Age and cytogenetics as predictors of event free survival in patients with acute non-lymphocytic leukemia receiving high dose cytosine arabinoside and daunorubicin as consolidation chemotherapy.

Between 1991 and 1999, 67 patients with acute non-lymphocytic leukemia (ANLL) in complete remission received high dose cytarabine (HiDAC) 3 gm/m2 q12h x 12 doses followed by daunorubicin 45 mg/m2/day x 3 days as consolidation therapy. Five year actuarial event free survival (EFS) was 34% +/- 6%. Age was significantly associated with EFS. EFS was 60% +/- 15% in patients age 20 to 29, 48% +/- 16% in patients age 30 to 39, 23% +/- 10% in patients age 40 to 49, 31% +/- 11% in patients age 50 to 59, and 0% in patients age > or = 60. Contrary to other reports which have used different HiDAC regimens, we found no relationship between cytogenetics and EFS. Cytogenetics were defined as favorable risk: t(8;21), inv (16), and del (16); neutral risk: normal or t(15;17); and unfavorable risk: any abnormality not included in favorable risk or neutral risk. EFS was 29% +/- 17% in patients with favorable cytogenetics, 37% +/- 14% in patients with neutral cytogenetics, and 31% +/- 12% in patients with unfavorable cytogenetics. These differences were not statistically significant. Because of the successful use of allogeneic transplantation at relapse in patients with matched related donors, five year actuarial survival (S) in this series was 40% +/- 6%. Five year actuarial survival was 57% +/- 9% for patients age < or = 44 and 25% +/- 8% for patients age > or = 45. This difference is statistically significant, p < .025. Clinicians should be cautious about making clinical decisions regarding consolidation therapy of ANLL on the basis of the presence or absence of cytogenetic abnormalities as the importance of cytogenetics may depend on the specific therapy which is employed.

Limited efficacy of intensified preparative regimens and autologous transplantation as salvage therapy in high grade non-Hodgkin's lymphoma.

Between 9/86 and 6/98, 22 patients with relapsed or refractory high grade lymphoma received intensified preparative therapy and underwent autologous transplantation at a single institution. Two intensified preparative regimens were used--cyclophosphamide, etoposide, total body irradiation (CY-VP-TBI) (N=17) and cyclophosphamide, BCNU, etoposide (CBV) (N=5). For all patients undergoing autologous transplantation, 5 year actuarial survival (S) and 5 year event free survival (EFS) were only 18% +/- 8%. Treatment related mortality was 14% overall but only 8% in patients receiving G-CSF or GM-CSF. Survival was significantly inferior to the survival observed in a concurrent series of patients with intermediate grade lymphoma, 34% +/- 6%, p < .05. Using high dose therapy in conjunction with autologous transplantation at the time of relapse may not be as valuable a strategy in high-grade lymphoma as in intermediate grade lymphoma although most studies combine the two disorders. Alternative strategies for the use of transplantation in high grade lymphoma, such as the use of transplantation as consolidation therapy, need to be investigated.

Is total body irradiation a necessary component of preparative therapy for autologous transplantation in non-Hodgkin's lymphoma.

Between September 1986 and June 1998, 157 patients with low grade, intermediate grade, or high grade lymphoma underwent autologous transplantation at a single institution. Two preparative regimens were used: cyclophosphamide, etoposide, total body irradiation (CY-VP-TBI) (N=110) and cyclophosphamide, BCNU, etoposide (CBV) (N=47). The two groups were not significantly different with respect to source of stem cells, gender, stage at presentation, incidence of prior bone marrow involvement, sensitivity to salvage therapy, or histologic grade of lymphoma. The CBV group was significantly older, 49% of patients over age 50, as compared to 26% of patients over age 50 for the CY-VP-TBI group. Response rates and the incidence of fatal toxicity were similar for the two groups. Five year actuarial survival was 31% +/- 9% for CBV and 38% +/- 5% for CY-VP-TBI, p =.85. In a multivariate analysis, in which preparative regimen, age, histologic grade of lymphoma, and sensitivity to salvage therapy were the independent variables, TBI was not significantly associated with survival, and the direction of the trend was for TBI to be less effective than CBV. TBI does not appear to be an essential component of preparative therapy for autologous transplantation in patients with lymphoma.

Intensified preparative regimens and allogeneic transplantation in refractory or relapsed intermediate and high grade non-Hodgkin's lymphoma.

Between September 1986 and June 1998, 32 patients with relapsed or refractory intermediate or high grade lymphoma received intensified preparative therapy and underwent allogeneic transplantation at a single institution. Patients were considered for allogeneic transplantation if they failed to respond to initial therapy, failed to respond to salvage therapy, relapsed after autologous transplantation, had bone marrow involvement, or failed attempts to harvest autologous stem cells. Patients had a median age of 39 years and had generally received at least two chemotherapy regimens. Five year actuarial survival (S) was 16% +/- 6%; median survival was 4 months. Survival was significantly worse in patients who had received high intensity brief duration chemotherapy prior to transplantation and was also significantly worse in patients who did not receive total body irradiation (TBI). This likely reflects the fact that the patients with the most resistant disease had required local radiotherapy and could not receive TBI. While treatment related mortality played a major role in limiting the effectiveness of allogeneic transplantation, in this heavily pre-treated population of patients with resistant disease, only 39% of patients achieved a complete response following allogeneic transplantation, and in only 40% of that group was long term disease free survival achieved.

Intensified preparative regimens and autologous transplantation in refractory or relapsed intermediate grade non-Hodgkin's lymphoma.

Between September 1986 and June 1998, 99 patients with relapsed or refractory IGL received intensified preparative therapy and underwent autologous transplantation at a single institution. Two intensified preparative regimens were used: cyclophosphamide, etoposide, total body irradiation (CY-VP-TBI) (n = 66) and cyclophosphamide, BCNU, etoposide (CBV) (n = 33). As clinical features and results were not different for the two preparative regimens, results were combined. For all patients undergoing autologous transplantation, 5-year actuarial overall survival (OS) was 34% +/- 6%; 5-year event-free survival (EFS) was 26% +/- 5%. For patients who responded to primary therapy, salvage therapy, or both, OS was 42% +/- 7%; for non-responders to prior therapy, OS was 14% +/- 7%, P < 0.025. OS was better among patients responding to salvage therapy (50% +/- 9%), than among patients who had a complete response to initial therapy, but failed to respond or were untested/unevaluable with respect to salvage therapy (26% +/- 10%; P < 0.025). On multivariate analysis, response to salvage therapy was associated with survival following autologous transplantation (P < 0. 005). Treatment related mortality was 9% overall and only 6% after G-CSF and GM-CSF were introduced into routine clinical practice. High-intensity preparative therapy is highly effective, with acceptable treatment-related mortality, in patients with IGL who have responded to induction therapy, salvage therapy, or both. The best responses are observed in patients responding to salvage therapy. Randomized prospective studies will be needed to further define the role of intensified preparative regimens. Bone Marrow Transplantation (2000) 25, 257-262.

Polymerase chain reaction amplification of three different Trypanosoma cruzi DNA sequences from human chagasic cardiac tissue.

Chagas' disease is caused by the hemoflagellate protozoan Trypanosoma cruzi. The most common, serious manifestation of Chagas' disease is a progressive inflammatory cardiomyopathy, which occurs decades after primary infection. The inability to consistently demonstrate T. cruzi by histologic techniques in inflammatory cardiac lesions has suggested that the parasites' persistence may not be required for the pathology of the chronic phase. In this report we further analyze the persistence and localization of T. cruzi DNA in the hearts of seven patients with chronic chagasic cardiomyopathy, along with four indeterminate patients and seven control patients seronegative for T. cruzi infection. In the seven patients with chronic chagasic cardiomyopathy, we extracted DNA from selected inflammatory foci-positive (IFP) and inflammatory foci-negative (IFN) areas of' hematoxylin and eosin-stained cardiac tissue. We then used polymerase chain reaction methodology to amplify three different T. cruzi sequences (a minicircle sequence [MCS], a satellite repetitive sequence [RS], and, a low copy number sequence within the gene coding for a flagellar protein [FPS]). The MCS was detected in approximately 100% of both the IFP and IFN areas analyzed. The RS was detected in 37.5% and 23% of the IFP and IFN areas, respectively (difference not statistically significant; P > 0.10, degrees of freedom = 1, G test of independence = 1.9522). The FPS was rarely detected (2%), and was only present in DNA extracted from IFP areas. The MCS was also detected in most indeterminate cases (none of whom had inflammatory lesions) although with a markedly diminished amplification signal relative to cardiomyopathy cases. The MCS was not amplified from the cardiac tissues from seronegative controls. These results suggest that the quantity of T. cruzi DNA persisting in hearts of patients with Chagas' disease correlates with cardiomyopathy, but may not be preferentially associated with inflammatory foci.

Detection of Trypanosoma cruzi with the polymerase chain reaction and in situ hybridization in infected murine cardiac tissue.

Chagas' disease is caused by the hemoflagellate protozoan Trypanosoma cruzi, which is predominantly found in South and Central America and Mexico. Although the parasite is present in the United States, confirmed cases of human disease are rare. The most serious manifestation of chronic Chagas' disease is a progressive inflammatory cardiomyopathy. However, T. cruzi has not been consistently demonstrated with histologic techniques in inflammatory cardiac lesions. In this study, we used both polymerase chain reaction (PCR) amplification of extracted DNA from hematoxylin and eosin-stained tissue scrapings, and in situ hybridization to detect the presence of T. cruzi in infected murine cardiac tissue sections. Three T. cruzi-specific DNA sequences were used: a 122-basepair (bp) sequence localized within the minicircle network (MCS), a 188-bp nuclear repetitive sequence (RS), and a 177-bp sequence within the open reading frame of a gene coding for a flagellar protein (FPS). We found that all three sequences are amplifiable from scrapings of murine cardiac tissue. The MCS and RS are detected at 0.167 and 0.24 amastigote DNA equivalents, while FPS is barely detected at 0.24 amastigote DNA equivalents. On the other hand, in situ hybridization with all three sequences allowed for the detection of T. cruzi amastigotes within the tissue. The MCS and FPS, however, consistently yielded a more intense signal. These results indicate that PCR and in situ hybridization may prove useful in establishing the prevalence of T. cruzi in human chagasic cardiomyopathy.

High-dose cytosine arabinoside and etoposide: an effective regimen without anthracyclines for refractory childhood acute non-lymphocytic leukemia.

The purpose of this report is to describe the tolerability and activity of the combination of high-dose cytosine arabinoside (Ara-C) given at the maximum tolerated dose of 36 g/m2, together with high doses of etoposide in relapsed and refractory childhood acute leukemias. Eighteen children with relapsed or refractory acute leukemia were treated with Ara-C 3 g/m2 every 12 h on days 1-6, followed by etoposide 400 mg/m2 on days 7-9 (HDAC/VP-16). Eight children with refractory disease received HDAC/VP-16 as salvage induction therapy after failing conventional induction regimens; four of five refractory ANLL patients (80%) had a complete response (CR) after HDAC/VP-16 therapy. Ten patients received HDAC/VP-16 as post-remission intensification therapy; five patients (four ANLL, one relapsed ALL) remain in second CR at 56, 26, 9, 5 and 2 months. Toxicities were primarily hematologic and dermatologic. Seven patients (39%) developed bacterial or fungal infections; four patients developed grade 3 or 4 acral erythema. No patient died of therapy-related toxicity. The combination of 36 g/m2 cytosine arabinoside and 1200 mg/m2 etoposide is an effective regimen for children with relapsed or refractory acute nonlymphocytic leukemia, with tolerable toxicities; the absence of anthracyclines makes this regimen suitable for patients who have previously received maximal doses of anthracyclines or who have evidence of cardiac dysfunction. Further evaluation of this regimen in acute nonlymphocytic leukemia is presently being investigated.

Cat scratch disease: detection of Bartonella henselae DNA in archival biopsies from patients with clinically, serologically, and histologically defined disease.

Serological and epidemiological studies suggest that Bartonella henselae is the etiological agent of cat scratch disease. We designed a study to detect B. henselae in archival biopsies by polymerase chain reaction amplification of the 16S rRNA gene followed by Southern blot hybridization. Forty-two histologically defined cat scratch disease biopsies and eighteen controls were selected for blinded analysis. After testing, charts were reviewed for clinical, immunological, and microbial evidence of infection. Results were correlated with duration of illness and antimicrobial therapy. B. henselae DNA was identified in 27 of 42 (64%) histologically defined patients and 23 of 34 (68%) patients defined both clinically and histologically. There were no false positives (0 of 18). A small subset (n = 14) had cat scratch disease serological tests performed. B. henselae was identified in 8 of 10 serologically positive patients. Polymerase chain reaction detected 50% of our DNA-positive cases (most of these early in the clinical course). Southern blotting of amplicons both doubled sensitivity (detecting patients > 4 weeks into illness) and confirmed B. henselae as the causative species. Our study strongly associates B. henselae with cat scratch disease, suggesting that it may be the most likely etiological agent in the majority of patients with cat scratch disease.

Clinical applications of flow cytometry in hematology and immunology.

OBJECTIVE: To review the major applications of flow cytometry in the diagnosis of hematologic and immunologic disease. DATA SOURCES: Professional literature and authors' experience. DATA SYNTHESIS: Flow cytometry is evolving as a major diagnostic tool for evaluating hematologic disease, monitoring HIV infection and assessing peripheral blood and bone marrow for CD34 positive stem cells in marrow transplantation. This review highlights the opportunities and pitfalls of this area of diagnostic laboratory medicine. CONCLUSION: The applications of flow cytometers in diagnostic hematology and immunology are expanding, providing new opportunities for enhanced precision in diagnosis. As in any relatively new field much work remains in order to optimize accuracy and efficiency while minimizing cost.

Cutting edge technologies in the evaluation of bone marrow samples.

OBJECTIVE: To review cutting edge technologies for evaluation of bone marrow samples. DATA SOURCES: Professional literature and authors' experience. DATA SYNTHESIS: Novel technologies for evaluating bone marrow specimens that have prognostic and therapeutic value in chronic and acute leukemia have been recently developed. Polymerase chain reaction-based assays that are useful in detecting chromosomal translocations in CML and AML are relevant to diagnosis and initial therapy. These assays can also be used to detect minimal residual disease after therapy and thus have prognostic value. In addition, a novel and elegant technique for determining leukemic cell resistance to chemotherapy agents (in vitro drug sensitivity) has prognostic and therapeutic value in AML. Use of these assays may lead to greater diagnostic precision, improved prognostication and more effective therapy. CONCLUSION: Cutting edge technologies that have utility in the evaluation of bone marrow specimens include polymerase chain reaction based assays for detecting specific chromosomal translocations, particularly useful in CML and AML, as well as in vitro drug sensitivity testing in AML.

Microsatellite analysis of posttransplant lymphoproliferative disorders: determination of donor/recipient origin and identification of putative lymphomagenic mechanism.

The genetic mechanisms that give rise to posttransplant lymphoproliferative disorders (PTLDs) are not well established, yet previous studies have focused or) the role of immunosuppression and EBV infection. We investigated whether microsatellite analysis could: (a) determine the recipient/donor origin of the tumor; and (b) document novel genetic alterations in PTLDs, i.e., microsatellite instability. We characterized seven cases of PTLD (five B-cell and two T-cell non-Hodgkin's lymphomas) in which donor allograft tissue, normal recipient tissue, and tissue from the PTLDs were available. In each case, six microsatellite loci were analyzed. Five cases were of host origin (three B-cell and two T-cell lymphomas). The two cases of donor origin were B-cell lymphomas. Multiple loci showed microsatellite instability in two cases of host-derived T-cell non-Hodgkin's lymphomas (28% of PTLDs). These findings show that microsatellite analysis may be used to determine the host or donor origin of PTLDs and suggest for the first time that defective DNA mismatch repair may be an underlying genetic mechanism of lymphomagenesis in some cases of PTLD.

CD4 predicts nonlymphocytic lineage in acute leukemia. Insights from analysis of 125 cases using two-color flow cytometry.

The classification of acute leukemia into lymphoid or nonlymphoid is of critical therapeutic importance. Two-color flow cytometric analysis has emerged as a valuable addition to morphology and cytochemistry for the distinction of acute lymphocytic leukemia (ALL) and acute nonlymphocytic leukemia (ANLL). By careful selection of monoclonal antibody (mAb) combinations, diagnostic accuracy, and cost effectiveness may be enhanced compared to flow cytometry using one-color analysis. The sensitivity and specificity of a mAb panel were assessed in the determination of nonlymphocytic lineage in acute leukemia. One hundred twenty-five consecutive cases of acute leukemia were analyzed in which Wright's-stained smears, cytochemical stains, and immunophenotyping studies had been performed. The antibody panel included the nonlymphoid markers CD13, CD33, CD14, and CD4 in combination with CD2, as well as a broad panel of lymphoid and nonlineage specific markers. Of the 125 cases of acute leukemia studied, 85 cases (68%) were nonlymphocytic and 32 cases (26%) were lymphocytic (28 cases B cell, 4 cases T cell). CD13 and CD33 were very sensitive in the detection of ANLL, being expressed on 94% and 93% of ANLL cases, respectively. Sixty-five percent of cases of ANLL were CD4+ (CD2-). However, CD4+ (CD2-) had a much higher specificity (91%) for ANLL than CD13 (75%) or CD33 (84%), which were expressed in a significant number of ALL. When leukemic cells were positive for CD4 (CD2-) and either CD13 or CD33, specificity and positive predictive value (PPV) for ANLL rose to 96% and 98%, respectively. The combination of CD4 positivity with either CD13 or CD33 has higher specificity and PPV than the traditional positivity for both CD13 and CD33 (specificity 89%, PPV 96%). Careful analysis of the sensitivity, specificity, and predictive values of mAbs using this method has also allowed us to establish a more cost-effective and diagnostically relevant mAb panel. Our studies show that CD4 is underappreciated as a very specific and moderately sensitive marker for ANLL.

Progression of childhood linear scleroderma to fatal systemic sclerosis.

An 8-year-old girl presented with linear scleroderma, no evidence of systemic disease, and a negative antinuclear antibody (ANA) test. Over the next 12 months, she functioned normally. However, over the subsequent 5 months, she developed dyspnea, progressive pulmonary hypertension, a positive ANA test, and died 17 months after presentation. At autopsy, diffuse pulmonary interstitial fibrosis, small pulmonary arterial fibroplasia, tricuspid and mitral valve subendocardial fibrosis, and distal esophageal fibrosis were seen. Contrary to suggestions in the rheumatology literature, childhood linear scleroderma, even when ANA negative at presentation, may progress to fatal systemic sclerosis.

t(5;14)(q33-34;q11), a new recurring cytogenetic abnormality in childhood acute leukemia.

A novel translocation, t(5;14)(q33-34;q11), was identified in the leukemic cells of four children presenting with acute lymphoblastic leukemia (ALL). The patients were 14 months, 2 years, 10 years, and 12 years old; each presented with one or more features of bulky disease, including lymphadenopathy, organomegaly or a mediastinal mass. The leukemic blasts were B lineage in two cases and T lineage in two cases. The patients with B-lineage ALL remain in continuous remission at 22 and 19 months following diagnosis. One patient with T-lineage ALL relapsed 6 months after diagnosis. The other patient with T-lineage ALL developed acute myelocytic leukemia (AML) 17 months after diagnosis; t(5;14)(q33-34;q11) was present in both the lymphoblasts at diagnosis and the myeloblasts at relapse, consistent with a lineage switch from ALL to AML. Rearrangement of the T-cell receptor delta chain (TCRD) gene at 14q11 was demonstrated in the three cases studied, suggesting its involvement in the pathogenesis of these leukemias by alteration of the structure or expression of an unidentified gene(s) on the long arm of chromosome 5.

Determination of B-cell clonality in paraffin-embedded endoscopic biopsy specimens of abnormal lymphocytic infiltrates and gastrointestinal lymphoma by polymerase chain reaction.

External and seminested polymerase chain reaction techniques were used to determine B-cell clonality in paraffin-embedded biopsy specimens of abnormal lymphocytic infiltrates and malignant lymphomas of the gastrointestinal tract. Using consensus primers for the variable and joining regions, the authors detected clonal immunoglobulin heavy-chain gene rearrangements in five of eight endoscopic biopsy specimens (62.5%) and six of eight resection specimens (75%) of well-characterized B-cell gastrointestinal lymphomas. No clonal rearrangements were detected in 21 negative controls including 7 cases of chronic gastritis and 7 cases of Crohn's disease. In endoscopic biopsy specimens of eight patients with abnormal lymphocytic infiltrates, clonal immunoglobulin heavy-chain gene rearrangement was found in three of six cases (50%) in whom gastrointestinal involvement by lymphoma was subsequently established. Therefore, polymerase chain reaction may be used to demonstrate B-cell clonality in paraffin-embedded endoscopic biopsy specimens of abnormal lymphocytic infiltrates and may circumvent the need for more invasive procedures.

Childhood acute lymphocytic leukemia with a preleukemic phase: report of an associated translocation and review of the literature.

A fourteen month old boy presented with hepatosplenomegaly and pancytopenia. An extensive evaluation, including bone marrow aspiration and biopsies of both liver and lymph nodes, revealed a polyclonal B cell proliferation consistent with a reactive process, with no evidence of leukemia. After receiving transfusions of red cells and platelets, his blood counts recovered. Five weeks later, he returned with an elevated white blood count and bone marrow findings diagnostic of acute lymphocytic leukemia. The leukemic blasts contained a novel chromosomal translocation, t(5;14) (q34;q12). We describe the clinical, immunophenotypic and cytogenetic features of this case, review the literature of acute lymphocytic leukemia associated with a preleukemic phase, and discuss the relationship of this clinical entity to the 5q-abnormality associated with myelodysplasia.

Detection of clonal immunoglobulin heavy chain gene rearrangements by polymerase chain reaction in scrapings from archival hematoxylin and eosin-stained histologic sections: implications for molecular genetic studies of focal pathologic lesions.

Using a simple scraping technique to obtain DNA directly from archival hematoxylin and eosin-stained slides, we successfully amplified clonal immunoglobulin heavy chain gene rearrangements (IGR) from lymphomatous infiltrates, as small as 1 mm2. The fragments amplified by the polymerase chain reaction (PCR) were identical in size to those from corresponding whole unstained sections freshly cut from the paraffin-embedded blocks. Using this technique, we detected clonal IGR from the scraping of a small lymphomatous nodule in the colon, although no amplified fragments were detected from the whole section. Furthermore, we demonstrated that two morphologically different areas in a case of B-cell lymphoma have identical amplified fragments. It is important that no amplified fragments were detected in scrapings from adjacent nonneoplastic areas. Although DNA recovered from scrapings was partially degraded, fragments as large as 725 base pairs were successfully amplified from a slide stored more than 11 years. This technique thus allows detection of clonal IGR in tissues focally involved by B-cell lymphoma and molecular genetic studies of focal pathologic lesions as an alternative to in situ hybridization or in situ PCR. Finally, this technique can be applied to archival slides when tissue blocks are not available.

Expression of major histocompatibility complex antigens and adhesion molecules in hearts of patients with chronic Chagas' disease.

We have previously reported that heart lesions in patients with chronic cardiac Chagas' disease are composed predominantly of granzyme A+, cytolytic CD8+ T lymphocytes. We now pursue this study in the immunopathology of chronic chagasic cardiomyopathy by investigation of the expression of HLA antigens, and adhesion molecules in the hearts of seven chagasic patients with cardiac disease, two asymptomatic chagasic patients, and seven normal controls. Comparative immunohistochemical analyses show that HLA-ABC antigen expression is enhanced on the myocardial cells of chagasic patients with chronic cardiomyopathy, suggesting a possible role for these cells as targets for the CD8+ cytolytic lymphocytes dominant in these lesions. The HLA-DR antigens are not observed on myocardial cells, but are consistently upregulated on the endothelial cells in the hearts of patients with chronic chagasic cardiomyopathy. Intercellular adhesion molecule is expressed by endothelial cells of both chagasic and nonchagasic individuals, but E-selectin was detected only on vessels of hearts from chagasic patients who had chronic cardiomyopathy. Most of the lymphocytes in these lesions express lymphocyte function antigen-1 (LFA-1), CD44, and very late antigen-4, and a few display weak expression of LFA-3. We propose that the expression of these adhesion molecules and major histocompatibility complex antigens by endothelial cells, myocardial cells, and lymphoid cells in these lesions contribute to the pathogenesis of chronic chagasic cardiomyopathy.