biopsy section (F) (A-D, Wright, ×1,000; E, Wright-Giemsa, ×1,000; F, H&E, ×200). © American Society for Clinical Pathology Am J Clin Pathol 2011;135:291-303 297 DOI: 10.1309/AJCPVFY95MAOBKRS 297 297 Wang et al / Pseudo–Pelger-Huët Anomaly Induced by Medications A B C D E F Image 3 (Case 1, Table 2) Pseudo–Pelger-Huët anomaly (PPHA) and associated other pathologic abnormalities in a patient with myelodysplastic syndrome (MDS). A and B, Note the hypolobated neutrophils with abnormally clumped chromatin and twisted or irregular nuclear contours in peripheral blood (A and B, Wright, ×1,000). C, D, and E, Many PPHA cells in the marrow aspirate smear (C-E), markedly dysplastic erythroid normoblasts (C-E, arrows), 1 dysplastic megakaryocyte (micromegakaryocyte) (D, arrowhead), and increased blasts (E, arrowheads) in aspirate smear (C-E, Wright-Giemsa, ×1,000). F, Hypercellular bone marrow with increased immature cells on bone marrow biopsy section (H&E, ×200). 298 298 Am J Clin Pathol 2011;135:291-303 DOI: 10.1309/AJCPVFY95MAOBKRS © American Society for Clinical Pathology Hematopathology / Original Article normalized neutrophilic segmentation a few months later, and, thus, the diagnosis was corrected to “reversible PPHA, probably related to transplant medications.” Clearly, recognition of the benign nature of iatrogenic PPHA in these cases is crucial to avoid unnecessary diagnostic intervention and medical treatment. Clinicopathologic studies comparing the peripheral blood and bone marrow findings in benign vs neoplastic PPHA are lacking in the literature. We investigated the features of 13 cases of iatrogenic PPHA and compared them with 9 cases of MDS-related PPHA. In contrast with MDS, we found that iatrogenic PPHA tends to have a higher proportion of circulating PPHA neutrophils (median, 33% vs 9%; mean, 47.4% vs 12.3%; P < .01), which show more homogeneous unilobed nuclei. The proportion of PPHA cells in the iatrogenic group is within the ranges previously reported in the literature,7,8,11 while that for the MDS group seems to be higher than what has been described in the literature,6,23 likely owing to a selection bias in this subset of MDS. However, the quantitative differences in PPHA cells between the iatrogenic and MDS groups is not as obvious in the bone marrow aspirate smear as in peripheral blood, which may be explained by intramedullary destruction (apoptosis) of neoplastic myeloid elements in MDS.23-25 As the timing of peripheral blood and bone marrow analyses can be quite variable in iatrogenic PPHA cases, a subset of these cases might be evaluated at periods beyond the peak of PPHA cell formation, eg, due to waning drug effects, and, thus, the proportion of circulating PPHA cells could be relatively low, as was seen in cases 2, 5, 6, and 13 of the iatrogenic group (Table 1). In these cases, the number of PPHA cells overlapped with those in the MDS group. Therefore, the proportion of circulating PPHA cells ❚Image 4❚ (Case 7, Table 2) Clonal cytogenetic abnormality detected by chromosomal karyotyping. Representative karyotype showing an apparently balanced translocation between the X chromosome and chromosome 5. The International System for Human Cytogenetic Nomenclature describing this clonal abnormality is as follows: 46,X,t(X;5) (q13;q32)[10]/46,XX[11]. In this case, mild dysplasia and hypercellularity were noted by morphologic examination of the bone marrow biopsy specimen, and the presence of a clonal cytogenetic abnormality confirmed the diagnosis of myelodysplastic syndrome. should be used with caution when distinguishing between iatrogenic PPHA and MDS. Of interest, the MDS cases in this study displayed highgrade features with increased blasts (3/9) or a high frequency of clonal cytogenetic abnormalities (7/9). In particular, all 6 ❚Table 2❚ Evaluation of MDS With Component of PPH Anomaly* CBC Marrow Case No./ Sex/Age (y) History WBCs Neutrophils Hemoglobin Platelets Blasts PPH Blasts (× 109/L) (× 109/L) (g/L) (× 109/L) (%) Cells (%) Cellularity (%) Dysplasia† Cytogenetics Classification 1/M/75 2/F/61 3/M/53 4/M/67 5/M/63 6/F/61 7/F/80 8/M/82 Cytopenia Anemia Anemia FL Cytopenia Anemia Anemia Anemia 2.5 6.0 3.5 5.6 4.8 6.0 8.3 8.5 0.52 NA 2.24 2.62 2.1 ND ND 5.36 100 69 116 126 106 99 78 90 59 143 229 88 71 210 145 291 — — — — — 1 — — 24 3 4 9 18 5 2 18 Hyper Hyper Norm Hyper Hyper Hyper Hyper Hyper 7 8 <5 <5 <5 12 <5 <5 46,XY Complex del(5q),+21 Complex del(3p) 46,XX t(X;5) del(11q) RAEB RAEB RCMD T-MDS RA RAEB RA RARS 9/M/81 CA 4.7 2.63 85 137 1 28 Hyper <5 Complex T-MDS Marked Marked Moderate Moderate No Mild Mild Mild/ moderate Marked CA, prostatic carcinoma; FL, follicular lymphoma; Hyper, hypercellular; MDS, myelodysplastic syndrome; Norm, normocellular; PPH, pseudo–Pelger-Huët; RA, refractory anemia; RAEB, RA with excess blasts; RARS, RA with ringed sideroblasts; RCMD, refractory cytopenia with multilineage dysplasia; T-MDS, therapy-related MDS. * Values for WBCs, neutrophils, hemoglobin, and platelets are given in Système International units; conversions to conventional units are as follows: WBCs and neutrophils (/μL), divide by 0.001; hemoglobin (g/dL), divide by 10.0; and platelets (× 103/μL), divide by 1.0. † Morphologic dysplasia other than PPH anomaly. © American Society for Clinical Pathology Am J Clin Pathol 2011;135:291-303 299 DOI: 10.1309/AJCPVFY95MAOBKRS 299 299 Wang et al / Pseudo–Pelger-Huët Anomaly Induced by Medications A AM… vWA 100 1,000 800 600 400 200 0 X D8S1179 140 14 180 TPOX 220 18 FGA 260 14 300 8 340 12 20 22 Y B AM… vWA 100 1,000 800 600 400 200 0 X D8S1179 140 180 18 TPOX 220 260 14 Y FGA 300 340 8 16 22 25 10 C AM… vWA 100 1,000 800 600 400 200 0 X D8S1179 140 180 TPOX 220 18 260 14 Y 8 16 FGA 300 340 22 10 25 ❚Figure 1❚ (Case 1, Table 1) Bone marrow engraftment study demonstrating donor cell engraftment in a hematopoietic stem cell recipient when 94% pseudo–Pelger-Huët anomaly cells were identified in the peripheral blood. Short tandem repeat (STR) genotype profile of pretransplantation/recipient (A), donor (B), and posttransplantation CD15+ cells (C). The letters and numbers in the boxes above each block indicate the names of STR markers, and the numbers below indicate the numbers of repeats for each STR marker. Note a complete match of 4 STR loci between donor’s (B) and posttransplant recipient’s CD15+ cells (C). AM indicates the amelogenin gene, which is used here for sex determination and DNA quality control. The X chromosome gene, AMELX, gives rise to a 106 base pair amplicon (indicated by “X” in the box) and the Y chromosome gene, AMELY, a 112 base pair amplicon (indicated by “Y” in the box). cases with morphologically low- to intermediate-risk MDS (cases 3, 4, 5, 7, 8, and 9; Table 2) had clonal cytogenetic abnormalities detected. Of 7 cases with clonal cytogenetic abnormalities, 3 were in a poor prognostic category and 4 were in an intermediate category according to the International Prognostic Scoring System for MDS.21 All but 1 case showed 300 300 Am J Clin Pathol 2011;135:291-303 DOI: 10.1309/AJCPVFY95MAOBKRS morphologic dysplasia other than PPHA. All cases demonstrated at least 2 additional bone marrow abnormalities that were diagnostic or suggestive of MDS (Table 2), whereas none of these additional pathologic features were present in the iatrogenic PPHAs except for 2 CLL cases (hypercellularity in case 10 and hypercellularity/complex cytogenetic abnormality in case 11; Table 1) in which the abnormalities were apparently related to the underlying CLL. Thus, our analysis shows a tendency for clustered pathologic abnormalities in MDS-related PPHA, which can aid in the distinction from iatrogenic PPHA. Nevertheless, our selective subset of MDS cases shows pathologic abnormalities more frequent than those in MDS in general.26,27 This selection bias may be explained by the fact that PPHA represents a severe degree of dysplasia in cases of MDS, and, thus, its presence tends to be associated with other pathologic abnormalities related to MDS. Based on this comparative study, we propose a diagnostic algorithm for workup of cases in which PPHA cells are identified in peripheral blood or bone marrow aspirate smears ❚Figure 2❚. In this algorithm, acquired or pseudo-PHA (PPHA) is suggested if the patient had normal neutrophilic segmentation in the past. PPHA with normal peripheral blood cell counts would suggest a reversible (benign) change, particularly in organ and bone marrow transplant recipients, or in patients currently taking medications known to induce PPHA and with a high proportion of PPHA cells. Otherwise, MDS or a related myeloid neoplasm should be considered and should be excluded by additional tests. In cases of bone marrow or hematopoietic stem cell transplantation, donor engraftment would suggest a donor origin of PPHA, which is likely to be reversible or benign but still needs to be confirmed by the absence of other pathologic abnormalities diagnostic or suggestive of a myeloid neoplasm. The optimal evidence of the benign nature of iatrogenic PPHA would be spontaneous resolution of PPHA cells, normalization of neutrophilic segmentation, or altered proportions of PPHA cells in correlation with dose adjustment of relevant medications. An isolated PPHA (without other pathologic abnormalities related to MDS or other myeloid neoplasms), particularly with high proportions of PPHA cells (typically >30%) escalating within a short time, is indicative of benign or reversible PPHA unless proven otherwise. A fairly extensive number of drugs has been associated with the occurrence of PPHA ❚Table 3❚. Of note, along with the rise in organ and hematopoietic stem cell transplantation in the past decade, cases of iatrogenic PPHA have become more frequent in hematology clinics and/or clinical laboratories, with some resulting in bone marrow biopsies to exclude MDS or related myeloid neoplasms. PPHA in transplant recipients has been related to 2 immunosuppressive drugs, mycophenolate mofetil7-9 and tacrolimus.7,10 Indeed, of the 13 iatrogenic PPHA cases in our series, 9 (69%) were treated © American Society for Clinical Pathology Hematopathology / Original Article with mycophenolate mofetil, and 8 of these patients received tacrolimus as well. Concomitant use of the antiviral drug gancyclovir9 or antifungal drug fluconazole10 has also been suggested to have a role in development of PPHA in transplant recipients, probably via drug-drug interactions or altered enzymatic activities involving the metabolic pathway of transplant medications. In all reported cases, the morphologic changes in neutrophils appear reversible because PPHA cells are decreased following dose adjustment of transplant medications or disappear after discontinuation of the relevant drugs.7-10 Generally, in transplant recipients, a resolution of neutrophilic abnormality occurs 2 to 6 weeks after adjustment or cessation of the relevant medications.7,10 In our series, 2 of 9 patients treated with transplant medications (cases 1 and 7 among cases 1-9, including case 6 of autoimmune disease treated with mycophenolate mofetil) resumed normal neutrophilic segmentation following dose adjustment of immunosuppressive drugs. The remaining 7 cases showed spontaneous resolution of PPHA cells without change of relevant medications, which may be explained by a drug desensitization or tolerance. This desensitization might not be permanent, as illustrated in case 8, in which following a transient and spontaneous decrease in PPHA cells, a recurrence of these cells to high numbers was noted during an episode of renal failure, perhaps related to increased serum drug levels due to lack of renal excretion. Why the 2 cases with changes of PPHA related to dose adjustment of medications had longer intervals for the resolution is unclear, but it may be due to failure to desensitize and the persistence of PPHA until adjustment of medications for other medical issues. Of 3 CLL cases, 1 patient (case 10) was receiving co-trimoxazole PHA No Prior normal segmentation? Work up for hereditary PHA Yes Acquired/ PPHA Cytopenia? No Yes No Transplant/other relevant medications? Possible MDS Yes No Yes BMT? Cytogenetics? No Yes No Engraftment? Yes Possible reversible PPHA Increased blasts? Needs confirming Yes No Other dysplasia? Yes No Confirmed Spontaneous decrease in PHA No ❚Figure 2❚ Diagnostic algorithm for pseudo–Pelger-Huët anomaly (PPHA) identified in a peripheral blood smear or/ and bone marrow aspirate smear. BMT, bone marrow transplantation; MDS, myelodysplastic syndrome; PPHA, Pelger-Huët anomaly. ❚Table 3❚ Medications Associated With Pseudo–Pelger-Huët Anomaly as Reported in the Literature Drug Trade or Other Name Pharmacologic Action Indication Effect Reference Mycophenolate mofetil Tacrolimus CellCept Immunosuppression Organ transplantation D Prograf; FK-506 Immunosuppression Organ transplantation D/I Valproate Depakote, Depacon or Stavzor Gantrisin; sulfafurazole Cytovene; Cymevene Diflucan; Trican Advil; Motrin; Nurofen Taxol Taxotere Neupogen Sargramostim; Leukine; Leucomax Colcrys Cuprimine; Depen Inhibition of GABA transaminase Antibiotic Antiviral DNA polymerase Antifungal cytochrome P450 Inhibition of cyclooxygenase Antimitosis Antimitosis Growth factor Growth factor Bipolar disorders, seizures Bacterial infection Viral infection Fungal infection Rheumatoid conditions; pain Cancer Cancer Neutropenia Leukopenia D Etzell and Wang,7 Asmis et al,8 Kennedy et al9 Etzell and Wang,7 Gondo et al10 May and Sunder15 D I I D D D D D Kaplan and Barrett14 Kennedy et al9 Gondo et al10 Moreira et al13 Juneja et al11 Juneja et al11 Teshima et al12 Teshima et al12 Antimitosis Immunosuppression Gout Rheumatoid conditions D D Clevenger et al17 Levin16 Sulfisoxazole Ganciclovir Fluconazole Ibuprofen Paclitaxel Docetaxel G-CSF (filgrastim) GM-CSF (molgramostim) Colchicine D-penicillamine D, direct; GABA, γ-aminobutyric acid; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-monocyte colony-stimulating factor; I, indirect or via drug-drug interaction. © American Society for Clinical Pathology Am J Clin Pathol 2011;135:291-303 301 DOI: 10.1309/AJCPVFY95MAOBKRS 301 301 Wang et al / Pseudo–Pelger-Huët Anomaly Induced by Medications bendamustine, ciprofloxacin, and other drugs, while the other 2 patients (cases 11 and 12) were treated with fludarabine and rituximab, in addition to several other drugs. While the sulfonamide component sulfamethoxazole in Bactrim may have a role in the formation of PPHA in the former (case 10), concomitant use of other medications complicates the analysis of the causative factors. No drug listed in Table 3 was noted to be used in the other 2 cases of CLL (cases 11 and 12). Although both patients were taking fludarabine and rituximab, suggesting possible causality, PPHA has been described historically in CLL, even before these 2 drugs were introduced.28,29 Clinical observations of change in neutrophilic segmentation in correlation with dose adjustment of certain drugs should be able to identify the causative regimens in these cases. Case 13 was a patient who was administered granulocyte colonystimulating factor (a drug listed in Table 3) at the time when circulating PPHA was noted in peripheral blood and aspirate smears. Owing to the complexity of numerous medications in each case (ranging from 10 to 26 drugs when PPHA was identified in the peripheral blood), it is difficult to ascertain the exact role of individual drug(s) or which one might be essential for the development of PPHA in our cases. Actually, in a clinical setting, it would not be practical to allow a complete cessation or even a dose reduction of relevant medications to test the causative effect strictly for investigative purposes owing to the risk of losing grafts or other potential complications. Therefore, pathologic exclusion of MDS or related myeloid neoplasms has a central role in defining the benign nature of iatrogenic PPHA in these cases. The underlying mechanism of PPHA induced by medications is unclear. Hoffmann et al30 discovered the linkage of hereditary PHA to the lamin B-receptor (LBR) gene located on the long arm of chromosome 1 (1q41-43) by using microsatellite-based genetic linkage analysis. They also found a gene dose-dependent reduction of LBR protein, the quantity of which is inversely correlated with severity of neutrophilic hypolobation or hyposegmentation. LBR is an integral protein component of the inner nuclear membrane and seems to interact with lamin B and heterochromatin to affect nuclear lobation. While the reversible nature of PPHA induced by medications does not suggest a permanent change or mutation of the LBR gene, certain drugs may have a role in down-regulation of LBR gene expression or may interact directly with LBR protein to block its function. In addition, future investigations regarding the mutation status of the LBR gene in cases of PPHA secondary to MDS or related myeloid neoplasms might provide an additional genetic marker for diagnosis of these neoplasms, which may also enable a more definitive distinction between myeloid neoplasia–related and iatrogenic PPHA. 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