PCR-based tissue identification: the UCLH experience.

The need to accurately identify tissue of an individual can arise in a variety of settings including mislabelled slides or sample carryover. Reported rates of carryover range from 0.6% to 2.9% of slides depending on the methods of evaluation. Carryover becomes particularity clinically important when malignant tissue is found in an otherwise benign sample. The suspicion of malignancy causes immense psychological stress to the patient and results in additional management costs due to the additional investigations required to rule out malignancy. Proving a negative can be difficult and many cases result in lifelong follow-up for the patient. Molecular techniques such as PCR amplification of simple tandem repeat (STR) sequences can be used to identify tissue and hence its provenance. At University College London Hospital, STR PCR analysis has been used since 2003. Here the authors report their experience with regard to the clinical scenarios, the technique used and the outcomes.

GNAS1 mutations occur more commonly than previously thought in intramuscular myxoma.

Mutation detection plays an important role in diagnostic pathology, not only in providing a tissue diagnosis, but also in predicting response to antitumourigenic agents. However, mutation detection strategies are often hampered by masking of mutant alleles by wild-type sequences. Coamplification at lower denaturation temperature PCR (COLD-PCR) reportedly increases the proportion of rare variant sequences in a wild-type background by using PCR cycles in which the denaturation temperature is reduced to favour product formation with lower melt temperatures and heteroduplexes arising from minor variants. Intramuscular myxoma is a rare benign soft tissue neoplasm that occurs sporadically and less commonly in association with fibrous dysplasia (Mazabraud's syndrome). Fibrous dysplasia results from activating GNAS1 mutations, and the same mutations have been identified in small numbers of intramuscular myxoma. The aim of the study was primarily to establish whether COLD-PCR is more sensitive than conventional PCR; this was achieved by testing for GNAS1 mutations in intramuscular myxomas using the two methodologies. Mutations were detected in 8 of 28 (29%) cases of intramuscular myxomas using conventional PCR followed by mutation-specific restriction enzyme digestion (PCR-MSRED) whereas 17 of 28 (61%) mutations were detected using COLD-PCR/MSRED. Mutations were detected in two cases where a diagnosis of low-grade myxofibrosarcoma had been favoured over intramuscular myxoma. No mutations were detected in an additional 9 low-grade and 19 high-grade myxofibrosarcomas, and another 40 control samples. This study shows the power of COLD-PCR compared with conventional PCR in mutation detection, and shows that GNAS1 mutation detection increases diagnostic accuracy when distinguishing between intramuscular myxoma and low-grade myxofibrosarcoma.

IgV H mutations in blastoid mantle cell lymphoma characterize a subgroup with a tendency to more favourable clinical outcome.

Mantle cell lymphoma (MCL) is associated with a very unfavourable clinical course. This is particularly true for mantle cell lymphoma of the blastoid subtype (MCL-b). In order to define prognostic factors, we analysed the impact of immunoglobulin heavy chain variable (IgV H) gene somatic hypermutations on clinical outcome in a series of 21 cases of morphologically, phenotypically, and genotypically well-characterized MCL-b. Testing and estimation were performed using log-rank statistics and displayed on Kaplan-Meier graphs. Thirteen of 21 cases of MCL-b revealed a homology rate of > or = 99% compared to IgV H germ-line sequences in the databases and were scored as non-mutated. Eight of 21 cases (38%) of MCL-b were mutated. In MCL-b the mutation frequency was usually low and the mutation pattern was only rarely antigen-selected, in contrast to a control group of 11 cases with morphologically almost identical, but phenotypically and genotypically clearly distinguishable, diffuse large B cell lymphoma, derived, most likely, from germinal centre B cells. In our series of 21 MCL-b, positive IgV H mutational status, irrespective of varying homology thresholds, had no statistically significant prognostic impact on event-free or overall survival. However, mutated MCL-b tended to present more frequently at an earlier stage and without bone marrow involvement and to show lower rates of relapse and death, resulting in a more favourable clinical outcome.

Distinct cellular origins of primary effusion lymphoma with and without EBV infection.

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with three distinct lymphoproliferative disorders: primary effusion lymphoma (PEL), multicentric Castleman's disease (MCD) and germinotropic lymphoproliferative disorder (GLD). KSHV positive lymphocytes in GLD and in most cases of PEL are co-infected by Epstein-Barr virus (EBV) and these viral double positive cells harbour mutated rearranged immunoglobulin (Ig) genes, suggesting that they originate from germinal centre or post-germinal centre B-cells. In contrast, KSHV positive cells in MCD are invariably negative for EBV, do not carry Ig gene mutation and are believed to originate from naïve IgMlambda expressing B-cells. Interestingly, one EBV negative PEL (BC3) also lacks Ig gene mutation, raising the question whether KSHV preferentially targets naïve B-cells in the absence of EBV. We compared the cellular origin of PEL with and without EBV infection by analysis of Ig gene mutation. High molecular weight DNA from 17 PELs was subjected to PCR of the rearranged Ig heavy and light chain genes. Successful amplification was achieved from eight cases (four EBV positive and four EBV negative) and the PCR products were sequenced. All four EBV positive PEL showed variable levels of mutation in their rearranged V(H) or V(L) genes, ranging from 4 to 7%. In contrast, two of the four EBV negative PELs including BC3 displayed absence of mutation in their rearranged Ig genes. Our results indicate that EBV positive PELs are derived from germinal centre or post-germinal centre B-cells, whereas EBV negative PELs may originate from either germinal/post-germinal centre or naïve B-cells.

Phenotype and genotype of interfollicular large B cells, a subpopulation of lymphocytes often with dendritic morphology.

We describe large B cells, many of which have a stellate or dendritic morphology, found in the interfollicular T-cell-rich areas of human peripheral lymphoid tissue. These B cells probably require CD40/CD40 ligand signaling for their generation and have a unique phenotype and a post-germinal center pattern of immunoglobulin gene mutation. The only comparable B cell is the "asteroid" cell of the thymic medulla. Their anatomic location and pattern of gene mutation suggest that these cells may represent the cell of origin of some human large-cell lymphomas. Studies in experimental animals should help to elucidate the role of these cells in the immune response.

Gastric marginal zone lymphoma is associated with polymorphisms in genes involved in inflammatory response and antioxidative capacity.

Gastric marginal zone lymphoma (GMZL) is strongly associated with Helicobacter pylori infection, which induces a chronic inflammatory response. Inflammation can result in DNA damage related to its severity, the cellular antioxidant capacity, and the integrity of DNA repair mechanisms. Interleukin-1 (IL-1) polymorphisms have been shown to be important mediators of inflammation, while glutathione S-transferase GST T1 and GST M1 polymorphisms are believed to affect cellular antioxidant capacity. We aimed to determine whether polymorphisms at the IL-1 and GST T1 and GST M1 loci modulate the risk of developing GMZL. Blood and biopsy samples were obtained for a historical series of 66 GMZL cases, whereas blood samples were available from 163 healthy controls. Genotypes were obtained for GST T1, GST M1, IL-1 RN, and IL-1B-31 using PCR-based techniques. H pylori infection was found in 86.0% of cases, whereas in the control population only 37.4% tested positive. The IL-1 RN 2/2 genotype was significantly associated with risk of GMZL (odds ratio [OR], 5.51; 95% confidence interval [CI] 2.16-14.07), but not the IL-1B-31 genotype. Likewise, the GST T1 null genotype was strongly associated with risk of GMZL (OR, 9.51; 95% CI 4.57-19.81), but not the GST M1 genotype. Evidence was found of effect modification between the IL-1 RN and GST T1 genotypes (P =.02). The combination of the IL-1 RN 2/2 and GST T1 null genotype was most strongly associated with risk of GMZL (OR, 32.29; 95% CI 6.92-150-63). These results support the hypothesis that the risk of developing GMZL is influenced by inter-individual variation in the cellular inflammatory immune responses to H pylori infection, and to antioxidative capacity.

High frequency of t(11;18) in gastric mucosa-associated lymphoid tissue lymphomas in Taiwan, including one patient with high-grade transformation.

t(11;18)(q21;q21), the most frequent chromosomal aberration of mucosa-associated lymphoid tissue (MALT) lymphoma, occurs in 30% of gastric patients. Although the translocation is often associated with an 'aggressive' course, it has not been described in transformed MALT lymphomas. We screened 15 gastric MALT lymphomas [three with concurrent or subsequent high-grade transformation and 11 diffuse large B-cell lymphomas (DLBCLs)] in Chinese patients for t(11;18). t(11;18) was found in 9/15 (60%) MALT lymphomas, but not in any DLBCLs. One patient, with subsequent high-grade transformation, showed the translocation in low- and high-grade lesions. t(11;18) was frequent in Chinese gastric MALT lymphomas and unusually one transformed lymphoma carried the translocation.

KSHV- and EBV-associated germinotropic lymphoproliferative disorder.

Kaposi sarcoma-associated herpesvirus (KSHV) is known to be associated with 3 distinct lymphoproliferative disorders: primary effusion lymphoma (PEL), multicentric Castleman disease (MCD), and MCD-associated plasmablastic lymphoma. We report 3 cases of a previously undescribed KSHV-associated lymphoproliferative disorder. The disease presented as localized lymphadenopathy and showed a favorable response to chemotherapy or radiotherapy. Histologically, the lymphoproliferation is characterized by plasmablasts that preferentially involved germinal centers of the lymphoid follicles, forming confluent aggregates. They were negative for CD20, CD27, CD79a, CD138, BCL6, and CD10 but showed monotypic kappa or lambda light chain. Clusters of CD10(+)CD20(+) residual follicle center cells were identified in some of the follicles. The plasmablasts were positive for both KSHV and EBV, and most of them also expressed viral interleukin-6 (vIL-6). Unexpectedly, molecular analysis of whole tissue sections or microdissected KSHV-positive aggregates demonstrated a polyclonal or oligoclonal pattern of immunoglobulin (Ig) gene rearrangement. The plasmablasts showed somatic mutation and intraclonal variation in the rearranged Ig genes, and one case expressed switched Ig heavy chain (IgA), suggesting that they originated from germinal center B cells. We propose calling this distinctive entity "KSHV-associated germinotropic lymphoproliferative disorder."

High incidence of Kaposi sarcoma-associated herpesvirus-related non-Hodgkin lymphoma in patients with HIV infection and multicentric Castleman disease.

Multicentric Castleman disease (MCD) is a distinct type of lymphoproliferative disorder associated with inflammatory symptoms and interleukin 6 (IL-6) dysregulation. In the context of human immunodeficiency virus (HIV) infection, MCD is associated with Kaposi sarcoma-associated herpesvirus, also called human herpesvirus type 8 (KSHV/HHV8). Within a prospective cohort study on 60 HIV-infected patients with MCD, and a median follow-up period of 20 months, 14 patients developed KSHV/HHV8-associated non-Hodgkin lymphoma (NHL): 3 "classic" KSHV/HHV8(+) Epstein-Barr virus-positive (EBV(+)) primary effusion lymphoma (PEL), 5 KSHV/HHV8(+) EBV(-) visceral large cell NHL with a PEL-like phenotype, and 6 plasmablastic lymphoma/leukemia (3/3 KSHV/HHV8(+) EBV(-)). The NHL incidence observed in this cohort study (101/1000 patient-years) is about 15-fold what is expected in the general HIV(+) population. MCD-associated KSHV/HHV8(+) NHL fell into 2 groups, suggesting different pathogenesis. The plasmablastic NHL likely represents the expansion of plasmablastic microlymphoma from the MCD lesion and progression toward aggressive NHL. In contrast, the PEL and PEL-like NHL may implicate a different original infected cell whose growth is promoted by the cytokine-rich environment of the MCD lesions.

Neoplastic T cells in angioimmunoblastic T-cell lymphoma express CD10.

Angioimmunoblastic T-cell lymphoma (AITL) is a systemic disease involving lymph nodes, spleen, and bone marrow. Although the histologic features have been well described, the diagnosis is often challenging, as there are no specific phenotypic or molecular markers available. This study shows that the neoplastic cells of AITL can be identified by aberrant CD10 expression. Archival material from 30 cases of AITL, 10 cases of peripheral T-cell lymphoma unspecified (PTL), and 10 cases of reactive lymphoid hyperplasia were reviewed. Single and double immunostaining for CD3, CD4, CD8, CD20, CD21, CD10, BCL6, Ki67, and LMP-1 in situ hybridization for Epstein-Barr early region and polymerase chain reaction (PCR) for T-cell receptor gamma chain gene and immunoglobulin heavy chain gene were performed. Three overlapping histologic patterns with hyperplastic follicles, depleted follicles, or without follicles were identified in AITL. Of the 30 cases of AITL, 27 contained CD10(+) T cells. No CD10(+) T cells were present in the cases of PTL or reactive hyperplasia. PCR confirmed a monoclonal or oligoclonal T-cell population in 29 of 30 cases of AITL and a monoclonal B-cell population in 6 cases. Analysis of microdissected CD10(+) single cells showed that they belonged to the neoplastic clone. In conclusion CD10 is a phenotypic marker that specifically identifies the tumor cells in 90% of AITL, including the early cases. The presence of these cells distinguishes AITL from other PTLs. This finding provides an objective criterion for accurate and early diagnosis of AITL.