Cost-effective approach to the diagnostic workup of B cell lymphoproliferative disorders via optimal integration of flow cytometric data.

INTRODUCTION: The workup of lymphoproliferative disorders (LPDs) involves the combined use of flow cytometry (FC) and immunohistochemistry (IHC). This often results in duplicate immunophenotypic testing and adds costs that may not be eligible for reimbursement based on the Medicare National Correct Coding Initiative. We aimed to establish a cost-effective diagnostic algorithm based on initial FC categorization to reduce repetitive immunophenotyping. METHODS: We retrospectively reviewed 242 cases of suspected LPDs with concurrent FC and IHC testing over a 12-month period. We correlated FC with surgical diagnoses and evaluated the frequency of repeat IHC testing. RESULTS: Repetitive immunophenotyping was common; overall, 85% of cases had at least one marker repeated. Concordant cases were significantly less likely to have markers repeated than discordant cases. Of concordant B cell malignancies, 57% represented recurrent disease; however, repeat marker usage was not decreased as compared to new diagnoses. The most frequently repeated markers were CD3, CD5, CD10, and CD20. CONCLUSIONS: We propose that in concordant cases, CD5 and CD10 should not be repeated by IHC; this would decrease the use of these markers by 80% and 76%, respectively. We developed an algorithmic approach to IHC usage that has improved incorporation of FC data at our institution and may reduce healthcare costs.

Clonal rearrangement of chromosome band 6p21 in the mesenchymal component of pulmonary chondroid hamartoma.

Pulmonary chondroid hamartomas (PCH) are biphasic benign tumors that contain both mesenchymal and epithelial populations. In this report we describe two PCH in which clonal translocations at chromosome band 6p21 were demonstrated in mesenchymal cells. One of these had a unique translocation, t(6;14)(p21;q24), that was also found in one of two PCH karyotyped previously. The t(6;14) has not been described in other varieties of benign or malignant neoplasia. The 6p21 aberrations are of particular interest because break points in this chromosomal region appear to be characteristic of endometrial polyps. Endometrial polyps, like PCH, are biphasic benign tumors in which mesenchymal clonality has been demonstrated.

Breakpoints of the t(11;18)(q21;q21) in mucosa-associated lymphoid tissue (MALT) lymphoma lie within or near the previously undescribed gene MALT1 in chromosome 18.

Lymphomas arising in mucosa-associated lymphoid tissue (MALT) are indolent B-cell tumors that have a predilection for epithelial sites and often develop in a setting of chronic inflammation or autoimmunity. As many as 50% of low-grade MALT lymphomas contain an (11;18)(q21; q21) chromosomal translocation. Using fluorescence in situ hybridization, we have analyzed the position of recombination within chromosome 18 DNA in three examples of MALT lymphoma bearing this translocation. In all three cases, the breakpoint maps to DNA in BAC b357H2, covering about 150 kb of sequence. A previously undescribed, ubiquitously expressed gene, which we refer to as MALT1, was identified within this sequence and was found to be broken in one case for which we have definitively located the position of recombination between chromosomes 18 and 11. The sequence of this gene indicates the presence of two immunoglobulin-like C2 domains and a region of partial homology to caspases, suggesting a possible role for MALT1 in the regulation of apoptosis.

Intermediate lymphocytic lymphoma: clinical and pathologic features of a recently characterized subtype of non-Hodgkin's lymphoma.

PURPOSE: We present a comprehensive review of clinical, pathologic, molecular, and prognostic features and therapy of intermediate lymphocytic (mantle cell) lymphoma (ILL/MCL), a recently characterized subtype that represents 2% to 8% of non-Hodgkin's lymphomas (NHLs), but which has not been included in most classification schemes, including the International Working Formulation. DESIGN: The English-language literature encompassing the above aspects, published between 1977 and 1992, is critically reviewed. RESULTS AND CONCLUSION: ILL/MCL is a disease of proliferating B lymphocytes that is characterized by generalized lymphadenopathy and frequent, often extensive, involvement of the spleen, bone marrow, and gastrointestinal tract. The malignant cells usually express the markers CD5 and IgM with or without IgD, but not CD10, on the cell surface, and grow in one of two dominant histologic patterns: mantle zone and diffuse. The characteristic cytogenetic abnormality is a t(11;14)(q13;q32) translocation, which juxtaposes the bcl-1 locus on chromosome 11 with the immunoglobulin (Ig) heavy-chain locus on chromosome 14, and appears to result in dysregulated expression of the gene encoding cyclin D1. Median survival is in the range of 2 to 5 years. While responses to chemotherapy may be seen in up to half the patients, relapses are the rule, and longterm survival is uncommon. The optimal treatment remains undefined, although therapy may be deferred until there are symptoms or complications, at which time judicious administration of alkylating agents and glucocorticoids may result in effective palliation.

Improved prognosis of diffuse histiocytic and undifferentiated lymphoma by use of high dose methotrexate alternating with standard agents (M-BACOD).

A new combination chemotherapy program (M-BACOD) was administered to 101 patients with advanced diffuse histiocytic and diffuse undifferentiated lymphoma (DHL and DUL). High dose methotrexate (M) 3 g/m2 with leucovorin factor rescue was given on day 14 between cycles of bleomycin (B), adriamycin (A), cyclophosphamide (C), oncovin (O), and dexamethasone (D) administered every 3 weeks for 10 cycles. The complete remission rate (CR) was 72% in all 101 patients or 77% in 95 evaluable patients. The median follow-up is 3 yr 2 mo with one-third of CR patients followed beyond 4 yr. Twenty-six percent of CR patients have relapsed with a projected 5-yr survival rate of 80% (5-yr disease-free rate 65%). The overall survival of all 101 study patients reaches a plateau at 59% projected out to 5 yr. Patients with prior therapy had a significantly lower CR rate than those without prior treatment (p = 0.001); however, no other unfavorable prognostic characteristics could be identified. Relapse in the central nervous system CNS occurred in only 5.4% of CR patients. M-BACOD results in prolonged survival and possible cure in a high proportion of all patients with DHL and DUL.

The m-BACOD combination chemotherapy regimen in large-cell lymphoma: analysis of the completed trial and comparison with the M-BACOD regimen.

One hundred thirty-four assessable patients with stage II-IV large-cell lymphoma (LCL) were treated with the combination chemotherapy regimen methotrexate with leucovorin, bleomycin, doxorubicin, cyclophosphamide, vincristine, and dexamethasone (m-BACOD) between July 1981 and May 1986. The m-BACOD regimen substituted moderate-dose methotrexate (200 mg/m2 x 2) for the high-dose methotrexate used in the preceding M-BACOD regimen; all other drugs were administered as with m-BACOD. Eighty-two patients (61%) in the completed m-BACOD trial achieved a complete response (CR). With a median follow-up of 3.6 years, 62 patients (76%) continue in CR. Predicted survivals of 1, 3, and 5 years for the entire m-BACOD group are 80%, 63%, and 60%, respectively, with a 5-year disease-free survival (DFS) of 74% for the patients who achieve CR. The results obtained with m-BACOD are comparable with those obtained in the preceding M-BACOD trial, which now has a median follow-up of 8.0 years. The reduction in methotrexate dosage in m-BACOD patients was not associated with an increased incidence of CNS relapse. Long-term follow-up of the 215 M/m-BACOD patients indicates that the regimens are not associated with an increased incidence of secondary malignancy. Prolonged follow-up also indicates that advanced-stage patients have a persistent rate of late relapse of about 7.0% per year for years 2 to 5 of their follow-up and that stage II patients have an approximate 2.1% per year rate of late relapse. Application of the previously described prognostic factor model to the 215 M/m-BACOD patients from the completed trials identifies a high-risk group of patients with a CR rate and predicted 5-year survival (38% and 24%, respectively) that are significantly worse than those of the group as a whole (65% and 57%, respectively).

Sequestered substernal goiter.

A young woman with a normally located and only subtly nodular thyroid gland in the neck was found to have a clinically distinct and radioisotopically "cold" anterior mediastinal mass, which proved to be a benign colloid adenoma. While this constellation of findings usually suggests the presence of a nonthyroidal neoplasm, eg, lymphoma, thymoma, or teratoma, our case illustrates that sequestered benign nodular goiter should also be considered in the differential diagnosis. Clinical clues, such as a nodular thyroid gland, movement of the mass with deglutition, and a family history of nodular goiter, should suggest this possibility. A characteristic computed tomographic appearance may also prove useful in recognition of this rare disorder.

Involucrin expression in normal and neoplastic human skin: a marker for keratinocyte differentiation.

Involucrin is a recently recognized structural component of mature squamous epithelial cells. We examined involucrin expression using an immunoperoxidase technique in normal skin and in a variety of epidermal hyperplasias and neoplasms to determine whether distinctive staining patterns existed within these lesions. Four patterns of reactivity were observed: diffuse intracellular staining typical of keratinocytes of the upper third of normal epidermis and epidermal hyperplasias and benign neoplasms; staining at cell borders, seen principally in benign epidermal neoplasms; patchy staining characteristic of squamous cell carcinoma in situ; and absence of staining in benign and neoplastic basaloid epithelium. Invasive nests of squamous cell carcinomas were negative for involucrin reactivity, whereas pseudoinvasive tongues of epithelium at the bases of keratoacanthomas were focally positive. These results suggest that immunoperoxidase staining for involucrin may be useful in distinguishing certain benign from malignant epidermal neoplasms as well as in understanding the altered maturation and kinetics of proliferative processes afflicting keratinocytes.

The m-BACOD combination chemotherapy regimen in the treatment of diffuse large cell lymphoma.

The m-BACOD regimen attempted to lower the dose of methotrexate in the M-BACOD (methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine, dexamethasone) program. Between July 1981 and January 1985, 87 previously untreated or minimally treated patients with diffuse large cell lymphoma were treated with the m-BACOD regimen (methotrexate 200 mg/m2 on days 8 and 15, bleomycin 4.0 mg/m2 on day 1, doxorubicin 45 mg/m2 on day 1, cyclophosphamide 600 mg/m2 on day 1, vincristine 1.0 mg/m2 on day 1, and dexamethasone 6 mg/m2 on days 1 to 5; leucovorin was given 24 hours after methotrexate at 10 mg/m2 every six hours for eight doses orally). Of 86 evaluable patients, 59 (68.5%) had a complete remission (CR). Partial response was seen in 21 patients with six still surviving (5 to over 15 months). Of the seven patients who had no change, all have died. The median duration of follow-up for the entire series was 30 months (range, 2 to 61). Relapse from CR occurred in 15 of 59 (25%). Currently, 56 of 87 patients (64%) survive; all but 12 are in their first remission. Overall survival was 84% for those achieving an apparent CR. The major toxic effect of the m-BACOD regimen was myelosuppression with severe leukopenia and fever, which required hospitalization for about 33% of patients. Mucositis occurred in 39 patients; 19 had severe mucositis. No significant difference in overall survival was seen between the high-dose methotrexate M-BACOD and the low-dose m-BACOD regimens.

Rheumatoid aortitis: a rarely recognized but clinically significant entity.

Aortitis as a feature of rheumatoid arthritis is considered rare. We have, however, identified 10 patients with aortitis from among 188 consecutive autopsy cases of rheumatoid arthritis. There were 5 men and 5 women with a mean duration of rheumatoid arthritis of 9.6 years. Nine were rheumatoid factor positive and had associated nodules. In addition to standard treatment regimens, 9 patients received corticosteroids. Although involvement of the thoracic aorta was most common, involvement of both the thoracic and abdominal aorta was present in 4 cases. Two patients had aneurysmal dilatation of the thoracic aorta and 1 of the abdominal aorta. Microscopic features of aortitis included necrosis of medial smooth muscle and elastica, with an inflammatory infiltrate comprising primarily lymphocytes and plasma cells. A panmural aortitis was seen in 3 cases. Rheumatoid granulomas were noted in the aortic wall in 5. The diagnosis of aortitis was not made until autopsy in any case. Aortitis was hemodynamically significant in 3 patients. Two had congestive heart failure secondary to thoracic aortitis and aortic valvulitis, and 1 had rupture of an abdominal aortic aneurysm at a site involved by aortitis. Seven patients had rheumatoid vasculitis with a mean of 10 organs involved. Six of these died of complications directly related to vasculitis, including 4 patients with coronary arteritis and associated myocardial infarction. Aortitis can be a feature of severe rheumatoid arthritis and is often associated with rheumatoid vasculitis. Hemodynamic compromise does occur and may be fatal.

Epstein-Barr virus in Hodgkin's disease: correlation of risk factors and disease characteristics with molecular evidence of viral infection.

Risk factors suggestive of relatively late exposure to EBV have been consistently associated with Hodgkin's disease (HD) in younger adults. In addition, evidence of EBV infection has been found in the Reed-Sternberg cells themselves in about one-third to one-half of all HD cases. However, no study yet published has correlated these childhood social environment risk factors with the presence of EBV in Hodgkin's tumor cells. We examined whether EBV-positive HD occurs in those patients whose childhood environment would predispose them to relatively late exposure to EBV. The study population consisted of 102 cases of mixed cellularity (MC; n = 25) or nodular sclerosing (n = 77) HD. Samples that tested positive for either EBV-encoded RNA or latent membrane protein or both were considered EBV-positive. Of the 102 cases, 83 completed a questionnaire regarding childhood social environment. The association with EBV-positivity was estimated by the odds ratio (OR) with 95% confidence intervals (CI). Twenty-two percent of the cases were EBV-positive. These cases were more likely to be MC (OR, 6.2; CI, 2.3-16.3) and male (OR, 3.4; CI, 1.3-9.0). History of infectious mononucleosis (IM) was not predictive of EBV-positivity, with only 3 of 14 such patients being EBV-positive (P = 0.82). Contrary to our hypothesis, no association between EBV and childhood environment risk factors was identified. The association of EBV with MC histology and male gender agrees with previous reports. The most intriguing finding was the dissociation between IM history and EBV-positivity, in that almost all of the cases with a history of IM were EBV-negative.

Intestinal anisakiasis. First case report from North America.

The first case of intestinal anisakiasis in North America is described. This parasitic disease is recognized as a public health hazard in Japan and Europe. Man becomes infected with a larval form of the nematode Anisakis by consuming raw or undercooked fish containing the parasite. Typically, patients present with acute abdominal syndromes. Clinical and reontgenographic features may cause confusion with regional enteritis. Histologically, a striking oesinophilic granulomatous reaction occurs. Anisakiasis is most effectively prevented by discouraging the consumption of raw fish.

Immunohistochemical demonstration of epithelial differentiation in adamantinoma of the tibia.

The nature of the neoplastic cells in a case of adamantinoma of the tibia was studied with an immunocytochemical method. The antigens investigated were factor VIII-related antigen and keratin, as markers for endothelial cells and epithelial cells, respectively. The tumor cells of adamantinoma stained strongly for keratin but were completely negative for factor VIII-related antigen. These results strongly suggest that cells of tibial adamantinomas are of epithelial rather than endothelial nature, thus confirming previous light-microscopic observations and electron-microscopic studies performed on this tumor. Although the negativity for factor VIII-related antigen does not rule out by itself the presence of an endothelial component, the fact that the tumor cells are positive for keratin makes this possibility highly unlikely. Additional cases of this entity should be studied with these cytoplasmic markers in order to confirm the findings here presented.

Extramammary Paget's disease--evidence for an apocrine origin. An immunoperoxidase study of gross cystic disease fluid protein-15, carcinoembryonic antigen, and keratin proteins.

The histogenesis of extramammary Paget's disease has long remained unresolved and controversial. In an attempt to delineate the origin of the neoplastic cells in this disease, the immunoperoxidase localization of gross cystic disease fluid protein (GCDFP-15), a marker of apocrine epithelium, carcinoembryonic antigen (CEA), and keratin proteins, was determined for seven cases of extramammary Paget's disease (five vulvar, one anogenital, and one axillary). Immunoreactivity for GCDFP-15 was localized within Paget cells in six of our seven cases, including five cases from the vulva and one case from the axilla. CEA was present in the Paget cells in all seven cases. None of the Paget cells exhibited immunoreactivity for keratin proteins. Within normal skin, eccrine glands were immunoreactive for both keratin and CEA, whereas GCDFP-15 localized only to apocrine ducts and glands. Our findings strongly support an apocrine cell derivation for extramammary Paget's disease.

Nodular lymphoid lesion of the liver: an immune-mediated disorder mimicking low-grade malignant lymphoma.

Three cases of unusual lymphoid infiltrate forming nodular macroscopic masses in the liver were studied in the authors' surgical pathology laboratory. These lesions posed difficulty in diagnosis, and their differentiation from low-grade lymphoma was not possible on histopathologic evaluation alone. The liver masses were analyzed histologically and immunohistochemically as well as for clonal immunoglobulin heavy chain (IgH) and T-cell receptor gamma (TCR-gamma) gene rearrangements. The lesions were seen as solitary grossly distinct firm nodules in all three patients, measuring 0.4, 0.7, and 1.5 cm, respectively, in their greatest dimensions. Two were found in livers removed because of end-stage primary biliary cirrhosis at the time of orthotopic liver transplantation, and the third was an incidental finding during laparotomy. Microscopically, these were nodules composed of small lymphocytes, plasma cells, and immunoblasts, with varying degrees of admixed acute inflammatory cells and scattered lymphoid follicles. By immunohistochemistry and molecular studies, these were found to be reactive lymphoid proliferations. All patients are alive and well at 2, 4, and 13 years, respectively. It is concluded that these cases represent a unique type of nodular lymphoid lesion, which is probably an immune-mediated benign reactive hyperplasia. It constitutes an entity by itself and must be distinguished from low-grade lymphoma. For a definitive diagnosis, immunohistochemistry and molecular studies are required.

Development of gastrointestinal beta2-microglobulin amyloidosis correlates with time on dialysis.

Dialysis-associated beta2-microglobulin (beta2m) amyloidosis affects predominantly musculoskeletal tissue, but visceral involvement also occurs. To evaluate the clinical significance and prevalence of gastrointestinal beta2m amyloidosis, we studied hemodialysis patients admitted for gastrointestinal-related complaints. Hemodialysis patients (excluding those with non-beta2m amyloidosis) who were admitted with gastrointestinal complaints from 1984 to 1994 were identified. Gastrointestinal tissues from patients with available autopsy or surgical specimens were examined using hematoxylin and eosin stain, Congo red stain, and beta2m immunostain. Each case was evaluated independently by two pathologists and scored for quantity and location of beta2m amyloid and associated pathology. Of 24 patients, eight (four men and 4 women) had beta2m amyloid deposits within the gastrointestinal tract. Acute clinical presentation ranged from abdominal pain to gastrointestinal bleeding and was not significantly different for patients with or without gastrointestinal beta2m amyloid deposits. However, the mean time on dialysis of 15.3 +/- 5.7 years (range 6-24 years) for patients with gastrointestinal beta2m amyloidosis was significantly greater than that of patients without gastrointestinal beta2m amyloidosis (10.5 +/- 7.0 years, range <1 to 22 years, p < 0.05). Vascular histopathology ranged from mild focal thickening of vessel walls to massive vascular beta2m amyloid deposition with thrombosis. Extravascular beta2m amyloid ranged from mild to severe with marked expansion of the submucosa. Mucosal pathology ranged from none to severe ulceration. The degree of beta2m amyloid and the associated pathology tended to increase in severity with time on dialysis. Gastrointestinal beta2m amyloid deposition is an underappreciated complication of chronic hemodialysis that is significantly associated with increased time on dialysis. Gastrointestinal beta2m amyloidosis should be considered in any patient on hemodialysis 10 years or more who has gastrointestinal symptoms and can be identified in resection specimens as well as some biopsy specimens. Congo red stain and beta2m immunostains may be necessary for sensitive histopathologic evaluation of gastrointestinal beta2m amyloidosis.

Antigenic separation of a nonkinin-generating TAMe esterase from human urinary kallikrein and immunohistochemical comparison of their localization in the kidney.

Two urinary enzymes that cleave alpha-N-p-tosyl-L-arginine methyl ester (TAMe) have been separated and utilized to elicit monospecific antisera; only one, urinary kallikrein (urokallikrein), had kinin-generating activity. The nonkinin-generating TAMe esterase and urokallikrein were antigenically unrelated. Immunoperoxidase studies of normal human kidney revealed localization of nonkinin-generating TAMe esterase to epithelial cells of the distal tubule, including the ascending thick limb, the macula densa region, and some areas of convoluted tubule. Immunoreactivity for urokallikrein was confined to reabsorption droplets of proximal tubules and to focal segments of the distal convoluted tubules. Electrophoretic, antigenic, and immunohistochemical studies have established that urokallikrein and a nonkinin-generating TAMe esterase represent two distinct renal distal tubule enzymes.

Localization of keratin proteins in the human epidermis by a postembedding immunoperoxidase technique.

Individual keratin species were localized ultrastructurally to the tonofilaments and tonofibrils of the human epider-misusing a postembedding immunoperoxidase method. Low molecular weight keratin (45 kD) was localized primarily to the stratum basale and displayed a fine cytoplasmic staining of individual tonofilaments and some tonofibrillar staining. Higher molecular weight keratins (55 and 63 kD) were found predominantly in the suprabasal, differentiated squamous epithelial cells, staining tonofibrils with a web-like pattern. The technique utilized is suitable for studying the distribution of keratin proteins in normal and disease states.

Immunohistochemical localization of smooth muscle myosin in normal human tissues.

Immunohistochemical localization of smooth muscle myosin, an immunologically distinct contractile protein, was achieved using rabbit anti-human uterine smooth muscle myosin antibodies. In immunodiffusion studies and in cryostat sections, these antibodies were highly specific and reacted with smooth muscle myosin but not with platelet, skeletal muscle, or cardiac muscle myosin. To evaluate comprehensively the structural profile of smooth muscle elements in normal human tissues, an indirect immunoperoxidase technique (peroxidase-antiperoxidase) was applied to a wide variety of specimens. Parallel studies comparing cryostat sections with fixed (10% formalin, B5, Bouin's, or Zenker's solution) paraffin-embedded tissues revealed optimal immunoreactivity, sensitivity, and specificity of staining for smooth muscle myosin using frozen tissues. Strong immunoreactivity was present in muscular tissues such as blood vessels and the muscularis of gastrointestinal and genitourinary tracts. Distinct delineation of smooth muscle elements, including individual smooth muscle cells, and their specific patterns of alignment and organization, were observed, e.g., cells comprising the muscularis mucosae and extending into the lamina propria of the gastrointestinal tract, and myoepithelial cells of skin, exocrine glands, and breast. This method provides excellent morphologic preservation and readily permits unambiguous identification of individual cells containing smooth muscle myosin.

Immuno-ultrastructural localization of B-cell-specific monoclonal antibodies B1 and B2.

Monoclonal antibodies B1 and B2 are thought to recognize B-lineage restricted antigens, and have been used to define stages of B-cell maturation and characterize B-cell lymphomas. Immunostaining on cryostat sections has revealed a puzzling dendritic or extracellular pattern of staining for B2 within germinal centers and neoplastic follicles. In this study B1 and B2 are localized precisely on hyperplastic and neoplastic lymphoid tissues using immuno-ultrastructural techniques on cryostat sections, cell suspensions, and cell monolayers. B1 and B2 were localized to cell surfaces, including microvillous surface projections, on small and large transformed normal and neoplastic B lymphocytes. B2, in addition to staining in lymphoid cells, was localized to anastomosing cytoplasmic processes of dendritic histiocytes. These findings explain the apparently extracellular localization of B2 in cryostat sections and indicate that patterns of staining for B2 may represent a combination of staining on lymphoid cells and dendritic histiocytes.