Paraffin-embedded Bone Marrow Biopsy Specimens Research Articles

PDL1 Positivity By FISH in Patients Not in Complete Remission at Transplantation

Introduction Failure to achieve morphological complete remission occurs in 30% AML patients resulting in dismal outcomes. Data addressing influence of pre-transplant PDL-1 status on post-transplant outcomes is limited. We hypothesize that PDL-1 expression by immunohistochemistry (IHC) or Fluorescent in situ hybridization (FISH) prior to transplantation may predict a poor outcome. Methods This was a retrospective, observational single center pilot study involving refractory AML patients who underwent allogeneic stem cell transplantation (ASCT) at Karmanos Cancer Institute from 01/01/2012 to 01/01/2018. A planned sample size of 24 patients were selected and assigned to two cohorts based on their respective responses to ASCT: a) poor responders: persistent leukemia by bone marrow biopsy within 30 days of ASCT, and b) good responders: no evidence of leukemia at 1-year post-transplant. Descriptive analyses evaluated differences in demographic, disease and treatment variables. FISH assay was used to evaluate PDL-1 alterations in the pre-transplant archival, formalin-fixed, paraffin-embedded bone marrow biopsy specimens including frequency and magnitude of 9p24.1 alterations— low-level disomy, polysomy, copy gain, and amplification. The expression of PD-L1 was evaluated by IHC in a semi-quantitative (0 through +3) manner. Results Of the total 24 patients, 12 patients were poor responders and 12 were good responders; among poor responders, median age was 58 years (range, 31- 72) and gender distribution was 1:1. Seven patients (58%) had prior MDS, Myelofibrosis or CMML, while five patients had de-novo AML. Treatment information is provided in table 1. At pre-transplant bone marrow biopsy assessment, median number of bone marrow and peripheral blood blast counts were 8.1% (1.7-37) and 2% (0-37), respectively. Bone marrow cytogenetics assessed prior to transplant included intermediate risk in 2 (17%) and high risk disease in 9 (75%) patients and one patient without cytogenetic evaluation. PD-L1 expression was negative by IHC in all samples, but FISH testing was positive in 3 of 12 (25%) poor responders, characterized by copy gain. In good responders, median age was 59 years (range, 43-70) with a 3:2 male predominance. Four patients (25%) had prior MDS or Myelofibrosis and 8 had de-novo AML. Five patients (42%) had intermediate risk and 7 (58%) had high-risk cytogenetics. At pre-transplant bone marrow biopsy assessment, median number of bone marrow and peripheral blast counts were 6% (1-40) and 1% (0-7), respectively. PD-L1 expression was negative by both IHC and FISH examination in this cohort. Conclusion Our study demonstrates that PDL-1 expression by FISH was specifically detected in 25% of patients with poor responders following ASCT, which could possibly provide an insight into the poor outcome. A larger sample will be required to validate this observation in this population.

CD71 (Transferrin Receptor)

Accurate analysis of the erythroid lineage is essential in evaluating bone marrow biopsy specimens and can be particularly challenging in the setting of dyserythropoiesis. Transferrin receptor (CD71) mediates the uptake of transferrin-iron complexes and is highly expressed on the surface of cells of the erythroid lineage. Although CD71 has been used for flow cytometric analysis, its usefulness in paraffin-embedded bone marrow biopsy specimens has not been examined. This study defined the immunohistochemical profile of CD71, as compared with glycophorin A (CD235a) and hemoglobin, in 65 bone marrow biopsy specimens, including normal marrow specimens and cases of myelodysplastic syndrome, acute myeloid leukemia, acute lymphoblastic leukemia, plasma cell neoplasm, and metastatic carcinoma. Immunoreactivity for CD71 was restricted to erythroid precursors in normal and dyspoietic marrow samples and exhibited a membranous and cytoplasmic staining pattern. The vast majority of mature erythrocytes lack expression of CD71, greatly facilitating interpretation. CD71 is a highly effective marker for the detection of cells of erythroid lineage in bone marrow biopsy specimens.

Contribution of IgH-PCR to the Evaluation of B-Cell Lymphoma Involvement in Paraffin-Embedded Bone Marrow Biopsy Specimens

We investigated whether the determination of clonality by polymerase chain reaction (PCR) analysis of immunoglobulin heavy chain (IgH) gene rearrangements could be helpful in the evaluation of B-cell lymphoma (BCL) involvement of bone marrow (BM) biopsy specimens. We evaluated 83 paraffin-embedded BM biopsy specimens from 26 patients with BCL. When BM biopsy specimens considered positive, “suspicious,” or negative by morphologic and immunohistochemical examination were evaluated by PCR, a monoclonal B-cell population was detected in 81% (39/48), 64% (9/14), and 11% (2/18), respectively. In most cases, a reproducible monoclonal IgH gene rearrangement was observed from BM and extramedullary sites. Nevertheless, in 4 cases, a different and independent monoclonal IgH rearrangement was observed during the disease course. PCR is efficient and complementary to morphologic and immunohistochemical examination for the evaluation of BCL involvement of BM biopsy specimens, especially when a reproducible rearrangement is found in 2 different samples.

Adult Hypocellular Acute Leukaemia with Lymphoid Differentiation

The rare hypocellular variants of acute leukemia (AL) previously also termed smouldering leukemia, almost always exhibit myeloid differentiation. Very rare cases of hypocellular AL with lymphoid differentiation have been reported, usually in children. This paper describes two cases (an 87-year-old woman and a 79-year-old man) in whom the blood findings were suggestive of AL. Paraffin-embedded bone marrow biopsy specimens revealed similar findings in both patients: there was severe hypocellularity, the cells of normal hemopoiesis were greatly reduced in number, and there was a diffuse increase in blast cells, which represented more than 50% of nucleated marrow cells. The blasts coexpressed TdT and CD34 and were negative for myeloperoxidase, CD117, CD68 and naphthol AS-D chloroacetate esterase. For the first time immunohistochemical Pax-5/CD34 doublestainings are provided, which revealed the blasts in one case to coexpress Pax-5 and CD34. All the blasts were CD79a-positive and 20% were also CD10-positive. In the other case, 20% of the blasts were CD79a-positive, 30% coexpressed Pax-5 and CD34 by doublestaining, and showed a clonal rearrangement of the immunoglobulin heavy chain gene. Thus a diagnosis of AL of lymphoid lineage, hypocellular variant, was made on the basis of immunohistochemical findings. The clinical course appears to be similar to that of hypocellular AML, as neither patient has developed overt leukemia during the one-year follow-up period.

Cyclin D1 overexpression in multiple myeloma. A morphologic, immunohistochemical, and in situ hybridization study of 71 paraffin-embedded bone marrow biopsy specimens.

Cyclin D1 expression was evaluated by immunohistochemical analysis and biotin-labeled in situ hybridization (ISH) in a series of 71 decalcified, paraffin-embedded bone marrow biopsy specimens from patients with multiple myeloma (MM). Cyclin D1 messenger RNA (mRNA) overexpression was detected by ISH in 23 (32%) of 71 cases, whereas cyclin D1 protein was identified by immunohistochemical analysis in 17 (24%) of 71 specimens. All cases that were positive by immunohistochemical analysis also were positive by ISH. Statistically significant associations were found between cyclin D1 overexpression and grade of plasma cell differentiation and between cyclin D1 overexpression and extent of bone marrow infiltration. Our findings demonstrate the following: (1) ISH for cyclin D1 mRNA is a sensitive method for the evaluation of cyclin D1 overexpression in paraffin-embedded bone marrow biopsy specimens with MM. (2) ISH is more sensitive than immunohistochemical analysis in the assessment of cyclin D1 expression. (3) Cyclin D1 overexpression in MM is correlated positively with higher histologic grade and stage.

PCR analysis of IgH-gene rearrangements in small lymphoid infiltrates microdissected from sections of paraffin-embedded bone marrow biopsy specimens

The differentiation of benign lymphoid infiltrates from nodular infiltrates of B-cell lymphoma is difficult in bone marrow (BM) biopsy specimens taken from patients with non-Hodgkin's lymphoma (NHL). We investigated whether the determination of clonality by polymerase chain reaction (PCR) analysis of the immunoglobulin heavy chain (IgH) genes could be of help for the distinction of benign and malignant lymphoid infiltrates. BM biopsy specimens of 28 patients were studied, comparing PCR of entire bone marrow sections with microdissected nodular lymphoid infiltrates. Patients were divided into 4 groups according to morphologic criteria: group 1 (n = 12), positive for B-NHL infiltration; group 2 (n = 5), suspicious for infiltration by known B-NHL; group 3 (n = 5), morphologically benign infiltrates in patients with B-NHL; group 4 (n = 6), benign lymphoid infiltrates in patients without history of B-NHL. PCR products were analyzed using polyacrylamide gels and a fragment length analysis system (Genescan). PCR of whole sections showed clonal amplification products in all cases of group 1 and 1 case of group 2. PCR analysis from microdissected nodular infiltrates showed the presence of a clonal B-cell population in 5 additional cases of groups 2 and 4. In 3 of these cases, clonal rearrangements of corresponding size were obtained from the primary lymphoma biopsy specimens. None of the cases of group 3 showed evidence of a clonal population with either technique. The results indicate that microdissection of small nodular lymphoid infiltrates from paraffin-BM sections increases the sensitivity of IgH gene rearrangement analysis. To avoid detection of biologically irrelevant clonal populations, comparison of PCR products obtained from the BM and the primary lymphoma biopsy is advisable. HUM PATHOL 31:847-853.

The Mi15 monoclonal antibody (anti-syndecan-1) is a reliable marker for quantifying plasma cells in paraffin-embedded bone marrow biopsy specimens

Plasmocyte selective monoclonal antibodies (MAb) recognizing syndecan-1 have recently been described. They belong to a new cluster, CD138. Using the MAb MI15, we investigated the expression of syndecan-1 in routinely paraffin-embedded tissues. Nontumoral lymph nodes (25 cases) and bone marrow biopsy specimens (63 cases) showed strong membrane staining of plasma cells only, allowing accurate analysis of the nuclear structure. The MI15 positivity correlated with kappa and lambda light chain expression in the cytoplasm. The percentages of plasma cells calculated in bone marrow biopsy specimens after MI15 staining were, respectively, 2.1% (range, 1% to 4%) in normal bone marrows, 8.5% (range, 5 to 17) in reactive plasmocytosis, and 4.66% in monoclonal gammapathy of undetermined significance (MGUS) patients (range, 1 to 13), in the same range but slightly higher than those obtained on smears or on hematoxylin and eosin (H&E)-stained sections. In multiple myeloma (40 cases), all plasma cell types were marked, and Mi15 MAb gave additional information in 8 of 40 (20%) patients. In lymph nodes, Mi15 MAb reacted with Reed-Sternberg cells of dassical Hodgkin's disease in 23 of 31 cases (74%) with variable intensity. In contrast, nodular lymphocyte predominance Hodgkin's disease (10 cases), most B cell lymphomas (88 of 107 cases) and all T cell lymphomas (30 cases) were negative. In B cell lymphomas, plasmocytomas (8 cases), plasmocytic lymphomas (2 cases), and 5 of 13 cases of immunoblastic lymphoma with plasmocytoid differentiation were stained. In lymphoplasmocytoid lymphomas (4 lymph nodes and 20 bone marrow biopsy specimens), only mature plasma cells were positive. Moreover, a wide distribution of syndecan-1 was observed in normal and tumoral epithelial tissues. Finally, Mi15 MAb appears to be a reliable marker for identifying and quantifying normal and tumoral plasma cells in paraffin-embedded bone marrow and lymph node samples.

Practical importance of routine paraffin-embedded bone marrow biopsy in multiple myeloma.

Paraffin-embedded bone marrow biopsy specimens obtained prior to (37) or after (25) therapy from 62 patients with multiple myeloma (MM) were analyzed with particular reference to infiltration pattern, extent of infiltration, and myeloid to myeloma tissue percentage ratio (MMR) to verify their mutual relationships and clinicopathologic relevance. Fifty-nine biopsies were evaluable for infiltration pattern (diffuse in 27, interstitial in 25, and nodular in 7). Diffuse and interstitial patterns were more common (P less than 0.025) in stage III and stage I patients, respectively. A higher (P less than 0.001) mean serum paraprotein level was found in patients with the diffuse pattern than in those with the interstitial pattern. The average extent of infiltration by myeloma cells in the residual myeloid tissue was higher (P less than 0.001) and a high extent (75% or more) was more frequently (P less than 0.005) seen in diffuse than in interstitial pattern cases. The average MMR value was lower (P less than 0.001) and a MMR value less than 1 was more frequently (P less than 0.005) seen in the diffuse pattern group than in the interstitial pattern group. All these differences were present also when a separate analysis was performed for treated and untreated patients. It seems that a diffuse histologic pattern, as opposed to interstitial, would significantly predict a bone marrow extent of infiltration of 75% or more, a MMR lower than 1, a higher serum paraprotein level, and a clinical stage III. Bone marrow biopsy appears thus to play a role in providing parameters of prognostic relevance in MM also in the course of the disease. Prospective studies are needed to establish whether histologic pattern has an independent prognostic value.

An immunohistochemical study of hemoglobin A, hemoglobin F, muramidase, and transferrin in erythroid hyperplasia and neoplasia.

The bone marrow biopsy specimens of 35 patients with benign and malignant erythroid hyperplasias were examined for the presence of hemoglobin A, hemoglobin F, muramidase (lysozyme), and transferrin, using an indirect immunoperoxidase method (PAP) on Zenker's-fixed paraffin-embedded bone marrow biopsy specimens and particles. Five cases of each of the following entities were studied: erythroleukemia and erythremic myelosis, acute granulocytic leukemia with maturation (FAB M2), polycythemia rubra vera, myeloproliferative syndrome in childhood, megaloblastic anemia (B12 and folate deficiency), erythroid hyperplasia (regenerating bone marrow and hemolytic anemia), and Ph' chromosome positive chronic granulocytic leukemia. Hemoglobin A was present in both the early and late erythroid precursors in all conditions. Hemoglobin F was the predominant hemoglobin in early erythroblasts of pernicious anemia and in both early and late erythroid elements in erythroleukemia and erythremic myelosis. Small quantities of hemoglobin F were present in a few isolated clusters in other conditions. Staining for hemoglobin F may be useful in identifying immature erythroid precursors and in distinguishing some cases of dysplastic erythroid hyperplasia from neoplasia. Additionally, these findings suggest that the maturational switch in hemoglobin synthesis operates with distinct pathways under different conditions.