Automated prognostic pattern detection shows favourable diffuse pattern of FOXP3(+) Tregs in follicular lymphoma.

BACKGROUND: Histopathological prognostication relies on morphological pattern recognition, but as numbers of biomarkers increase, human prognostic pattern-recognition ability decreases. Follicular lymphoma (FL) has a variable outcome, partly determined by FOXP3 Tregs. We have developed an automated method, hypothesised interaction distribution (HID) analysis, to analyse spatial patterns of multiple biomarkers which we have applied to tumour-infiltrating lymphocytes in FL. METHODS: A tissue microarray of 40 patient samples was used in triplex immunohistochemistry for FOXP3, CD3 and CD69, and multispectral imaging used to determine the numbers and locations of CD3(+), FOXP3/CD3(+) and CD69/CD3(+) T cells. HID analysis was used to identify associations between cellular pattern and outcome. RESULTS: Higher numbers of CD3(+) (P=0.0001), FOXP3/CD3(+) (P=0.0031) and CD69/CD3(+) (P=0.0006) cells were favourable. Cross-validated HID analysis of cell pattern identified patient subgroups with statistically significantly different survival (35.5 vs 142 months, P=0.00255), a more diffuse pattern associated with favourable outcome and an aggregated pattern with unfavourable outcome. CONCLUSIONS: A diffuse pattern of FOXP3 and CD69 positivity was favourable, demonstrating ability of HID analysis to automatically identify prognostic cellular patterns. It is applicable to large numbers of biomarkers, representing an unsupervised, automated method for identification of undiscovered prognostic cellular patterns in cancer tissue samples.

Presence of monoclonal T-cell populations in B-cell post-transplant lymphoproliferative disorders.

As has been previously shown, the lack of immune surveillance plays a major role in the unchecked proliferation of Epstein-Barr virus (EBV)-infected B cells in the pathogenesis of B-cell post-transplant lymphoproliferative disorders. We hypothesised that the lack of immune surveillance should possibly also affect T cells, and this should lead to subsequent emergence of T-cell clones. The presence of both B- and T-cell clones in post-transplant lymphoproliferative disorders samples has rarely been demonstrated in the past. We systematically evaluated 26 B-cell post-transplant lymphoproliferative disorder, 23 human immune deficiency virus-associated B-cell lymphoma and 10 immune-competent diffuse large B-cell lymphoma samples for B- and T-cell clonality (polymerase chain reaction and heteroduplex analysis using BIOMED-2 protocol), T-cell subsets (immunohistochemistry) and EBV association (in situ hybridisation using EBER). One-half of B-cell post-transplant lymphoproliferative disorders showed evidence of monoclonal T-cell expansion, and among the T cells present in the tissue samples, CD8-positive cells predominated. Although 9/13 (69%) B-cell post-transplant lymphoproliferative disorders with the presence of monoclonal T-cell population had a CD4:CD8 ratio of ≤0.4, 0/13 of the cases without monoclonal T-cell expansion had a ratio ≤0.4 (P = 0.002). Only 2/26 (8%) demonstrated significant cytological atypia in the CD3/CD8-positive cells. There was no association between EBV and presence of T-cell clones. T-cell clones were not identified in lymphomas other than B-cell post-transplant lymphoproliferative disorders. Among 53.8% cases of EBV-positive B-cell post-transplant lymphoproliferative disorders with associated clonal expansion of T-cells tested, none had EBV-positive T cells. We conclude that half of B-cell post-transplant lymphoproliferative disorders are associated with clonal expansion of CD8-positive T cells, most of which do not amount to the coexistence of a T-cell post-transplant lymphoproliferative disorders.

E-Cadherin Expression in Blastic Plasmacytoid Dendritic Cell Neoplasms: An Unrecognized Finding and Potential Diagnostic Pitfall.

E-cadherin is expressed in hematopoietic erythroid precursors, but to our knowledge, its expression in blastic plasmacytoid dendritic cell neoplasm (BPDCN) has not been described. We report a case of BPDCN showing strong expression of E-cadherin, arising in a patient with history of primary myelofibrosis. Four more cases of BPDCN tested all showed strong expression of E-cadherin. Lack of awareness of this pattern of expression may lead to erroneous diagnosis of acute erythroid leukemia. It is increasingly becoming important to correctly identify this group of neoplasms, as approved new anti-CD123-targeted therapies are becoming available.

Clinical quantitation of immune signature in follicular lymphoma by RT-PCR-based gene expression profiling.

Microarray gene expression profiling studies have demonstrated immune response gene signatures that appear predictive of outcome in follicular lymphoma (FL). However, measurement of these marker genes in routine practice remains difficult. We have therefore investigated the immune response in FL using real-time polymerase chain reaction (PCR) to measure expression levels of 35 candidate Indicator genes, selected from microarray studies, to polyA cDNAs prepared from 60 archived human frozen lymph nodes, in parallel with immunohistochemical analysis for CD3, CD4, CD7, CD8, CD10, CD20, CD21, and CD68. High levels of CCR1, a marker of monocyte activation, were associated with a shorter survival interval, and high levels of CD3 with better survival, while immunohistochemistry demonstrated association of high numbers of CD68(+) macrophages with a shorter survival interval and of high numbers of CD7(+) T cells with a longer survival interval. The results confirm the role of the host immune response in outcome in FL and identify CCR1 as a prognostic indicator and marker of an immune switch between macrophages and a T cell-dominant response. They demonstrate the utility of polyA DNA and real-time PCR for measurement of gene signatures and the applicability of using this type of "molecular block" in clinical practice.

Epstein-Barr virus (EBV) genotypes among human immunodeficiency virus (HIV)-related B-cell lymphomas and B-cell post-transplant lymphoproliferative disorders (B-PTLD)--late-onset lymphomas, especially in the HIV setting, are associated with type-B-EBV.

We investigated 26 B-cell post-transplant lymphoproliferative disorders (B-PTLD) and 15 human immunodeficiency virus-related aggressive B-cell lymphomas (HIV-BCL) from England that were associated with Epstein-Barr virus (EBV) for the polymorphic sequences of the EBV-encoded nuclear antigen 3C (EBNA3C) gene to distinguish the two different EBV strains. Type-A-EBV was identified in 92% of B-PTLDS and in 53% of HIV-BCL (P = 0.003). Among HIV-BCL, patients associated with type-B-EBV had been HIV positive for significantly longer when compared to those associated with type-A (P = 0.037) although there were no correlations with ethnicity, CD4 cell counts or plasma HIV viral load.

Alveolar rhabdomyosarcoma with neuroendocrine/neuronal differentiation: report of 3 cases.

The aim of this study is to report the clinicopathologic characteristics of 3 cases of alveolar rhabdomyosarcoma with neuroendocrine/neuronal differentiation. Specimens of 3 cases of alveolar rhabdomyosarcoma were studied using histologic, immunohistochemical, ultrastructural, and molecular genetic techniques. The patients were a 19-year-old man with metastatic alveolar rhabdomyosarcoma in a groin lymph node, a 16-year-old girl with alveolar rhabdomyosarcoma of the perineum, and a 20-year-old man with recurrent orbital alveolar rhabdomyosarcoma. Microscopically, case 1 was composed of compact sheets of medium to large tumor cells. Cases 2 and 3 were small blue round cell tumors. Cases 1 and 3 were solid throughout, whereas case 2 demonstrated alveolar and solid architecture. By immunohistochemistry, the following markers were positive: desmin (3/3), myogenin (3/3), synaptophysin (3/3), and chromogranin (2/3). Ultrastructurally, sarcomeric filaments were seen in all cases, while neuroendocrine granules were detected only in case 1. PAX:FKHR fusion transcript was identified in case 2, case 3 had a variant PAX3 transcript, and case 1 was negative. The data presented expands the known differentiation of alveolar rhabdomyosarcoma.

Ultrastructurally confirmed myofibrosarcoma: a series of 10 new cases, with a discussion on diagnostic criteria.

Some view ultrastructure as key to myofibrosarcoma diagnosis, whereas others argue that electron microscopy is too little used in contemporary practice to be considered an important diagnostic tool. These views are discussed in the context of 10 ultrastructurally confirmed cases of myofibrosarcoma, some occurring at rare sites such as skin and penis. Patient age ranged from 21 to 83 years, with a 6:4 male to female ratio. Size ranged from 2 to 7.5 cm and all had infiltrative margins. Histologically, all consisted of variably cellular fascicles of spindle cells with mild to moderately pleomorphic nuclei, small punctate nucleoli, and eosinophilic cytoplasm. All cases showed α-smooth muscle actin positivity and 2 showed very focal weak positivity for desmin. Ultrastructurally, the tumor cells contained rough endoplasmic reticulum, mainly peripheral smooth-muscle myofilaments, and fibronectin fibrils or fibronexus junctions at the cell surface. The most confident diagnosis of myofibrosarcoma is provided by ultrastructural examination. However, given the right histological appearance, use of a panel of antibodies that includes α-smooth muscle actin, desmin, and h-caldesmon, serves as an acceptable practical way of diagnosing myofibrosarcoma.

PLK1 and YY1 interaction in follicular lymphoma is associated with unfavourable outcome.

AIMS: Ying Yang 1 (YY1) is a transcription factor involved in both proliferation and apoptosis. It is prognostic in follicular lymphoma (FL), increased protein levels being associated with favourable outcome. PLK1 is a critical regulator of mitosis, playing a role in spindle formation and in regulation of the G2/M cell cycle checkpoint. PLK1 phosphorylates YY1 at the G2/M checkpoint with activation of YY1 and resultant progression from G2 into mitosis. METHODS: This study aims to investigate possible molecular coexpression and interaction of YY1 with PLK1 in FL using Duolink II in situ proximity ligation assay (PLA) in 51 FL samples in a tissue microarray. RESULTS: Positive PLA signals were present at variable frequency and Kaplan-Meier analysis showed association of signal frequency above the median with unfavourable outcome (p=0.0270). PLA signals were localised to the nuclear edge, with only one signal per cell, suggesting PLK1 and YY1 coexpression at the centrosome. In a minority of cells, two very close PLA signals were present in a single cell, and occasionally, there was a strong ring of semi-confluent fluorescent PLA signals round the nucleus of non-dividing cells, while rarely events were observed in the cytoplasm surrounding dividing cells. CONCLUSIONS: The results confirm association of YY1 and PLK1 with outcome in FL and suggest coexpression at the centrosome. Given the reported interaction of YY1 with PLK1 at the centriole and promotion of cell division at the G2/M checkpoint, the results would concord with the known association of higher proliferation with poor outcome in FL.

Cribriform-morular variant of papillary thyroid carcinoma: molecular characterization of a case with neuroendocrine differentiation and aggressive behavior.

We describe an especially aggressive case of cribriform-morular variant (C-MV) of papillary thyroid carcinoma (PTC) in a 42-year-old man with familial adenomatous polyposis who died with lung and brain metastases 17 months after thyroidectomy. The angioinvasive neoplasm combined a mixture of trabecular, solid, cribriform, and follicular patterns of growth with CD10+ morules. Follicles were devoid of colloid, and the nuclear features typical of PTC were present in some areas and missing in others. Tumor cells were positive for thyroid transcription factor-1 and, in 40% of the tumoral mass, also were positive for chromogranin and synaptophysin and were negative for thyroglobulin and calcitonin. Strong nuclear staining for beta-catenin was found in all tumor cells, as was positivity for p53 and cyclin D1. In addition to the germline heterozygous APC Ex 2-3 duplication mutation, a somatic homozygous silent p. Thr1493Thr gene variant was found in the neoplastic cells along with RET/PTC rearrangement. This tumor represents the first case of C-MV of PTC showing neuroendocrine differentiation.

Clinical quantitation of diagnostic and predictive gene expression levels in follicular and diffuse large B-cell lymphoma by RT-PCR gene expression profiling.

Recent microarray gene expression profiling studies have identified gene signatures predictive of outcome, so-called "indicator" genes, for diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). However, measurement of these genes in routine practice remains difficult. We applied real-time polymerase chain reaction (PCR) to polyA cDNAs prepared from 106 archived human frozen lymph nodes (63 of FL, 25 of DLBCL, 10 reactive lymph nodes, and cases with paired samples of FL [4] and subsequent DLBCL [4]). Reverse transcription and polyA reverse transcriptase (RT)-PCR was performed, and resultant cDNA was probed by real-time PCR for 36 candidate indicator genes, selected from microarray studies. Nine genes showed statistically significant different expression between FL and DLBCL, including cyclin B, COL3A1, NPM3, H731, PRKCB1, OVGL, ZFPC150, HLA-DQ-a, and XPB. Of these, cyclin B, NPM3, and COL3A1 were higher in DLBCL. Six genes showed statistically significant higher expression in the neoplastic nodes compared with reactive nodes, namely PRKCB1, BCL-6, EAR2, ZFX, cyclin B, YY1. High levels of YY.1 were associated with a shorter survival interval in both FL and DLBCL. The method is simple, sensitive, and robust, facilitating routine use and may be used as a platform for clinical measurement of prognostic gene signatures.

Transformed diffuse large B-cell lymphomas with gains of the discontinuous 12q12-14 amplicon display concurrent deregulation of CDK2, CDK4 and GADD153 genes.

Transformation of the indolent follicular lymphoma (FL) to the aggressive diffuse large B-cell lymphoma (DLBCL) results in resistance to therapy with shortened survival. It has been demonstrated that the 12q12-14 region was mainly amplified in DLBCL cases but not in their FL counterparts. Therefore, we examined the DNA copy number and protein expression profiles for CDK2, CDK4 and GADD153, three genes that map to 12q12-14, in a set of 44 paired FL/DLBCL samples from 22 patients. The concordant amplification of these genes occurred in seven of 22 (32%) of FL cases, compared with 15 of 22 (68%) of DLBCL cases. At the protein level, 15 of 22 of the DLBCL samples (68%) showed strong staining for the CDK2 protein, compared with five of 21 of FL samples (24%). The majority of the DLBCL samples (16/22, 72%) expressed the CDK4 protein, whereas the majority of the FL samples (12/21, 57%) showed no expression of this protein. Except for one DLBCL case, no expression of the GADD153 protein could be detected. The deregulation of the CDK2 and CDK4 genes at the genetic and protein levels suggest a functional role for these genes in the transformation process and could potentially provide targets for prognostic tests or therapeutic interventions.

Contribution of electron microscopy to understanding cellular differentiation in mesenchymal tumors of the gastrointestinal tract: a study of 82 tumors.

Eighty-two mesenchymal tumors of the gastrointestinal tract were examined by electron microscopy for the purposes of subtyping for diagnostic precision and of understanding cellular differentiation. Tumors were subclassified into leiomyoma/leiomyosarcoma, tumors of the interstitial cell of Cajal (equivalent to traditionally defined GISTs [Miettinen et al. Hum Pathol. 1999; 30:1213-1220; Mod Pathol. 2000; 13:1134-1142]), gastrointestinal autonomic nerve tumors (GANTs), and fibroblastic and myofibroblastic tumors, using criteria from the literature. Leiomyoma/leiomyosarcoma were diagnosed by myofilaments, attachment plaques, plasmalemmal caveolae, and lamina; GIST by processes or cell bodies full of intermediate filaments, solitary focal densities amid intermediate filaments, attachment plaques with incomplete lamina, scarce myofilaments, and smooth endoplasmic reticulum; GANTs by neuroendocrine granules, cell bodies/processes full of intermediate filaments (more rarely microtubules), and smooth endoplasmic reticulum; fibroblastic/myofibroblastic tumors by abundant rough endoplasmic reticulum, myofilaments, and fibronexuses. Seventy-three tumors (89%) were successfully subclassified, as 5 leiomyoma/leiomyosarcoma (6%), 36 GISTs (44%), 22 GANTs (27%), 10 fibroblastic and myofibroblastic tumors (12%). Results indicated overlap between poorly differentiated leiomyosarcoma and GIST, and between GIST and GANT. GANT is emphasized as a neuronal tumor identifiable by electron microscopy, and thereby distinguishable from GIST.