Sheron C. Lear

Transient T cell depletion causes regression of melanoma metastases

BackgroundCognate immunity against neoplastic cells depends on a balance between effector T cells and regulatory T (Treg) cells. Treg cells prevent immune attack against normal and neoplastic cells by directly suppressing the activation of effector CD4+ and CD8+ T cells. We postulated that a recombinant interleukin 2/diphtheria toxin conjugate (DAB/IL2; Denileukin Diftitox; Ontak) may serve as a useful strategy to deplete Treg cells and break tolerance against neoplastic tumors in humans.MethodsWe administered DAB/IL2 (12 μg/kg; four daily doses; 21 day cycles) to 16 patients with metastatic melanoma and measured the effects on the peripheral blood concentration of several T cell subsets and on tumor burden.ResultsWe found that DAB/IL2 caused a transient depletion of Treg cells as well as total CD4+ and CD8+ T cells (< 21 days). T cell repopulation coincided with the de novo appearance of melanoma antigen-specific CD8+ T cells in several patients as determined by flow cytometry using tetrameric MART-1, tyrosinase and gp100 peptide/MHC conjugates. Sixteen patients received at least one cycle of DAB/IL2 and five of these patients experienced regressions of melanoma metastases as measured by CT and/or PET imaging. One patient experienced a near complete response with the regression of several hepatic and pulmonary metastases coupled to the de novo appearance of MART-1-specific CD8+ T cells. A single metastatic tumor remained in this patient and, after surgical resection, immunohistochemical analysis revealed MART1+ melanoma cells surrounded by CD8+ T cells.ConclusionTaken together, these data indicate that transient depletion of T cells in cancer patients may disrupt the homeostatic control of cognate immunity and allow for the expansion of effector T cells with specificity against neoplastic cells. Several T cell depleting agents are clinically available and this study provides strong rationale for an examination of their efficacy in cancer patients.Trial registrationNCT00299689 (ClinicalTrials.gov Identifier).

Tartrate-Resistant Acid Phosphatase as an Immunohistochemical Marker for Inflammatory Macrophages

Human serum contains 2 isoforms of type-5 tartrate-resistant acid phosphatase (TRACP): 5a and 5b. TRACP-5b is osteoclastic. Our goal was to determine if serum TRACP-5a could originate from inflammatory macrophages (MPhi). We stained 246 paraffin-embedded tissue samples for TRACP using monoclonal antibody 9C5 (mab9C5) to isoforms 5a and 5b and a novel mab220 specific to isoform 5a. CD68 and lysozyme were also stained. MPhi of chronic and granulomatous inflammation and in tissues that undergo strong antigenic stimulation were strongly positive for TRACP, more so with mab220 than with mab9C5. Noninflammatory MPhi in lymph node sinuses or germinal centers and red pulp MPhi of spleen were weak or negative for TRACP. Marginal zone lymphocytes and sebaceous glands of skin were weakly positive for TRACP. Tissue mast cells displayed strong TRACP staining. Neuroendocrine cells of gastrointestinal tissues were strongly immunoreactive with mab9C5 but negative with mab220. Restricted expression of TRACP primarily in inflammatory MPhi supports our hypothesis that circulating TRACP-5a could be a biomarker of chronic inflammatory disease activity.

CD10 Expression in Cutaneous Adnexal Neoplasms and a Potential Role for Differentiating Cutaneous Metastatic Renal Cell Carcinoma

CONTEXT Recent investigations have demonstrated the utility of CD10 as a marker for renal cell carcinoma (RCC). Cutaneous metastases occur in up to 11% of patients with RCC and may be the presenting sign of widespread disease. The differential diagnosis in histopathologic evaluation of these cases includes cutaneous adnexal neoplasms, and describing the expression of CD10 in these tumors may be helpful in delineating the differential diagnosis. OBJECTIVE To determine CD10 expression in a variety of adnexal lesions and to determine the diagnostic utility of CD10 in an immunohistochemical panel differentiating metastatic cutaneous renal cell carcinoma from cutaneous adnexal neoplasms. DESIGN We studied 57 primary adnexal neoplasms of eccrine (n = 31), apocrine (n = 16), and sebaceous (n = 10) differentiation as well as normal skin (n = 3) and RCC metastatic to the skin (n = 4). A CD10 monoclonal antibody was applied to formalin-fixed, paraffin-embedded tissue. Specimens were randomized and categorized as immunopositive or immunonegative by a pathologist with expertise in immunohistochemistry who was blinded to the diagnoses. RESULTS Two (6.5%) of 31 eccrine, 1 (6%) of 16 apocrine, and 4 (40%) of 10 sebaceous neoplasms demonstrated CD10 immunopositivity. Four (100%) of 4 RCC were CD10 immunopositive. CD10 expression was significant for eccrine and apocrine neoplasms (P < .001) compared to metastatic RCC, but not for sebaceous neoplasms (P = .08). CONCLUSION Based on these results, CD10 is a useful additional immunostain for the discrimination of RCC metastatic to the skin and cutaneous adnexal neoplasms with eccrine and apocrine differentiation, but not with sebaceous differentiation.

Complement component 9 activation, consumption, and neuronal deposition in the post-hypoxic-ischemic central nervous system of human newborn infants.

The role of complement in neonatal hypoxic-ischemic brain injury is not known. Therefore, cerebral spinal fluid (CSF) and post-mortem cerebral tissue were analyzed to determine whether complement is activated and complement component 9 (C9) is deposited on neurons in the central nervous systems (CNS) of newborn infants who developed moderate to severe hypoxic-ischemic encephalopathy (HIE). Control CSF samples were obtained during routine evaluation for possible sepsis from infants who were not depressed at birth. In ELISA assays of CSF obtained from 16 infants with HIE, compared to CSF from 7 control infants, the mean concentration of terminal complement complexes was elevated and the mean C9 concentration was diminished. Immunofluorescence microscopy of post-mortem frozen brain tissue obtained from two infants who expired at 4-5 days of life after severe HIE revealed that activated C9 was deposited on cells in all lobes. Double label immunofluorescence microscopy demonstrated that nearly all of the C9-positive cells were neurons and essentially all of the neurons were C9-positive. Immunoperoxidase immunohistochemistry of formalin-fixed tissue also confirmed the presence of many C9-positive cells, particularly in the hippocampus. The C9-positive cells usually manifested morphology consistent with neurons, most of which contained fragmented nuclei. In summary, complement was activated in the CNS of newborn infants who developed moderate to severe HIE. C9 was deposited on neurons, including morphologically apoptotic neurons. Further investigations into a possible role of complement in the pathogenesis of neonatal hypoxic-ischemic cerebral injury are warranted.

Localization of tartrate-resistant acid phosphatase in human placenta

SummaryTartrate-resistant acid phosphatase is an inducible marker of cell differentiation and activation expressed by specialized cells of macrophage lineage and some activated lymphocytes. Clinically, this phosphatase is a diagnostic marker for hairy cell leukaemia and osteoclast activity. The cDNA for this enzyme has been cloned from a placental expression library, yet the cell(s) expressing the enzyme protein has not been determined with certainty. Our laboratories have developed a monoclonal antibody, 9C5, suitable for immunohistochemical localization of tartrate-resistant acid phosphatase in paraffin sections. The purpose of this study was to use antibody 9C5 to identify cells expressing tartrate-resistant acid phosphatase in sections of paraffin-embedded, normal, full-term placenta and to determine if those cells expressed other macrophage markers including CD68(PG-M1 antibody), LN5, lysozyme α1-antitrypsin and α1-antichymotrypsin. Histochemical localization of activity in frozen sections was compared with immunohistochemical localization in paraffin sections of the same tissue specimens. The activity and antigenicity of this enzyme were detected in decidual cells, syncytiotrophoblast, and some macrophages distributed throughout maternal and embryonic tissues, but not in neutrophils. Unlike other tissues previously examined, placenta contains significant numbers of the phosphate-positive cells that are not of macrophage origin.

IL-4 production by CD8+ lymphomatoid papulosis, type C, attracts background eosinophils

There are two subsets of CD8+ T cells: Tc1 and Tc2. INF‐γ production by Tc1 cells causes granulomatous inflammation. IL‐4 production by Tc2 cells attracts eosinophils. A 76‐year‐Caucasian female presented with CD8+ lymphomatoid papulosis (LyP), type C. We hypothesized that the LyP cells belonged to the Tc2 subset because of abundant background eosinophils. Hematoxylin and eosin and immunohistochemical stains were carried out on tissue sections from a skin punch biopsy. Antibodies for immunohistochemical stains included CD3, CD4, CD5, CD7, CD8, CD30, CD56, ALK‐1, clusterin and IL‐4. There was involvement of the dermis by a dense lymphoid infiltrate composed of large atypical cells and numerous eosinophils. The LyP cells expressed CD5, CD8, CD30 and IL‐4. Keratinocytes showed a membranous pattern of immunoreactivity for IL‐4. IL‐4 production by CD8+ LyP, type C indicates that it belongs to the Tc2 subset. The cytokine milieu produced by the LyP cells attracted eosinophils. The IL‐13R complex on keratinocytes bound IL‐4 and produced a membranous staining pattern. Although CD8+ LyP is rare, we believe that this CD30+ lymphoproliferative disorder should be included in the World Health Organization‐European Organization for Research and Treatment of Cancer classification of cutaneous T‐cell lymphomas.