Serum antibodies to the HPV16 proteome as biomarkers for head and neck cancer.

BACKGROUND: Human papillomavirus (HPV) type 16 is associated with oropharyngeal carcinomas (OPC). Antibodies (Abs) to HPV16 E6 and E7 oncoproteins have been detected in patient sera; however, Abs to other early HPV-derived proteins have not been well explored. METHODS: Antibodies to the HPV16 proteome were quantified using a novel multiplexed bead assay, using C-terminal GST-fusion proteins captured onto Luminex beads. Sera were obtained from untreated patients with OPC (N=40), partners of patients with HPV16+ OPC (N=11), and healthy controls (N=50). RESULTS: Oropharyngeal carcinomas patients with known virus-like capsid particle+ Abs had elevated serum Abs to HPV16 E1, E2, E4, E6, and E7, and L1 antibody levels, but not E5. The ratios of specific median fluorescence intensity to p21-GST compared with controls were E1: 50.7 vs 2.1; E4: 14.6 vs 1.3; E6: 11.3 vs 2.4; E7: 43.1 vs 2.6; and L1: 10.3 vs 2.6 (each P≤0.01). In a validation cohort, HPV16 E1, E2, and E7 antibody levels were significantly elevated compared with healthy control samples (P≤0.02) and partners of OPC patients (P≤0.01). CONCLUSION: Patients with HPV16+ OPC have detectable Abs to E1, E2, and E7 proteins, which are potential biomarkers for HPV-associated OPC.

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.

Tumor biology: use of tiled images in conjunction with measurements of cellular proliferation and death in response to drug treatments.

Tumor growth is dependent on the balance between cell proliferation and cell death, and these events occur heterogenously within an individual tumor. We present a methodology that provides integrative information about cell kinetics, cell death, and cell growth within individual tumors in animals treated with cytotoxic chemotherapeutic agents. Using HCT-116 and NCI-H460 cells, human colonic adenocarcinoma and non-small cell lung cells, respectively, traditional xenograft studies were performed. The tumor-bearing animals were treated with cyclophosphamide (Cytoxan), gemcitabine (Gemzar), or mitomycin C, and extensive analysis of the tumors was studied. Cell kinetics were evaluated by measuring the apoptotic and proliferation indices. The ability to image an entire tumor section using "tiling" by creating a large montage from many high-resolution images makes it possible to identify regional differences within areas of tumor and to demonstrate differences in these tumor regions after treatment with selected chemotherapeutic agents. Two specific areas within tumors have been identified: (a) areas of viable cells within the cell cycle, determined by bromodeoxyuridine and/or morphological characteristics determined by hematoxylin staining; and (b) areas of necrosis determined by the absence of bromodeoxyuridine and proliferating cell nuclear antigen-labeled cells coupled with morphological changes. By standardizing the tumor size to 100 mm2, different patterns of tumor responses to chemotherapeutic agents were determined. By creating such tiled images and by quantitating cell cycle kinetics, it is possible to gain a more complete understanding of tumor growth and response to treatment, leading to the development of more reliable methods for assessing the clinical behavior of anticancer drugs.

Peripheral T-cell lymphoma with follicular involvement and a CD4+/bcl-6+ phenotype.

A truly follicular pattern is thought to be restricted to B-cell lymphomas. We observed a prominent follicular growth pattern in three cases of nodal peripheral T-cell lymphomas, of which two were initially diagnosed as follicular lymphomas. All three patients were male, ranged in age from 50 to 70 years, and had generalized lymphadenopathy at the time of diagnosis. The follicles were sharply demarcated in two cases and large and vague in one case; in all cases, they contained abundant follicular dendritic cells. Neoplastic cells were small to medium, with irregular cleaved or round nuclei and clear cytoplasm, which was abundant in one case. Lymphoma cells in all cases were CD4+ CD8- CD57- bcl-6, with CD10 coexpression in 2 cases. Clonal rearrangement of the gamma chain of the T-cell receptor gene was demonstrated in each case. These cases expand the differential diagnosis of lymphomas with a follicular growth pattern and suggest that neoplastic T cells may have the capacity to induce or home to follicular structures.

Diffuse colonic mantle cell lymphoma in a patient with presumed ulcerative colitis: detection of a precursor monoclonal lymphoid population using polymerase chain reaction and immunohistochemistry.

Primary lymphoma of the colon, a rare and typically late complication of ulcerative colitis, exhibits high-grade morphology and behavior when it occurs. Recently, several reports of colonic lymphoma masquerading as ulcerative colitis have been described. These previous reports described inflammatory mucosal changes typical of ulcerative colitis as being present in superficial biopsies, leading to the initial diagnosis of ulcerative colitis; however, further workup resulted in a diagnosis of primary colonic lymphoma within several months in these cases, and all symptoms and mucosal changes resolved after treatment of the lymphoma. Herein we report a case of mantle cell lymphoma arising in the colon and rectum in a 71-year-old woman with a 4-year history of ulcerative colitis. Immunoglobulin heavy-chain gene rearrangements were detected using the polymerase chain reaction procedure in fixed tissue in the lymphoma as well as in a prior resection specimen that histologically appeared to show only changes of severe ulcerative colitis. This finding suggests that an indolent lymphoid proliferation may have been the underlying disease in this patient and raises questions about the role of colonic lymphoma in causing mucosal injury.

Cutaneous b-cell lymphomas of follicular and marginal zone types: use of Bcl-6, CD10, Bcl-2, and CD21 in differential diagnosis and classification.

Cutaneous follicular lymphomas (FLs) and cutaneous B-cell lymphomas of extranodal marginal zone (MZL)/mucosal-associated lymphoid tissue (MALT) type may have morphologic overlap, despite the fact that they are thought to be of distinct derivation (germinal center vs. postgerminal center). The problem is compounded by the reported absence of bcl-2 expression by many cutaneous FLs, leading to speculation that cutaneous FL may be unrelated to nodal FL. The authors analyzed the expression of the germinal center-associated antigens bcl-6 and CD10 and of bcl-2 in 18 cutaneous B-cell lymphomas (10 FLs and eight MZLs), in relationship to CD21+ follicular structures, to clarify the relationship of nodal to cutaneous FLs and to explore the value of these antigens in differential diagnosis. The authors studied 10 cutaneous FLs (seven primary and three secondary) and eight MZLs (six primary and two secondary). The FLs (found in six men and four women age 45-75 years) involved the trunk (n = 3) and scalp, face and neck (n = 7). The MZLs (found in five women and three men age 34-81 years) involved the trunk (n = 4), face and neck (n = 2), and arm (n = 2). Immunostaining for CD21, bcl-6, CD10, and bcl-2 allowed the delineation of compartments within the tumors and yielded distinct patterns of staining in FL and MZL. In both follicular and interfollicular/diffuse areas of FL the neoplastic cells were bcl-6+ (10 of 10), often CD10+ (seven of 10, four of seven primary), and bcl-2+ (nine of 10, six of seven primary). Only three of seven cases (one of five primary) had bcl-2 rearrangement detectable by polymerase chain reaction. In the MZLs, the neoplastic B-cells were bcl-6-, CD10-, and bcl-2+ (eight of eight). Three patterns of CD21+ follicles were identified in MZL: reactive germinal centers, uniformly bcl-6+, CD10+, and bcl-2- (five of eight MZLs); colonized follicles, both bcl-6-, bcl-2+, and L26+ cells, and bcl-6+ and bcl-2- cells (five of eight MZLs); and expanded/colonized follicular dendritic cell meshworks, bcl-6- and bcl-2+ B cells with rare residual bcl-6+ and bcl-2- cells (four of eight MZLs). The authors conclude that cutaneous FLs express bcl-6 uniformly, usually express CD10 and bcl-2, and have a follicular pattern similar to nodal FL and consistent with a germinal center origin. The immunophenotype of cutaneous FL is distinct from that of cutaneous MZL, which is negative for bcl-6 and CD10. Colonized follicles in MZL, identified by CD21+ follicular dendritic cell meshworks, contained numerous bcl-6- and bcl-2+ B cells, and were readily distinguished from neoplastic follicles in FL. Conversely, CD21- interfollicular and diffuse areas in FLs contained bcl-6+ and CD10+ cells, which were not seen in diffuse areas of MZLs. Thus, the combination of bcl-2, bcl-6, and CD21 staining is useful for the distinction of cutaneous MZL from cutaneous FL.

Immunoblastic lymphoma of donor origin in the allograft after lung transplantation.

Posttransplant lymphoproliferative disorders (PTLD) are EBV-associated lymphoid neoplasms that are caused by the uncontrolled growth of EBV-infected B lymphocytes. The clinical presentation of PTLD can range from benign polygonal lymphoproliferative disorders to aggressive monoclonal immunoblastic lymphomas. In this report, we describe a seronegative lung transplant recipient who developed an immunoblastic lymphoma 4 months after lung transplantation from a seropositive donor. The neoplastic cells expressed B lymphocyte markers (CD19+, CD20+, sIgM+, kappa+) as well as the EBV antigen EBNA-2. A cell line with similar cytologic features spontaneously grew from in vitro cultures of the patient's peripheral blood mononuclear cells. The cell line and the lymphoma were EBV+, expressed a similar spectrum of B cell surface proteins, and had the donor's HLA haplotype. Analysis of immunoglobulin gene rearrangements and viral terminal repeat sequences revealed that the cell line and the tumor represented distinct B cell clones. Cultured peripheral blood mononuclear cells were restimulated in vitro with the EBV transformed cell line and tested for cytolytic activity. The host T cells demonstrated high levels of cytolytic activity against the tumor cell line that was abrogated by the addition of a anti-monomorphic HLA class I monoclonal antibody (mAb) (W6/32). These studies indicate that cells of donor origin can persist in the transplanted organ and may lead to an EBV-associated posttransplant lymphoma.

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.

Frequency of factor V(Leiden) and prothrombin G20210A in placentas and their relationship with placental lesions.

The most common hereditary hypercoaguable states are factor V(Leiden) (FVL) and prothrombin mutations (PRO). FVL and PRO present with an incidence of approximately 5% in a heterogeneous population, and 45% to 63% of the thrombophilic population. The frequency of these mutations in the fetal population and their clinical importance is unknown. Fetal side thromboembolic events (FST) include congenital stroke and renal vein thromboses. In some cases, FST can be diagnosed by placental histopathology when avascular (infarcted) villi are present in a patent maternal vascular space. FST can present as placenta-fetal-vascular or fetal-visceral-vascular lesions. Causes include vascular damage from cord compression or inflammation, but most remain unclear. Potential causes of FST include FVL and PRO. We describe the incidence of FVL and PRO from a prospective group of 169 consecutive placentas and in a retrospective group of archived placentas diagnosed with placental FST. One each of FVL and PRO heterozygosity was found in the prospective set (< 1% incidence for each). Five prospective placentas were diagnosed with placental FST, for an incidence of 3%; all were wild-type for FLV and PRO. Twenty-seven of 65 archived FST cases had analyzable DNA to find 5 FVL heterozygotes (18.5%); all were wild-type for PRO. Twenty-one of 65 retrospective archived controls analyzable found 1 case of FVL heterozygosity (< 5%). We find that the frequency of FVL and PRO may be decreased in the pregnant population but increased in cases of placental FST. Because factor V Leiden heterozygosity carries an increased risk for thrombotic complications, we suggest placental diagnosis of fetal side thromboemboli warrants clinical evaluation for FVL in infant and potentially the parents.

Silicone lymphadenopathy in a long distance runner: complication of a silastic prosthesis.

A 33-year-old male runner, who had undergone a Swanson silastic prosthetic implant for degenerative joint disease of the first metatarsal head and proximal phalanx of the right great toe, presented with unilateral inguinal lymphadenopathy. Biopsy revealed confluent, non-caseating granulomas containing silastic material. Silicone lymphadenopathy is unusual and most frequently presents as axillary adenopathy in patients with rheumatoid arthritis as a sequelae of prosthetic surgery. This case is clinically distinctive for its site of presentation in a healthy athlete and is histologically remarkable for the marked granulomatous response to the silastic elastomers.

Small lymphocytic non-Hodgkin's lymphoma of suppressor/cytotoxic T-cell phenotype [CD4(-), CD8(+)].

An unusual case of small lymphocytic non-Hodgkin's lymphoma of suppressor/cytotoxic T-cell phenotype is described. The patient presented with significant involvement of the spleen, lymph nodes, and bone marrow, but without a leukemic phase. Most small lymphocytic lymphomas are of B-cell phenotype, and those of T-cell origin are overwhelmingly of T-helper/inducer phenotype. Furthermore, T-cell lymphoproliferative lesions of suppressor/cytotoxic phenotype generally present as leukemias comprising large granular lymphocytes. This case reveals that suppressor/cytotoxic T lymphocytes may give rise to a lymphoproliferative disorder with a distinctive phenotype and presentation.

Telepathology diagnosis by means of digital still images: an international validation study.

Telepathology affords the means to provide pathological diagnosis and consultation to remote sites. However, before telepathology can become an acceptable medical tool, it will be vital to determine the diagnostic accuracy of this technology. We report the results of a single-blind study of the accuracy of diagnosis performed using computerized still images obtained from a telepathology workstation used in a French telepathology network. Four pathologists, each working alone, reviewed a total of 200 cases of routine surgical pathology (50 cases each), and performed diagnosis based on both computer CD-ROM still images (CD) and conventional glass slides (GS). Concordance between GS and CD diagnosis, as well as accuracy, were determined. Other factors related to performance were also measured, including diagnostic certainty, reasons for uncertainty, and causes of diagnostic error. Overall, there was good agreement between CS and CD diagnosis. There was 87.5% concordance between CS and CD diagnosis, and comparison to consensus (correct) diagnosis showed accuracy of 95.5% and 88.5% for GS and CD diagnosis, respectively. Marked variability in accuracy of CD diagnosis was observed among the four pathologists, and issues related to image selection and/or quality appeared responsible for 60% of the diagnostic errors. The lack of sufficient images and clinical information were frequently cited as reasons for diagnostic uncertainty, as well as feelings of insufficient expertise. It is likely that the opportunity for interaction with the referring pathologist and the use of subspecialty consultants would likely improve the performance of telepathology.

T-cell blast crisis in chronic myelogenous leukemia. Immunophenotypic and molecular biologic findings.

T-cell blast crisis in chronic myelogenous leukemia is rare. We examined three patients (ages 35 to 72 years) in whom T-cell blast crisis developed 11 to 36 months (mean, 25 months) after diagnosis of chronic myelogenous leukemia and who died 4 to 12 months (mean, 7 months) thereafter. Two patients had diffuse lymphadenopathy, and the third had marked lymphocytosis (white blood cell count 217,000/microL, with 90% circulating blasts). In all three patients, neoplastic cells had the appearance of lymphoblasts and were immunoreactive for T-cell markers by immunohistochemical or flow cytometric analysis or both. Molecular diagnostic studies revealed the presence of a bcr-abl oncogene rearrangement in all three cases, but none exhibited a clonal T-cell receptor delta, beta, or gamma chain gene rearrangement. One case exhibited deletion of the J delta 1 region of both delta chain genes. The significance of these findings is discussed, and they are compared with those of other reported cases of T-cell blast crisis in chronic myelogenous leukemia.

Comparison of functional testing for resistance to activated protein C and molecular biological testing for factor V R506Q in 370 patients.

OBJECTIVE: To compare functional and molecular biological tests for resistance to activated protein C (APC)/factor V R506Q, the most common cause of familial thrombosis. METHODS: We developed functional and molecular biological tests for resistance to APC/factor V R506Q at our institution and correlated the results for 370 patients studied by both methods. The functional method is based on addition of exogenous APC to an activated partial thromboplastin time-based assay. The molecular biological method is based on polymerase chain reaction followed by endonuclease digestion. RESULTS: Considering the molecular biological test as definitive for detecting the factor V R506Q mutation, the sensitivity of the functional assay was 100%, and the specificity was 74%. The prevalence of the factor V mutation in the population studied was 12% (41 heterozygotes, two homozygotes), and the positive predictive value of the functional assay was 34%. Although a normalized sensitivity ratio (nAPC-SR) less than 0.84 is considered evidence of resistance to APC by functional testing, we found that all patients with factor V R506Q had an nAPC-SR less than or equal to 0.71. When this alternative positive cutoff was used, the specificity of the functional test for factor V R506Q increased to 87%, and the positive predictive value increased to 52%, which constituted a significant improvement. We compared clinical findings from patients with resistance to APC with or without the presence of factor V R506Q, and found that as a group, those with factor V R506Q had a higher incidence of hypercoagulability, but fewer additional risk factors for hypercoagulability. The mechanism of resistance to APC in factor V R506Q-negative individuals is unclear, but may be related to other risk factors for hypercoagulability. CONCLUSIONS: The functional assay for resistance to APC is an excellent screening test for factor V R506Q, but confirmatory molecular biological testing is necessary when the functional test is positive, because of the high false-positive rate.

A low-temperature embedding colloidal gold technique for immunoelectron microscopy.

A low-temperature embedding technique using the polar resin Lowicryl K4M with protein A gold as a marker was utilized to localize a variety of antigens at the ultrastructural level. Carcinoembryonic antigen immunoreactivity was noted over the microvilli, secretory vacuoles, and the Golgi apparatus of normal colonic epithelium. Epithelial membrane antigen was localized to the luminal plasma membrane and cytoplasmic vesicles of glandular epithelium. The lymphoid antigens leucocyte common antigen and HLA-DR were both found on the membranes of lymphocytes and histiocytes. In addition, HLA-DR immunoreactivity was noted in the Golgi apparatus of these cells. The method described appears suitable for the localization of both surface and intracellular antigens with excellent preservation of both morphology and antigenicity.

The ultrastructural localization of prostatic specific antigen and prostatic acid phosphatase in hyperplastic and neoplastic human prostates.

A low temperature embedding, protein A-gold technique was used to localize prostatic specific antigen and prostatic acid phosphatase at the ultrastructural level in hyperplastic and neoplastic human prostates. Prostatic specific antigen immunoreactivity was localized over the endoplasmic reticulum, cytoplasmic vesicles and vacuoles, and within the lumina of prostatic glands. In contrast, prostatic acid phosphatase immunoreactivity was localized to lysosomal granules. The pattern of labelling was similar in both hyperplastic glands and adenocarcinomas. This is the first localization of prostatic specific antigen at the ultrastructural level. The localization of prostatic acid phosphatase by an immunochemical technique confirms and expands previous histochemical observations.

Molecular analysis of DNA rearrangements in leukemias and non-Hodgkin's lymphomas.

Genetic markers for leukemias and lymphomas include chromosomal translocations and antigen-receptor gene rearrangements. Clonal rearrangements of immunoglobulin or T cell receptor (TCR) genes reflect clonal proliferations of lymphocytes, a characteristic feature of lymphoid neoplasia. These rearrangements can be detected as described in this unit by Southern blot hybridization or, in many instances, the polymerase chain reaction (PCR). Specific chromosomal translocations can also serve as markers for clonality, for malignant transformation, and for various defined subtypes of hematopoietic cancers. PCR protocols are described for detection of the two most commonly assayed translocations, t(9;22) of chronic myelogenous leukemia or acute lymphoblastic leukemia, and t(14;18) of follicular lymphomas.

Restriction of expression of an integrated recombinant retrovirus in primary but not immortalized murine hematopoietic stem cells.

A recombinant retrovirus (DHFR*-SVADA) in which human adenosine deaminase (ADA) cDNA is transcribed from an internal SV40 promoter was used to infect murine hematopoietic stem and progenitor cells. Human ADA enzyme was not expressed in infected primary murine pluripotent stem cell-derived spleen or progenitor colonies (CFU-GM, CFU-Mix, BFU-E). In contrast, human ADA enzyme activity was readily detected in progenitor colonies derived from immortalized multipotent factor-dependent cells. The level of human enzyme was near endogenous murine enzyme levels and was equivalent in undifferentiated stem cells and differentiated myeloid, erythroid, and mixed colonies. These results indicate that cellular properties other than the stage of differentiation are important in determining the expression of foreign sequences introduced by retroviruses. Cell lines that are immortalized but still capable of induced differentiation may contain factors that abrogate blocks to expression that are manifested in primary hematopoietic stem cells.

Ki-1-positive large cell lymphomas, a heterogenous group of neoplasms. Morphologic, immunophenotypic, genotypic, and clinical features of 24 cases.

Clinical and pathologic features of 24 patients with large cell lymphomas that expressed the activation antigen Ki-1 are described. Phenotypic and/or genotypic studies characterized these neoplasms as T-cell (16 cases), B-cell (six cases), or null cell (two cases) type. Males predominantly were affected. Age of patients ranged from 19 to 73 years, with a bimodal distribution, with peaks in the third and seventh decades. Lymphadenopathy was present in nearly all patients. Extranodal involvement, including skin, soft tissue, bone, central nervous system, lung, or small intestine was observed in a total of 54% of the patients, either at presentation or during the course of disease. "Prototypic" features of large cell anaplastic lymphomas were observed for eight T-cell lymphomas, with morphologic heterogeneity noted for the remainder. Eight patients, all with T-cell neoplasms (only one with prototypic morphology), have died of lymphoma (median survival, 5 months). An antecedent history of a lymphoproliferative disorder (mycosis fungoides, B-cell lymphoma, immunoblastic lymphadenopathy) was apparent in seven patients. An 8-year history of Crohn's disease occurred in one patient with a T-cell lymphoma involving small intestine. Phenotypically, loss of one or more markers was typically noted for T-cell neoplasms. Leukocyte common antigen was detected in all cases, although partial loss of immunoreactivity was noticed in some cases. Nearly all cases evaluated for Ia antigen or alpha-1-antichymotrysin were reactive. Eleven of 16 T-cell, two of six B-cell, and two null cell lymphomas expressed epithelial membrane antigen. Ki-1-positive large cell lymphomas are characterized by clinical, morphologic, and immunophenotypic heterogeneity.