Konnie M. Hebeda

A Dominant-Negative GFI1B Mutation in the Gray Platelet Syndrome

The gray platelet syndrome is a hereditary, usually autosomal recessive bleeding disorder caused by a deficiency of alpha granules in platelets. We detected a nonsense mutation in the gene encoding the transcription factor GFI1B (growth factor independent 1B) that causes autosomal dominant gray platelet syndrome. Both gray platelets and megakaryocytes had abnormal marker expression. In addition, the megakaryocytes had dysplastic features, and they were abnormally distributed in the bone marrow. The GFI1B mutant protein inhibited nonmutant GFI1B transcriptional activity in a dominant-negative manner. Our studies show that GFI1B, in addition to being causally related to the gray platelet syndrome, is key to megakaryocyte and platelet development.

FISH analysis of MALT lymphoma-specific translocations and aneuploidy in primary cutaneous marginal zone lymphoma.

Primary cutaneous marginal zone lymphomas (PCMZL) share histological and clinical characteristics with mucosa‐associated lymphoid tissue (MALT) lymphomas suggesting a common pathogenesis. A number of recurrent structural and numerical chromosomal aberrations have been described in MALT lymphoma, but their incidence in PCMZL is largely unknown, as is their relation with clinical and pathological data. In this study, the incidence of t(11;18)(q21;q21), t(1;14)(p22;q32), two different t(14;18)(q32;q21), involving either IGH/MALT1 or IGH/BCL2, and numerical aberrations of chromosomes 3, 7, 12 and 18 were analysed in 12 patients with PCMZL, with follow‐up of up to 10 years. Nuclei were isolated from paraffin wax sections for dual‐colour interphase fluorescence in situ hybridization (FISH) using various probe sets either flanking or spanning the involved genes. T(14;18)(q32;q21), with breakpoints in IGH and MALT1, was found in three cases. All three had partly monocytoid histological appearances and lacked blastic transformation. An additional trisomy of chromosome 3 was detected in one of these cases. Trisomy 18 was present in two lymphomas without monocytoid morphology. No definite correlation was seen with any clinical feature, including Borrelia serology. Neither t(11;18)(q21;q21), nor t(1;14)(p22;q32) or any other translocation involving IGH, BCL10, MALT1, BCL2 and API2, amplification or deletion of chromosomal region 11q21, 18q21, 1p22, and 14q32 was detected. These results indicate that a subgroup of PCMZL with partly monocytoid morphology is genetically related to MZL at other extranodal sites. Copyright

In vivo targeting of DC-SIGN-positive antigen-presenting cells in a nonhuman primate model.

In vivo targeting of antigen-presenting cells (APCs) with antigens coupled to antibodies directed against APC-specific endocytic receptors is a simple and a promising approach to induce or modulate immune responses against those antigens. In a recent in vitro study, we have shown that targeting of APCs with an antigen coupled to an antibody directed against the endocytic receptor DC-SIGN effectively induces a specific immune response against that antigen. The aim of the present study was to determine the ability of the murine antihuman DC-SIGN antibody AZN-D1 to target APCs in a cynomolgus macaque model after its administration in vivo. Immunohistochemical analysis demonstrated that macaques injected intravenously with AZN-D1 have AZN-D1–targeted APCs in all lymph nodes (LNs) tested and in the liver. DC-SIGN–positive cells were mainly located in the medullary sinuses of the LNs and in the hepatic sinusoids in the liver. No unlabeled DC-SIGN molecules were found in the LN of AZN-D1–injected macaques. Morphologic criteria and staining of sequential LN sections with a panel of antibodies indicated that the DC-SIGN–targeted cells belong to the myeloid lineage of APCs. In conclusion, this is the first study that shows specific targeting of APCs in vivo by using antibodies directed against DC-SIGN.

Photodynamic Effectiveness and Vasoconstriction in Hairless Mouse Skin after Topical 5‐Aminolevulinic Acid and Single‐ or Two‐fold Illumination

Several options were investigated to increase the efficacy of photodynamic therapy (PDT) using protoporphyrin IX (PpIX) induced by topically applied 5‐aminolevulinic acid (ALA). Hairless mice with normal skin or UVB‐light‐induced skin changes were used as a model. In the first part of the study animals were illuminated immediately (t = 4) or 6 h (t = 10, PpIX fluorescence maximum) after the end of a 4 h ALA application. A total incident light fluence of 100 J/cm2 (514.5 nm) was delivered at a fluence rate of 100 or 50 mW/cm2. The PDT‐induced damage to normal skin was more severe after treatment at t = 10 than at t = 4. Illumination at 50 mW/ cm2 caused significantly more visible damage than the same light fluence given at 100 mW/cm2. For UVB‐illu‐minated skin, different intervals or fluence rates made no significant difference in the severity of damage, although some qualitative differences occurred. In situ fluence rate measurements during PDT indicated vasoconstriction almost immediately after the start of the illumination. A fluorescein exclusion assay after PDT demonstrated vasoconstriction that was more pronounced in UVB‐treated skin than in normal skin. The second part of the study examined the effect of two illuminations. The first illumination bleaches the PpIX fluorescence. At the start of the second illumination, new PpIX had been formed. Light of 514.5 nm was delivered at 100 mW/cm2 to a total incident light fluence of 200 J/cm2 at t = 4 (single illumination) or 100 J/cm2 at t = 4 plus 100 J/cm2 at t = 10. There was no visual difference in skin damage between 100 and 200 J/cm2 single illumination. Two‐fold illumination (100 + 100 J/cm2) caused significantly more skin damage, indicating a potentially successful option for increasing the efficacy of topical ALA‐PDT.

Light fractionation does not enhance the efficacy of methyl 5-aminolevulinate mediated photodynamic therapy in normal mouse skin

Previous work demonstrated that fractionated illumination using two fractions separated by a dark interval of 2 h, significantly enhanced the clinical efficacy of photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA). Considering the increasing clinical use of methyl 5-aminolevulinate (MAL) and the expected gain in efficacy by light fractionation we have investigated the response to MAL-PDT using a single and a two-fold illumination scheme and compared that with ALA-PDT. Our results show that fractionated illumination does not enhance the efficacy of PDT using MAL as it does using ALA despite the comparable fluorescence intensities at the end of the first light fraction and at the start of the second light fraction. Only the initial rate of photobleaching was slightly greater during ALA-PDT although the difference was small. Previously we hypothesized that cells surviving the first fraction are more susceptible to the second fraction. Since this is not true for MAL-PDT our data suggest that the distribution of MAL and ALA in tissues, and therefore the site of PDT induced damage, is an important parameter in the mechanism underlying the 2-fold illumination scheme.

Hypermutation in mantle cell lymphoma does not indicate a clinical or biological subentity

Mantle cell lymphoma is a prime example of a well-defined entity based on morphology, phenotype, genetics and also clinical features. Although most patients have an adverse clinical course, some have a better survival than others. The most consistently reported adverse prognostic parameter is a high mitotic rate. Recently, it has been shown that hypermutation in the immunoglobulin heavy-chain gene occurs in a subset of mantle cell lymphomas. It is, however, unclear whether the mutational status is stable over time within a given case, whether hypermutation might be influenced by therapy and how it is related to other relevant biological features of mantle cell lymphoma. In this study, we analyzed 23 typical mantle cell lymphoma cases with respect to mutational status and compared the results with clinicopathological and genetic data to determine whether the presence of mutation indicates a subentity with clinical or pathological relevance. We found somatic hypermutation in 26% of our cases and, interestingly, one case showed ongoing somatic hypermutation. In tumor cells of both mutated and unmutated cases, we found a preferential usage of VH3-21 (23%) and VH4-34 (19%). No significant correlations were found between mutation status and the other morphological and genetic features analyzed. In conclusion, our results provide additional evidence that mutation status in mantle cell lymphoma is better interpreted as a feature within the spectrum of disease that seems to have little clinical or pathological relevance.

Sequential immunohistochemistry: a promising new tool for the pathology laboratory

Current immunohistochemical methods to study the expression of multiple proteins in a single tissue section suffer from several limitations. In this article, we report on sequential immunohistochemistry (S‐IHC), a novel, easy method that allows the study of numerous proteins in a single tissue section, while requiring very limited optimization.

Tetraspanin CD37 protects against the development of B cell lymphoma

Worldwide, B cell non-Hodgkin lymphoma is the most common hematological malignancy and represents a substantial clinical problem. The molecular events that lead to B cell lymphoma are only partially defined. Here, we have provided evidence that deficiency of tetraspanin superfamily member CD37, which is important for B cell function, induces the development of B cell lymphoma. Mice lacking CD37 developed germinal center-derived B cell lymphoma in lymph nodes and spleens with a higher incidence than Bcl2 transgenic mice. We discovered that CD37 interacts with suppressor of cytokine signaling 3 (SOCS3); therefore, absence of CD37 drives tumor development through constitutive activation of the IL-6 signaling pathway. Moreover, animals deficient for both Cd37 and Il6 were fully protected against lymphoma development, confirming the involvement of the IL-6 pathway in driving tumorigenesis. Loss of CD37 on neoplastic cells in patients with diffuse large B cell lymphoma (DLBCL) directly correlated with activation of the IL-6 signaling pathway and with worse progression-free and overall survival. Together, this study identifies CD37 as a tumor suppressor that directly protects against B cell lymphomagenesis and provides a strong rationale for blocking the IL-6 pathway in patients with CD37- B cell malignancies as a possible therapeutic intervention.

Very low prevalence of germline MSH6 mutations in hereditary non-polyposis colorectal cancer suspected patients with colorectal cancer without microsatellite instability

Hereditary non-polyposis colorectal cancer (HNPCC) is caused by mutations in one of the mismatch repair genes MLH1, MSH2, MSH6, or PMS2 and results in high-level microsatellite instability (MSI-high) in tumours of HNPCC patients. The MSI test is considered reliable for indicating mutations in MLH1 and MSH2, but is questioned for MSH6. Germline mutation analysis was performed in 19 patients with an MSI-high tumour and absence of MSH2 and/or MSH6 protein as determined by immunohistochemistry (IHC), without an MLH1 or MSH2 mutation, and in 76 out of 295 patients suspected of HNPCC, with a non-MSI-high colorectal cancer (CRC). All 295 non-MSI-high CRCs were analysed for presence of MSH6 protein by IHC. In 10 patients with an MSI-high tumour without MSH2 and/or MSH6 expression, a pathogenic MSH6 mutation was detected, whereas no pathogenic MSH6 mutation was detected in 76 patients with a non-MSI-high CRC and normal MSH6 protein expression. In none of the 295 CRCs loss of MSH6 protein expression was detected. The prevalence of a germline MSH6 mutation is very low in HNPCC suspected patients with non-MSI-high CRC. Microsatellite instability analysis in CRCs is highly sensitive to select patients for MSH6 germline mutation analysis.

Immunohistochemical differentiation between follicular lymphoma and nodal marginal zone lymphoma – combined performance of multiple markers

Although many lymphomas can be reliably classified according to the World Health Organization Classification of 20081, the differentiation between nodal marginal zone lymphoma (NMZL) and follicular lymphoma (FL) is problematic in some cases. In fact, NMZL is often diagnosed by exclusion, resulting in heterogeneity in the diagnostic category of NMZL. New markers for NMZL have been described, but they have not yet been tested in combination.2,3 In this study, we compared multiple immunohistochemical markers for their use in distinguishing NMZL from FL. From the results obtained, we constructed an algorithm that combines these markers to help distinguish between FL and NMZL. Notably, this algorithm also contains a category of “B-cell lymphoma, unclassifiable”, thus underlining the difficulty that remains in distinguishing NMZL from FL. For the initial test series, we selected 47 patients with FL with a chromosomal rearrangement of BCL2, and 44 patients with a diagnosis of NMZL or probable NMZL, from the archive of the Department of Pathology at the Radboud university medical center (Nijmegen, the Netherlands). For all NMZLs, BCL2 translocations were excluded using fluorescent in-situ hybridization with split-signal probes. Patient characteristics are described in Online Supplementary Table S1. For a diagnosis of NMZL, the following diagnostic criteria were used: 1) effaced architecture of the lymph node, due to a small B-cell proliferation with a follicular, marginal zone, or diffuse growth pattern, 2) either centrocytoid or more round cells with intermingled centroblasts, 3) a mature B-cell immunophenotype with expression of BCL2, 4) in cases with a follicular/ nodular growth pattern, signs of follicular colonization (presence of BCL2 negative cells and high Ki67 staining), 5) not fitting a diagnosis of chronic lymphocytic leukemia/small lymphocytic lymphoma, mantle cell lymphoma, or lymphoplasmacytic lymphoma. Expression of germinal center markers was not considered an exclusion criterion for a diagnosis of NMZL in this study. As expected, immunohistochemistry showed significant differences between NMZL and FL (Table 1). Overall, FLs were positive for germinal center markers (CD10, BCL6, LMO2, HGAL) and negative for MNDA and IRTA1 (Online Supplementary Figure S1). NMZLs mostly showed an opposite pattern with positivity for MNDA in approximately two thirds of cases, IRTA1 staining in approximately one fifth of cases and usually no staining with germinal center markers. However, all germinal center markers were positive in a subset of NMZLs, and similarly, FLs with expression of MNDA were also identified (Online Supplementary Figure S2). Table 1. Immunohistochemistry results. Based on the immunohistochemistry results, a combination of markers were used to design an algorithm that helped to distinguish NMZL from FL (Figure 1). This algorithm was built empirically, allowing inclusion of a category of “B-cell lymphoma, unclassifiable” to prevent contamination of the NMZL category. As expected, this algorithm classified most lymphomas according to their original diagnosis (Table 2). However, in the initial test series, one case of FL was classified as NMZL, and 6 cases of NMZL were classified as FL by the algorithm; a significant proportion of cases (13%) were considered “B-cell lymphoma, unclassifiable” by the algorithm. Most (75%) of these unclassifiable cases had an original diagnosis of NMZL. Figure 1. Immunohistochemical algorithm for separation of nodal marginal zone lymphoma (NMZL) from follicular lymphoma (FL). The algorithm starts at the top with a lymphoma that is considered to be either FL or NMZL. If all four germinal center markers (BCL6, CD10, ... Table 2. Algorithm results. To validate the algorithm, a second validation group of 21 FLs and 13 NMZLs, collected from the archive of the Department of Pathology at the Hospital del Mar (Barcelona, Spain) was stained for the same markers as the initial group. Overall, staining results were comparable to those in the test group, with a high sensitivity of BCL6 for FL and a high specificity of IRTA1 for NMZL (Table 1); CD10 expression had a higher sensitivity in comparison to the test group, but a lower specificity. LMO2 and HGAL were less sensitive but more specific. In this validation group, the algorithm gave a concordant classification as either NMZL or FL in 85% of cases (Table 2). No follicular lymphoma was misclassified as NMZL, and only one NMZL was misclassified as FL. Four cases (12%) were considered unclassifiable, three of which had an original diagnosis of FL and one with an original diagnosis of NMZL. The algorithm was designed based on a comparison of NMZLs with FLs with a translocation involving BCL2. However, because a BCL2 translocation can be demonstrated relatively easily, the actual problem we are faced with in daily practice is the separation of NMZL from FL without a BCL2 translocation. FLs with and without a BCL2 translocation might be different from each other, as has been suggested by one gene expression study4, and also by a recent comparative genomic hybridization study.5 In the latter study, genetic aberrations in FLs without a BCL2 translocation bore more resemblance to those in NMZL than those in FLs with a BCL2 translocation. To address this problem, we tested a small series of 6 FLs without a BCL2 translocation, which were all classified as FL by the algorithm. This supports the idea that this algorithm also applies to FLs without a BCL2 translocation, but confirmation of this theory will require the study of a larger series. Thus far, the majority of the markers in the algorithm has only been described in single studies. Kanellis and colleagues reported expression of MNDA in 75% of NMZLs versus 5% of FLs.2 In our series, a less pronounced, but similar difference was observed, with MNDA expression in NMZL and FL in 70% and 15%, respectively. In accordance with Falini and colleagues6, IRTA1 also discriminated between NMZL and FL in our series. However, in the study carried out by Falini et al.,73% of NMZLs expressed IRTA1, compared to only 21% in our study. This difference could be caused by a difference in interpretation. In our case, faint IRTA1 expression was quite frequently observed in NMZLs, but also in some FLs. As the reproducibility of the scores assigned to these cases proved to be very poor amongst different observers, a case was only considered positive if 30% or more of the cells showed moderate or strong expression of IRTA1. This aforementioned approach produced a strong improvement in the diagnostic value of IRTA1 in our series, and explains the small proportion of NMZLs positive for IRTA1. Expression of the germinal center markers HGAL and LMO2 in lymphomas has been described by Natkunam et al., whose work showed expression in the majority of FLs and only very rare expression in NMZL.7–10 For both markers, they detected only a single case of NMZL that was positive.7,8 In our series, we observed more frequent expression of both LMO2 and HGAL in NMZL. We believe this could be caused by differences in inclusion criteria between our study and previous studies. In the study effected by Salama et al., which contains the largest majority of NMZLs previously studied for LMO2 and HGAL, cases were excluded from the NMZL group if they expressed germinal center markers9, which explains why expression of germinal center markers was not detected in NMZLs in their study. A recent study by Dyhdalo and colleagues reported LMO2 staining in 2 out of 25 NMZLs, of which one case also expressed BCL6.11 In our study, expression of germinal center markers was not considered an exclusion criterion. We made this choice because, in our experience, typical cases of NMZL do occasionally express germinal center markers and expression of CD10 and BCL6 in NMZL has been previously reported.12–14 The FLs that were used to build the algorithm were all required to have a BCL2 break, which, together with morphology, ensured that the diagnosis of FL was correct. Thus, misclassification of FL with a BCL2 break as NMZL has been excluded. Unfortunately, no markers for NMZL can compare with the BCL2 translocation for FL. Therefore, the NMZL group can still be expected to be more heterogeneous than the FL group, with some cases representing FL or other lymphomas rather than NMZL. Indeed, when looking at the initial and validation series together, ‘NMZLs’ were quite frequently considered FL (in 12%) or unclassifiable (in 18%) by the algorithm. This illustrates the difficulty that remains in the definition and diagnosis of NMZL; the lack of both an accurate definition and of positive diagnostic markers for NMZL result in a heterogeneous diagnostic category. The ultimate question is: what is the gold standard? For this study we have used the combination of morphology and phenotyping for follicular colonization as defining criteria for NMZL. The addition of extensive immunohistochemistry, including new markers, might help to create a better gold standard for NMZL. At present, however, it is very difficult to compare different strategies to diagnose NMZL, due to the fact that as of yet no perfect positive marker for NMZL is available. Hopefully, elucidation of the pathogenesis of NMZL will provide us with better positive markers for NMZL. The results from this study could assist in achieving this goal; the addition of extensive immunohistochemistry to conventional criteria for NMZL will help to establish smaller, but potentially more homogeneous study groups, facilitating studies into the pathogenesis of NMZL.