Platelet satellitism as presenting finding in mantle cell lymphoma. A case report.

Platelet satellitism surrounding polymorphonuclear neutrophils has been observed almost exclusively in EDTA-treated blood at room temperature. The mechanism underlying this phenomenon is not understood fully. We report a case of platelet rosetting around atypical lymphocytes in peripheral blood smears made from EDTA-treated and untreated blood. Flow cytometry of the peripheral blood sample and immunohistochemical stains of the subsequent bone marrow biopsy specimen revealed a monoclonal B-cell population positive for CD5, CD20, and cyclin D1 and negative for CD3 and CD23; cytogenetic findings revealed a complex karyotype that included t(11;14). These findings were consistent with mantle cell lymphoma. To our knowledge, the finding of platelet satellitism involving mantle cell lymphoma cells in peripheral blood has not been reported previously.

Can polymerase chain reaction help distinguish benign from malignant lymphoid aggregates in bone marrow aspirates?

OBJECTIVE: Although morphologic and immunologic clues are helpful in distinguishing benign from malignant lymphoid aggregates in bone marrow biopsies, there remain some cases in which it is not possible to arrive at a definitive diagnosis. Since the malignant aggregates are monoclonal B-cell proliferations, we sought to determine whether performing polymerase chain reaction for the immunoglobulin heavy-chain locus would be helpful in distinguishing these 2 entities. METHODS AND RESULTS: Scrapings from unstained bone marrow aspirate smears or touch preparations of bone marrow biopsies from 15 patients with benign bone marrow lymphoid aggregates and 18 patients with malignant lymphoid infiltrates were analyzed for rearrangements of the FR3 region of the immunoglobulin heavy-chain gene locus by a heminested polymerase chain reaction procedure. All specimens had amplifiable DNA, as shown by amplification of the ras proto-oncogene. None of the 15 cases of benign bone marrow lymphoid aggregates demonstrated clonality upon amplification of the immunoglobulin heavy-chain gene locus. In contrast, 8 of the 18 malignant samples were positive (P =.01 by chi(2) test; sensitivity, 44%; specificity, 100%; positive predictive value, 100%; negative predictive value, 60%). There was a tendency for there to be more lymphocytes in stained bone marrow aspirate smears from the cases of malignant lymphoid aggregates with a positive polymerase chain reaction result than in those without demonstrable clonality (36.0 +/- 35.4% vs 9.8 +/- 8.0%, P =.13). CONCLUSIONS: Polymerase chain reaction for the immunoglobulin heavy-chain gene locus may help distinguish benign from malignant bone marrow lymphoid aggregates. Although the presence of false-negative samples may be related to the relative lack of lymphocytes in the bone marrow aspirates, other factors, such as the lack of amplification of the FR3 region of the immunoglobulin heavy-chain gene locus in particular tumors, cannot be ruled out with certainty.

Evaluation of the Sysmex UF-100 automated urinalysis analyzer.

Urinalysis is a high-volume procedure that currently requires significant labor to examine microscopic sediment. We evaluated the Sysmex UF-100 automated urinalysis analyzer for performing this task. Instrument accuracy was assessed by comparing continuous counts of microscopic elements from the UF-100 with ranges of cells (per low-power field or high-power field) from manual microscopy performed on centrifuged urines. Counts showed good agreement between methods (gamma statistic: 0.880-0.970) for all microscopic elements in 252 urine samples. Within-run imprecision of cell counts expressed as CV (mean cell count/microL) was for erythrocytes (RBC) 31% (5), 18% (50), 2.4% (800); for leukocytes (WBC) 14% (10), 11% (100), 8.5% (400); for squamous epithelial cells (SEC) 18% (5), 12% (30), 7.0% (100); for casts 45% (1), 17% (4); for bacteria 2-12% (entire range of 40-2500). Between-run imprecision on quality-control cell suspensions expressed as CV (mean cell count/microL) was for RBC 6.1% (50), 2.7% (256); for WBC 26.9% (54), 4.9% (228). Cells counted on dilution were 99.1% of expected for RBC, 102.0% for WBC, and 121.8% for bacteria. Carryover was <0.04% for RBC, <0.03% for WBC, <0.14% for SEC, <0.29% for bacteria. We conclude that the UF-100 can automatically perform reliable quantitative microscopic urinalysis in batches without operator interaction.

Ploidy Analysis and Ki-67 Expression in Myelodysplastic Syndromes.

BACKGROUND: The Myelodysplastic Syndromes (MDS) are disorders involving clonal proliferative activity in the bone marrow that can lead to marrow failure and acute leukemia. This study was undertaken to evaluate the relationships between clinical factors, type of MDS, DNA ploidy, and Ki-67 expression. DESIGN: Air-dried bone marrow smears from 27 patients were Feulgen stained for DNA and analyzed using the CAS 200 image analyzer. Ki-67 expression was also examined by image analysis in 25 of these cases in bone marrow core biopsy specimens using the monoclonal antibody MIB-1. Age, sex, bone marrow cellularity, and MDS grade of each patient were also recorded. Percent S-phase was assessed only for the diploid samples. RESULTS: There were 16 diploid and 11 aneuploid cases on analysis of Feulgen-stained bone marrow aspirate smears. The percentage of MIB-1+ cells ranged from 6.4%-61.7% (mean-37.7 ± 6.4%). Among the 16 diploid cases, 18.2 ± 5.7% of the cells were in S-phase. High grade MDS (RAEB-T, RAEB, CMML) was associated with younger age and male sex (p = 0.03), lower percentage of cells in S-phase (p = 0.04), greater bone marrow cellularity (p = 0.005), and greater MIB-1 expression (p = 0.04). With increasing age, there were more females (p = 0.03), more low grade MDS (RA, RARS), and a lower percentage of cells in S-Phase (p = 0.04). Female patients tended to be older, have less cellular bone marrows (p = 0.003), less MIB-1 expression (p = 0.03), low grade MDS (p = 0.02), and increased percentage of cells in S phase (p = 0.008). CONCLUSION: Ki-67 expression, percent cells in S-phase, clinical parameters and subtype of MDS tend to distinguish two separate groups of MDS patients, and may explain in part the difference in biologic behaviour between high and low grade MDS.

Malignant lymphoma can present as hepatobiliary disease.

BACKGROUND: Liver involvement by non Hodgkin's lymphoma usually represents systemic progression of a low grade lymphoma. Sixteen patients without palpable lymphadenopathy who presented with features suggesting hepatic inflammation were found to have non Hodgkin's lymphoma involving the liver. METHODS: Tissue sections stained with hematoxylin and eosin were examined by light microscopy. Immunohistochemical studies were performed on formalin-fixed paraffin embedded tissue to determine phenotype. Patients' medical records were reviewed for clinical details. RESULTS: Clinical presentation included fever, jaundice, and elevated liver enzymes. Peripheral lymphadenopathy was absent. Hepatomegaly and splenomegaly were common. Imaging studies revealed a homogeneous pattern of liver involvement without a discrete mass. Histologically, infiltration of the liver was predominantly portal consisting of large, atypical cells scattered within a background of small lymphoid cells. By immunohistochemistry, it was determined that eight were T-cell and eight were B-cell lymphomas. Of the eight patients who had lymph node sampling, three showed involvement by diffuse mixed, small cleaved, and large cell lymphomas. Each of these three involved lymph nodes was located near the liver. Bone marrow infiltration was demonstrated in 10 of the 11 patients in whom it was sampled. Eleven of the 16 patients died 2 days to 31 months after diagnosis, and at last follow-up, 5 patients were alive 2 weeks to 13 months after diagnosis. CONCLUSIONS: Patients with non-Hodgkin's lymphoma can present with hepatic dysfunction and without peripheral lymphadenopathy. This presentation of lymphoma is associated with extensive disease and an aggressive course.

Rhabdoid cells in peritoneal fluid. A case report.

BACKGROUND: Rhabdoid tumor is an aggressive, malignant renal neoplasm of infants. Many extrarenal sites have been documented. CASE: A poorly differentiated carcinoma of the ovary with rhabdoid features occurred in a 36-year-old woman. The peritoneal fluid contained numerous malignant cells with rhabdoid features. Electron microscopy and immunocytochemistry corroborated the rhabdoid nature of the cells. CONCLUSION: Rhabdoid cells can be distinguished from other neoplasms with similar cytologic features by ancillary studies and clinical history.

Polymyositis as a manifestation of chronic graft-versus-host disease.

A syndrome indistinguishable from idiopathic polymyositis occurred in 11 patients as a manifestation of chronic GVHD. All patients had elevation of creatine phosphokinase (CPK). Immunohistology demonstrated the effector cells in the muscle infiltrates as cytotoxic T cells, a finding similar to idiopathic polymyositis. Polymyositis is a rarely reported complication of chronic graft-versus-host disease (GVHD) with only 8 cases described in the literature. We encountered this syndrome in a small but significant percentage of our patients with chronic GVHD. Polymyositis associated with chronic GVHD does not affect the overall prognosis for the patient. Moreover, polymyositis can be the only manifestation of chronic GVHD. Awareness of this complication is important because it can be confused with other causes of muscle weakness after bone marrow transplantation. Finally, prompt initiation of corticosteroid therapy results in a rapid improvement of the associated symptoms.

Antibody NCL-CD5 fails to detect neoplastic CD5+ cells in paraffin sections.

Many low grade B-cell lymphomas, and most T-cells lymphomas, express CD5 on their surface. This expression has been demonstrated in fresh cells or frozen sections. Recently, a new monoclonal antibody to CD5, NCL-CD5, has been introduced that detects CD5 in paraffin-embedded tissues. Paraffin-embedded tissues from five patients with small lymphocytic lymphoma (SL), four patients with chronic lymphocytic leukemia (CLL), and one patient each with large granular lymphocytic leukemia, diffuse large cell lymphoma (T cell), and mycosis fungoides were stained with the NCL-CD5 antibody after unmasking the antibody with the steam/citrate technique. All these cases had CD5 positivity demonstrated by flow cytometry or on cytospin or frozen section preparations. In addition, one case of angiofollicular lymph node hyperplasia (CD5+), two cases of SL/CLL (CD5-), and one case CD5- T-cell lymphoma were also investigated. Of the 12 CD5+ malignancies, only 1, an SL, was positive with the NCL-CD5 antibody. In seven of these cases, both B-5 and formalin-fixed tissues were tested; the one positive case was positive only in the formalin tissue. The three CD5- malignancies were also negative in paraffin sections (P = .001; McNemar test). However, reactive T cells did stain in these biopsy sections. The case of Castleman's disease showed many CD5+ cells with the NCL-CD5 antibody. Although NCL-CD5 does indeed stain reactive T cells in paraffin sections, it does not appear to stain neoplastic CD5+ cells.

Induction of long-term graft tolerance and donor/recipient chimerism.

We compared the effect of different immunosuppressive drug regimens on both mean and long-term allograft survival and the histologic appearance of donor/recipient chimeras in the ACI to Lewis rat heterotopic cardiac transplant model. Intraabdominal cardiac transplantation was performed in standard fashion and microchimerism was assessed using immunohistochemical staining of cut sections of recipient spleen and lymph nodes. All of the drug regimens studied resulted in excellent mean allograft survival. Furthermore, long-term (> 120 day) allograft survival was consistently observed in each group. In fact, many of the recipients continue to have functioning heterotopic grafts even as they approach the end of the natural life span of a Lewis rat. Mixed allogeneic chimerism, however, was not observed with 100% frequency despite the consistent induction of graft tolerance. This finding may be related to the immunosuppressive agents used or the sensitivity of the immunohistochemical assay. Therefore, the exact role, if any, of chimerism in the induction of graft tolerance remains unanswered.

Amplification methods in the molecular diagnosis of genetic diseases.

The ability to amplify DNA by the polymerase chain reaction (PCR) technique has revolutionized our ability to test for genetic mutations. Many different assay systems are available for analyzing the amplified DNA and RNA. These techniques can be performed easily on material from adults, children, fetuses, and even single cells from blastomeres and polar bodies. The detection rate of the screening test, as well as the frequency of the mutation in the study population and the technical limitations of the procedure, will determine the usefulness of a positive or negative result. Preimplantation testing provides a paradigm for the ease of use of PCR-based testing, yet also underscores the problems encountered with genetic screening because of the multitude of possible mutations and the possible misinterpretation of results.

Condyloma and cervical intraepithelial neoplasia of the endometrium.

Many studies have shown the presence of squamous metaplasia, dysplasia, carcinoma in situ and squamous cell carcinoma of the endometrium, whether they arose de novo or from direct extension from the cervix. Condyloma associated with squamous metaplasia or dysplasia of the endometrium is rare. We report a case of cervical intraepithelial neoplasia (CIN I) and condyloma of the endometrium. A 58-year-old woman presented with high-grade dysplasia on two successive pap smears. A total vaginal hysterectomy showed extensive CIN I and condyloma involving the entire endometrium. DNA in situ hybridization and polymerase chain reaction documented the presence of condyloma.

Small cell carcinomas of the lung express the Bcl-2 protein.

In our laboratory, we observed that a case of small cell carcinoma of the lung (SCLC) stained with a monoclonal antibody (Dako Corp., Bcl-2-124) against the Bcl-2 protein. To determine how common this reaction was, and whether it was specific for SCLC, we stained formalin-fixed, paraffin-embedded tissues from 23 cases of SCLC and 20 cases of squamous cell carcinoma of the lung for Bcl-2. Fifteen of the 23 SCLC cases stained positively for the Bcl-2 oncogene protein. In contrast, weak staining was observed in only three of the squamous cell carcinomas (chi 2-test; P = 0.001). In addition, four small cell carcinoma cell lines (H69, H146, H209, and WB) were tested by immunohistochemistry and flow cytometry for expression of this protein; all four were positive. In these and three other (H128, H432, and H510) SCLC lines, Northern blots of polyadenylated RNA revealed expression of the 6-kb Bcl-2 mRNA. Moreover, Western blots of extracts from these cell lines revealed the characteristic 26-kd band for Bcl-2 protein. In a single cell line, H82, which has previously been characterized as a variant SCLC, we failed to detect expression of the Bcl-2-specific mRNA and protein. We therefore conclude that most cases of SCLC of the lung express the Bcl-2 oncoprotein, which could play a role in the pathogenesis of this disease.

Comparison of the clinic microscopy laboratory with the cytopathology laboratory in the detection of malignant cells in body fluids.

The clinical microscopy (fluids) laboratory evaluates almost every body fluid that is obtained in the hospital. Because the fluids laboratory functions at all hours, it is often the first laboratory to receive a body fluid. In addition to its primary purpose of quantitating categories of cells, the medical technologist in this laboratory has an opportunity to identify malignant cells. To our knowledge, no formal study has ever been undertaken to evaluate the performance of the fluids laboratory in detecting malignancy. The authors therefore retrospectively identified 928 body fluids (pleural, peritoneal, cerebrospinal, and miscellaneous) over a 2-year period that had undergone simultaneous cytologic examination in our cytopathology laboratory and body fluid analysis in our fluids laboratory. Of these, a cytologic diagnosis of malignancy was made by the cytopathology laboratory in 107 cases; 821 were considered to be benign. No false-positive results were rendered by the fluids laboratory (100% specificity), but only 26 of the 107 malignant cases were identified (24% sensitivity); the overall accuracy was 93%. Factors contributing to the inability of the fluids laboratory to identify malignant cells included (1) too few cells to warrant a cytocentrifuge preparation, especially in cerebrospinal fluid specimens; (2) differences in the processing of specimens; (3) differences in staining procedures; and (4) differences in the training of personnel. The authors conclude that although the fluids laboratory correctly identifies neoplastic cells in approximately one fourth of the cases in which they are present, it should not be expected to detect malignant cells in every cytologically malignant case.(ABSTRACT TRUNCATED AT 250 WORDS)

CD3+, CD56+ aggressive variant of large granular lymphocyte leukemia.

Clonal expansions of CD3+ large granular lymphocytes (LGL) have been classified as T-LGL leukemia. The majority of patients with T-LGL leukemia have a chronic disease (years) manifested often by severe neutropenia, rheumatoid arthritis, and mild-to-moderate splenomegaly. The characteristic phenotype of the leukemic LGL is CD3+, CD8+, CD16+, CD57+, and CD56-. In this report we describe an aggressive variant of T-LGL leukemia in which leukemic LGL also expressed CD56, as identified by two-color flow-cytometry analysis. In contrast to the chronic nature typical of T-LGL leukemia, these patients presented with a severe systemic illness that was rapidly progressive and resistant to treatment. Atypical clinical features included rapidly increasing spleen size to massive proportions, extensive lymphadenopathy, and the presence of B symptoms (fever, nightsweats, weight loss). Hematologic and pathologic features were also unusual for T-LGL leukemia. These patients had very high LGL counts at diagnosis (range 11,692 to 26,312 microL), which increased rapidly despite treatment. Histopathologic examination of splenic sections showed extensive infiltration of red pulp cords and sinuses by leukemic cells with atrophy of the white pulp. These clinicopathologic features are similar to those described for patients with natural killer cell (NK)-LGL leukemia, whose cells are also CD56+. However, unlike NK-LGL leukemia, we could not show a direct pathogenic role for Epstein-Barr virus (EBV), as Southern-blot analyses using an EBV-joined termini probe were negative in these patients. Our findings suggest that CD3+, CD56+ LGL leukemia is a distinct clinicopathologic entity separate from the usual CD3+, CD56- T-LGL leukemia. The expression on leukemic LGL of CD56, an adhesion molecule, may determine the aggressive biologic nature of this newly described disease.

Staining for Bcl-2 protein helps to distinguish benign from malignant lymphoid aggregates in bone marrow biopsies.

Lymphoid nodules in a bone marrow biopsy may be either benign or malignant. Morphological clues may help to differentiate the benign from the malignant nodules. However, it is sometimes difficult, if not impossible, to make this distinction, especially in patients with a known low-grade lymphocytic malignancy. This study was undertaken to determine whether staining bone marrow biopsies with an antibody to the bcl-2 protein might aid in making this differentiation. Using a monoclonal antibody to bcl-2, we stained 26 bone marrows with benign lymphoid aggregates, 19 with a follicular lymphoma, 10 with small lymphocytic lymphoma/chronic lymphocytic leukemia, three with other non-Hodgkin's lymphomas, and three with other miscellaneous hematopoietic lesions. Only one of the 26 benign lymphoid aggregates had moderate to intense staining with this antibody; in contrast, 79% of the follicular lymphomas stained positively. Eight of the 10 small lymphocytic lymphoma/chronic lymphocytic leukemia cases stained with moderate to intense intensity; the other two cases had weak staining. No consistent pattern was seen with the other six lesions. Based on this data, we conclude that lack of staining of small lymphoid aggregates within the bone marrow with the antibody to the bcl-2 protein is suggestive of a benign aggregate, whereas moderate to strong staining intensity is most consistent with a malignant process.

Bone marrow changes associated with recombinant granulocyte-macrophage and granulocyte colony-stimulating factors. Discrimination of granulocytic regeneration.

The hematopoietic growth factors recombinant human granulocyte-macrophage colony-stimulating factor and recombinant human granulocyte colony-stimulating factor are associated with changes of the bone marrow. To evaluate the morphologic features and to differentiate them from leukemia, bone marrow specimens from 12 patients who had been treated with one of these agents were evaluated. The bone marrow displayed marked promyelocytic hyperplasia and a less striking increased percentage of myeloblasts. In each of the 11 patients without leukemia at the time of bone marrow biopsy, the percentage of promyelocytes in the bone marrow was greater than that of myeloblasts. Cytologic features of stimulated regeneration included diffuse cytoplasmic hypergranulation of immature neutrophilic precursors that had prominent perinuclear spherical clear areas representing the Golgi zones. With consideration of bone marrow composition and careful attention to cytologic detail, the distinction of bone marrow regeneration from acute leukemia can be made in most patients who are being treated with recombinant hematopoietic growth factors.

Acute monoblastic leukemia presenting with pericardial effusion and cardiac tamponade.

A previously healthy young child presented with a large pericardial effusion and cardiac tamponade. The chest radiography was key to the recognition of the pericardial effusion. Cytologic examination of the pericardial fluid ultimately established the diagnosis of acute monoblastic leukemia in the absence of associated clinical or laboratory findings. The pericardial fluid was vital for leukemic cell classification because the bone marrow has hypocellular and non-diagnostic. This presentation of acute monoblastic leukemia is very rare, and in the three previously reported pediatric cases has been associated either with peripheral blasts or a history of preleukemia. When the cardiac configuration suggests pericardial effusion in a previously healthy young child, the diagnosis of new onset leukemia should be considered.

Breast conservation therapy: local tumor control in patients with pathologically clear margins who receive 5000 cGy breast irradiation without local boost.

A retrospective study was performed to determine the value of pathological evaluation of inked primary tumor specimen margins in the local control of patients with stage I and II breast cancer. In 150 patients with 153 invasive breast cancers, treatment involved surgical resection of the primary tumor, pathological determination of tumor-free inked specimen margins, and 5000 cGy whole breast radiation therapy (RT) without tumor bed RT local boost. This approach yielded an actuarial five-year local control rate of 95%. The local control rate was 96% for T-1 cases and 93% for T-2 cases. The local control rate was 96% for patients with clear margins achieved at initial resection and 94% for patients with clear margins achieved at re-excision. Among patients with clear margins at re-excision, the local control rate was 97% for those with no residual cancer and 88% for those with residual cancer. Patients with surgical margins clear by 3 mm or less had a local control rate of 92% at five years. Local control rates appear to be comparable to other breast conservation approaches which routinely employ local RT boosts. In omitting the local RT boost in patients with clear margins, the overall RT course will be briefer and the cosmetic changes associated with high-dose, large volume local RT boosts can be avoided.

Evidence that a leukocyte type of 12-lipoxygenase is expressed and regulated by angiotensin II in human adrenal glomerulosa cells.

The 12-lipoxygenase (LO) pathway of arachidonic acid plays an important role in angiotensin II (AII)-mediated aldosterone synthesis. Several distinct isoforms of 12-LO have been cloned. However, in humans only the platelet form of 12-LO has been reported to be present. Western immunoblotting analysis in cultured human adrenal glomerulosa cells using polyclonal antibodies to porcine leukocyte 12-LO enzyme or peptide showed a specific 72-kilodalton band, which is identical to the molecular size of the porcine leukocyte form of 12-LO. In addition, AII (10(-7)) increased the intensity of the 72-kilodalton band nearly 2-fold over basal. In situ hybridization analysis indicated a strong positive reaction with the porcine leukocyte type of 12-LO antisense riboprobe in the zona glomerulosa of the adrenal cortex. The 12-LO probe also recognized a near 4-kilobase messenger RNA (mRNA) from human glomerulosa cells in Northern blots. Since the leukocyte type of 12-LO is highly homologous to human 15-LO, a reverse transcriptase and polymerase chain reaction was used to analyze the specific type of 12-LO mRNA in human cells. The mRNA for a porcine leukocyte type of 12-LO was detected in human adrenal glomerulosa cells, and the level of 12-LO transcripts was increased approximately 60-fold by AII (10(-7) M). The leukocyte type of 12-LO also was detected in human monocyte-like U937 cells, but not in IM-9 lymphocytes or human erythroleukemia cells. These results suggest that human adrenal glomerulosa cells and human monocyte-like U937 cells express a 12-LO which has immunological and molecular biological characteristics similar to the porcine leukocyte 12-LO.