IL-10 induces the development of immunosuppressive CD14(+)HLA-DR(low/-) monocytes in B-cell non-Hodgkin lymphoma.

The biological role of monocytes and macrophages in B-cell non-Hodgkin lymphoma (NHL) is not fully understood. We have previously reported that monocytes from patients with B-cell NHL have an immunosuppressive CD14(+)HLA-DR(low/-) phenotype that correlates with a poor prognosis. However, the underlying mechanism by which CD14(+)HLA-DR(low/-) monocytes develop in lymphoma is unknown. In the present study, we found that interleukin (IL)-10, which is increased in the serum of patients with B-cell NHL, induced the development of the CD4(+)HLA-DR(low/-) population. Using peripheral blood samples from patients with B-cell NHL, we found that absolute numbers of CD14(+) monocytic cells with an HLA-DR(low/-) phenotype were higher than healthy controls and correlated with a higher International Prognostic Index score. IL-10 serum levels were elevated in lymphoma patients compared with controls and were associated with increased peripheral monocyte counts. Treatment of monocytes with IL-10 in vitro significantly decreased HLA-DR expression and resulted in the expansion of CD14(+)HLA-DR(low/-) population. We found that lymphoma B cells produce IL-10 and supernatants from cultured lymphoma cells increased the CD14(+)HLA-DR(low/-) population. Furthermore, we found that IL-10-induced CD14(+)HLA-DR(low/-) monocytes inhibited the activation and proliferation of T cells. Taken together, these results suggest that elevated IL-10 serum levels contribute to increased numbers of immunosuppressive CD14(+)HLA-DR(low/-) monocytes in B-cell NHL.

PD-1 expression defines two distinct T-cell sub-populations in follicular lymphoma that differentially impact patient survival.

To determine the biological and clinical relevance of programmed death 1 (PD-1) in follicular lymphoma (FL), we characterized PD-1(+) T-cell subsets and assessed their biological function as well as potential clinical impact. We found that PD-1 is expressed on intratumoral CD4(+) T cells with both bright and dim intensity, representing two different sub-populations of cells. By immunohistochemistry, we found that CD4(+)PD-1(high) T cells predominantly reside in the lymph node follicles, while PD-1(low) T cells are mainly located in an interfollicular pattern. Intratumoral CD4(+)PD-1(high) T cells have a TFH cell phenotype, express CXCR5, secrete IL-21 and are BCL-6 positive with no TIM-3 expression. In contrast, CD4(+)PD-1(low) T cells have an exhausted phenotype, express TIM-3 and do not express BCL-6 and CXCR5. Functionally, CD4(+)PD-1(high) T cells actively supported B-cell growth, while CD4(+)PD-1(low) T cells displayed a reduced cytokine production and cell-signal transduction. Clinically, we observed that the numbers of CD4(+) or CD8(+)PD-1(low) T cells significantly correlate with a reduced overall survival in FL patients (P=0.007 and 0.04 respectively; n=32). In contrast, the number of CD4(+)PD-1(high) T cells was not associated with patient outcome. Taken together, these results indicated that PD-1 expression defines two sub-populations with distinct functions that differentially impact patient outcome in FL.

Activation of TAK1 by MYD88 L265P drives malignant B-cell Growth in non-Hodgkin lymphoma.

Massively parallel sequencing analyses have revealed a common mutation within the MYD88 gene (MYD88L265P) occurring at high frequencies in many non-Hodgkin lymphomas (NHLs) including the rare lymphoplasmacytic lymphoma, Waldenström's macroglobulinemia (WM). Using whole-exome sequencing, Sanger sequencing and allele-specific PCR, we validate the initial studies and detect the MYD88L265P mutation in the tumor genome of 97% of WM patients analyzed (n=39). Due to the high frequency of MYD88 mutation in WM and other NHL, and its known effects on malignant B-cell survival, therapeutic targeting of MYD88 signaling pathways may be clinically useful. However, we are lacking a thorough characterization of the role of intermediary signaling proteins on the biology of MYD88L265P-expressing B cells. We report here that MYD88L265P signaling is constitutively active in both WM and diffuse large B-cell lymphoma cells leading to heightened MYD88L265P, IRAK and TRAF6 oligomerization and NF-κB activation. Furthermore, we have identified the signaling protein, TAK1, to be an essential mediator of MYD88L265P-driven signaling, cellular proliferation and cytokine secretion in malignant B cells. Our studies highlight the biological significance of MYD88L265P in NHL and reveal TAK1 inhibition to be a potential therapeutic strategy for the treatment of WM and other diseases characterized by MYD88L265P.

TGF-ß upregulates CD70 expression and induces exhaustion of effector memory T cells in B-cell non-Hodgkin's lymphoma.

Transforming growth factor beta (TGF-ß) has an important role in mediating T-cell suppression in B-cell non-Hodgkin lymphoma (NHL). However, the underlying mechanism responsible for TGF-ß-mediated inhibition of effector memory T (Tm) cells is largely unknown. As reported here, we show that exhaustion is a major mechanism by which TGF-ß inhibits Tm cells, and TGF-ß mediated exhaustion is associated with upregulation of CD70. We found that TGF-ß upregulates CD70 expression on effector Tm cells while it preferentially induces Foxp3 expression in naive T cells. CD70 induction by TGF-ß is Smad3-dependent and involves IL-2/Stat5 signaling. CD70+ T cells account for TGF-ß-induced exhaustion of effector Tm cells. Both TGF-ß-induced and preexisting intratumoral CD70+ effector Tm cells from B-cell NHL have an exhausted phenotype and express higher levels of PD-1 and TIM-3 compared with CD70- T cells. Signaling transduction, proliferation and cytokine production are profoundly decreased in these cells, and they are highly susceptible to apoptosis. Clinically, intratumoral CD70-expressing T cells are prevalent in follicular B-cell lymphoma (FL) biopsy specimens, and increased numbers of intratumoral CD70+ T cells correlate with an inferior patient outcome. These findings confirm TGF-ß-mediated effector Tm cell exhaustion as an important mechanism of immune suppression in B-cell NHL.

Denileukin diftitox in combination with rituximab for previously untreated follicular B-cell non-Hodgkin's lymphoma.

Follicular lymphoma exhibits intratumoral infiltration by non-malignant T lymphocytes, including CD4+CD25+ regulatory T (T(reg)) cells. We combined denileukin diftitox with rituximab in previously untreated, advanced-stage follicular lymphoma patients anticipating that denileukin diftitox would deplete CD25+ T(reg) cells while rituximab would deplete malignant B cells. Patients received rituximab 375 mg/m(2) weekly for 4 weeks and denileukin diftitox 18 mcg/kg/day for 5 days every 3 weeks for 4 cycles; neither agent was given as maintenance therapy. Between August 2008 and March 2010, 24 patients were enrolled. One patient died before treatment was given and was not included in the analysis. Eleven of 23 patients (48%; 95% confidence interval (CI): 27-69%) responded; 2 (9%) had complete responses and 9 (39%) had partial responses. The progression-free rate at 2 years was 55% (95%CI: 37-82%). Thirteen patients (57%) experienced grade ≥3 adverse events and one patient (4%) died. In correlative studies, soluble CD25 and the number of CD25+ T cells decreased after treatment; however, there was a compensatory increase in IL-15 and IP-10. We conclude that although the addition of denileukin diftitox to rituximab decreased the number of CD25+ T cells, denileukin diftitox contributed to the toxicity of the combination without an improvement in response rate or time to progression.

A role for IFN-lambda1 in multiple myeloma B cell growth.

Multiple myeloma (MM) is a progressive disease that results from dysregulated proliferation of plasma cells. Although, causative factors such as genetic events and altered expression of anti-apoptotic factors have been described in a number of patients, the mechanistic details that drive myeloma development and continued growth of malignant cells remain largely undefined. Numerous growth factors, including interleukin (IL)-6, Insulin-like growth factor-1 and IL-10 have been shown to promote growth of MM cells suggesting a significant role for cytokines in this disease. Interferon (IFN)-lambda1 is a new member of the Class II cytokine family that, similar to IFN-alpha, has been shown to mediate viral immunity. In light of data supporting a role for cytokines in myeloma, we investigated the significance of IFN-lambda1 on myeloma cell biology. Our studies show for the first time that myeloma cells bind to soluble IFN-lambda1, and that IFN-lambda1 induces myeloma cell growth and protects against dexamethasone-induced cell death. Our data also show that IFN-lambda1 induces phosphorylation of STAT1, STAT3 and Erk. Taken together, our results suggest that IFN-lambda1 may regulate myeloma cell biology and could prove to be therapeutically important.

Adenoma-specific alterations of protein kinase C isozyme expression in Apc(MIN) mice.

Members of the protein kinase C (PKC) family appear to play important roles in colorectal carcinogenesis. To investigate the potential involvement of PKC isozymes in adenomatous transformation induced by inactivation of the adenomatous polyposis coli (APC) gene product, we examined protein levels and localizations of ten PKC isozymes by immunohistochemistry in normal and adenomatous ileal epithelium of ApcMIN mice. Compared with surrounding normal epithelium, adenomas showed dramatically reduced staining for PKCs a, beta1, and zeta, as well as dysplasia-specific punctate nuclear staining of PKC mu. We conclude that reduced protein expression of PKC alpha, beta1, and zeta, and nuclear localization of PKC mu are markers of, and are perhaps involved in, adenomatous transformation induced by APC inactivation in ApcMIN mice.

Monoclonal antibodies against the human sodium iodide symporter: utility for immunocytochemistry of thyroid cancer.

The recent cloning of the thyroidal protein that is responsible for iodide transport, the sodium iodide symporter (hNIS), has made possible studies designed to characterize its structure, function and expression in thyroidal tissues. Using a mannose binding protein (MBP)-hNIS fusion protein as antigen, we have developed mouse monoclonal antibodies against hNIS to utilize as tools in such studies. Twenty-four clones were initially recovered which recognized the MBP-hNIS fusion protein, but only two of them were specific for hNIS while the others recognized MBP alone. Both antibodies were found to be immunoglobulin G (IgG) 1kappa (kappa). The specificity of antibodies was tested by Western blotting using membranes prepared from COS-7 cells transiently transfected with the pcDNA3 plasmid containing the full-length hNIS cDNA, or cells transfected with the pcDNA3 vector. A major band with a molecular weight (MW) of approximately 97 kDa, and several minor bands with MW of approximately 160 kDa, approximately 68 kDa, approximately 30 kDa and approximately 15 kDa, were detected specifically in the hNIS-transfected cells. After enzymatic deglycosylation, the major band was present at 68 kDa, as expected based upon the amino acid sequence of hNIS. Immunohistochemistry was performed with several different types of thyroid tissue and non-thyroidal tissues, using the monoclonal antibodies. Strong immunostaining was observed in Graves' tissue, with intermediate staining in papillary and follicular thyroid cancers and an absence of staining in Hürthle cell cancer. The staining was specific for the follicular epithelium and was concentrated in the basolateral portion of the cell membrane. These monoclonal hNIS antibodies should prove useful in the characterization of NIS expression in benign and malignant thyroid tissue and in studies characterizing its structure and function.

Demonstration of distinct antigenic profiles of small B-cell lymphomas by paraffin section immunohistochemistry.

The immunoperoxidase technique was used with antibodies against B-cell-associated antigens, including CD20, CD79a, CD10, CD23, CD43, cyclin D1, bcl-2, and kappa and lambda immunoglobulin light chains on formalin-fixed and B5-fixed tissue sections of follicular, small lymphocytic, mantle cell, and marginal zone lymphomas. Results obtained with paraffin section immunohistochemistry for CD20, CD10, CD23, and kappa and lambda light chains were compared with results obtained with flow cytometry or frozen section immunohistochemistry. Cells in all of the lymphoma types were positive for CD20 and CD79a. The antigenic profiles of the B-cell lymphomas demonstrated in paraffin sections were lymphoma type distinctive. Intrafollicular lymphocytes in follicular lymphomas were positive for CD10 and bcl-2. Small lymphocytic lymphomas expressed CD43 and CD23 and were negative for CD10 and cyclin D1. Mantle cell lymphomas characteristically expressed CD43 and cyclin D1 and were negative for CD23 and CD10. Marginal zone lymphomas were negative for CD23, CD10, and cyclin D1. All of the antibodies performed better in B5-fixed tissues, but formalin-fixed tissue immunophenotypes were always similar to those obtained on the B5-fixed tissue. These results were possible using well-fixed tissue, various antigen retrieval strategies, paraffin section reactive primary antibodies, and sensitive detection systems. Paraffin section immunohistochemistry on sections of routinely fixed tissue can be used similarly to flow cytometry and frozen section immunohistochemistry when classifying the lymphomas of small B lymphocytes.

In vivo analysis of Fas/FasL interactions in HIV-infected patients.

Recent insights into the pharmacological control of HIV replication and the molecular mechanisms of peripheral T cells homeostasis allowed us to investigate in vivo the mechanisms mediating T cell depletion in HIV-infected patients. Before the initiation of highly active antiretroviral therapy (HAART), a high degree of lymphoid tissue apoptosis is present, which is reduced upon HAART initiation (P < 0.001) and directly correlates with reduction of viral load and increases of peripheral T lymphocytes (P < 0.01). Because Fas/FasL interactions play a key role in peripheral T lymphocyte homeostasis, we investigated the susceptibility to Fas-mediated apoptosis in peripheral T lymphocytes and of FasL expression in lymphoid tissue before and during HAART. High levels of Fas-susceptibility found in peripheral CD4 T lymphocytes before HAART were significantly reduced after HAART, coinciding with decreases in viral load (P = 0.018) and increases in peripheral CD4 T lymphocyte counts (P < 0.01). However, the increased FasL expression in the lymphoid tissue of HIV-infected individuals was not reduced after HAART. These results demonstrate that lymphoid tissue apoptosis directly correlates with viral load and peripheral T lymphocyte numbers, and suggest that HIV-induced susceptibility to Fas-dependent apoptosis may play a key role in the regulation of T cell homeostasis in HIV-infected individuals.

The expression of Fas Ligand by macrophages and its upregulation by human immunodeficiency virus infection.

Fas/Fas Ligand (FasL) interactions play a significant role in peripheral T lymphocyte homeostasis and in certain pathological states characterized by T cell depletion. In this study, we demonstrate that antigen-presenting cells such as monocyte-derived human macrophages (MDM) but not monocyte-derived dendritic cells express basal levels of FasL. HIV infection of MDM increases FasL protein expression independent of posttranslational mechanisms, thus highlighting the virus-induced transcriptional upregulation of FasL. The in vitro relevance of these observations is confirmed in human lymphoid tissue. FasL protein expression is constitutive and restricted to tissue macrophages and not dendritic cells. Moreover, a significant increase in macrophage-associated FasL is observed in lymphoid tissue from HIV (+) individuals (P < 0.001), which is further supported by increased levels of FasL mRNA using in situ hybridization. The degree of FasL protein expression in vivo correlates with the degree of tissue apoptosis (r = 0.761, P < 0. 001), which is significantly increased in tissue from HIV-infected patients (P < 0.001). These results identify human tissue macrophages as a relevant source for FasL expression in vitro and in vivo and highlight the potential role of FasL expression in the immunopathogenesis of HIV infection.

Expression of nm23/nucleoside diphosphate kinase-A protein in endometrial carcinoma.

A metastatic tumor suppressor role for the nm23 gene product in breast carcinoma has been proposed. The biologic significance of nm23/NDP kinase-A (NDPK-A) expression in endometrial carcinoma remains undetermined. We sought to (1) characterize the pattern and intensity of nm23 protein expression in endometrial carcinoma and (2) assess the relationship between intensity/pattern of nm23 protein immunostaining and treatment response assessed by progression-free survival and survival to death. Formalin-fixed paraffin-embedded sections from 234 patients with endometrial cancer were immunostained with a mouse monoclonal IgG to nm23/NDPK-A protein. In most specimens of endometrial carcinoma (67.5%), nm23 expression was strongly upregulated. No association was found between either intensity (0 vs 1, 2, 3) or pattern (nuclear membrane vs cytoplasmic) of immunostaining and FIGO stage, ploidy status, histologic subtype, myometrial invasion, progression-free survival, or survival to death. Absence of nm23 staining (0 vs 1, 2, 3) was significantly associated with lower tumor grades (P = 0.02). For stage I patients, moderate to strong nm23 immunostaining intensity (2, 3) was associated with a trend toward diminished progression-free survival (P = 0.08). Our data imply a heterogeneity of nm23 protein expression and possible distinct biologic roles for nm23 in endometrial compared with breast or ovarian carcinoma.

Monoclonal antibodies specific for peptide epitopes of the epidermal growth factor receptor's extracellular domain.

The ErbB tyrosine kinase receptor family plays an important role in normal cellular growth and differentiation. In addition, ErbB receptor family members are commonly amplified and overexpressed in various human neoplasms and tumor-derived cell lines, where it is believed that increased signalling as a result of receptor overexpression may play an important role in oncogenesis. Consequently, ErbB receptor family members are being investigated rigorously as potential biomarkers of cancer and as therapeutic targets in malignant tissues. Numerous studies now demonstrate the existence of "soluble" ErbB (sErbB) analogs in normal and cancerous tissues. These sErbB proteins embody the extracellular domain (ECD) of the receptor only; they are generated by either proteolytic cleavage or from truncated, alternatively spliced mRNA transcripts. Recently, we have identified an alternate transcript of the human c-erbB1 (Epidermal Growth Factor Receptor) proto-oncogene from placenta that encodes a sErbB1 protein of 60-kDa. This protein, p60 sErbB1, is glycosylated and secreted when expressed in transfected tissue culture cells in vitro. Although "soluble" receptor analogs may play important physiological roles in intercellular communication, tissue morphogenesis, tissue regeneration and repair, and embryogenesis by inhibiting or stimulating specific mitogenic and pattern forming signals, their mechanism of action has not been thoroughly elucidated. To further characterize sErbB1 expression in human tissues and cell lines and to better understand their role in carcinogenesis and normal development, we have generated monoclonal antibodies (MAbs) toward specific peptide epitopes of ErbB1 extracellular subdomains III and IV. These antibody reagents are described here and should be useful experimental, preparative, analytical, diagnostic, and therapeutic reagents for the study of sErbB1 molecules in normal development and cancer.

Absence of mutations in DNA mismatch repair genes in sporadic endometrial tumors with microsatellite instability.

DNA mismatch repair genes have been reported to play a role in the pathogenesis of hereditary nonpolyposis colorectal cancer (HNPCC). Mutations of DNA mismatch repair genes have accounted for 90% of HNPCC-related colon and endometrial tumors. These mutations have been associated with microsatellite instability (MIN). Because endometrial cancer (EC) is the most common extracolonic malignancy associated with HNPCC, we hypothesized that similar molecular alterations may occur in sporadic endometrial tumors exhibiting MIN. Mutational analysis of the MSH2 and MLH1 genes was undertaken in sporadic EC that demonstrate MIN to determine the role of these genes in the pathogenesis of sporadic ECs. Established microsatellite markers were used to determine the incidence of MIN from 28 patients with sporadic EC. MIN was observed in 32% (9 of 28) of the tumor specimens analyzed. Mutational analysis of MSH2 and MLH1 genes was performed by immunohistochemical analysis and direct sequencing of tumor specimens that exhibited MIN. All 28 tumor specimens exhibited strong nuclear staining with both MSH2 and MLH1 antibodies, suggesting the absence of mutations. Sequencing of all exons of both the MSH2 and MLH1 genes in the nine MIN-positive tumor specimens demonstrated no mutations. We conclude that the MSH2 and MLH1 genes do not play a role in the pathogenesis of sporadic endometrial cancer.

p53 expression in neurofibroma and malignant peripheral nerve sheath tumor. An immunohistochemical study of sporadic and NF1-associated tumors.

Malignant peripheral nerve sheath tumors (MPNST) are highly malignant sarcomas arising either de novo or in transition from neurofibroma. Although relatively little is known of the molecular genetic alterations that underlie their formation, recent DNA sequencing studies have demonstrated the presence of p53 mutations in some MPNST. This tumor-suppressor gene has been implicated in the progression of a variety of human malignancies, including sarcomas. Employing the anti-p53 monoclonal antibody Do-7, this retrospective immunohistochemical study of p53 gene overexpression in MPNST found reactivity to be present in 68% and to be significant in degree in 57%. In contrast, although some degree of p53 overexpression was present in 48% of neurofibromas, none stained strongly and only 1 of the 27 (4%), a cellular example, showed significant staining. No difference in the frequency or degree of p53 staining was noted between MPNSTs from patients with or without neurofibromatosis 1. The observed overexpression of the gene product, possibly the reflection of a p53 gene mutation, suggests a role for p53 in the progression of neurofibroma to MPNST. Although the prognostic of p53 overexpression in MPNST remains to be confirmed, in the present series immunopositive tumors were associated with a shorter median patient survival (18 months) than were tumors showing no reactivity (82 months) (P = .02).

Chronic increases in blood flow upregulate endothelin-B receptors in arterial smooth muscle.

Experiments were designed to characterize endothelin receptors in arteries after chronic increases in blood flow. A fistula was created between the femoral artery and vein in one hindlimb of dogs; contralateral blood vessels were sham operated. Sham- and fistula-operated arteries were removed 6 wk postoperatively. Some arteries were prepared for measurement of isometric force or for isolation of membrane proteins. Other arteries were used for histological staining with an endothelin-B (ETB) receptor antibody. In arteries suspended for the measurement of isometric force, endothelin-1 produced concentration-dependent increases in tension that were significantly greater in fistula- than in sham-operated arteries without endothelium. The ETB-receptor-selective peptide sarafotoxin S6c produced concentration-dependent increases in tension only in fistula-operated arteries. In receptor-binding studies of membrane proteins, Scatchard analysis of saturation binding with 125I-labeled endothelin-1 (125I-endothelin-1) indicated that the total number of receptors was greater in fistula-operated arteries; affinity was threefold less in fistula- than in sham-operated arteries. Competitive displacement of 125I-endothelin-1 by endothelin-3 was significant for a two-site model in membranes prepared from sham-and fistula-operated arteries. Competitive inhibition of 125I-endothelin-1 binding by sarafotoxin S6c was significant for a one-site binding model in all arteries. Sarafotoxin S6c binding sites were elevated significantly in fistula-operated arteries. Immunohistochemical staining for the ETB receptor was significantly greater in both the endothelium and smooth muscle of fistula- than in sham-operated arteries. These results suggest that chronic increases in blood flow upregulate endothelin receptors, including ETB receptors in arterial smooth muscle.

Changes in proliferating cell nuclear antigen expression in glioblastoma multiforme cells along a stereotactic biopsy trajectory.

Proliferating cell nuclear antigen, an auxiliary protein of deoxyribonucleic acid polymerase-delta, has been shown to be a reliable marker of nuclear deoxyribonucleic acid synthetic activity. We applied a monoclonal antibody to proliferating cell nuclear antigen to a series of serial stereotactic biopsies from patients with glioblastoma multiforme and found the proliferative activity to vary relative to biopsy location within or surrounding the solid tissue component of the tumor. Twenty-seven trajectories in 26 patients were analyzed, each consisting of two to five sequential 10 x 1.5 mm core biopsies (mean = 3). The proliferative index (PI) was greatest in those cells located at the solid tumor-infiltrated parenchyma interface. PI values were significantly lower in those biopsy cores located proximal (within infiltrated parenchyma) and distal (within solid tumor tissue) to the solid tumor-infiltrated parenchyma interface (median PI values, proximal to distal: 0.38, 0.66, 5.45 solid tumor-infiltrated parenchyma interface], 0.39, 0.09%). The mean PI values were significantly lower in neoplastic cells samples from regions of peripheral hypodensity on computed tomographic scans compared with those sampled from contrast-enhancing regions (0.9 and 3.91%, respectively). There was no significant difference in the mean PI values of neoplastic cells sampled from regions of contrast enhancement or central hypodensity (3.91 and 4.31%, respectively).

Marjolin's ulcer: immunohistochemical study of 17 cases and comparison with common squamous cell carcinoma and basal cell carcinoma.

Formalin-fixed, paraffin-embedded biopsy specimens of 17 cases of squamous cell carcinoma of Marjolin's ulcer (SCC-MU), 6 cases of common SCC (SCC), and 5 cases of basal cell carcinoma (BCC) were stained with three monoclonal antikeratin antibodies (CAM 5.2, MAK-6, and MA-903), a monoclonal antivimentin antibody (V9), and a polyclonal anticarcinoembryonic antigen antiserum (A115). Neoplastic cells of SCC-MU, SCC, and BCC showed consistently negative staining for CAM 5.2. A wide range of reactivity, from negative to diffuse strong positivity, among neoplastic cells of SCC-MU and SCC was noted with MAK-6. Alternatively, neoplastic cells of SCC-MU, SCC, and BCC consistently showed diffuse moderate to strong reactivity with MA-903. These findings imply that SCC-MU has largely high-molecular-weight keratins. They also showed a wide range of reactivity with V9. However, neoplastic cells of five of the six SCC and five cases of BCC were negative for V9. These findings suggest that neoplastic cells of SCC-MU contain vimentin in higher frequency than in the more usual SCC.

Value of immunohistochemistry in the diagnosis of leukemia cutis: study of 54 cases using paraffin-section markers.

A grave prognosis is usually associated with leukemic skin infiltrates (leukemia cutis). However, some leukemic skin infiltrates are clinically similar to reactive non-leukemic infiltrates in patients with leukemia; thus it is of great importance to distinguish them. Fifty-four cases which were thought clinically to be leukemia cutis underwent immunophenotyping with a panel of nine T, B, monocytic, and macrophage markers using paraffin sections. Immunohistochemistry helped identify 44 cases with leukemia cutis and 10 with reactive infiltrates. In all cases of leukemia cutis, the staining patterns of skin infiltrates were concordant with cell type in the bone marrow. Furthermore, the panel of markers was usually helpful in distinguishing reactive from leukemia infiltrates, especially in cases with chronic lymphatic leukemia. Immunohistochemistry is a valuable adjunct in histopathologic differentiation of skin infiltrates in most cases of leukemia. With formalin-fixed, paraffin-embedded biopsies, we recommend that CD45 (LCA), CD45RO (UCHL-1), CD3, CD20 (L-26), CD43 (Leu-22), CD68 (KP-1), lysozyme, and chloroacetate esterase be considered in cases of systemic leukemia with cutaneous papules and nodules that prove difficult to interpret with routine section.

Methods for measuring plasma hemoglobin in micromolar concentration compared.

Of eight methods examined for measuring plasma hemoglobin in micromolar concentration, all exhibited acceptable linearity, reproducibility, and concurrence except when specimens were icteric or lipemic or contained methemoglobin or methemalbumin. Measurement of absorbance at 578 nm with an Allen correction permits precise assay of plasma oxyhemoglobin concentration as low as 0.01 g/L (1 mg/dL, 0.16 mumol/L), unaffected by hyperlipidemia or hyperbilirubinemia. Discrepancies between methods occurred in 11.6% of a consecutive series of 50 nonicteric patients' plasma specimens. Examination of absorption spectra is helpful when discrepancies are observed between methods. The presence of methemalbumin or methemoglobin in plasma is not recognized by methods that measure only oxyhemoglobin. Increased ceruloplasmin or beta-carotene does not significantly affect results.