Molecular Pathogenesis of Primary Gastrointestinal Tract Lymphomas.

Primary gastrointestinal lymphomas are rare though the incidence is significantly increased among adult patients in recent years. The majority of the patients present with symptoms overlapping with other gastrointestinal disorders and imaging findings are not specific. Therefore, histologic examination is necessary to establish the diagnosis. Insight into etiologies, molecular pathogenesis and critical signaling pathways in lymphomas including gastrointestinal lymphomas has significantly expanded within the last 3 decades. Given the increasing demand for incorporation of genetic data, the appropriate handling and processing of small endoscopic gastrointestinal biopsy samples of suspected lymphoma is becoming extremely crucial and at times challenging. The use of next generation sequencing with analysis of genes relevant to diagnosis, prognosis, and therapeutic targets continues to have a significant promising impact on management of patients in lymphoid malignancies. In particular, the identification of constitutively activated pathways and the emergence of novel targeted medications predict that more effective therapies will be identified for these disorders in the coming years.

Integration of ruxolitinib into dose-intensified therapy targeted against a novel JAK2 F694L mutation in B-precursor acute lymphoblastic leukemia.

A 17-year-old girl with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) with persistent minimal residual disease (MRD) who underwent standard chemotherapy was found to have a BCR-ABL1-like gene expression pattern. Genome sequencing revealed a JAK2 mutation not previously described in BCP-ALL and a potential therapeutic target. Due to concern for an on-therapy relapse, the JAK2 inhibitor ruxolitinib was incorporated into a modified chemotherapy backbone to achieve complete remission prior to stem cell transplant. Treatment was well tolerated and she had undetectable MRD prior to a matched allogeneic stem cell transplant and remained in remission at day +100.

Scoring of MYC protein expression in diffuse large B-cell lymphomas: concordance rate among hematopathologists.

Recent studies have shown that immunohistochemical evaluation of MYC protein expression in diffuse large B-cell lymphoma is a useful prognostic tool with high concordance rate among pathologists. Concordance in these studies was assessed among few pathologists from one institution by scoring tissue microarrays. In daily practice, MYC evaluation is performed on entire tumor sections by a diverse group of pathologists. In our study, nine hematopathologists from two institutions scored whole-tissue sections of two sets of cases. The training set included 13 cases of diffuse large B-cell lymphoma and 4 cases of Burkitt lymphoma. The validation set included 18 cases of diffuse large B-cell lymphoma and 1 case of Burkitt lymphoma. MYC positivity was defined as ≥40% of tumor cells demonstrating nuclear staining similar to prior studies. The mean score for each case was used to determine MYC status with discrepant cases defined as having any score causing a different MYC status designation. Discrepant cases from the training set were characterized by staining heterogeneity, extensive necrosis or crush artifact and had mean scores within 15 percentage points of 40%. Cases from the validation set that demonstrated any of these features were scored twice on two different days. Overall concordance was moderate (Kappa score: 0.68, P-value<0.001) with no significant change between the two sets (Kappa scores: 0.69 vs 0.67). Thirty-nine percent of cases were discrepant. The findings indicate that a significant number of diffuse large B-cell lymphomas are inherently difficult to score due to staining heterogeneity. The effect of heterogeneity can be under-represented when concordance is measured among few pathologists scoring tissue microarrays. Careful scoring strategy in our study failed to improve concordance. In the absence of specific instructions on how to deal with heterogeneity, caution is advised when evaluating MYC expression in diffuse large B-cell lymphoma.

Recommendations for gross examination and sampling of surgical specimens of the spleen.

This review examines handling and processing of spleen biopsies and splenectomy specimens with the aim of providing the pathologist with guidance in optimizing examination and diagnosis of splenic disorders. It also offers recommendations as to relevant reporting factors in gross examination, which may guide diagnostic workup. The role of splenic needle biopsies is discussed. The International Spleen Consortium is a group dedicated to promoting education and research on the anatomy, physiology, and pathology of the spleen. In keeping with these goals, we have undertaken to provide guidelines for gross examination, sectioning, and sampling of spleen tissue to optimize diagnosis (Burke). The pathology of the spleen may be complicated in routine practice due to a number of factors. Among these are lack of familiarity with lesions, complex histopathology, mimicry within several types of lesions, and overall rarity. To optimize diagnosis, appropriate handling and processing of splenic tissue are crucial. The importance of complete and accurate clinical history cannot be overstated. In many cases, significant clinical history such as previous lymphoproliferative disorders, hematologic disorders, trauma, etc, can provide important information to guide the evaluation of spleen specimens. Clinical information helps plan for appropriate processing of the spleen specimen. The pathologist should encourage surgical colleagues, who typically provide the specimens, to include as much clinical information as possible.

Mutually exclusive driver mutations identifies 2 separate primaries in a collision tumor initially interpreted as a solitary lung adenocarcinoma with tumor heterogeneity.

Distinction of histologically heterogenous, single primary tumor from two or more collision tumors with different primaries could represent a challenge to practicing pathologists. Histologic variations including differences in degree of differentiating within a tumor, are typically interpreted as tumor heterogeneity in a contiguous small size tumor biopsy. The authors report a case of adult former smoker female who presented with lung mass and a metastatic lytic lesion of acetabulum. A needle biopsy of a lung mass revealed an adenocarcinoma with well and moderately differentiated components. Next generation sequencing studies proved 2 different primaries in this small needle biopsy.

Risk factors for non-Hodgkin lymphoma subtypes defined by histology and t(14;18) in a population-based case-control study.

The t(14;18) chromosomal translocation is the most common cytogenetic abnormality in non-Hodgkin lymphoma (NHL), occurring in 70-90% of follicular lymphomas (FL) and 30-50% of diffuse large B-cell lymphomas (DLBCL). Previous t(14;18)-NHL studies have not evaluated risk factors for NHL defined by both t(14;18) status and histology. In this population-based case-control study, t(14;18) status was determined in DLBCL cases using fluorescence in situ hybridization on paraffin-embedded tumor sections. Polytomous logistic regression was used to evaluate the association between a wide variety of exposures and t(14;18)-positive (N=109) and -negative DLBCL (N=125) and FL (N=318), adjusting for sex, age, race, and study center. Taller height, more lifetime surgeries, and PCB180 exposure were associated with t(14;18)-positivity. Taller individuals (third tertile vs. first tertile) had elevated risks of t(14;18)-positive DLBCL (odds ratio [OR] = 1.8, 95% confidence interval [CI] 1.1-3.0) and FL (OR=1.4, 95%CI 1.0-1.9) but not t(14;18)-negative DLBCL. Similar patterns were seen for individuals with more lifetime surgeries (13+ vs. 0-12 surgeries; t(14;18)-positive DLBCL OR=1.4, 95%CI 0.7-2.7; FL OR=1.6, 95%CI 1.1-2.5) and individuals exposed to PCB180 greater than 20.8 ng/g (t(14;18)-positive DLBCL OR=1.3, 95%CI 0.6-2.9; FL OR=1.7, 95%CI 1.0-2.8). In contrast, termite treatment and high alpha-chlordane levels were associated with t(14;18)-negative DLBCL only, suggesting that these exposures do not act through t(14;18). Our findings suggest that putative associations between NHL and height, surgeries, and PCB180 may be t(14;18)-mediated and provide support for case-subtyping based on molecular and histologic subtypes. Future efforts should focus on pooling data to confirm and extend previous research on risk factors for t(14;18)-NHL subtypes.

Etiologic heterogeneity among non-Hodgkin lymphoma subtypes.

Understanding patterns of etiologic commonality and heterogeneity for non-Hodgkin lymphomas may illuminate lymphomagenesis. We present the first systematic comparison of risks by lymphoma subtype for a broad range of putative risk factors in a population-based case-control study, including diffuse large B-cell (DLBCL; N = 416), follicular (N = 318), and marginal zone lymphomas (N = 106), and chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL; N = 133). We required at least 2 of 3 analyses to support differences in risk: (1) polytomous logistic regression, (2) homogeneity tests, or (3) dichotomous logistic regression, analyzing all 7 possible pairwise comparisons among the subtypes, corresponding to various groupings by clinical behavior, genetic features, and differentiation. Late birth order and high body mass index (>/= 35) kg/m(2)) increased risk for DLBCL alone. Autoimmune conditions increased risk for marginal zone lymphoma alone. The tumor necrosis factor G-308A polymorphism (rs1800629) increased risks for both DLBCL and marginal zone lymphoma. Exposure to certain dietary heterocyclic amines from meat consumption increased risk for CLL/SLL alone. We observed no significant risk factors for follicular lymphoma alone. These data clearly support both etiologic commonality and heterogeneity for lymphoma subtypes, suggesting that immune dysfunction is of greater etiologic importance for DLBCL and marginal zone lymphoma than for CLL/SLL and follicular lymphoma.

Comparison of Tissue Molecular Biomarker Testing Turnaround Times and Concordance Between Standard of Care and the Biocartis Idylla Platform in Patients With Colorectal Cancer.

OBJECTIVES: Management of colorectal cancer warrants mutational analysis of KRAS/NRAS when considering anti-epidermal growth factor receptor therapy and BRAF testing for prognostic stratification. In this multicenter study, we compared a fully integrated, cartridge-based system to standard-of-care assays used by participating laboratories. METHODS: Twenty laboratories enrolled 874 colorectal cancer cases between November 2017 and December 2018. Testing was performed on the Idylla automated system (Biocartis) using the KRAS and NRAS-BRAF cartridges (research use only) and results compared with in-house standard-of-care testing methods. RESULTS: There were sufficient data on 780 cases to measure turnaround time compared with standard assays. In-house polymerase chain reaction (PCR) had an average testing turnaround time of 5.6 days, send-out PCR of 22.5 days, in-house Sanger sequencing of 14.7 days, send-out Sanger of 17.8 days, in-house next-generation sequencing (NGS) of 12.5 days, and send-out NGS of 20.0 days. Standard testing had an average turnaround time of 11 days. Idylla average time to results was 4.9 days with a range of 0.4 to 13.5 days. CONCLUSIONS: The described cartridge-based system offers rapid and reliable testing of clinically actionable mutation in colorectal cancer specimens directly from formalin-fixed, paraffin-embedded tissue sections. Its simplicity and ease of use compared with other molecular techniques make it suitable for routine clinical laboratory testing.

Instances of mantle cell lymphoma morphologically mimicking other subtypes of B-cell lymphoid proliferation.

Mantle cell lymphoma is a well-characterized category of mature B-cell lymphoma with aberrant coexpression of CD5 antigen. This subtype of lymphoma is genetically defined by t(11;14) resulting in upregulation of cyclin D1 protein. In clinical practice, mantle cell lymphoma is typically diagnosed based on combination of morphology, CD20/CD5 coexpression, and nuclear staining of cyclin D1 protein by immunohistochemistry. Although other neoplastic processes can also be cyclin D1 positive, documentation of cyclin D1 positivity in a CD5-positive B-cell process is virtually diagnostic of mantle cell lymphoma. However, on morphologic grounds, it is well known that mantle cell lymphoma can mimic other subtypes of B-cell lymphoid neoplasm. We identified several unusual examples of immunohistochemically confirmed cyclin D1-positive mantle cell lymphoma with morphologic features overlapping with a wide variety of other subtypes of mature B-cell lymphomas including follicular, marginal zone, small lymphocytic and Burkitt lymphoma.

Her-2/neu expression in salivary duct carcinoma: an immunohistochemical and chromogenic in situ hybridization study.

Salivary duct carcinoma (SDC) shares significant morphologic and immunophenotypic overlap with ductal carcinoma of the breast, including HER-2/neu expression. Previous studies have detected HER-2/neu at the protein level in SDCs; however, no study, to date, has assayed whether this expression is related to gene amplification detected by chromogenic in situ hybridization (CISH). Formalin-fixed, paraffin-embedded tissue sections from 12 previously diagnosed SDCs were evaluated by immunohistochemistry (IHC) and CISH for HER-2/neu status. Result concordance was seen in all 12 cases. A total of 4 SDCs were positive by IHC; all 4 cases showed amplification with CISH. The remaining 8 cases were negative by IHC and showed no gene amplification with CISH. SDCs in this study show HER-2/neu overexpression on both the protein and gene levels in approximately 30% of cases. These findings suggest a role may exist for Herceptin (trastuzumab) based therapy in some SDC patients.

BRAF V600E Mutation Across Multiple Tumor Types: Correlation Between DNA-based Sequencing and Mutation-specific Immunohistochemistry.

The B-Raf proto-oncogene (BRAF) encodes a cytoplasmic serine/threonine kinase with a key role in regulating the mitogen-activated protein kinase signal transduction pathway. An activating missense mutation in codon 600 of exon 15 (V600E) of BRAF gene has been identified in multiple neoplasms including melanoma, colorectal carcinoma, papillary thyroid carcinoma, hairy cell leukemia, and Langerhans cell histiocytosis. Patients with BRAF V600E-mutated melanoma respond to FDA-approved BRAF inhibitors. In addition, subsets of other BRAF V600E-mutated tumors may also benefit from BRAF inhibitor therapy. Currently, clinical laboratories typically use molecular-based methods for mutation analysis. However, recently a BRAF V600E mutation-specific antibody has become available as a cost-effective alternative method to DNA-based molecular testing. We analyzed multiple tumor types including melanoma, colorectal carcinoma, papillary thyroid cancer, hairy cell leukemia, and Langerhans cell histiocytosis using both DNA-based sequencing and the BRAF V600E mutation-specific antibody. Our results show a high degree of concordance between the 2 methods. However, the high concordance seems to be limited only to the V600E mutation since variant V600 mutations are missed by V600E mutation-specific immunohistochemistry.

HER-2 gene amplification in Paget disease of the nipple and extramammary site: a chromogenic in situ hybridization study.

Patients with human epidermal growth factor receptor 2 (HER-2) overexpressing breast carcinomas have a more aggressive clinical behavior and their tumors are often hormone receptor negative. However, the recently introduced anti-HER-2 antibody trastuzumab has been proven to improve the survival and controls the disease in a significant proportion of these patients. Therefore, the analysis of HER-2 in patients with breast cancer has become an important and routine test to select those who may benefit from the gene-based targeted therapy trastuzumab (herceptin). There is good correlation between HER-2/neu protein overexpression and HER-2 gene amplification in breast cancer. However, inconsistent results have been reported in the rate of HER-2/neu protein overexpression in other malignant neoplasms. Furthermore, only rare studies have investigated the correlation between the HER-2/neu protein overexpression and the status of HER-2 gene in these tumors. We investigated the HER-2 gene and protein status in several cases of Paget disease of the nipple and vulva by using a chromogenic in situ hybridization assay and immunohistochemistry. We find that the majority of the Paget disease of the breast demonstrate HER-2 gene amplification, whereas most of the extramammary Paget disease lack HER-2 gene amplification. In addition, our results show a good correlation between HER-2/neu protein overexpression and HER-2 gene amplification in Paget disease of the nipple, but we were unable to confirm this correlation in HER-2/neu protein overexpressing Paget disease of the vulva.

Regulating the tumor suppressor gene maspin in breast cancer cells: a potential mechanism for the anticancer properties of tamoxifen.

PURPOSE: Mammary epithelial cells and the majority of breast cancer tumors require estrogen for continued growth. Antiestrogen therapy alone, or in combination with other drugs, has long been a common procedure for breast cancer treatment and prophylaxis. Thus, there is a critical need to elucidate the mechanism(s) of action of antiestrogen treatment, especially for patients who are at risk of breast cancer development or who are currently receiving hormone therapy. In this study, we examined the ability of hormones to regulate the expression of a tumor suppressor gene, maspin, which is a serine protease inhibitor (serpin) that plays an important role in mammary gland development and is silenced during breast cancer progression. Specifically, our hypothesis tested the clinical efficacy of tamoxifen to regulate maspin expression. EXPERIMENTAL DESIGN: We used maspin promoter luciferase reporter plasmids that were transfected into normal human mammary epithelial (HMEC1331) and MCF-7 breast cancer cells, followed by determination of the effect of hormones and their antagonists on maspin promoter activity. At the protein level, cytosolic fractions from both cell types before and after hormone treatment were subjected to Western blot analysis to determine maspin level. RESULTS AND CONCLUSIONS: Our studies revealed that the antiestrogen tamoxifen induces maspin promoter activity. Interestingly, antiandrogen flutamide could also induce maspin in both cell lines tested. These observations were further confirmed in patient tissues. These novel findings provide a new mechanism of action for tamoxifen under normal and pathological conditions. More significantly, these findings could have a potential impact on future therapeutic intervention strategies for breast cancer.

Detection of factor V leiden and prothrombin gene mutations in patients who died with thrombotic events.

CONTEXT: Individuals with factor V or prothrombin gene mutations are at increased risk for thrombotic events. Furthermore, the risk of recurrent deep venous thrombosis in heterozygous carriers of both factor V Leiden and prothrombin gene mutations is high enough that some investigators suggest lifelong warfarin prophylaxis for these individuals, even with a single spontaneous thrombotic event. OBJECTIVES: To assess the incidence of factor V Leiden and prothrombin gene mutations in an autopsy population and to determine if these tests can prove useful in identification of at-risk family members. DESIGN: We analyzed factor V Leiden and prothrombin gene mutations in 45 patients who died with or of thrombotic events, using archival tissue and multiplex allele-specific polymerase chain reaction amplification. The wild-type factor V gene was amplified in all 45 patients, whereas the wild-type prothrombin gene was amplified in 29 patients. RESULTS: Two patients (4.4%) who died with thrombotic events at the ages of 35 and 92 years were heterozygous for factor V gene mutation. Two additional patients (6.7%), who died with thrombotic events at the ages of 26 and 39 years, were heterozygous for prothrombin gene mutation. Patients homozygous for either factor V or prothrombin gene or simultaneously heterozygous for both genes were not detected in our study. CONCLUSIONS: Our findings suggest that screening the relatives of elderly patients who die with thrombotic events would not be cost-effective because of the low incidence of these mutations in the autopsy population. However, because the incidence of these mutations appeared significantly more frequently among individuals who died at 39 years or younger, testing the relatives of this subset of patients may prove useful for detection of at-risk individuals who would benefit from preventive anticoagulation therapy.

Fluorescence immunophenotyping and interphase cytogenetics (FICTION) detects BCL6 abnormalities, including gene amplification, in most cases of nodular lymphocyte-predominant Hodgkin lymphoma.

CONTEXT: BCL6 translocations are a frequent finding in B-cell lymphomas of diverse subtypes, including some cases of nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL). However, reliable analysis of BCL6 rearrangements using fluorescence in situ hybridization is difficult in NLPHL because of the relative paucity of neoplastic cells. Combined immunofluorescence microscopy and fluorescence in situ hybridization, or fluorescence immunophenotyping and interphase cytogenetics as a tool for the investigation of neoplasms (FICTION), permits targeted analysis of neoplastic cells. OBJECTIVE: To better define the spectrum of BCL6 abnormalities in NLPHL using FICTION analysis. DESIGN: We performed an optimized FICTION analysis of 24 lymph nodes, including 11 NLPHL, 5 follicular hyperplasia with prominent progressive transformation of germinal centers, and 8 follicular hyperplasia without progressive transformation of germinal centers. RESULTS: BCL6 rearrangement was identified in 5 of 11 cases of NLPHL (46%). In addition, BCL6 gene amplification, with large clusters of BCL6 signals in the absence of chromosome 3 aneuploidy, was detected in 3 of 11 cases of NLPHL (27%). One NLPHL showed extra copies of BCL6 present in conjunction with multiple copies of chromosome 3. Altogether, we detected BCL6 abnormalities in 9 of 11 cases of NLPHL (82%). None of the progressive transformation of germinal centers or follicular hyperplasia cases showed BCL6 abnormalities by FICTION. CONCLUSIONS: To our knowledge, this is the first report of BCL6 gene amplification in NLPHL. Our optimized protocol for FICTION permits detection of cytogenetic abnormalities in most NLPHL cases and may represent a useful ancillary diagnostic technique.

Pyrosequencing for classification of human FcγRIIIA allotypes: a comparison with PCR-based techniques.

BACKGROUND: Surface-specific antigens expressed by hematopoietic cells are attractive targets for antibody-mediated immunotherapy. Monoclonal antibodies (mAbs) involve various mechanisms to eliminate target cells, including antibody-dependent cellular cytotoxicity (ADCC)- and phagocytosis (ADCP)-mediated killing through natural killer (NK) and macrophage effector cells bearing FcγRIIIA (CD16). The clinical efficacy of ADCC is particularly impacted by a single nucleotide polymorphism (SNP) found in the gene encoding FcγRIIIA (FCGR3A), which generates a variable distribution of the 158 V/V, F/V or F/F CD16 allotypes (F = phenylalanine, V = valine) in the normal human population. Currently, most patients are not screened for CD16 allotypes, creating the potential to include in their treatment a mAb-based therapy that may have limited benefit. Therefore, it is important to identify CD16 allotypes when considering mAb therapies that require ADCC/ADCP. OBJECTIVE: The objective of this study was to develop a reliable PCR-based assay for classification of human FcγRIIIA allotypes. METHODS: We studied 42 normal human subjects for the incidence of FcγRIIIA-158 polymorphisms using comparative molecular approaches. RESULTS: The results of our study showed 100% accuracy in genotyping by pyrosequencing. In contrast, nested PCR-based allele-specific restriction assay and quantitative PCR techniques proved to be relatively less sensitive and less specific in distinguishing variant genotypes. CONCLUSION: Since the efficacy of the mAb-based targeted immunotherapy may be highly dependent upon the CD16 polymorphism in a given individual, we recommend pyrosequencing for CD16 allotype testing.

LIM domain only 2 protein expression, LMO2 germline genetic variation, and overall survival in diffuse large B-cell lymphoma in the pre-rituximab era.

Both LMO2 (LIM domain only 2) mRNA and protein expression in diffuse large B-cell lymphoma (DLBCL) have been associated with superior survival. However, a role for germline genetic variation in LMO2 has not been previously reported. Immunohistochemistry (IHC) for LMO2 was conducted on tumor tissue from diagnostic biopsies, and 20 tag single nucleotide polymorphisms (SNPs) from LMO2 were genotyped from germline DNA. LMO2 IHC positivity was associated with superior survival (hazard ratio [HR] = 0.55; 95% confidence interval [CI] 0.31-0.97). Four LMO2 SNPs (rs10836127, rs941940, rs750781, rs1885524) were associated with survival after adjusting for LMO2 IHC and clinical factors (p < 0.05), and one of these SNPs (rs941940) was also associated with IHC positivity (p = 0.02). Compared to a model with clinical factors only (c-statistic = 0.676), adding the four SNPs (c-statistic = 0.751) or LMO2 IHC (c-statistic = 0.691) increased the predictive ability of the model, while inclusion of all three factors (c-statistic = 0.754) did not meaningfully add predictive ability above a model with clinical factors and the four SNPs. In conclusion, germline genetic variation in LMO2 was associated with DLBCL prognosis and provided slightly stronger predictive ability relative to LMO2 IHC status.

Immunostaining to identify molecular subtypes of diffuse large B-cell lymphoma in a population-based epidemiologic study in the pre-rituximab era.

Gene expression profiling studies have distinguished diffuse large B-cell lymphomas (DLBCLs) by cell of origin, with distinct pathogenetic mechanisms and prognosis. We attempted to identify DLBCL molecular subtypes in an epidemiologic study of 214 DLBCL patients diagnosed during 1998-2000 with archival tissues to investigate etiology. Immunohistochemical staining for CD10, BCL6, LMO2, MUM1/IRF4, and BCL2 and fluorescence in situ hybridization for t(14;18) were conducted, with ≥93% blinded duplicate agreement. CD10, LMO2, and BCL2 expression was similar to previous reports (32%, 44%, and 44% of DLBCLs, respectively), but BCL6 and MUM1/IRF4 expression was lower than expected (29% and 5%, respectively). We classified 112/214 (52%) cases as germinal center B-cell-like DLBCL (GCB-DLBCL; Hans et al., Blood 2004; CD10+ or CD10-/BCL6+/MUM1-), with no difference in prognosis compared with non-GCB-DLBCL (Cox regression, P=0.48). Comparing other GCB correlates, LMO2 expression and t(14;18) were more common but not exclusive to GCB-DLBCL as defined in our study, whereas BCL2 expression did not differ between DLBCL molecular subtypes. We could not confidently identify patients with GCB-DLBCL using these immunohistochemistry-based markers on archival tissues.