A HRM assay for identification of low level BRAF V600E and V600K mutations using the CADMA principle in FFPE specimens.

Melanoma patients with BRAF V600E and V600K mutations show complete or partial response to vemurafenib. Detection assays often scan for the common V600E mutation rather than the rare V600K variant, although this mutation can be found in a high proportion of melanoma patients in the South Pacific. Herein, we describe a BRAF high resolution melting (HRM) assay that can differentiate low level of V600E and V600K mutations using formalin fixed, paraffin embedded (FFPE) reference standards for assay validation. The assay is based on the competitive amplification of differentially melting amplicons (CADMA principle) and has a limit of detection of 0.8% mutant allele for V600K and 1.4% mutant allele for V600E. A differentiation between the two mutations based on the melting profile is possible even at low mutation level. Sixty FFPE specimens were scanned and mutations could be scored correctly as confirmed by castPCR. In summary, the developed HRM assay is suitable for detection of V600K and V600E mutations and proved to be reliable and cost effective in a diagnostic environment.

Identification of germinal centres in the lymph node of a patient with hyperimmunoglobulin M syndrome associated with congenital rubella.

BACKGROUND: The hyper immunoglobulin M syndrome (HIM) associated with congenital rubella infection (rHIM) is an extremely rare disorder, where patients have elevated serum IgM in association with reduced IgG and IgA. We have previously shown that in contrast to X-linked HIM (XHIM), a patient with well-characterised rHIM is able to express functional CD40 ligand, undergo immunoglobulin isotype switching and to generate memory B cells. Here we describe the ultrastructural features of an excised lymph node from this patient. METHODS: An inguinal lymph node was surgically removed and examined histologically as well as by immunohistochemistry. It was then stained with multiple fluorescent dyes to visualize the cellular interactions within the node. Flow cytometry was undertaken on a cellular suspension from the node. FINDINGS: Our patient has normal lymph node architecture by light microscopy. Immunohistochemistry studies showed the presence of scattered germinal centres. Polychromatic immunofluorescence staining showed disruption of the architecture with mostly abnormal germinal centres. A small number of relatively intact germinal centres were identified. Both IgM and IgG bearing cells were identified in germinal centres. INTERPRETATION: In contrast to XHIM where germinal centres are absent, the presence of small numbers of relatively normal germinal centres explain our previous identification of isotype switched memory B cells in rHIM.

IgG4-related sclerosing mastitis: description of a new member of the IgG4-related sclerosing diseases.

Immunoglobulin G (IgG)4-related sclerosing disease is a recently described syndrome characterized by mass-forming lesions in various organs due to dense lymphoplasmacytic infiltrates and stromal sclerosis, elevated serum IgG4 titer, increased tissue IgG4 plasma cells, and favorable clinical outcome. We describe 4 patients with IgG4-related sclerosing mastitis, which represents a new member of this family of diseases. All patients were female with a mean age of 47.5 years, presenting with painless masses in 1 or both breasts. One patient had concurrent IgG4-related lymphadenopathy, and another had eyelid swelling of undetermined cause. The serum IgG4 titer was elevated in 1 tested patient, and circulating autoantibodies were found in 3 tested patients. All patients were well with no recurrence after excision or biopsy of the mass. Histologically, the breast masses featured dense lymphoplasmacytic infiltrates, prominent stromal sclerosis and loss of breast lobules. Phlebitis was present in 1 case. IgG4 cells ranged from 272 to 495 per high-power field, constituting 49% to 85% of all IgG cells. IgG4 cells were scarce in 9 of 9 cases of lymphocytic mastitis and 6 of 7 cases of granulomatous mastitis studied as controls. In summary, IgG4-related sclerosing mastitis appears to be a distinctive form of mastitis, sometimes accompanied by other components of IgG4-related sclerosing disease, and shows a favorable clinical outcome.