Bleomycin, lomustine, cyclophosphamide, vincristine, procarbazine and prednisone (BLEO-CCVPP) in patients with Hodgkin's disease who relapsed after radiotherapy alone: a long-term follow-up study of the Eastern Cooperative Oncology Group (E3481).

Thirty-three evaluable patients with Hodgkin's disease who failed radiotherapy were treated on this phase II study with bleomycin, lomustine, cyclophosphamide, vincristine, procarbazine and prednisone given every 28 days for a minimum of eight courses. Twenty-five patients (76%; 95% CI=55.6-87.1%) achieved a complete remission, the median duration of which cannot yet be determined, but the probability of remaining in continuous complete remission at 10 years is.64. The median survival from entry on this study for all evaluable patients is 10 years, and 12 patients were alive at the time of this analysis with a median follow-up for them of 15.5 years. Of the 22 patients who died, 11 died of progressive or recurrent Hodgkin's disease and 11 died of other causes including 7 second primary neoplasms and at least one myocardial infarction. Both are now well known late complications of Hodgkin's disease treatment.

Immunohistochemistry can be used to subtype acute myeloid leukemia in routinely processed bone marrow biopsy specimens. Comparison with flow cytometry.

Flow cytometry (FC) is the preferred method of immunophenotyping acute myeloid leukemia (AML). However, there are situations in which FC is unavailable and in which immunohistologic staining of bone marrow biopsy specimens can be used to provide immunophenotypic information. To evaluate immunohistologic staining and to confirm its value, we selected 80 newly diagnosed cases of AML that were classified according to French-American-British (FAB) criteria and confirmed by flow cytometric analysis for this study. Paraffin-embedded bone marrow specimens were stained using a panel of antibodies that included CD34 (QBEND10), antimyeloperoxidase (anti-MPO), antihemoglobin, factor VIII-related antigen, and 3 epitopes of CD68 (HAM56, KP1, and PG-M1). Our findings suggest that with the use of the paraffin-reactive antibodies CD34 (QBEND10), MPO, CD68 (PG-M1), antihemoglobin, and factor VIII-related antigen, immunohistochemistry can be used to subclassify AML. Comparison of immunohistochemical results with FC immunophenotyping suggests that there is significant concordance in the results for markers that can be used with both techniques, indicating that the sensitivity and specificity of both methods is comparable (P > .53 in all cases).

Mediastinal non-seminomatous germ cell tumours: their association with non-germ cell malignancies.

Mediastinal non-seminomatous germ cell tumors are unique tumors in that they are associated with both sarcomatous and hematologic neoplasms. This paper relates our experience at Indiana University with these tumors, and discusses the possible mechanisms of their occurrence, especially with respect to the hematologic neoplasms.

Marked granulocytic proliferation induced by granulocyte colony-stimulating factor in the spleen simulating a myeloid leukemic infiltrate.

The authors describe a patient with a long-standing history of systemic lupus erythematosus and leukopenia who received multiple intermittent doses of recombinant granulocyte colony-stimulating factor (G-CSF) and who underwent splenectomy because of a clinical impression of sequestration of granulocytes by the spleen. Histologic evaluation of the spleen revealed marked granulocytic hyperplasia with an increase in immature myeloid precursors, morphologically indistinguishable from a myeloid leukemic infiltrate. A postsplenectomy bone marrow aspirate and biopsy revealed a normocellular bone marrow with active hematopoiesis and trilineage maturation. The bone marrow aspirate cultured cells showed no numeric or structural chromosomal abnormality. Extramedullary hematopoiesis after receipt of G-CSF was previously reported, but, to our knowledge, ours is the first report of morphologic changes virtually identical to a leukemic infiltrate in spleen after G-CSF treatment. We describe the histologic and immunohistochemical findings in the spleen, compare our observations with those of others reported in the literature, and postulate a possible mechanism for this phenomenon.

Posttransplantation lymphoproliferative disorders in bone marrow transplant recipients are aggressive diseases with a high incidence of adverse histologic and immunobiologic features.

Posttransplantation lymphoproliferative disorders (PT-LPDs) occurring in T-cell depleted (TCD) allogeneic bone marrow transplant recipients seem to be different from those that arise in solid organ recipients in their early development, the high incidence of extensive dissemination at presentation, and their aggressive course and high fatality rate. We report a series of 10 patients with PT-LPDs after TCD allogeneic bone marrow transplant. We studied the correlation between the morphology of the lesions; their clonality based on immunoglobulin (Ig) heavy chain gene rearrangement analysis and immunohistochemistry; their proliferative activity as measured by immunoperoxidase staining for the proliferating cell nuclear antigen (PCNA) and the presence of p53 gene product overexpression. Histologically, our cases corresponded to the two morphologic categories of polymorphic B-cell lymphoma (PBCL, seven cases) and malignant lymphoma immunoblastic (ML-IB, three cases). Ig light-chain staining showed monoclonality in a minority of the cases, whereas Ig gene rearrangement analysis by polymerase chain reaction revealed B-cell clonality in three of seven cases of PBCL and in all three cases of ML-IB. The Epstein-Barr virus (EBV) genome, the expression of EBV latent membrane protein or both were found in all 10 specimens. High proliferative activity (PCNA > or = 66%) was found in all cases, with a mean PCNA value of 56% in PBCL and 84% in ML-IB. Five specimens were p53+ (two of seven PBCL and three of three ML-IB). Two of four PBCL cases resolved with the administration of donor leukocytes. All of the remaining patients died of the PT-LPD within a short time from admission. Our results show that the PT-LPDs after TCD bone marrow transplantation are characterized by a high frequency of high-grade histologic subtypes, frequent monoclonality, high proliferative activity, frequent overexpression of p53 gene product, and poor prognosis. These characteristics observed in only a minority of cases of PT-LPDs occurring after solid organ transplantation may account for the less aggressive clinical behavior observed in those diseases.

013 (CD99) positivity in hematologic proliferations correlates with TdT positivity.

The monoclonal antibody 013, which detects the cell surface glycoprotein p30/32mic2 (CD99) is a characteristic, if nonspecific, marker for peripheral neuroepithelioma and Ewing's sarcoma. 013 was first produced against a human thymus leukemia antigen and has also been found in immature terminal deoxynucleotidyl transferase (TdT)-positive T cells and in a small group of hematopoietic precursor cells in the bone marrow. 013 is reactive in lymphoblastic lymphomas and acute leukemias, as well as in a variety of other hematologic malignancies. Because the distribution of 013 positivity in hematopoietic proliferations is similar to that of TdT, we hypothesized that 013 might correlate with TdT positivity. We studied 67 lymphoblastic lymphomas and acute lymphoblastic leukemias, 6 chronic myelogenous leukemias in blast crisis, a variety of acute myeloid leukemias, 5 granulocytic sarcomas, and a spectrum of 94 diffuse lymphomas other than lymphoblastic type. With the use of heat-induced epitope retrieval and automated immunostaining, we compared the results obtained with 013 and TdT, a well-established marker of lymphoblastic lymphomas and acute lymphoblastic leukemias that can also be successfully demonstrated in tissue sections by use of a similar technique. In our study, all of the 013-positive cases were also TdT positive. 013 reacted with 44 (71%) of 62 of the TdT-positive lymphoblastic lymphomas and acute lymphoblastic leukemias cases studied. We also found 013 to be positive in one case of TdT-positive acute myeloid leukemia, in two cases of chronic myelogenous leukemia in blast crisis, and in three TdT-positive granulocytic sarcomas. 013 was negative in all of the other high-grade malignant lymphomas and in TdT-negative leukemias. With use of our technique, 013 positivity appears to be restricted to hematologic proliferations that demonstrate TdT positivity. 013 may be a helpful additional marker in the diagnosis of TdT-positive leukemias and lymphomas in conventionally processed tissue sections.

Hypoplastic myelodysplastic syndromes can be distinguished from acquired aplastic anemia by CD34 and PCNA immunostaining of bone marrow biopsy specimens.

Hypoplastic myelodysplastic syndromes (h-MDSs) are difficult to distinguish from acquired aplastic anemia (AA) because of the considerable clinical, cytologic, and histologic similarities between these two disorders. Recent studies have suggested that the bone marrow (BM) in AA is characterized by a decreased number of CD34+ cells and reduced expression of proliferating cell nuclear antigen (PCNA), features that have not been associated with MDS. To determine the potential importance of these markers in the differential diagnosis of hypoplastic BM disorders, we immunostained 50 BM biopsy specimens of cytogenetically characterized cases of AA (27) and h-MDS (23). Immunohistochemical staining for CD34 was performed with QBEND10 (Vector, Burlingame, Calif), a monoclonal antibody (MoAb) reactive in routinely processed specimens, while PCNA was assessed by the PC10 MoAb (Dako, Carpinteria, Calif) using a microwave over-based antigen retrieval technique. Bone marrow specimens of h-MDS cases showed statistically higher values of PCNA and CD34 than did those of the AA cases: mean values (+/- SD) of CD34-positive cells in h-MDS, 0.94% +/- 1.1; AA, 0.04% +/- 0.1 (P = .0002); PCNA-positive cells in h-MDS, 43.59% +/- 13.3; AA, 14.80% +/- 6.4 (P < .0001). Our study confirms that AA is characterized by low expression of PCNA in BM and reduced CD34 frequency compared with h-MDS and supports the concept of an early deficiency of stem cells in the former disorder. The results also illustrate how immunostaining permits a simple distinction of these conditions in routinely processed BM biopsy specimens.

Dicentric (9;20)(p11;q11) identified by fluorescence in situ hybridization in four pediatric acute lymphoblastic leukemia patients.

Four children with acute lymphocytic leukemia (ALL) and a dic(9;20) are described. All four patients were diagnosed with pre-B-cell All, and the three for whom information was available were CD10+. Age at diagnosis ranged from 23 months to 12 years. All patients achieved remission, with two in continuous remission for 2 years 6 months and 3 years, one patient relapsed, dying 3 years 2 months after diagnosis, and one patient was lost to follow-up. These four patients were initially diagnosed as having a deletion of 9p and loss of one chromosome 20. Re-examination of the karyotypes indicated a possible dic(9;20). The dicentric chromosome was verified using dual-color fluorescence in situ hybridization (FISH) with centromeric probes for chromosomes 9 and 20 on interphase nuclei. Three of the four patients had multiple chromosomal abnormalities in addition to the translocation; one was hypodiploid, one was pseudodiploid, and two were hyperdiploid. This dicentric chromosome was recently described in four adult and nine pediatric patients with ALL [8, 9]. All reported patients had CD10+ pre-B-cell All, and achieved remission, as was the case for our four pediatric dic(9;20) patients. Two of our three patients for whom follow-up is available are in continuous remission as were two adults and five pediatric patients in the previous reports. These studies confirm the dic(9;20) as a recurring abnormality in ALL. Due to the subtle nature of the translocation, FISH is very useful in confirming the chromosomal abnormality.

Prognostic implications of ploidy and proliferative activity in the diffuse, aggressive non-Hodgkin's lymphomas.

The International Index is a powerful predictor of outcome in the aggressive non-Hodgkin's lymphomas that is based solely on clinical features. Proliferative activity (% S-phase) measured by flow cytometry has been reported to have prognostic significance in many series and may represent a biologic correlate of clinical behavior that further defines prognosis. Flow cytometric analysis of cellular DNA content and proliferative activity (% S-phase) was performed on fixed paraffin-embedded biopsy specimens from 242 previously untreated patients with diffuse, aggressive non-Hodgkin's lymphomas entered on phase III intergroup clinical trials. The International Index was calculated for each patient based on stage, lactate dehydrogenase, performance status, number of extranodal sites, and age, as previously reported. The International Index consistently predicted response to therapy (P = .027) and survival (P = .007) in this series. DNA aneuploidy was shown in 57% of cases, but was not predictive of clinical outcome. The median % S-phase was 9.9 (median coefficient of variation, 3.6%), which was highly correlated with mitotic index (P = .0001). Although a trend associating low proliferative activity with good early survival and very high S-phase with a shortened survival was shown, International Index risk was the only significant predictor of survival in the multivariate analysis. Although proliferative activity quantitated by flow cytometric analysis of nuclei extracted from paraffin-embedded specimens is probably predictive of survival, it is a less powerful prognostic indicator than clinical parameters represented by the International Index and provides no additional prognostic information.

mdm-2 oncoprotein expression associated with deletion of the long arm of chromosome 12 in a case of mantle cell lymphoma with blastoid transformation [corrected].

We report a unique case of mantle cell lymphoma in blastoid transformation associated with deletion of the long arm of chromosome 12 and with 90 kDa mdm-2 protein overexpression. Neither the mantle cells nor their blastoid counterparts expressed p53 gene product by immunohistochemical analysis. This seems to be the first reported case of this subtype of lymphoma associated with these specific cytogenetic and molecular genetic abnormalities.

p53 overexpression in myeloid leukemic disorders is associated with increased apoptosis of hematopoietic marrow cells and ineffective hematopoiesis.

Recent evidence has shown that p53 overexpression in leukemic cells may be a consequence of p53 gene mutation or can occur via posttranslational modification mechanisms. While mutant forms of p53 may stimulate cell proliferation and transformation, wild-type p53 may inhibit DNA synthesis and cause leukemic cells to enter apoptosis. Nine bone marrow biopsies of acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) with known p53 overexpression were analyzed for evidence of apoptosis of both leukemic blasts and of background hematopoietic cells. This was quantified and compared with that seen in p53- AML and MDS and in normal control marrows. The rate of cell death due to apoptosis was measured by in situ end-labeling of fragmented DNA with the following results: mean values of apoptotic cells/mm2 of BM, p53+ AML and MDS (9 cases), 2.41 +/- 1.7; p53- AML and MDS (10 cases), 0.16 +/- 0.1; control marrows (20 samples), 0.05 +/- 0.0. Our results showed a significant association (P < 0.001) between p53 overexpression and increased apoptosis in all cases studied. The difference was entirely caused by the high rate of cell death observed in the erythroid and myeloid precursor cells of these marrows. These findings suggest that p53+ AML and MDS are a distinct group of marrow disorders characterized by a high rate of intramedullary cell death. This may explain why patients with p53+ leukemic disorders show excessive marrow sensitivity to chemotherapy with prolonged marrow suppression associated with the presence of cytogenetically abnormal blasts that display great drug resistance.

Correlation between presence of clonal rearrangements of immunoglobulin heavy chain genes and B-cell antigen expression in Hodgkin's disease.

Southern blot analysis of Hodgkin's disease (HD), although often compromised by the small number of abnormal cells present in the tissue, have tended to favor a B-cell derivation of the Hodgkin's and Reed-Sternberg (HRS) cells in cases of nodular sclerosis (NS) and mixed cellularity (MC) Hodgkin's disease. Eighteen frozen and 29 paraffin-embedded sections of lymph node specimens from 29 patients with pretreatment HD (22 NSHD and 7 MCHD) were studied by molecular analysis and immunohistochemistry to determine the phenotype of HRS cells. All cases were reviewed and showed typical morphology and CD45-, CD30+, CD15+, BLA.36+ HRS cells. In 11 of 29 (38%) cases, HRS cells were reactive with at least one B-cell marker (CD20, CD79a, MB2), 7 of 29 (24%) cases showed reactivity with the T-cell marker CD3, and 11 of 29 (38%) cases displayed a "null" phenotype. By using a polymerase chain reaction (PCR) and consensus primers for the V and J regions of the immunoglobulin heavy chain (IgH) gene, the authors were able to detect B-cell clonality in 9 of 18 (50%) frozen samples of HD analyzed. IgH gene rearrangement was present in 8 of 15 (53%) NSHD and in 1 of 3 (33%) MCHD. In five of nine (56%) of these cases, HRS cells were reactive with at least one B-cell marker, whereas one case expressed the T-cell marker CD3. The other three cases with IgH gene rearrangement showed a "null" immunophenotype. IgH gene analysis was negative in all remaining CD3+ cases and in two other cases that expressed B-cell markers by immunohistology. Southern blotting failed to detect rearrangement of the T-cell receptor beta-chain gene and immunoglobulin heavy and light genes in any of these cases. The results show that PCR represents a specific and sensitive technique for the detection of IgH gene rearrangements in cases of Hodgkin's disease. The results also suggest a lymphoid B-cell derivation of HRS cells in a high proportion of the cases.

Functional hyposplenism due to a primary epithelioid hemangioendothelioma of the spleen.

We present a unique case of functional hyposplenism due to massive involvement of the spleen by a rare tumor, an epithelioid hemangioendothelioma, in a 9-year-old girl. To our knowledge, three previous cases of this disorder involving the spleen have been reported, but this is the first associated with functional hyposplenism.

Aggressive chemotherapy in the treatment of Burkitt's and non-Burkitt's undifferentiated lymphoma.

Because of the aggressive nature and frequent recurrence of malignant lymphomas of the undifferentiated type, we used a multi-drug induction chemotherapy regimen that has met with some success in children with similar type of histopathology followed by intensification and 8 cycles of consolidation chemotherapy in an attempt to prolong the duration of remission and survival in adult patients with this diagnosis. Fifty-one patients (median age 35 years) with undifferentiated malignant lymphoma were collected over a 4 year period (1984-1988) and entered into a phase III protocol done under the auspices of the Eastern Cooperative Oncology Group (ECOG). Six patients who had their diagnosis made at surgery and had resection of their tumor were excluded from analysis of response to therapy. Sixty percent of the patients had Stage IV disease. Sixteen patients had marrow involvement and five had central nervous system (CNS) disease. None of the patients received CNS radiation therapy. The 45 patients evaluated for response showed a response rate of 67% (30/45) and a complete response rate of 40% (18/45). Thirteen responders continue disease-free with a median follow-up of > 40 months and have an estimated 5 year survival of 80%. Only two treatment related deaths were reported for the entire group. Patients with undifferentiated non-Burkitt's lymphoma had a longer survival than those with undifferentiated Burkitt's. We concluded that adult patients with undifferentiated lymphomas could be treated successfully with an aggressive multi-drug chemotherapy regimen, consisting of multiple alternating cycles of non-crossed-resistant chemotherapy. Toxicity with this aggressive prolonged regimen was acceptable.

In vivo effects of recombinant human stem cell factor treatment. A morphologic and immunohistochemical study of bone marrow biopsies.

Bone marrow (BM) aspirate and biopsy specimens from seven female patients with advanced or metastatic breast cancer and preserved marrow function treated on a phase I trial of recombinant methionyl human stem cell factor (r-metHuSCF; SCF) were evaluated by immunohistochemical staining before and after treatment with SCF. Doses of SCF included 10 g/kg/day in 2 patients, 25 micrograms/kg/day in 2 patients, and 50 micrograms/kg/day in 3 patients administered as subcutaneous bolus injections for 14 days. Following treatment, bone marrow cellularity increased up to 1.6-fold (P = NS), with an increased frequency of promyelocytes (P < .002), but an unchanged relative frequency of other marrow hematopoietic cells. The mean relative frequency of BM CD34+ progenitor cells increased from 0.9% to 1.8% (P < .001). The mean proportion of BM cells stained by Ki-67/MIB 1 and PCNA/PC10, monoclonal antibodies (MoAb) recognizing proliferation-associated nuclear proteins, increased from 18.6% to 35.4% (P < .003) and from 32.4% to 49.4% (P < .01), respectively. Most of the Ki-67 and PCNA positive cells were represented by promyelocytes, proerythroblasts, and myeloblasts. SCF therapy was not associated with marrow fibrosis or increases in the number of macrophages. Peripheral white blood cell counts increased 1.3- to 3.6-fold following SCF. The mean absolute neutrophil counts increased from 3.9 x 10(9)/L (range 2.6-5.3) to 7.2 x 10(9)/L (range 4.7-12.3), and reticulocyte counts increased by a mean of 1.5 fold (range 1.2-fold to 2.0-fold). No consistent difference in platelet counts was seen. These results suggest that SCF given in vivo is effective in increasing the frequency of CD34+ BM progenitor cells, and has the capacity to increase the proliferation and differentiation rate of hematopoietic precursor cells. These effects indicate that SCF may represent a cytokine capable of affecting multiple hematopoietic lineages.

Splenic marginal zone cell lymphoma: report of an indolent variant without massive splenomegaly presumably representing an early phase of the disease.

Splenic marginal zone (MRZ) cell lymphoma is a recently described neoplasm arising in a unique compartment of splenic white pulp, producing massive splenomegaly and spreading to bone marrow and distant lymph nodes. We report three cases of splenic lymphoma that morphologically and immunohistochemically appear to originate from MRZ cells that presented as indolent neoplasms involving the spleen but with no or only moderate enlargement of the organ, presumably representing an early clinical stage of this disorder. Despite the evidence of involvement of the liver in one case, lymph nodes and bone marrow proved to be uninvolved. Histologically, the three spleens showed similar features, being characterized by the involvement of white pulp follicles and periarteriolar lymphoid sheaths by medium-sized lymphoid cells with slightly irregular nuclei and ample cytoplasm. Immunohistochemically, all the specimens expressed a series of B-lineage markers that, in contrast to specimens of monocytoid B cell lymphoma (MBCL) and hairy cell leukemia (HCL) studied for comparison, did not react with KiB3, LN1, and DBA.44 monoclonal antibodies.

Terminal deoxynucleotidyl transferase staining in acute leukemia and normal bone marrow in routinely processed paraffin sections.

Terminal deoxynucleotidyl transferase (TdT) is a nuclear protein widely used as a marker for the diagnosis and classification of acute leukemia. The usual methods for detecting TdT require smears, imprints, or cryostat sections of unfixed tissue. A polyclonal rabbit anti-TdT serum was used to immunostain 54 routinely processed bone marrow sections from patients with acute leukemic disorders, using a recently described antigen-unmasking technique based on microwave oven heating. The specificity of this method of TdT analysis was confirmed by comparing the results obtained with conventional TdT analysis by indirect immunofluorescence. Terminal deoxynucleotidyl transferase reactivity was also evaluated in 44 nonmalignant and normal bone marrow specimens. All cases that were TdT-positive by immunofluorescence (41 of 42 "pre-B" and T-cell acute lymphoblastic leukemia, 2 of 5 acute myeloid leukemia, and 1 of 5 chronic myeloid leukemia in blast crisis) were also positive in paraffin sections. The percentage fluorescence positivity correlated with the percentage of immunoperoxidase stained cells in 44 of 45 cases. The remaining nonneoplastic and normal bone marrow biopsy specimens were TdT-negative. These results show that TdT immunoperoxidase staining of conventionally processed bone marrow specimens can be readily achieved by the use of a simple antigen-unmasking technique and may provide useful diagnostic information particularly in cases in which fresh tissue samples are unavailable.