Maximizing the diagnostic yield from bone marrow aspirate material using the cell block technique on clot sections.

BACKGROUND: Paraffin section of bone-marrow aspirate (clot section) is one of several components of bone marrow biopsy. Improper acquisition of aspirate material results in lack of diagnostic tissue and a waste of resources. OBJECTIVE: To detail a novel cell block method of aspirated marrow as a way to ensure maximum yield. This is of particular value when the material gathered via core biopsy is inadequate. METHOD: We used the cell block method to evaluate paraffin-embedded sections of hematopoietic tissue from bone marrow aspirate. RESULTS: The range of diagnoses possible from an adequate clot section parallels those from a comparable core biopsy. Examples of an adequate clot section include lesions assessed by routine hematoxylin-eosin (H&E) staining, special and immunohistochemistry (IHC) stains, and molecular diagnostic studies such as fluorescent in situ hybridization (FISH) and polymerase chain reaction (PCR). CONCLUSION: A bone marrow clot section comprised of sinusoidal blood is inadequate for morphological interpretation and is a waste of resources. Hence, we recommend the cell block technique for procurement; this method ensures maximum capture of material needed to establish a diagnosis.

The effects of high dose interferon-ß1a on plasma microparticles: correlation with MRI parameters.

OBJECTIVES: We previously reported a correlation between levels of micro particles carrying CD31 (PMP(CD31+)) and disease activity in MS. However, the effects of long term (12 month) treatment with high dose, high frequency interferon-ß1a (Rebif™) on plasma levels of PMP(CD31+), PMP(CD146+), and PMP(CD54+) and MRI measures of disease activity have not yet been assessed. METHODS: During this prospective 1-year study, we used flow cytometry to measure changes in plasma micro particles (PMP) bearing CD31 (PMP(CD31+)), CD146 (PMP(CD146+)), and CD54/ICAM-1 (PMP(CD54+)) in 16 consecutive patients with relapsing-remitting MS (RRMS) before and after 3, 6, and 12 months of subcutaneous therapy with interferon-beta1a (44 micrograms, 3X weekly). At each visit, clinical exams and expanded disability status scale (EDSS) scores were recorded. RESULTS: Plasma levels of PMP(CD31+), and PMP(CD54+) were significantly reduced by treatment with IFN-ß1a. PMP(CD146+) appeared to decrease only at 3 months and did not persist at 6 and 12 months (p = 0.0511). In addition, the decrease in plasma levels of PMP(CD31+) and PMP(CD54+) levels at 12 months were associated with a significant decrease in the number and volume of contrast enhancing T1-weigthed lesions. CONCLUSION: Our data suggest that serial measurement of plasma micro particles (PMP), particularly in the initial stages of MS (when neuro-inflammatory cascades are more intense), may serve as reliable and reproducible surrogate markers of response to IFN-ß1a therapy for MS. In addition, the progressive decline in plasma levels of PMP(CD31+) and PMP(CD54+) further supports the concept that IFN-ß1a exerts stabilizing effect on the cerebral endothelial cells during pathogenesis of MS.

Tracheal non-Hodgkin's lymphoma masquerading as benign granulation tissue: a report of two cases.

Primary tracheal non-Hodgkin's lymphoma (NHL) is rare, with fewer than 30 cases reported to date. We review the clinical presentation, evaluation, and treatment of 2 cases of tracheal NHL mimicking granulation tissue. The first patient was a 67-year-old man with myelodysplastic syndrome and Crohn's disease who had a recurring lesion of the proximal trachea causing significant airway obstruction. The second patient was a 47-year-old man with a history of multiple intubations who presented with dyspnea and stridor due to circumferential tracheal stenosis. In both cases, bronchoscopy revealed abundant granulation tissue, and the initial biopsy results indicated benign disease. However, after requests from the diagnostic team to rule out lymphoma, additional immunohistochemical stains and polymerase chain reaction testing confirmed NHL. Radiotherapy was initiated. The first patient responded well and remains disease-free after 4 years. The second patient died of airway obstruction due to severe distal tracheal stenosis. Recurrent granulation tissue should raise the suspicion of malignancy and prompt further tissue evaluation for evidence of lymphoma. Steroids for airway compromise may cause progression to mature stenosis as prednisone is used in the treatment of lymphoma. Localized disease involving the central airways may be treated successfully with limited chemotherapy and radiotherapy.

Underexpression and overexpression of Fas and Fas ligand: a double-edged sword.

OBJECTIVE: To compare autoimmune lymphoproliferative syndrome (ALPS) and Stevens-Johnson syndrome (SJS) with respect to the defects in Fas- and Fas ligand (FasL)-mediated apoptosis. DATA SOURCES: Selected reviews, case reports, and original studies were searched in PubMed and MEDLINE for the keywords ALPS, SJS, Fas, FasL, and apoptosis. STUDY SELECTION: Case reports of ALPS and SJS were selected as examples of Fas- and FasL-mediated diseases. In addition, we selected articles that examined the pathophysiology of apoptosis in the context of Fas-FasL interaction. RESULTS: Failure to initiate apoptosis of abnormal T lymphocytes occurs in such diseases as ALPS, leading to the accumulation of double negative T cells with an increase in autoimmunity. In contrast to apoptotic failure, SJS is associated with a pathological increase in programmed keratinocyte cell death. CONCLUSION: The consequences of dysregulated Fas- and FasL-mediated apoptosis leads to self-reactivity, malignant transformation, and immune dysfunction. An understanding of underlying mechanisms and qualitative assessment of Fas and FasL may have clinical benefits when control of these homeostatic mechanisms is in question.

Pharmacodynamic evaluation of temsirolimus in patients with newly diagnosed advanced-stage head and neck squamous cell carcinoma.

BACKGROUND: Activation of the mammalian target of rapamycin (mTOR) pathway in surgical margins of head and neck squamous cell carcinoma (HNSCC) is a predictor of recurrence and patients with minimal residual disease may benefit from adjuvant therapy with temsirolimus, an mTOR inhibitor. METHODS: The effects of 3 weekly doses of 25 mg of temsirolimus on Akt/mTOR pathway biomarkers were evaluated in tumor and peripheral blood mononuclear cells (PBMCs) of patients with HNSCC. Adverse events were assessed. RESULTS: Temsirolimus significantly decreased pS6 and p4E-BP1 in tumors, and pS6 and pAkt in PBMCs (p < .05). There was no significant upregulation of pAkt(Ser(473)) in tumor tissue. Side effects were minimal and reversible. CONCLUSION: Significant inhibition of the mTOR pathway was noted in both tumors and PBMCs of HNSCC with minimal side effects. The mTOR inhibitors can potentially be used as adjuvant therapy for patients with minimal residual disease and PBMCs are potential surrogate markers in this setting.

The role of fluorescence in situ hybridization and polymerase chain reaction in the diagnosis and classification of lymphoproliferative disorders on fine-needle aspiration.

BACKGROUND: Fine-needle aspiration (FNA) has been used in the evaluation of lymphadenopathy for a long time and is highly reliable in the identification of metastatic malignancies. However, the role of FNA in the assessment of new lymphoproliferative disorders continues to be a subject of debate. The objective of the current study was to evaluate the role of molecular cytogenetic studies in FNA diagnoses of lymphoproliferative disorders. METHODS: A retrospective, computer-based search for lymph node FNAs from 2006 to 2007 was performed. Cases with either fluorescence in situ hybridization (FISH) and/or polymerase chain reaction (PCR) studies were subjected to further analysis. RESULTS: In total, 243 lymph node FNAs were performed during the period, including 104 that were positive/suspicious for metastatic malignancies, 16 that were positive/suspicious for lymphomas, 15 that demonstrated atypical lymphoid proliferation, 73 that were reactive, 14 that were deemed granulomas, and 21 that were determined to be nondiagnostic. Molecular analysis included combined FISH/PCR in 4 cases, FISH only in 7 cases, and PCR only in 4 cases. By using multiplex PCR, 6 cases with atypical/negative flow cytometry results were diagnosed as 4 B-cell lymphomas, 1 T-cell lymphoma, and 1 reactive lymph node; and 4 cases that had atypical T cells determined by flow cytometry were diagnosed as reactive. One CD10-negative follicular lymphoma and 2 cases with suspicious flow cytometry results were positive for t(14;18)(q32;q21) by FISH. Forty-five cases had follow-up histology with 3 false-negative findings and no false-positive results. CONCLUSIONS: In this study, multiplex PCR studies for immunoglobulin heavy-chain or T-cell receptor gene rearrangements were useful for demonstrating clonality, and FISH studies were able to detect translocations or gene rearrangements that allowed for the subclassification of B-cell non-Hodgkin lymphomas.

Characterization of the lymphoid stroma in Warthin's tumor of salivary gland by immunohistochemistry, heavy chain gene and Bcl-2 gene rearrangement.

Warthin's tumor is rarely associated with malignant lymphoma. Only 18 cases were reported in the literature so far. In most cases the latter is a low grade process, including Marginal zone/Mucosa associated lymphoid tissue (MALT) type lymphoma, follicular lymphoma, and rarely diffuse large cell lymphoma which may arise de novo or secondary to low grade lymphoma. This study was conducted to determine the prevalence of occult B cell monoclones and genetic alterations in Warthin's tumor. Fourteen cases of Warthin's tumor were stained with antibodies to CD3, CD20, kappa and lambda light chains. On six cases of randomly selected Warthin's tumor, polymerase chain reaction (PCR) of IgH gene rearrangement (IgH-GR) was performed on genomic DNA extracted from formalin-fixed paraffin embedded tissue. One case of primary salivary gland indolent B-cell lymphoma and 3 cases of sialadenitis were analyzed by the same methods for comparison. In all Warthin's tumor and sialadenitis cases most of lymphoid stroma was B cell phenotype and concentrated in germinal centers. T cells were mostly located between germinal centers. No light chain restriction was demonstrable by kappa and lambda immunostains. Molecular genetic studies failed to show IgH-GR by FISH and showed polyclonal by IgH PCR. In contrast, the lymphoma case showed a diffuse proliferation of small B cells with light chain restriction and a minor component of reactive T cells. FISH showed IgH-GR and bcl-2 gene translocation with monoclonality by IgH PCR. Our study concludes that the lymphoid stroma of Warthin's tumor is reactive.

Eukaryotic initiation factor 4E overexpression in triple-negative breast cancer predicts a worse outcome.

BACKGROUND: Triple-negative (estrogen receptor [ER], progesterone receptor [PR], and HER2/neu receptor negative) breast neoplasms are typically high grade and portend a higher risk for relapse. Not being amendable to ER, PR, or HER2-targeted therapy, adjuvant cytotoxic chemotherapy remains the only option. High eukaryotic Initiation Factor 4E (eIF4E) overexpression in tumor specimens is an independent predictor for relapse in breast cancer, perhaps secondary to tousled-like kinase 1B upregulation and subsequent doxorubicin resistance. In this prospective study, eIF4E elevation in triple-negative breast cancer (TNBC) specimens was studied to determine its effect on cancer outcome. METHODS: A prospective study of 103 TNBC patients was initiated. Tumor specimens were quantified for eIF4E expression using Western blots. Clinical outcomes data were collected after standardized adjuvant treatment and surveillance protocols. Primary end points were cancer recurrence and cancer-related death. The eIF4E levels in cancer specimens were quantified as x-fold over benign samples from noncancer patients. Statistical procedures performed include survival analysis by Kaplan-Meier method, log-rank test, Cox proportional hazards regression model, and the chi-square test. RESULTS: Levels of eIF4E were categorized into 3 tertiles. Among 103 patients, 36 were in the low group (< or =7.5-fold), 40 were in the intermediate group (7.5- to 15-fold), and 27 were in the high group (> or =15-fold). Patients with triple-negative neoplasms that were in the high eIF4E group had greater rates of cancer recurrence (P = .04) and cancer-related death (P = .02) than the low eIF4E group. Among patients with node-negative disease, high eIF4E overexpression in tumor specimens continues to portend a greater rate of cancer recurrence (P = .02), and a higher rate of cancer death (P = .03) than those in the low eIF4E group. CONCLUSION: TNBC patients with high eIF4E overexpression are more likely to recur and die from cancer recurrence. High eIF4E seems to be a significant prognostic marker, even in TNBC patients.

Follow-up study of immune defects in patients with dysmorphic disorders.

BACKGROUND: In a previous study, we noted immunologic abnormalities in 46 (54.8%) of 84 individuals with dysmorphic disorders. OBJECTIVE: To reevaluate patients with dysmorphic disorders and immunologic abnormalities 2 to 3 years after an initial study to determine any changes in those abnormalities. METHODS: Information was gathered regarding significant infections during the previous 12 months. Blood samples were drawn for the immunologic tests that were previously performed (IgG, IgA, and IgM level determinations; complete blood cell count; and lymphocyte subset enumeration) and for determination of IgG subclasses and T-cell activation by CD69 expression. RESULTS: In the 21 patients available, 26 (63.4%) of the previously noted 41 low immunologic values were still present. In 5 patients, all previously noted immunologic abnormalities resolved. Of the 17 low values noted in 6 patients with Down syndrome, 12 (70.6%) were still present. Also, the 2 patients with Turner syndrome continued to have low IgA and IgM levels. Two patients had a low IgG4 level. A history of significant clinical infections within the previous 12 months was noted in 10 (58.8%) of 17 patients; 8 (47%) had current immune defects. There was a significantly lower T-cell response to staphylococcal enterotoxin B than in healthy controls. The T-lymphocyte activation response was low in 8 (38.1%) of the 21 patients. CONCLUSIONS: Our study revealed a high rate of immune defects in patients with dysmorphic disorders, both during the initial study and 2 to 3 years later, which may contribute to their increased susceptibility to infections. This association was most obvious in patients with Down syndrome and Turner syndrome. The findings should alert for early immunologic evaluation when such patients have infections.

Tissue microarray analysis of eIF4E and its downstream effector proteins in human breast cancer.

BACKGROUND: Eukaryotic initiation factor 4E (eIF4E) is elevated in many cancers and is a prognostic indicator in breast cancer. Many pro-tumorigenic proteins are selectively translated via eIF4E, including c-Myc, cyclin D1, ornithine decarboxylase (ODC), vascular endothelial growth factor (VEGF) and Tousled-like kinase 1B (TLK1B). However, western blot analysis of these factors in human breast cancer has been limited by the availability of fresh frozen tissue and the labor-intensive nature of the multiple assays required. Our goal was to validate whether formalin-fixed, paraffin-embedded tissues arranged in a tissue microarray (TMA) format would be more efficient than the use of fresh-frozen tissue and western blot to test multiple downstream gene products. RESULTS: Breast tumor TMAs were stained immunohistochemically and quantitated using the ARIOL imaging system. In the TMAs, eIF4E levels correlated strongly with c-Myc, cyclin D1, TLK1B, VEGF, and ODC. Western blot comparisons of eIF4E vs. TLK1B were consistent with the immunohistochemical results. Consistent with our previous western blot results, eIF4E did not correlate with node status, ER, PR, or HER-2/neu. CONCLUSION: We conclude that the TMA technique yields similar results as the western blot technique and can be more efficient and thorough in the evaluation of several products downstream of eIF4E.

Interleukin-2 treatment for persistent cryptococcal meningitis in a child with idiopathic CD4(+) T lymphocytopenia.

We report a 16-year-old male patient who presented with headache, behavior changes, and fever. His cerebral spinal fluid and blood cultures grew Cryptococcus neoformans. His laboratory evaluation was negative for human immunodeficiency virus infection but flow cytometry revealed low CD4(+) count of 39 cells/mm(3) and CD4:CD8 ratio of 0.43. He was initially treated with antifungal agents with only partial clinical improvement, and he was discharged to home on oral fluconazole and prophylactic co-trimoxazole. After discharge, he continued to have persistent headache and recurrent episodes of vomiting. He was readmitted several times because of worsening of meningitis symptoms and received prolonged courses of multiple antifungal therapy, with clearance of infection from the central nervous system. He was subsequently placed on prophylactic therapy with fluconazole. His peripheral CD4(+) cell count remained low after resolution of his meningitis. Eight months after the initial diagnosis, recombinant IL-2 therapy was initiated and within a few months, his CD4(+) cell count started to increase. Treatment with rIL-2 and prophylactic antifungal therapy continued and he has been asymptomatic for almost 20 months so far. This case is the first reported pediatric idiopathic CD4(+) T-lymphocytopenia case with cryptococcal meningitis that was successfully treated by the addition of rIL-2 therapy to antifungal therapy.

Coexpression of EGFR and HER-2 in pancreatic ductal adenocarcinoma: a comparative study using immunohistochemistry correlated with gene amplification by fluorescencent in situ hybridization.

Pancreatic ductal adenocarcinoma (PDAC) is the major pancreatic tumor and carries an extremely poor prognosis. Coexpression of epidermal growth factor receptor (EGFR) and the HER-2 oncoprotein has been reported to be related to the invasion and an adverse clinical outcome of human pancreatic ductal adenocarcinomas. HER-2 amplification, as determined by fluorescent in situ hybridization (FISH) analysis, has been identified as a positive predictor of response to EGFR tyrosine kinase inhibitor treatment in some other cancers. The aim of this study was to investigate the coexpression rate and amplification status of HER-2 oncogene in EGFR positive pancreatic ductal adenocarcinoma (PDAC) by immunohistochemistry and FISH. Overexpression of EGFR (>or=2+) was seen in 65% (21/32) of the study cases. EGFR gene amplification was not detected in any of the 32 PDACs. Overexpression of HER-2 protein (>or=2+) was seen in 17% (5/28) of the study cases and in 24% (5/21) of EGFR positive cases. None of the EGFR negative tumors showed HER-2 overexpression or gene amplification. The HER-2 gene locus was amplified in 11% (3/28) of the study cases and in 14% (3/21) of EGFR positive PDACs. There was 60% concurrence between HER-2 gene amplification and HER-2 protein expression in this study. These results suggest that HER-2 is an important cooperating member of the EGFR signal pathway in a subset of PDAC.

Serratia marcescens osteomyelitis in an infant.

Neutrophil dysfunction can result from oxidative burst defect or from glucose-6-phosphate dehydrogenase (G6PD) deficiency; we noted both in the same patient. A 4-month-old male infant with G6PD deficiency presented with swelling of the left middle finger, left leg, and right big toe. At 5 weeks of age he was hospitalized for fever for 2 days. A maternal uncle died at 5 years of age and a male maternal cousin died at the age of 21 months, both reportedly diagnosed with chronic granulomatous disease (CGD). On physical examination, he had a swollen erythematous left third finger, left distal leg swelling, and right big toe abscess. None of these areas was significantly tender. WBC was 18.7 x 10(3)/mm(3) with 37% PMN and 5% bands. The x-ray films showed osteomyelitis in the left third proximal phalanx and the distal right first metatarsal. Culture from the toe abscess grew Serratia marcescens. His neutrophil oxidative burst was tested by the dihydrorhodamine-123 assay and was markedly suppressed, typical of CGD. The mother and maternal grandmother were found to be CGD carriers. He was treated with i.v. antibiotics for 4 weeks and was discharged on prophylactic trimethoprim, itraconazole and interferon gamma, with substantial reduction in infections. Infection in this infant was unusual in its nature, in affecting multiple sites, and in its causative organism. Immune deficiency was suspected, particularly of the phagocytic component, but could not be attributed to his moderate degree of primary G6PD deficiency. Additional immunologic evaluation and the family history led to the diagnosis of X-linked CGD.

Iron chelators reduce chromosomal breaks in ataxia-telangiectasia cells.

Ataxia-telangiectasia (A-T) is characterized by ataxia, genomic instability, and increased cancer incidence. Previously, iron chelator concentrations which suppressed normal cell colony formation increased A-T cell colony formation. Similarly, iron chelators preferentially increased A-T cell colony formation following peroxide exposure compared to normal cells. Last, A-T cells exhibited increased short-term sensitivity to labile iron exposure compared to normal cells, an event corrected by recombinant ATM (rATM) expression. Since chromosomal damage is important in A-T pathology and iron chelators exert beneficial effects on A-T cells, we hypothesized that iron chelators would reduce A-T cell chromosomal breaks. We treated A-T, normal, and A-T cells expressing rATM with labile iron, iron chelators, antioxidants, and t-butyl hydroperoxide, and examined chromosomal breaks and ATM activation. Additionally, the effect of ATM-deficiency on transferrin receptor (TfR) expression and TfR activity blockage in A-T and syngeneic A-T cells expressing rATM was examined. We report that (1) iron chelators and iron-free media reduce spontaneous and t-butyl hydroperoxide-induced chromosomal breaks in A-T, but not normal, or A-T cells expressing rATM; (2) labile iron exposure induces A-T cell chromosomal breaks, an event lessened with rATM expression; (3) desferal, labile iron, and copper activate ATM; (4) A-T cell TfR expression is lowered with rATM expression and (5) blocking TfR activity with anti-TfR antibodies increases A-T cell colony formation, while lowering chromosomal breaks. ATM therefore functions in iron responses and the maintenance of genomic stability following labile iron exposure.

Increased transferrin receptor expression following 11q23 deletion as a mechanism of malignant progression in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a common adult leukemia characterized by the accumulation of mature neoplastic B-lymphocytes. Typically, CLL follows an indolent course, with most patients surviving for many years. However, 10-20% of CLL patients carry 11q23 chromosomal deletions and often exhibit a more severe disease course, with earlier onset of symptoms, shortened lymphocyte doubling time, poor response to therapy, and shortened survival. The molecular basis for 11q23 deletions resulting in a poor prognosis is currently poorly understood. The tumor suppressor gene, ataxia-telangiectasia mutated (ATM, 11q22.3-23.1), is considered a likely candidate gene whose loss could result in the poor prognosis associated with 11q23 deletion and is mutated in a significant percentage of CLL cases. Recently, recombinant ATM expression in ATM-deficient cells was found to decrease transferrin receptor (TfR) expression, suggesting that deletion of the chromosomal region carrying ATM results in increased TfR expression. TfR imports iron into cells, an event necessary for DNA synthesis and cell growth. Additionally, rapidly growing malignant cells, including lymphomas and CLL, often express high TfR levels. Based on this, we propose that one molecular mechanism by which 11q23 deletions confer a poor prognosis in CLL is via increased TfR expression secondary to ATM loss, resulting in the increased cellular iron import, and hence increased capacity for malignant growth. Our hypothesis may also partially explain why gallium, an atomically iron-like toxic metal that binds to transferrin and the TfR is incorporated into cells and was previously demonstrated to have anti-tumor activity in patients with lymphomas refractory to other chemotherapeutic treatments.

Immune defects in subjects with dysmorphic disorders.

Patients with dysmorphic disorders seem to have frequent respiratory infections that may be attributed to associated anatomic or neurological abnormalities, but immune defects may contribute to their susceptibility to infections. We screened subjects with dysmorphic conditions for major hematologic, B-cell and T-cell defects. We studied 84 subjects with dysmorphic disorders: 29 with chromosomal disorders, 27 with single gene disorders, and 28 with unclassified dysmorphic disorders. They were evaluated by physical examination; medical history suggestive of possible immune deficiency; complete blood count; serum immunoglobulin G (IgG), IgA, and IgM levels; and lymphocyte subsets. Low laboratory values (less than fifth percentile for age) were detected in 54.8%; was highest in the chromosomal disorder group (79.3%) followed by the single gene disorder group (55.6%) and was lowest in the unclassified dysmorphic disorder group (28.6%). The most common low values were in the CD19 and CD16/56 lymphocyte subpopulations followed by IgG and IgA levels. None of the subjects had neutropenia or thrombocytopenia. History of significant recurrent infections was noted in five subjects, all of whom had abnormal laboratory values. The highest frequency of abnormal laboratory values was in Down syndrome followed by Turner syndrome and chromosome deletions. We concluded that patients with dysmorphic disorders, particularly those with chromosomal disorders, have a high frequency of various B-cell and T-cell defects that may be contributing to their susceptibility to infection. Studies are needed to further delineate the immunologic defects in that population and to investigate a possible genetic basis at the molecular level.