Original Articles

Morphologic Examination of Sequential Bone Marrow Biopsies after Nonmyeloablative Stem Cell Transplantation Complements Molecular Studies of Donor Engraftment Anand S. Lagoo, MD, PhD; Jerald Z. Gong, MD; Timothy T. Stenzel, MD, PhD; Barbara K. Goodman, PhD; Patrick J. Buckley, MD, PhD; Nelson J. Chao, MD, PhD, MBA; Cristina Gasparetto, MD; Gwynn D. Long, MD; David A. Rizzieri, MD

● Context.—Nonmyeloablative stem cell transplantation (NMSCT) is a mode of immunotherapy increasingly employed in treating hematologic, lymphoid, and solid tumors. Patients are monitored principally by molecular analysis of donor engraftment. Objective.—To determine the role of morphologic examination of bone marrow after NMSCT. Design.—Seventy-three patients undergoing NMSCT under the Campath 1H (humanized anti-CD52 antibody) protocol were studied. Pretransplant and sequential posttransplant bone marrow specimens were evaluated and the findings were correlated with corresponding engraftment data. Results.—Pretransplant bone marrow specimens from 43% of the patients were involved by disease, and these marrow specimens were significantly more cellular than those that were free of disease. Morphologically detectable disease was still present in day 14 posttransplant marrow specimens in more than one half of these patients, but there was no difference in engraftment in those with or

without marrow disease. Early posttransplant marrow in nearly one half of the patients showed myeloid hyperplasia and atypical localization of immature myeloid precursors. Marrow cellularity for the first 2 months after NMSCT was significantly lower in those patients receiving stem cells mismatched at 1 to 3 loci as compared with those who received fully matched grafts (mean cellularity, 38.1% vs 54.1% at day 14). Marrow failure without recurrent disease at 3 to 6 months after transplant was detected by engraftment study in only approximately 15% of cases. Similarly, early recurrence of disease was detected first by morphologic examination in 4 of 13 cases before a decline in donor engraftment occurred. Conclusion.—Morphologic examination of bone marrow provides additional information that is complementary to donor engraftment analysis for optimal management after NMSCT. (Arch Pathol Lab Med. 2006;130:1479–1488)

A

largely the result of a graft-versus-tumor effect1–3 has led to wider use of reduced intensity or nonmyeloablative conditioning in preparation for allogeneic SCT during the past few years.4–13 Nonmyeloablative allogeneic stem cell transplantation (NMSCT) is also the preferred method for treating relatively indolent diseases in which the risks associated with a conventional myeloablative transplant would be considered unacceptable14–16 as well as in some cases of failed autografts.17,18 A recent study comparing myeloablative and nonmyeloablative SCT in patients more than 50 years of age19 found that the overall outcome of the NMSCT approach was at least as good as that of the myeloablative approach. Successful engraftment after conventional myeloablative conditioning is indicated by recovery of peripheral blood counts, which is accompanied by normal bone marrow morphology.20 Engraftment of donor cells and donor-derived effective hematopoiesis has been documented by cytogenetic analysis in sex-mismatched donors21 or by the use of genetically determined isoenzyme patterns.22,23 For more than a decade, fluorescent in situ hybridization has largely replaced conventional cytogenetics as a method for

llogeneic stem cell transplantation (SCT) or bone marrow transplantation is a potentially curative procedure for patients with malignant and nonmalignant hematologic conditions, and appears to have at least a palliative role in controlling nonhematologic malignancies as well. It provides an alternative to the conventional, myeloablative conditioning regimen with high-dose chemotherapy and total-body radiation, which is poorly tolerated by older patients, those receiving second transplants, and those with comorbid conditions. The realization that the curative potential of any stem cell transplantation was Accepted for publication March 24, 2006. From the Departments of Pathology (Drs Lagoo, Gong, Stenzel, Goodman, and Buckley) and Medicine (Drs Chao, Gasparetto, Long, and Rizzieri), Duke University Medical Center, Durham, NC. Dr Stenzel is currently medical director at Abbott Molecular, Des Plaines, Ill. Dr Rizzieri is on the speaker’s bureau for Berlex Laboratories. All other authors have no relevant financial interest in the products or companies described in this article. Reprints: Anand Shreeram Lagoo, MD, PhD, Associate Professor of Pathology, Department of Pathology, Duke University Medical Center, Box 3712 Durham, NC 27710 (e-mail: [email protected]). Arch Pathol Lab Med—Vol 130, October 2006

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determining donor cell engraftment in sex-mismatched transplants,24 and molecular methods based on DNA heterogeneity in donor and recipient have replaced the biochemical methods of isoenzyme determination.25 Periodic engraftment analysis to monitor patients is recommended by the American Society for Blood and Marrow Transplantation.26 Such analysis is useful to monitor early graft failure27 and possibly for early detection of disease recurrence,28,29 which may help in the timely administration of additional immunotherapy.30 In NMSCT, recipient hematopoiesis is preserved to a variable extent. Therefore, recovery of blood cell counts or bone marrow cellularity may not parallel engraftment status. It is recommended that molecular studies for engraftment be carried out more frequently in these patients because early patterns of chimerism may predict either graft-versus-host disease or graft loss.26 Because most emphasis is placed on the evaluation of molecular studies of engraftment when following patients after NMSCT, the role of morphologic examination of the bone marrow has not been systematically addressed. Morphologic studies of the bone marrow after conventional bone marrow transplantation have been published,31–40 but such studies after NMSCT are lacking. We evaluated the serial bone marrow specimens obtained after NMSCT and compared the findings with the engraftment studies. We found that morphologic examination yields information about persistent disease, early marrow failure, and recurrence of disease in some cases that are not apparent from the engraftment studies alone. These findings indicate that morphologic examination of bone marrow should remain an integral part of periodic assessment of patients who have undergone NMSCT. MATERIALS AND METHODS Patients We studied 73 consecutive patients who underwent NMSCT for hematologic, lymphoid, or solid tumors from December 1999 to May 2002 at Duke University Medical Center, Durham, SC. Thirty-two patients had hematologic malignancies (14 cases of acute myeloid leukemia, 8 cases of myelodysplastic syndrome, 4 chronic myeloproliferative disorders, 3 cases of plasma cell myeloma, 2 cases of acute lymphoblastic leukemia, and 1 case of chronic lymphocytic leukemia), 19 had lymphomas (9 cases of diffuse large B-cell lymphoma, 4 cases of mantle cell lymphoma, 3 cases of Hodgkin lymphoma, 2 cases of follicular lymphoma, and 1 case of small lymphocytic lymphoma), and 22 had metastatic solid tumors (14 cases of breast carcinoma, 6 cases of renal cell carcinoma, and 2 melanomas).

Nonmyeloablative Conditioning After obtaining institutional review board approval and informed consent, selected patients were treated with a nonmyeloablative conditioning regimen consisting of fludarabine 30 mg/ m2 and cyclophosphamide 500 mg/m2 for 4 days. For prophylaxis of acute graft-versus-host disease, patients received 5 daily infusions of a humanized anti-CD52 antibody, Campath 1H, at 20 mg/d in 250 mL of D5 normal saline or normal saline that was infused during 3 hours.41

stimulating factor with a goal of infusing at least 5 ⫻ 106 (preferably 10–15 ⫻ 106) CD34⫹ cells per kilogram of donor body weight. The T-cell depletion in the concentrated mononuclear cells was achieved with 20 mg of the anti-CD52 antibody, Campath 1H. The patients received the T-cell–depleted stem cells in 3 or more daily infusions.

Bone Marrow Examination Serial bone marrow biopsies and bone marrow aspirates were performed pretransplant and 14 days, 1 month, 2 months, 3 months, 6 months posttransplant, and later if appropriate. The bone marrow biopsies were examined after staining with hematoxylin-eosin and reticulin. The bone marrow aspirates were examined after staining with Wright stain. A 200-cell differential was performed on the aspirate smears. The smears were examined by 1 of the 3 hematopathologists (A.S.L., J.Z.G., P.J.B.) for evidence of dysplasia or other hematologic disease, as appropriate. Storage iron was evaluated from smears stained with the Prussian blue stain. The core biopsies and clot sections were evaluated for marrow cellularity, myeloid and erythroid ratio, distribution of the hematopoietic cells, number of megakaryocytes, presence of granulomas, lymphoid aggregates, or metastatic disease, edema, or other abnormality of the stroma, and reticulin fibrosis. The semiquantitative findings were normalized among the 3 observers through a biweekly quality assurance conference. When required, additional immunohistochemical stains and/or flow cytometry examinations were performed.

Assessment of Donor Engraftment Level of engraftment was quantitated by polymerase chain reaction of short tandem repeats or fluorescent in situ hybridization for sex chromosomes. The short tandem repeat analysis was performed as previously described,43 with minor modifications. Briefly, genomic DNA was isolated from the peripheral blood or bone marrow samples using the PureGene system (Gentra Systems, Minneapolis, Minn). The PowerPlex 1.2 system (Promega, Madison, Wis), was used to amplify genomic DNA following the manufacturer’s directions. Amplifications were set up using 2 ng of input DNA for pretransplant, donor, and posttransplant samples, and 100 ng for each posttransplant sample as well. Use of 100 ng of input DNA increased the sensitivity of the assay so that it could routinely detect 1% mixtures. Amplified products were detected by using an Applied Biosystems 310 capillary genetic analyzer (Foster City, Calif) with a 47 cm ⫻ 50 mm capillary, and performance-optimized polymer 4. Allele designations were determined with the PowerPlex 1.2 file template using Genotyper Software (Applied Biosystems). The peak areas of donor alleles were divided by the total peak areas for the donor and recipient alleles to determine the donor percentage in each sample. Dual-color interphase fluorescent in situ hybridization analysis was performed using the X centromere and Y satellite III probe set (DXZ1, DYZ1, Abbott Molecular, Inc, Des Plaines, Ill) to detect the percentage of male (XY) cells and/or female (XX) cells present in each posttransplant total sample of 200 interphase nuclei.

Statistical Analysis The median and mean ⫾ standard deviation were calculated; an unpaired, one-tailed Student t test was applied (unless indicated otherwise) using the Microsoft Excel (Microsoft Corp, Redmond, Wash) software.

Details of donor selection, protocol for T-cell depletion, and method of stem cell infusion were previously described elsewhere.42 Both HLA-matched sibling donors and unrelated donors who had up to 3 (of a possible 6) locus HLA mismatches were used. Donor peripheral blood stem cells were harvested by priming the donors with 3 to 5 daily injections of granulocyte colony

RESULTS Findings in Pretransplant Marrow Pretransplant bone marrow was available in 68 of 73 patients. These specimens were obtained an average of 28 days (median, 14 days; range, 6–367) before the transplant. In 8 patients, the pretransplant biopsy specimen available for examination was obtained more than 30 days before transplantation. In 4 of these 8 patients, interval chemo-

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Harvest, Treatment, and Infusion of Donor Stem Cells

Table 1. Disease Status of Pretransplant and Day 14 Posttransplant Bone Marrows Pretransplant Marrow With Disease

Day 14 Marrow

Category

Diagnosis*

No Disease

No Marrow Available

No Disease

With Disease

Hematologic

AML T-ALL MDS CML Other MPD Plasma cell myeloma CLL Total hematologic

7 2 0 0 0 1 0 10

6 0 9 1 2 2 1 21

0 0 0 1 0 0 0 1

10 1 5 0 0 1 ⫹ 1‡ 1 19

3 1† 4 2 2 1 0 13

Total

13 2 9 2 2 3 1 32

Lymphomas

DLBCL MCL Refractory B-NHL SLL B-LBL FCL Hodgkin Total lymphoma

4 2 3 1 1 1 1 13

0 0 0 0 0 1 0 1

1 2 1 0 0 0 1 5

4 4 2 ⫹ 1† ⫹ 1§ 1 1 2 2 17 ⫹ 1§

1 0 0 0 0 0 0 1

5 4 4 1 1 2 2 19

Solid tumors

Renal cell cancer Breast cancer Melanoma Total solid tumors

5 7 1 13

0 6 0 6

1 1 1 3

4 ⫹ 2‡ 10 2 18

0 4 0 4

6 14 2 22

Total 36 28 9 54 ⴙ 1§ 18 73 * AML indicates acute myeloid leukemia; T-ALL, T-cell acute lymphoblastic leukemia; MDS, myelodysplastic syndrome; CML, chronic myelogenous leukemia; MPD, myeloproliferative disorder; CLL, chronic lymphocytic leukemia; DLBCL, diffuse large B-cell lymphoma; MCL, mantle cell lymphoma; B-NHL, B-cell nonHodgkin lymphoma; SLL, small lymphocytic lymphoma; B-LBL, B-cell lymphoblastic lymphoma; FCL, follicular lymphoma. † Poor engraftment until 3 months posttransplant; no marrow obtained for morphologic examination prior to 3 months. ‡ Results of marrow obtained 1 month posttransplant; day 14 marrow not available. § One patient died on the day after transplant.

therapy was administered and the available marrow findings may not represent the true condition of the marrow at the time of transplantation. Thus, in 64 patients, the available pretransplant marrow is deemed representative of the actual conditions under which the transplantation was carried out. The status of the pretransplant marrow by pretransplant diagnosis is shown in Table 1. There was involvement by the disease for which the transplant was being carried out in 28 (44%) of 64 patients; these diseases were 21 hematologic malignancies, 1 lymphoma, and 6 solid tumors. Of the 36 bone marrow specimens that were free of disease, 10 patients had diagnoses of hematologic malignancies, 13 had lymphomas, and 13 had solid tumors. The cellularity of marrow of those patients involved by the disease process was significantly higher (mean, 54.8%) compared with that of patients without disease (mean, 36%, P ⫽ .004). This reflects the high marrow cellularity seen in several patients with myelodysplastic syndrome and myeloproliferative processes; some patients with metastatic breast carcinoma also showed effectively very high marrow cellularity. The difference in cellularity was not related to a difference in the age of patients because there was no difference in this regard (mean age in the 2 groups was 45.9 and 47.7 years, P ⫽ .25). Except in rare cases, these pretransplant marrow specimens showed normal myeloid to erythroid (M/E) ratio in both groups. The ratio was high in a patient with continued presence of chronic myelogenous leukemia. A low (1:1 or lower) M/E ratio that was seen in 13 patients was indicative of early regenerating marrow following chemotherapy in most cases. Arch Pathol Lab Med—Vol 130, October 2006

Early Posttransplant Marrow One patient died the day after the transplant. Of the remaining 72 patients, marrow was first obtained on day 14 after NMSCT in 67 patients for morphologic examination and engraftment studies. Bone marrow aspirates from 3 additional patients were submitted for engraftment studies only, and engraftment was assessed by examination of the peripheral blood in 2 cases. Engraftment was assessed by sex chromosome fluorescent in situ hybridization in 13 cases and by short tandem repeat analysis in the remainder. The mean level of engraftment was 68.2% for the entire population. When the patients were divided according to whether their pretransplant marrow was involved by disease or not, engraftment at this time was similar at 70.3% and 65.5%, respectively (P ⫽ .29). The average marrow cellularity declined from the pretransplant levels of 54.8% to 52.1% in those patients with disease in pretransplant marrow, and increased from the pretransplant level of 36% to 45.2% in those patients without disease, and the 2 groups had statistically similar cellularity at day 14 (P ⫽ .14). The cellularity was less than 20% in 11 patients, but the donor engraftment was not significantly lower in these patients. Detectable disease was present in 18 patients (Table 1), and 10 patients with demonstrable pretransplant marrow involvement showed morphologic remission from disease at this time (Figure 1, A through F). In patients with continued presence of marrow disease at day 14 and those without marrow disease, the average cellularity (51.3% and 46.6% respectively, P ⫽ .26) or the level of engraftment (70.6% and 67.5%, respectively, P ⫽ .37) was statis-

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Figure 1. Comparison of morphology in representative cases that showed the presence of disease in pretransplant bone marrow (left), but not in day 14 posttransplant marrow (right). A and B, Myelodysplastic syndrome. Erythroid dysplasia is seen in A as binucleate normoblast (long arrow) and megaloblastoid change (short arrow). Increased myeloid blasts are noted (arrowheads). These changes are absent in B (Wright stain, original magnification ⫻600). C and D, Chronic lymphocytic leukemia. A lymphoid aggregate composed of small lymphocytes is demarcated by arrows in C, but there is no lymphocytosis in D (hematoxylin-eosin, original magnifications ⫻100 [C] and ⫻200 [D]). E and F, Acute myeloid leukemia. E, Increased myeloid blasts (arrows) are present in the pretransplant marrow (Wright stain, original magnification ⫻1000). Not shown: 21% abnormal blasts were detected by flow cytometry. F, In contrast, trilineage hematopoiesis with complete and orderly maturation is seen in this core biopsy taken on day 14 after transplantation (hematoxylin-eosin, original magnification ⫻400).

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Figure 2. Average cellularity (A) and donor engraftment (B) in bone marrow specimens obtained at indicated intervals after nonmyeloablative stem cell transplantation (NMSCT). The values for all specimens are shown along with that of those patients who received HLAmatched and HLA-unmatched stem cells. The values for HLA-matched and HLA-unmatched groups were compared at each time point using an unpaired t test. * indicates P ⫽ .01; †, P ⫽ .004. Figure 3. Average cellularity (A) and donor engraftment (B) in bone marrow specimens obtained at indicated intervals after nonmyeloablative stem cell transplantation (NMSCT). The values for marrows with detectable disease and those without disease are compared using an unpaired t test. * indicates P ⫽ .05; †, P ⫽ .01; ‡, P ⫽ .008; §, P ⫽ .005; ll, P ⫽ .001; ¶, P ⬍ .001.

tically similar. Indeed, in 1 case of metastatic breast carcinoma, the level of metastatic marrow involvement was higher in day 14 marrow compared with the pretransplant marrow, but 93% engraftment was still observed by molecular methods. In general, in patients showing continued presence of marrow disease at day 14 posttransplant, the pretransplant level of marrow involvement by metastatic disease was high and the marrow was often hypercellular. Average cellularity at this time was significantly lower in the 31 patients who had received stem cells that were mismatched at 1 or more of the 6 HLA loci compared with the 42 patients who received fully matched stem cells. The mean (and median) cellularity in the 2 groups was 38.8% (40%) and 54.1% (60%), respectively (P ⫽ .006). Engraftment in the 2 groups, on the other hand, was similar: mean, 70.8 and 64.7, respectively; (P ⫽ .22). The day 14 marrows showed a brisk myeloid response in many cases, with the M/E ratio exceeding 3:1 in 35 cases. In 24 cases there was atypical localization of immature myeloid precursors. All but 5 cases of atypical localization of immature myeloid precursors also showed high M/E ratio, and the average cellularity in this group was significantly higher than the specimens without atypical localization of immature myeloid precursors (55.7% vs 43.3%, P ⫽ .02). These cases included not only cases of myelodysplastic syndrome and acute myeloid leukemia, but also lymphoma and solid tumor. The frequency of high M:E ratio or occurrence of atypical localization of immature myeloid precursors was not different in those patients receiving HLA-matched or HLA-mismatched stem cells. In 11 cases the number of megakaryocytes was increased, with only approximately one third of these being associated with the presence of high M/E ratio and Arch Pathol Lab Med—Vol 130, October 2006

high cellularity. Reticulin fibrosis did not accompany the hypercellularity or increased megakaryocytes. Subsequent Posttransplant Changes in the Bone Marrow Figure 2, A, shows average bone marrow cellularity in all available marrow specimens at various posttransplant times and compares the cellularity in patients receiving HLA-matched and HLA-unmatched stem cells. The average cellularity of the marrow decreased slightly at 1 month from the day 14 value (43% from 47.8%, P ⫽ .15) and more significantly at 2 months (35.4% from 47.8%, P ⫽ .002). The cellularity in those patients receiving HLAmismatched stem cells remained significantly lower at 1 month after transplant but was similar in the 2 groups in which marrow was obtained 2 or 3 months posttransplant. The cellularity in the unmatched stem cell group was lower again at 6 months. As seen in Figure 2, B, there was no difference in the level of donor engraftment between the 2 groups at any time. The myeloid hyperplasia noted in day 14 marrow also gradually subsided during this time. For example, only 4 cases showed atypical localization of immature myeloid precursors in marrow obtained 2 months posttransplant, compared with 24 cases in day 14 posttransplant marrow. Cellularity declined below 20% in 11 cases at 3 months after transplant, but in only 2 of these cases was there a significant decline (below 75%) in donor engraftment. Similarly, at 6 months, only 2 of the 9 hypocellular marrow specimens were associated with low donor engraftment. Thus, a failing marrow could not be reliably detected by following only the donor engraftment. Conversely, marrow cellularity did not always accurately predict graft fail-

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Table 2. Number and Proportion of Bone Marrow Biopsies Showing Morphologically Detectable Disease at Subsequent Time Points After Nonmyeloablative Stem Cell Transplant (NMSCT) in Different Disease Categories Time after NMSCT, mo Disease Category

Hematologic (n ⫽ 32) Lymphomas (n ⫽ 19) Solid tumors (n ⫽ 22)

1

2

3

6

⬎6

8/26* (30.8%) 0/18 (0) 5/17 (29.5%)

10/24 (41.7%) 3/14 (21.4%) 3/15 (20%)

6/18 (30%) 1/6 (16.6%) 2/13 (15.4%)

6/17 (35.7%) 1/4 (25%) 3/6 (50%)

7/11 (63.6%) 1/3 (33%)

Total (N ⫽ 73)

13/61 16/53 9/37 10/27 (21.3%) (30.2%) (24.3%) (37%) * Fewer bone marrows were available at later time points because of attrition from death of patients.

ure; in 4 cases, the marrow remained normocellular at 3 months despite clearly declining donor engraftment. Persistent Disease and Recurrent Disease in Posttransplant Marrow As seen in Table 2, at 1 month posttransplant, 13 patients showed detectable bone marrow disease. All of these patients had marrow involvement in pretransplant marrow, but day 14 posttransplant marrows in 2 of these patients (1 each of acute myeloid leukemia and metastatic breast carcinoma) had been free of disease. In the latter 2 cases, the presence of disease in the 1-month posttransplant marrow can be viewed as early recurrence, and in others it represents refractory disease. At 2 months posttransplant, marrow from 16 patients was involved by disease; 3 of these patients did not have disease in pretransplant marrow and thus this represents recurrent disease. In the subsequent period, some additional patients developed marrow disease; some patients died in the intervening period, as seen from the declining number of total marrow specimens obtained. As seen in Figure 3, A, marrow cellularity was significantly higher in diseased marrow, presumably reflecting the involvement by conditions such as myelodysplastic syndrome, acute myeloid leukemia, and lymphoma. The level of engraftment shown in Figure 3, B, was lower in diseased marrow at some time points, especially after more than 6 months posttransplantation, but, surprisingly, this was not always the case. With more detailed analysis, we found that, of the 54 patients without demonstrable disease in the day 14 marrow, 13 patients developed recurrent bone marrow disease from 2 to 20 months posttransplantation (Table 3). Although the average donor engraftment at the time of recurrence declined significantly from the prerecurrence levels (from 89.7% to 66.6%, P ⫽ .005, paired t test), in 4 of the 13 cases there was no decline in donor cell percentage in the marrow, and in another 4 cases the decline was only modest. Representative photomicrographs and a flow cytometry histogram are shown in Figure 4, A through H. The difference in declining engraftment appears to be related to the amount of bone marrow involvement. In 4 of the 5 cases with significant decline in engraftment, more than 20% abnormal cells were detected in the marrow. On the other hand, 7 of the 8 cases that showed only modest or no decline in engraftment had less than 10% marrow involvement. However, 1 case showed no decline in donor cells despite nearly complete involvement by abnormal lymphoid cells (Figure 4, B). These data suggest that decline in donor engraftment as detected by 1484 Arch Pathol Lab Med—Vol 130, October 2006

8/14 (57.1%)

Table 3. Donor Engraftment in Bone Marrow Before and After Relapse of Original Disease

Diagnosis*

AML T-ALL AML MDS MDS AML Breast cancer AML MCL AML DLBCL Hodgkin lymphoma MDS

Months Posttransplant at Relapse

6 3 3 8 11 20 6 4 11 2 2 2 9

Prerelapse Donor, %

29 75 81 90 99 99 99 99 99 99 99 99 100

Relapse Donor, %

ND 10 33 13 14 80 81 84 90 99 99 99 98

Median 99 82.5 Mean 89.769231 66.667 SD 19.946403 37.349 95% CI 8.8198325 17.712 Paired t test 0.005 Unpaired t test 0.03 * AML indicates acute myeloid leukemia; T-ALL, T-cell acute lymphoblastic leukemia; MDS, myelodysplastic syndrome; MCL, mantle cell lymphoma; DLBCL, diffuse large B-cell lymphoma; CI, confidence interval; ND, not done.

molecular analysis alone, in this series of patients, is not a reliable criterion of disease recurrence, particularly during early recurrence, when intervention with chemotherapy or immunotherapy (ie, donor lymphocyte infusion) may be most valuable. COMMENT Serially obtained bone marrow specimens from 73 patients who underwent NMSCT were examined and the results of molecular or fluorescent in situ hybridization studies of engraftment were analyzed in parallel. The morphologic examination was found to be valuable in identifying disease refractory to pretransplant chemotherapy, and also in some cases of early recurrence of disease. Some key morphologic features of bone marrow after NMSCT were found to be dependent on the degree of donor-recipient HLA match, and others appear to be part of the engraftment process. The definition of morphologic features related to each category will prevent possible confusion with recurrent hematologic disease in patients treated with NMSCT. Marrow Examination and Engraftment in Mini Transplant—Lagoo et al

Morphologic examination of the marrow is most valuable in detecting refractory disease. In our series, we noted marrow involvement by disease in 28 patients immediately before transplantation, and in 18 of these patients there was continued refractory disease in early posttransplant marrow (Table 1). It is interesting that the level of donor engraftment in these 2 groups of patients was similar. A marrow harboring hematologic or other malignant disease would be expected to be a worse environment for establishment of successful donor engraftment. However, we did not find any correlation between the level of engraftment obtained and the presence or absence of morphologically detectable disease. In most published studies of NMSCT, bone marrow morphology is not specifically evaluated9,14,44,45 and the molecular level of engraftment is often used to monitor success of the transplant.44,46 In morphologic studies of bone marrow following conventional SCT for hematologic malignancies,32–35,37 persistent disease was not detected in the early posttransplant period, possibly as a reflection of the more aggressive preparative regimen. A recent study of NMSCT in plasma cell myeloma has shown that disease that was resistant to chemotherapy was the single most significant predictor of poor outcome.47 Our preliminary analysis also suggests that the presence of detectable disease in pretransplant or early posttransplant marrow is associated with shorter survival (A.S. Lagoo et al, unpublished data, 2006). Because engraftment is not significantly different in those patients with or without detectable marrow disease, as shown herein, morphologic examination appears to provide valuable additional information related to prognosis. We found that there was apparent morphologic remission in early posttransplant marrow in some patients with demonstrable disease in pretransplant marrow (Table 1). The beneficial effect of allografts, including NMSCT, partly derives from the graft-versus-leukemia/lymphoma effect or, more generally, the graft-versus-tumor effect.1,7,30,44,45,48 Evidence suggests that the graft-versus-leukemia effect is mainly caused by the activity of T cells in the graft, as T-cell depletion in the graft leads to an increased risk of disease relapse following transplantation.49 In this study, T-cell reduction of at least 2 orders of magnitude was achieved with the use of anti-CD52 antibody in the donor stem cells, and a T-cell–mediated antitumor effect seems unlikely at this early time point. But natural killer (NK) cells may survive through this treatment with anti-CD52 antibody.50,51 It has been shown that the NK cells are important mediators of graft-versus-tumor effects following bone marrow transplant52,53 and may contribute to the morphologic remission in early posttransplant marrow. It is also likely that the fludarabine used in the conditioning regimen produces the desired antitumor effect. Fludarabine is effective against hematologic malignancies54 but has no therapeutic effect on epithelial malignancies55–58 unless combined with radiotherapy.59 It is of interest to note that 8 of the 10 patients who achieved morphologic remission in day 14 marrow had hematologic malignancies. This early antitumor effect of fludarabine may be enhanced by the graft-versus-tumor effect of the transplanted NK cells. A minor contribution from the residual T cells cannot be ruled out. In any case, the patients who did not show morphologic remission in early posttransplant marrow tend to show worse survival in our series (not shown). These trends need further analysis to design treatment regimens that include additional immuArch Pathol Lab Med—Vol 130, October 2006

notherapy or other options in these cases of refractory disease. Histopathology of the bone marrow after conventional (myeloablative) SCT is notable for significant marrow injury effect in the form of proteinaceous edema and lipogranulomas in the early posttransplant period.34,37 These changes are not conspicuous after NMSCT, which may be a reflection of the less toxic preparative regimen. Similarly, the dyserythropoiesis noted in the early posttransplant period after conventional (myeloablative) SCT60 was not seen after NMSCT, except in cases with persistent myelodysplastic syndrome. We observed mixed trilineage hematopoiesis in contrast to the observed emergence of pure erythroid islands in some cases after conventional (myeloablative) SCT.34 In approximately one half of the patients in our series, myeloid hyperplasia was evident in the form of a high M/E ratio (3:1 or higher). It is well documented that administration of recombinant granulocyte-macrophage colony stimulating factor61 or granulocyte colony stimulating factor62 leads to significant increase in the M/E ratio in relatively uninjured marrow and leads to increased yields of myeloid progenitor cells.63 All patients in this study received daily granulocyte colony stimulating factor after NMSCT until the peripheral blood absolute neutrophil count reached 1500, so it is not clear why some patients did not respond with myeloid hyperplasia. However, an M/E ratio of 2:1 or less was not associated with any delay in reaching an absolute neutrophil count of 500 compared with those with an M/E ratio of 3:1 or higher. Our data suggest that a failure to observe the expected myeloid hyperplasia in early posttransplant marrow should not raise concerns about eventual myeloid recovery. There was nonparatrabecular or atypical localization of immature myeloid precursors in 24 cases at day 14 posttransplant, as has been noted previously in conventional (myeloablative) SCT.34,37 The presence of atypical localization of immature myeloid precursors was associated with a slight increase in the time to reach an absolute neutrophil count of 500, a delay that approached, but did not reach, statistical significance. The presence of atypical localization of immature myeloid precursors may be a consequence of disorganized hematopoiesis, as observed following conventional (myeloablative) SCT.31,34 Marrow cellularity in the early posttransplant period was significantly lower in those patients receiving stem cells that were HLA-mismatched at 1 to 3 loci. This effect lasted for 2 months posttransplant and at no time was accompanied by a lower level of donor engraftment judged by molecular methods (Figure 2, B). Our analysis of clinical outcome in these patients shows results comparable with those patients receiving HLA-matched stem cells (D.A. Rizzieri et al, unpublished data, 2006). To avoid misinterpretation as failing marrow in these cases, the HLA matching status should be taken into account when examining the posttransplant marrow. Bone marrow histology in HLA-mismatched recipients in conventional (myeloablative) SCT does not differ significantly from that of those receiving matched donor cells.34 Morphologic examination also detected recurrence of disease in some cases earlier than was seen by a decline in donor engraftment. In other cases, however, declining donor engraftment was a more sensitive indicator of recurrent disease. Additional immunotherapy in the form of donor lymphocyte infusion may be most valuable during

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early recurrence, when presumably the tumor burden is low.64–66 Detection of a new disease process, such as posttransplant lymphoproliferative disease, in these patients, as described elsewhere,67 also requires morphologic examination. Thus, a combined morphologic and molecular diagnostic approach will provide optimal monitoring of patients following NMSCT. This work was supported in part by research grants to Dr David Rizzieri from Amgen Corp (Thousand Oaks, Calif) and Berlex Laboratories (Montville, NJ). A gift of anti-CD52 antibody, Campath 1H, was received from ILEX Oncology, Inc (San Antonio, Tex) and Berlex Laboratories. References 1. Weiden PL, Sullivan KM, Flournoy N, Storb R, Thomas ED. Antileukemic effect of chronic graft-versus-host disease: contribution to improved survival after allogeneic marrow transplantation. N Engl J Med. 1981;304:1529–1533. 2. Sullivan KM, Weiden PL, Storb R, et al. Influence of acute and chronic graftversus-host disease on relapse and survival after bone marrow transplantation from HLA-identical siblings as treatment of acute and chronic leukemia. Blood. 1989;73:1720–1728. 3. Kolb HJ, Schattenberg A, Goldman JM, et al. European Group for Blood and Marrow Transplantation Working Party Chronic Leukemia: graft-versus-leukemia effect of donor lymphocyte transfusions in marrow grafted patients. Blood. 1995; 86:2041–2050. 4. Childs RW, Clave E, Tisdale J, Plante M, Hensel N, Barrett J. Successful treatment of metastatic renal cell carcinoma with a nonmyeloablative allogeneic peripheral-blood progenitor-cell transplant: evidence for a graft-versus-tumor effect. J Clin Oncol. 1999;17:2044–2049. 5. Craddock C. Nonmyeloablative stem cell transplants. Curr Opin Hematol. 1999;6:383–387. 6. Grigg A, Seymour JF, Roberts A, Szer J. Mini-allografts for haematological malignancies: an alternative to conventional myeloablative marrow transplantation. Aust N Z J Med. 1999;29:308–314. 7. Khouri IF, Lee MS, Romaguera J, et al. Allogeneic hematopoietic transplantation for mantle-cell lymphoma: molecular remissions and evidence of graftversus-malignancy. Ann Oncol. 1999;10:1293–1299. 8. Barrett J, Childs R. Non-myeloablative stem cell transplants. Br J Haematol. 2000;111:6–17. 9. Childs R, Chernoff A, Contentin N, et al. Regression of metastatic renal-cell carcinoma after nonmyeloablative allogeneic peripheral-blood stem-cell transplantation. N Engl J Med. 2000;343:750–758. 10. Sandmaier BM, McSweeney P, Yu C, Storb R. Nonmyeloablative transplants: preclinical and clinical results. Semin Oncol. 2000;27:78–81. 11. Hermann S, Klein SA, Jacobi V, et al. Older patients with high-risk fungal infections can be successfully allografted using non-myeloablative conditioning in combination with intensified supportive care regimens. Br J Haematol. 2001; 113:446–454. 12. Blaise D, Bay JO, Faucher C, et al. Reduced-intensity preparative regimen and allogeneic stem cell transplantation for advanced solid tumors. Blood. 2004; 103:435–441. 13. Kanda Y, Komatsu Y, Akahane M, et al. Graft-versus-tumor effect against advanced pancreatic cancer after allogeneic reduced-intensity stem cell transplantation. Transplantation. 2005;79:821–827. 14. Sykes M, Preffer F, McAfee S, et al. Mixed lymphohaemopoietic chimerism and graft-versus-lymphoma effects after non-myeloablative therapy and HLA-mismatched bone-marrow transplantation. Lancet. 1999;353:1755–1759. 15. Champlin R, Khouri I, Shimoni A, et al. Harnessing graft-versus-malignancy: non-myeloablative preparative regimens for allogeneic haematopoietic transplantation, an evolving strategy for adoptive immunotherapy. Br J Haematol. 2000;111:18–29. 16. van Besien K, Keralavarma B, Devine S, Stock W. Allogeneic and autologous transplantation for chronic lymphocytic leukemia. Leukemia. 2001;15: 1317–1325.

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← Figure 4. Representative cases comparing the morphology in day 14 posttransplant marrow (left) and at later times, showing disease recurrence in the marrow (right). A, Engraftment at day 14 was 99% when the marrow was free of previously diagnosed diffuse large B-cell lymphoma (hematoxylin-eosin [H&E] original magnification ⫻400). B, When precursor B-cell acute lymphoblastic leukemia was detected in the marrow of this patient 2 months posttransplantation, the donor cell engraftment was 100% by molecular analysis (H&E, original magnification ⫻600). C, Engraftment on day 14 was 81% in a marrow free of disease (H&E, original magnification ⫻100) and D, increased to 90% at 11 months posttransplant in spite of the fact that abnormal B cells (CD5⫹, circled) of mantle cell lymphoma were detected by flow cytometry. E, In this patient with Hodgkin lymphoma, donor engraftment was 92% in day 14 marrow when no disease was detectable (H&E, original magnification ⫻200). F, At 2 months posttransplantation, a granuloma was seen, suggesting reappearance of Hodgkin lymphoma (H&E, original magnification ⫻400) but the engraftment remained high at 99%. G, Donor engraftment of 87% that was detected at day 14 in disease-free marrow (H&E, original magnification ⫻400) and H, increased to 99% at 2 months posttransplantation in spite of the recurrence of acute myeloid leukemia (H&E, original magnification ⫻600). Presence of abnormal blasts was confirmed by flow cytometry in this case. Arch Pathol Lab Med—Vol 130, October 2006

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