Penny Wright

High resolution melting analysis for detection of BRAF exon 15 mutations in hairy cell leukaemia and other lymphoid malignancies

The BRAF V600E mutation has recently been described in all cases of hairy cell leukaemia (HCL). We have developed and validated a rapid and sensitive high‐resolution melting analysis (HRMA) assay that detects BRAF exon 15 mutations when hairy cells are as low as 5–10% in a sample. All 48 HCL patients were positive for the BRAF V600E mutation, while 114 non‐HCL cases were all V600E negative. Interestingly, we detected a novel BRAF D594N mutation in one patient with multiple myeloma. The HRMA assay offers a useful tool to aid the laboratory diagnosis of HCL.

BIOMED‐2 PCR assays for IGK gene rearrangements are essential for B‐cell clonality analysis in follicular lymphoma

B‐cell clonality analysis is commonly performed by polymerase chain reaction (PCR) targeting the IGH genes although a high false‐negative rate is recognized for germinal centre/post‐germinal centre B‐cell malignancies, especially follicular lymphoma. We assessed the diagnostic value of BIOMED‐2 IGK assays and investigated the cause of IGH PCR failure in 77 patients with follicular lymphoma. Using the full set of BIOMED‐2 reactions, clonal immunoglobulin gene rearrangements were detected in 74 (96%) cases. The clonality detection rate was 86% by two IGK reactions but only 68% by five IGH reactions (Pu2003<u20030·001). Sequencing of the clonal PCR products showed significantly fewer somatic mutations in the rearranged IGKV (9/27 cases, 33%, mean mutation rate 0·5%) than IGHV (17/17 cases, 100%, rate 11·0%) (Pu2003<u20030·01). All IGHV‐IGHJ PCR failures occurred in cases with at least one mutation at the corresponding IGHV primer binding sites. t(14:18)(q32:q21)/IGH‐BCL2 was detected in 50 of 71 (70%) cases and the presence of the translocation was not associated with the poor performance of IGH assays. Our results showed that BIOMED‐2 IGK assays are significantly more sensitive than IGH assays in follicular lymphoma due to the fact that the rearranged IGKV is less frequently targeted by somatic hypermutation than IGHV, and therefore, are essential in routine clonality analysis of these lymphomas.

British Lung Foundation/United Kingdom Primary Immunodeficiency Network Consensus Statement on the Definition, Diagnosis, and Management of Granulomatous-Lymphocytic Interstitial Lung Disease in Common Variable Immunodeficiency Disorders

A proportion of people living with common variable immunodeficiency disorders develop granulomatous-lymphocytic interstitial lung disease (GLILD). We aimed to develop a consensus statement on the definition, diagnosis, and management of GLILD. All UK specialist centers were contacted and relevant physicians were invited to take part in a 3-round online Delphi process. Responses were graded as Strongly Agree, Tend to Agree, Neither Agree nor Disagree, Tend to Disagree, and Strongly Disagree, scoredxa0+1,xa0+0.5, 0,xa0-0.5, andxa0-1, respectively. Agreement was defined as greater than or equal to 80% consensus. Scores are reported as mean ± SD. There was 100% agreement (score, 0.92 ± 0.19) for the following definition: GLILD is a distinct clinico-radio-pathological ILD occurring in patients with [common variable immunodeficiency disorders], associated with a lymphocytic infiltrate and/or granuloma in the lung, and in whom other conditions have been considered and where possible excluded. There was consensus that the workup of suspected GLILD requires chest computed tomography (CT) (0.98 ± 0.01), lung function tests (eg, gas transfer, 0.94 ± 0.17), bronchoscopy to exclude infection (0.63 ± 0.50), and lung biopsy (0.58 ± 0.40). There was no consensus on whether expectant management following optimization of immunoglobulin therapy was acceptable: 67% agreed, 25% disagreed, score 0.38 ± 0.59; 90% agreed that when treatment was required, first-line treatment should be with corticosteroids alone (score, 0.55 ± 0.51).

The prognosis of MYC translocation positive diffuse large B‐cell lymphoma depends on the second hit

A proportion of MYC translocation positive diffuse large B‐cell lymphomas (DLBCL) harbour a BCL2 and/or BCL6 translocation, known as double‐hit DLBCL, and are clinically aggressive. It is unknown whether there are other genetic abnormalities that cooperate with MYC translocation and form double‐hit DLBCL, and whether there is a difference in clinical outcome between the double‐hit DLBCL and those with an isolated MYC translocation. We investigated TP53 gene mutations along with BCL2 and BCL6 translocations in a total of 234 cases of DLBCL, including 81 with MYC translocation. TP53 mutations were investigated by PCR and sequencing, while BCL2 and BCL6 translocation was studied by interphase fluorescence in situ hybridization. The majority of MYC translocation positive DLBCLs (60/81u2009=u200974%) had at least one additional genetic hit. In MYC translocation positive DLBCL treated by R‐CHOP (nu2009=u200967), TP53 mutation and BCL2, but not BCL6 translocation had an adverse effect on patient overall survival. In comparison with DLBCL with an isolated MYC translocation, cases with MYC/TP53 double‐hits had the worst overall survival, followed by those with MYC/BCL2 double‐hits. In MYC translocation negative DLBCL treated by R‐CHOP (nu2009=u2009101), TP53 mutation, BCL2 and BCL6 translocation had no impact on patient survival. The prognosis of MYC translocation positive DLBCL critically depends on the second hit, with TP53 mutations and BCL2 translocation contributing to an adverse prognosis. It is pivotal to investigate both TP53 mutations and BCL2 translocations in MYC translocation positive DLBCL, and to distinguish double‐hit DLBCLs from those with an isolated MYC translocation.

Clonal antigen receptor gene PCR products outside the expected size range

Polymerase chain reaction (PCR) of the antigen receptor genes has clinical utility in establishing clonality in lymphoproliferations, which is an important correlate of lymphoid neoplasia. The most frequently used procedures for this purpose were developed by the BIOMED-2 consortium. One of the criteria for establishing monoclonality using PCR of the antigen receptor genes is the finding of an abundant amplicon within a size range determined by the positions of the PCR primers and the known variability in size inherent in the recombination events that assemble a functional antigen receptor gene. However, several cases have been reported in which an amplicon outside this size range has been shown to be a valid indicator of clonality after DNA sequence analysis. In this paper, we will report and discuss several additional cases in which an amplicon outside the accepted size range was consistent with a monoclonal lymphoproliferation. We conclude that oversized and undersized amplicons may indeed represent evidence for a monoclonal lymphoproliferation, but that this interpretation should preferably be confirmed by sequence analysis to avoid a false-positive result.

Early loss of Crebbp confers malignant stem cell properties on lymphoid progenitors.

Loss-of-function mutations of cyclic-AMP response element binding protein, binding protein (CREBBP) are prevalent in lymphoid malignancies. However, the tumour suppressor functions of CREBBP remain unclear. We demonstrate that loss of Crebbp in murine haematopoietic stem and progenitor cells (HSPCs) leads to increased development of B-cell lymphomas. This is preceded by accumulation of hyperproliferative lymphoid progenitors with a defective DNA damage response (DDR) due to a failure to acetylate p53. We identify a premalignant lymphoma stem cell population with decreased H3K27ac, which undergoes transcriptional and genetic evolution due to the altered DDR, resulting in lymphomagenesis. Importantly, when Crebbp is lost later in lymphopoiesis, cellular abnormalities are lost and tumour generation is attenuated. We also document that CREBBP mutations may occur in HSPCs from patients with CREBBP-mutated lymphoma. These data suggest that earlier loss of Crebbp is advantageous for lymphoid transformation and inform the cellular origins and subsequent evolution of lymphoid malignancies.

Richter transformation: clonal identity does not indicate a linear disease progression.

Approximately, 2–8% of patients with chronic lymphocytic leukaemia (CLL)/small lymphocytic lymphoma (SLL) develop diffuse large B-cell lymphoma (DLBCL), known as Richter transformation. Of these patients, 80% of DLBCL are clonally related to the CLL, and the majority of these lymphomas carry unmutated IG genes. The remaining 20% of DLBCL are not related to the CLL, and the majority of these CLL harbour mutated IG genes (Timar et al, 2004; Smit et al, 2006; Mao et al, 2007; Scandurra et al, 2010). Demonstration of identical IG gene rearrangements has been used as a proof of clonal evolution from CLL to DLBCL. However, such identical clonal relationship does not necessarily indicate a linear disease progression. Case 1 was a 75-year-old man who presented with a lump in his left axilla for 5 weeks without any systemic symptoms. Chest and abdominal computerized tomography (CT) scan showed extensive lymphadenopathy in his left axilla. Full blood count and lactate dehydrogenase (LDH) were normal. Two lymph nodes were removed. Haematoxylin and eosin (H&E) stained slides showed a total effacement of the nodal architecture by a diffuse infiltrate of relatively monomorphic, medium to large-sized lymphocytes (Fig 1A). The lymphoma cells displayed rounded nuclear outlines, stippled chromatin and occasional small nucleoli, and expressed CD20, CD79a, CD10, BCL6 and BCL2, but not CD5, CCND1 and TdT (Fig 1A). MIB1 was positive in 95% of cells. Interphase fluorescence in situ hybridization (FISH) with Vysis breakapart probes showed chromosome translocation involving the MYC, BCL2 and IGH loci and gain of an extra BCL6 copy (Fig 1A). Further FISH with the MYC/IGH dual-fusion probe demonstrated that the MYC translocation was not associated with the IGH locus, while polymerase chain reaction (PCR) confirmed fusion between the BCL2 and IGH joining region (Fig 1A). Based on the above findings, DLBCL was diagnosed. H&E slides of the bone marrow biopsy showed a prominent, non-paratrabecular infiltrate of small lymphocytes (Fig 1B), which expressed CD20, CD79a, BCL2, CD5 and CD23, but not CD10 and CCND1. MIB1 was positive in <10% of the lymphoid cells. There was no evidence of abnormality in myeloid or erythroid maturation. The above features supported a diagnosis of SLL. Interphase FISH showed no evidence of 17p13/TP53 deletion and trisomy 12. BIOMED-2 IGHV FR3-IGHJ and IGKV-IGKJ PCR showed identical clonal patterns between the DLBCL and CLL (Fig 1B). Further cloning and sequencing of the FR3-IGHJ PCR products demonstrated an identical CDR3 sequence, indicating clonal identity between the two lymphomas. Interphase FISH on the bone marrow biopsy showed that CLL was negative for MYC and BCL2 translocations. TP53 inactivation is frequently associated with Richter transformation (Rossi & Gaidano, 2009). TP53 immunohistochemistry showed nuclear staining in DLBCL, but not SLL, cells. PCR and sequencing of TP53 exons 5–10 identified a point mutation (c.904 A>G; p.Y236C) in the DLBCL but not in the CLL. A review of the H&E and immunohistochemical slides of the lymph nodes revealed several small areas in one of the nodes, where CD20 was diffusely positive, but MIB1 and TP53 positivity only in scattered large cells (Fig 2). The majority of the cells in these areas were small B-cells expressing CD5 and CD23 (Fig 2), indicating nodal involvement by SLL. The patient was given three cycles of R-CHOP (rituximabcyclophosphamide, doxorubicin, vincristine, and prednisone) and a follow up CT scan showed no evidence of lymphoma. He was then further treated with local radiotherapy (a total of 30 Gy in 15 fractions), and remains in complete remission 10 months post-chemotherapy. Case 2 was a 74-year-old man with a 12-year history of stable stage-A CLL who presented with diminished hearing and excess lacrimation, and subsequently developed a right VIth nerve palsy. Systemic symptoms were absent. Full blood count showed WBC 30Æ4 · 10/l, Hb 129 g/l, platelets 284 · 10/l, neutrophils 5Æ71 · 10/l, and lymphocytes 23Æ8 · 10/l. LDH was normal. A head CT scan revealed a mass in the right nasal cavity invading the medial right orbit. H&E slides of the mass biopsy showed a diffuse infiltrate of pleomorphic large lymphoid cells with prominent nucleoli, which expressed CD20, CD10, BCL6, and MUM1, but not CD5, CD23 and TP53. MIB1 was positive in 50% of cells. A diagnosis of DLBCL was made. H&E slides of the bone marrow biopsy showed an extensive infiltrate of small lymphocytes, which expressed CD20, CD5, CD23 and BCL2, but not CCND1, CD10, BCL6 and TP53. Immunoglobulin light chain staining demonstrated kappa light chain restriction. There was no evidence of abnormality in the myeloid or erythroid lineages. CLL was diagnosed. IGKV-IGKJ PCR showed an identical clonal pattern between the DLBCL and CLL, indicating clonal identity between the two lymphomas. Interphase FISH with MYC, BCL2, BCL6 and IGH break apart probe displayed no abnormalities in both lymphomas. Staging CT revealed no disease elsewhere. The patient was treated with six cycles of R-CHOP, with central nervous system prophylaxis (intrathecal methotrexate). A positron emission Correspondence

Pitfalls in the Diagnosis of Anaplastic Large Cell Lymphoma with a Small Cell Pattern

Anaplastic large cell lymphoma with a small cell pattern is a rare T-cell lymphoma. This condition is more frequently seen in younger patients and should be considered when patients present with leucocytosis and constitutional symptoms. In this report, we describe our diagnostic work-up for one such case using blood, lymph node, and bone marrow aspirate samples, highlighting the variability of antigen expression seen in different sample types and methodologies. This case shows the importance of having a high index of suspicion and assessing CD30 and anaplastic lymphoma kinase expression in all suspected T-cell neoplasms even though this rare condition is not necessarily expected.

The aberrant intraepithelial T cells in refractory coeliac disease show epidermotropism.

Coeliac disease is a gluten-mediated autoimmune disorder that occurs in genetically predisposed individuals carrying the HLA-DQ2 and/or HLA-DQ8 alleles. Most patients with coeliac disease can be effectively managed by a strict gluten-free diet. Approximately, 5% of adult patients with coeliac disease lose their clinical and histological response to a strict gluten-free diet and develop refractory coeliac disease (RCD). Based on the clonal and immunophenotypical features of intraepithelial lymphocytes (IELs), RCD is divided into two subtypes. In RCD-I, IELs are polyclonal and show a normal phenotype, while in RCD-II, IELs are monoclonal and immunophenotypically aberrant, expressing cytoplasmic CD3e but not surface CD3e, CD8 and T cell receptor (TCR) antigens.1–3 nnCoeliac disease mainly causes enteropathy of the small intestine, but can involve the entire gastrointestinal tract. Apart from the classic intestinal syndrome, patients with coeliac disease also show a wide spectrum of extraintestinal manifestations and 10–20% of patients with coeliac disease present with dermatitis herpetiformis. Here, we report a case of RCD with subsequent presentation of skin lesions and show that the neoplastic intraepithelial T cells of the gut also preferentially invade the epidermis.nnThe patient, a 47-year-old man, presented with a 2-year history of epigastric pain, occasional nausea and vomiting, intermittent light and bulky stool, and weight loss. Oesophagogastroduodenoscopy showed erosion and inflammation in the body and antrum of the stomach and proximal duodenum, and mild erythema in the distal duodenum. Campylobacter-like organism (CLO) test …

MRI Changes associated with Bone Marrow Reconversion can Mimic Infiltration with Multiple Myeloma

We describe a case of a fit 40-year-old who was referred for investigations to rule out Multiple Myeloma on the basis of an abnormal bone signal on MRI scanning. Haematological investigations including a bone marrow biopsy were normal and upon extended MRI re-scanning, bone changes were identified as those of marrow reconversion and attributed to his intensive exercise regime.