Polymerase chain reaction-based clonality testing in tissue samples with reactive lymphoproliferations: usefulness and pitfalls. A report of the BIOMED-2 Concerted Action BMH4-CT98-3936.

Lymphoproliferations are generally diagnosed via histomorphology and immunohistochemistry. Although mostly conclusive, occasionally the differential diagnosis between reactive lesions and malignant lymphomas is difficult. In such cases molecular clonality studies of immunoglobulin (Ig)/T-cell receptor (TCR) rearrangements can be useful. Here we address the issue of clonality assessment in 106 histologically defined reactive lesions, using the standardized BIOMED-2 Ig/TCR multiplex polymerase chain reaction (PCR) heteroduplex and GeneScan assays. Samples were reviewed nationally, except 10% random cases and cases with clonal results selected for additional international panel review. In total 75% (79/106) only showed polyclonal Ig/TCR targets (type I), whereas another 15% (16/106) represent probably polyclonal cases, with weak Ig/TCR (oligo)clonality in an otherwise polyclonal background (type II). Interestingly, in 10% (11/106) clear monoclonal Ig/TCR products were observed (types III/IV), which prompted further pathological review. Clonal cases included two missed lymphomas in national review and nine cases that could be explained as diagnostically difficult cases or probable lymphomas upon additional review. Our data show that the BIOMED-2 Ig/TCR multiplex PCR assays are very helpful in confirming the polyclonal character in the vast majority of reactive lesions. However, clonality detection in a minority should lead to detailed pathological review, including close interaction between pathologist and molecular biologist.

Powerful strategy for polymerase chain reaction-based clonality assessment in T-cell malignancies Report of the BIOMED-2 Concerted Action BHM4 CT98-3936.

Polymerase chain reaction (PCR) assessment of clonal T-cell receptor (TCR) and immunoglobulin (Ig) gene rearrangements is an important diagnostic tool in mature T-cell neoplasms. However, lack of standardized primers and PCR protocols has hampered comparability of data in previous clonality studies. To obtain reference values for Ig/TCR rearrangement patterns, 19 European laboratories investigated 188 T-cell malignancies belonging to five World Health Organization-defined entities. The TCR/Ig spectrum of each sample was analyzed in duplicate in two different laboratories using the standardized BIOMED-2 PCR multiplex tubes accompanied by international pathology panel review. TCR clonality was detected in 99% (143/145) of all definite cases of T-cell prolymphocytic leukemia, T-cell large granular lymphocytic leukemia, peripheral T-cell lymphoma (unspecified) and angioimmunoblastic T-cell lymphoma (AILT), whereas nine of 43 anaplastic large cell lymphomas did not show clonal TCR rearrangements. Combined use of TCRB and TCRG genes revealed two or more clonal signals in 95% of all TCR clonal cases. Ig clonality was mostly restricted to AILT. Our study indicates that the BIOMED-2 multiplex PCR tubes provide a powerful strategy for clonality assessment in T-cell malignancies assisting the firm diagnosis of T-cell neoplasms. The detected TCR gene rearrangements can also be used as PCR targets for monitoring of minimal residual disease.

Chemosensitization effects of XIAP downregulation in K562 leukemia cells.

The effect of downregulation of the expression of the antiapoptotic protein XIAP with antisense oligonucleotides was evaluated in the K562 chronic myeloid leukemia (CML) cell line. This was carried out by studying the effects of downregulation of XIAP expression on cellular viability, cellular apoptosis and on the response to two chemotherapeutical drugs, etoposide and doxorubicin. We document that downregulation of XIAP expression decreased cellular viability, increased cellular apoptosis and enhanced the effects of doxorubicin.

Limited synergistic effect of antisense oligonucleotides against bcr-abl and transferrin receptor mRNA in leukemic cells in culture.

The synergistic use of antisense oligonucleotides (ASOs) towards the bcr-abl and the transferrin receptor (TfR) mRNA was studied in a chronic myeloid leukemia (CML) cell line, aiming to improve the efficiency of individual ASO treatment. At 20 microM concentration, bcr-abl ASOs reduced cell growth by 40% and was specific for cells that have the translocation: there was a 34% reduction of BCR-ABL protein. The TfR ASO reduced cell growth by 20% and decreased TfR protein by 24%. The ASOs were more potent at reducing cell growth when used in combination (respectively, -20 and -17% than bcr-abl ASO and TfR ASO when used individually at the 10 microM concentration), thus we postulate that there is synergism of action. Cell cycle analysis also revealed that the sub-G1 peak was bigger in the synergistic treatment.

Solid cell nests of the thyroid: light microscopy and immunohistochemical profile.

The morphological, histochemical, and immunohistochemical findings of seven cases of solid cell nests (SCNs) of the thyroid are described. Light microscopy showed two cell types forming the SCNs, which we refer to as "main cells" and "C cells." In all cases "mixed thyroid follicles" (a unique structure lined by follicular epithelium and epidermoidlike cells) were observed in which the histochemical study confirmed the presence of intraluminal acid mucins. Adult adipose tissue and cartilage were found in one case and foci of cartilage were observed in another case in association with the SCN. Immunohistochemical studies showed positivity of "main cells" for carcinoembryonic antigen (CEA), high- and low-molecular weight keratins, neurotensin, and somatostatin. "C cells" were positive for calcitonin, calcitonin gene-related peptide (CGRP), and chromogranin. The two cell types in SCNs were consistently negative for thyroglobulin. Neuron-specific enolase (NSE)-positive cells were found in the vicinity of the SCN. The unusual association of adipose tissue and cartilage as well as the results of the extended immunohistochemical study in this series provides further support to the belief that SCNs and "mixed thyroid follicles" represent remnants of the ultimobranchial body and should be considered normal components of the thyroid gland.

Molecular pathology in the diagnosis of hematologic neoplasia. Review article.

Over the past decade molecular genetic methods have played an increasingly important role in the diagnosis of hematologic malignancies. Moreover, they have provided a tool to analyze many of the non-random cytogenetic anomalies associated with hematologic neoplasias, contributing considerably to our understanding of several of those diseases, and to improving diagnostic accuracy. The rapid development of molecular genetics progressively allows the replacement of time-consuming and technically demanding procedures. Even more relevant are the new clinical applications that already include the search for valuable prognostic information and ways of evaluating minimal residual disease or recognizing early relapsing disease. This paper is a critical but necessarily simplified overview of the main contributions of molecular genetics to the field of hematopathology. We discuss the information provided by several molecular methods within different clinical contexts, covering common problems in diagnostic pathology as well as prognostic evaluation and therapy monitoring.

Follicular and papillary variants of medullary carcinoma of the thyroid.

Two medullary carcinomas of the thyroid (MCT) with relatively unusual patterns are reported. The first was an aggressive tumour which occurred in a 66-year-old man and displayed in most areas follicular structures. The second tumour occurred in a 36-year-old woman, followed a benign course and showed papillary infoldings lined by multilayered neoplastic cells. The search for thyroglobulin yielded negative results whereas calcitonin immunoreactivity could be found in most neoplastic cells of both tumours. The diagnosis of MCT was further substantiated by the presence of scarce amyloid deposits and typical neuro-secretory granules by electron microscopy. These cases demonstrate once more that follicular and papillary structures can be a prominent feature of some MCTs reinforcing therefore the major role of immunocytochemistry in the differential diagnosis of thyroid carcinomas. Papillary MCT seems to carry a good prognosis in contrast to follicular MCT if one takes into account the follow-up data of the present cases together with those of similar cases reported in the literature; the whole series is nevertheless too small to allow for definite conclusions on this matter.

Lectin histochemistry and ultrastructure of medullary carcinoma of the thyroid gland.

Lectin histochemistry and electron microscopy were used to study a series of 13 solid or microacinar medullary carcinomas of the thyroid (MCTs) and compare them with four follicular MCTs and other forms of thyroid cancer. Lectin histochemistry was not found to be of diagnostic value, since the MCT did not display any distinct lectin-binding pattern. This approach demonstrated that all MCTs, irrespective of their histologic appearance, consist of polarized cells, arranged into microfollicles that can be demonstrated readily by electron microscopy. We conclude that all MCTs form follicles, some visible by light microscopy, while others are submicroscopic and apparent only on histochemical staining. Because of the histochemical and ultrastructural similarities between solid MCTs and tumors with a follicular pattern, the latter should not be considered a distinct variant of C-cell neoplasia.

Primary lymphomas of the thyroid gland: a review with emphasis on diagnostic features.

The information about primary lymphomas of the thyroid--a rare form of thyroid tumor and a rare form of lymphoma as well--is dispersed in numerous scientific publications. Recent developments in the field of lymphoid neoplasias, in particular the recognition of mucosa-associated lymphoid tissue (MALT) and the characterization of its distinctive type of lymphomas, has drawn new attention to primary lymphomas of the thyroid and has provided new insights into this field of thyroid pathology. In this paper we review the pathogenesis and the clinicopathologic features of primary lymphomas of the thyroid, we discuss the diagnostic basis and the strategies for differential diagnosis, and we address some recent data that may provide additional pathways to further our understanding of those rare thyroid diseases.

[Implications of electron microscopy and immunohistochemistry data in the concept of mixed tumors of the thyroid]. / Implications des données de la microscopie électronique et de l'immunohistochimie dans le concept des tumeurs "mixtes" de la thyroïde.

The role played by the ultimobranchial body in the histogenesis of thyroid tumors is discussed in the light of the results of studies of several types of thyroid carcinomas: poorly differentiated intermediate carcinomas, medullary carcinomas with thyroglobulin immunoreactivity, and mucoepidermoid carcinomas with "follicular" and "parafollicular" differentiation.

Medullary thyroid carcinoma with thyroglobulin immunoreactivity. A special entity?

Fourteen medullary carcinomas of the thyroid (MCT) immunoreactive for both thyroglobulin and calcitonin were studied by light microscopy and immunohistochemistry. Thyroglobulin immunoreactivity was seen in neoplastic follicles and/or in solid foci in the lymph node metastases of two cases. Colocalization of thyroglobulin and calcitonin was found in the same neoplastic cells of eight cases using a double immunostaining method; in three of these (including one with metastases), thyroglobulin was found to be colocalized with calcitonin gene related peptide as well. Our histological and immunohistochemical results support the assumption that MCT with thyroglobulin immunoreactivity is an unusual variant of the multihormone producing MCT and strengthen the hypothesis that a common stem cell is the origin of these tumors. The available clinical data suggest that thyroglobulin-positive MCT carry a better prognosis than thyroglobulin-negative MCT.

Solitary fibrous tumour of the thyroid: clinicopathological, immunohistochemical and ultrastructural study of three cases.

We describe three cases of solitary fibrous tumour (SFT) arising from thyroid stroma. Grossly, the tumours were clearly delimited but only partly encapsulated. The following histomorphological growth patterns were observed: bundles of cells in storiform configuration; non-structured bundles; prevalence of fibrous matrix; highly cellular, non-structured; prevalence of loose, non-structured extracellular substance; cellular proliferation and vascular spaces in a haemangiopericytic configuration and a lipomatous component. Immunohistochemical investigation demonstrated intense, diffuse vimentin positivity and focal, less intense actin positivity in all three cases. At electron microscopy we observed a primitive cell of mesenchymal type, with cytoplasm poor in organelles and rich in filaments; this cell sometimes presented differentiation characteristics. SFT is at present the most correct term for the lesions presented here despite some morphological characteristics which differ from cases reported in the literature.

The effects on growth and survival of IL-6 can be dissociated in the U-266-1970/U-266-1984 and HL407E/HL407L human multiple myeloma cell lines.

Several studies have documented IL-6-dependent growth promotion of murine and human neoplastic plasma cells. However, it is well known that human multiple myeloma (MM) cells in vitro show a considerable degree of heterogeneity concerning growth and survival requirements. This heterogeneity, which probably reflects overlapping effects of feeder cells, interleukin 6 (IL-6) and components of fetal calf serum (FCS) as well as tumour heterogeneity in vivo, has hampered the elucidation of molecular mechanisms underlying the effects of IL-6. In an attempt to dissociate growth and survival promotion of IL-6, we have studied two pairs of human MM cell lines, HL407E/HL407L and U-266-1970/U-266-1984, selected to represent different stages of in vitro tumour progression and dependence of feeder cells and exogenous IL-6. We demonstrated that exogenous IL-6, in the presence of FCS, conveyed: (a) a strong growth stimulatory effect with weak or no survival promotion in HL407L and U-266-1970 cells; (b) promotion of survival with no effects on growth in HL407E cells; (c) no growth or survival promotion to U-266-1984. Moreover, our results suggested that IL-6 may enhance apoptosis in U-266-1970/U-266-1984 cells, and that FCS may interfere with IL-6 in its growth stimulatory effect. The relative dissociation of growth, survival and apoptotic effects of IL-6 leads to the conclusion that the HL407E/HL407L and U-266-1970/U-266-1984 pairs of cell lines provide a useful human model system to study molecular mechanisms underlying these separate events.

A novel human B-cell line (U-2904) bearing t(8;14) and t(14;18) translocations.

Sequential activation of bcl-2 and c-myc appears to be involved in the pathogenesis of rare de novo acute lymphoid leukemias bearing both t(8;14) and t(14;18). Acquisition of t(8;14) by follicular-lymphoma cells with t(14;18) has also been related to the clinical transformation into an overt acute lymphoid leukemia in rare cases reported, and a role for c-myc involvement in the progression of some follicular lymphomas with t(14;18) has been suggested by the detection of c-myc rearrangements in association with histologic transformation. However, c-myc abnormalities different from those observed in Burkitt's lymphoma have been reported in diffuse large-cell lymphomas with breakpoints in 8q24, and a t(8;14) molecularly different from the classical one has been found in follicular lymphomas without t(14;18). We report the preliminary characterization of the EBV-negative cell line U 2904 established from a transformed follicular lymphoma that carries both t(8;14) (q24;q32) and t(14;18) (q32;q21) translocations. U 2904 cells have a mature B-cell phenotype, grow in agarose and are tumorigenic in nude mice. Rearrangements of both c-myc and bcl-2 confirmed the involvement of both oncogenes in the translocations which lead to abundant c-myc and bcl-2 transcripts. Two JH rearrangements and one C alpha rearrangement were observed which did not co-migrate with either c-myc or bcl-2 rearrangements. This is the first report of a cell line bearing both t(8;14) and t(14;18) derived from a follicular lymphoma after documented transformation into a centroblastic lymphoma without leukemic features. U 2904 provides further evidence for the involvement of c-myc in the progression of follicular lymphomas.