Association of Broad-Based Genomic Sequencing With Survival Among Patients With Advanced Non-Small Cell Lung Cancer in the Community Oncology Setting.

Importance: Broad-based genomic sequencing is being used more frequently for patients with advanced non-small cell lung cancer (NSCLC). However, little is known about the association between broad-based genomic sequencing and treatment selection or survival among patients with advanced NSCLC in a community oncology setting. Objective: To compare clinical outcomes between patients with advanced NSCLC who received broad-based genomic sequencing vs a control group of patients who received routine testing for EGFR mutations and/or ALK rearrangements alone. Design, Setting, and Participants: Retrospective cohort study of patients with chart-confirmed advanced NSCLC between January 1, 2011, and July 31, 2016, and who received care at 1 of 191 oncology practices across the United States using the Flatiron Health Database. Patients were diagnosed with stage IIIB/IV or unresectable nonsquamous NSCLC who received at least 1 line of antineoplastic treatment. Exposures: Receipt of either broad-based genomic sequencing or routine testing (EGFR and/or ALK only). Broad-based genomic sequencing included any multigene panel sequencing assay examining more than 30 genes prior to third-line treatment. Main Outcomes and Measures: Primary outcomes were 12-month mortality and overall survival from the start of first-line treatment. Secondary outcomes included frequency of genetic alterations and treatments received. Results: Among 5688 individuals with advanced NSCLC (median age, 67 years [interquartile range, 41-85], 63.6% white, 80% with a history of smoking); 875 (15.4%) received broad-based genomic sequencing and 4813 (84.6%) received routine testing. Among patients who received broad-based genomic sequencing, 4.5% received targeted treatment based on testing results, 9.8% received routine EGFR/ALK targeted treatment, and 85.1% received no targeted treatment. Unadjusted mortality rates at 12 months were 49.2% for patients undergoing broad-based genomic sequencing and 35.9% for patients undergoing routine testing. Using an instrumental variable analysis, there was no significant association between broad-based genomic sequencing and 12-month mortality (predicted probability of death at 12 months, 41.1% for broad-based genomic sequencing vs 44.4% for routine testing; difference -3.6% [95% CI, -18.4% to 11.1%]; P = .63). The results were consistent in the propensity score-matched survival analysis (42.0% vs 45.1%; hazard ratio, 0.92 [95% CI, 0.73 to 1.11]; P = .40) vs unmatched cohort (hazard ratio, 0.69 [95% CI, 0.62 to 0.77]; log-rank P < .001). Conclusions and Relevance: Among patients with advanced non-small cell lung cancer receiving care in the community oncology setting, broad-based genomic sequencing directly informed treatment in a minority of patients and was not independently associated with better survival.

Dosing clopidogrel based on CYP2C19 genotype and the effect on platelet reactivity in patients with stable cardiovascular disease.

CONTEXT: Variants in the CYP2C19 gene influence the pharmacologic and clinical response to the standard 75-mg daily maintenance dose of the antiplatelet drug clopidogrel. OBJECTIVE: To test whether higher doses (up to 300 mg daily) improve the response to clopidogrel in the setting of loss-of-function CYP2C19 genotypes. DESIGN, SETTING, AND PATIENTS: ELEVATE-TIMI 56 was a multicenter, randomized, double-blind trial that enrolled and genotyped 333 patients with cardiovascular disease across 32 sites from October 2010 until September 2011. INTERVENTIONS: Maintenance doses of clopidogrel for 4 treatment periods, each lasting approximately 14 days, based on genotype. In total, 247 noncarriers of a CYP2C19*2 loss-of-function allele were to receive 75 and 150 mg daily of clopidogrel (2 periods each), whereas 86 carriers (80 heterozygotes, 6 homozygotes) were to receive 75, 150, 225, and 300 mg daily. MAIN OUTCOME MEASURES: Platelet function test results (vasodilator-stimulated phosphoprotein [VASP] phosphorylation and VerifyNow P2Y(12) assays) and adverse events. RESULTS: With 75 mg daily, CYP2C19*2 heterozygotes had significantly higher on-treatment platelet reactivity than did noncarriers (VASP platelet reactivity index [PRI]: mean, 70.0%; 95% CI, 66.0%-74.0%, vs 57.5%; 95% CI, 55.1%-59.9%, and VerifyNow P2Y(12) reaction units [PRU]: mean, 225.6; 95% CI, 207.7-243.4, vs 163.6; 95% CI, 154.4-173.9; P < .001 for both comparisons). Among CYP2C19*2 heterozygotes, doses up to 300 mg daily significantly reduced platelet reactivity, with VASP PRI decreasing to 48.9% (95% CI, 44.6%-53.2%) and PRU to 127.5 (95% CI, 109.9-145.2) (P < .001 for trend across doses for both). Whereas 52% of CYP2C19*2 heterozygotes were nonresponders (≥230 PRU) with 75 mg of clopidogrel, only 10% were nonresponders with 225 or 300 mg (P < .001 for both). Clopidogrel, 225 mg daily, reduced platelet reactivity in CYP2C19*2 heterozygotes to levels achieved with standard clopidogrel, 75 mg, in noncarriers (mean ratios of platelet reactivity, VASP PRI, 0.92; 90% CI, 0.85-0.99, and PRU, 0.94; 90% CI, 0.84-1.04). In CYP2C19*2 homozygotes, even with 300 mg daily of clopidogrel, mean VASP PRI was 68.3% (95% CI, 44.9%-91.6%) and mean PRU, 287.0 (95% CI, 170.2-403.8). CONCLUSION: Among patients with stable cardiovascular disease, tripling the maintenance dose of clopidogrel to 225 mg daily in CYP2C19*2 heterozygotes achieved levels of platelet reactivity similar to that seen with the standard 75-mg dose in noncarriers; in contrast, for CYP2C19*2 homozygotes, doses as high as 300 mg daily did not result in comparable degrees of platelet inhibition. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01235351.

HHV8-positive, EBV-positive Hodgkin lymphoma-like large B-cell lymphoma and HHV8-positive intravascular large B-cell lymphoma.

Human herpesvirus type 8 (HHV8), also known as Kaposi's sarcoma-associated herpesvirus, is a human gamma herpesvirus that underlies the pathogenesis of Kaposi's sarcoma, primary effusion lymphoma and multicentric Castleman's disease. We recently encountered two cases of HHV8-positive large B-cell lymphoma with features not previously described. The first patient was a 61-year-old immunocompetent man with an enlarged cervical lymph node containing scattered large, bizarre cells in a reactive background of lymphocytes, plasma cells and scattered regressed follicles resembling those of hyaline-vascular Castleman's disease. The appearance suggested classical Hodgkin's lymphoma, but the large cells were negative for CD15, CD30, CD20 and CD3, and positive for MUM1/IRF4, EMA, HHV8, EBER and dim IgM lambda. The second patient was a 59-year-old HIV-positive man who died after several weeks of fever, night sweats, anemia, thrombocytopenia, hepatosplenomegaly and multiorgan failure. At autopsy an intravascular large B-cell lymphoma that was positive for MUM1/IRF4, HHV8 and IgM lambda, and negative for CD20 and EBER involved multiple organs, including lung, heart, kidney, liver and spleen. On the basis of the histologic features in these two cases, the presence of HHV8 was unexpected. These cases expand the spectrum of lymphoproliferative disorders that can be associated with HHV8.

Phase II study of temozolomide, thalidomide, and celecoxib for newly diagnosed glioblastoma in adults.

We conducted a phase II study of the combination of temozolomide and angiogenesis inhibitors for treating adult patients with newly diagnosed glioblastoma. Patients who had stable disease following standard radiation therapy received temozolomide for 5 days in 28-day cycles, in combination with daily thalidomide and celecoxib. Patients were treated until tumor progression or development of unacceptable toxicity. Four-month progression-free survival (PFS) from study enrollment was the primary end point, and overall survival (OS) was the secondary end point. In addition, we sought to correlate response with O(6)-methylguanine-DNA methyltransferase promoter methylation status and serum levels of angiogenic peptides. Fifty patients with glioblastoma were enrolled (18 women, 32 men). Median age was 54 years (range, 29-78) and median KPS score was 90 (range, 70-100). From study enrollment, median PFS was 5.9 months (95% confidence interval [CI]: 4.2-8.0) and 4-month PFS was 63% (95% CI: 46%-75%). Median OS was 12.6 months (95% CI: 8.5-16.4) and 1-year OS was 47%. Of the 47 patients evaluable for best response, none had a complete response, five (11%) had partial response, four (9%) had minor response, 22 (47%) had stable disease, and 16 (34%) had progressive disease. Analysis of serial serum samples obtained from 47 patients for four angiogenic peptides failed to show a significant correlation with response or survival for three of the peptides; higher vascular endothelial growth factor levels showed a trend toward correlation with decreased OS (p=0.07) and PFS (p=0.09). The addition of celecoxib and thalidomide to adjuvant temozolomide was well tolerated but did not meet the primary end point of improvement of 4-month PFS from study enrollment.

A novel method for interpretation of T-cell receptor gamma gene rearrangement assay by capillary gel electrophoresis based on normal distribution.

T-cell receptor gamma (TRG) gene rearrangement status is useful for the differential diagnosis of T-cell lesions. The BIOMED-2 protocol that uses two sets of Jgamma and four sets of Vgamma primers in a multiplex, two-tube reaction followed by capillary gel electrophoresis is emerging as a standard assay for this application. Here, we report a computer-aided method to evaluate the significance of a peak in this TRG clonality assay. A best-fit normal distribution (ND) curve and the chi(2) error for each peak are used to determine whether a peak is significantly taller than the background (cutoff for Vgamma(1-8) is 1). Eighty clinical samples that have been previously analyzed by a GC-clamped primer polymerase chain reaction/denaturing gradient gel electrophoresis assay were reanalyzed with the BIOMED-2 assay and scored by the ND method and four previously published methods: relative peak height (RPH), relative peak ratio (RPR), height ratio (HR), and peak height ratio (Rn). A greater than 90% concordance rate was observed between RPH and ND analysis, whereas RPR, Rn, and HR had a lower threshold to call a peak positive. The advantage of the ND method is that it is more objective, reproducible, and can be automated.

Laboratory practice guidelines for detecting and reporting JAK2 and MPL mutations in myeloproliferative neoplasms: a report of the Association for Molecular Pathology.

Recurrent mutations in JAK2 and MPL genes are genetic hallmarks of BCR-ABL1-negative myeloproliferative neoplasms. Detection of JAK2 and MPL mutations has been incorporated into routine diagnostic algorithms for these diseases. This Special Article summarizes results from a nationwide laboratory survey of JAK2 and MPL mutation analysis. Based on the current practice pattern and the literature, this Special Article provides recommendations and guidelines for laboratory practice for detection of mutations in the JAK2 and MPL genes, including clinical manifestations for prompting the mutation analysis, current and recommended methodologies for testing the mutations, and standardization for reporting the test results. This Special Article also points to future directions for genomic testing in BCR-ABL1-negative myeloproliferative neoplasms.

No evidence for interference of h&e staining in DNA testing: usefulness of DNA extraction from H&E-stained archival tissue sections.

Although histochemical staining has been believed to inhibit the DNA amplification reaction, no previous study has systematically evaluated the influence of histochemical staining on downstream molecular assays. To evaluate an influence of H&E staining on DNA testing, we isolated DNA from 10 unstained, 10 hematoxylin-stained, 10 eosin-stained, and 10 H&E-stained tissue sections (ie, 4 groups), from each of 5 colon cancers. Among the 4 groups, we did not observe any significant or appreciable difference in DNA fragmentation by agarose gel electrophoresis, in DNA amplification by real-time polymerase chain reaction (PCR), in microsatellite PCR fragment analyses, or in a PCR-pyrosequencing assay. As a proof-of-principle study, we successfully performed microsatellite instability analysis and sequencing of KRAS and BRAF on more than 1,300 colorectal cancers using DNA extracted from H&E-stained tissue sections. Our data provide no evidence for an interfering effect of H&E staining on DNA testing, suggesting that DNA from H&E-stained sections can be effectively used for routine DNA testing.

Molecular ontogeny of donor-derived follicular lymphomas occurring after hematopoietic cell transplantation.

UNLABELLED: The relative timing of genetic alterations that contribute to follicular lymphoma remains unknown. We analyzed a donor-recipient pair who both developed grade 2/3A follicular lymphoma 7 years after allogeneic transplantation and donor lymphocyte infusions. Both patients harbored identical BCL2/IGH rearrangements also present in 1 in 2,000 cells in the donor lymphocyte infusion, and the same V(D)J rearrangement, which underwent somatic hypermutation both before and after clonal divergence. Exome sequencing of both follicular lymphomas identified 15 shared mutations, of which 14 (including alterations in EP300 and KLHL6) were recovered from the donor lymphocyte infusion by ultra-deep sequencing (average read coverage, 361,723), indicating acquisition at least 7 years before clinical presentation. Six additional mutations were present in only one follicular lymphoma and not the donor lymphocyte infusion, including an ARID1A premature stop, indicating later acquisition during clonal divergence. Thus, ultrasensitive sequencing can map clonal evolution within rare subpopulations during human lymphomagenesis in vivo. SIGNIFICANCE: For the first time, we define the molecular ontogeny of follicular lymphoma during clonal evolution in vivo. By using ultrasensitive mutation detection, we mapped the time-course of somatic alterations after passage of a malignant ancestor by hematopoietic cell transplantation.

Translocation t(1;9) is a recurrent cytogenetic abnormality associated with progression of essential thrombocythemia patients displaying the JAK2 V617F mutation.

A cohort of 338 patients diagnosed with myeloproliferative neoplasms was investigated by conventional cytogenetics and evaluated for the presence of the JAK2 V617F mutation. A t(1;9)(p10;q10) in addition to two extra der(1;9)(q10;p10) chromosomes was observed in two patients of essential thrombocythemia that transformed to acute myelogenous leukemia or to myelofibrosis. These findings suggest that the presence of extra derivative chromosomes der(1q;9p) in combination with the JAK2 V617F mutation may play a role in the progression of myeloproliferative neoplasms and supports the use of cytogenetics in the follow-up of the disease.

Detection of KIAA1549-BRAF fusion transcripts in formalin-fixed paraffin-embedded pediatric low-grade gliomas.

Alterations of BRAF are the most common known genetic aberrations in pediatric gliomas. They frequently are found in pilocytic astrocytomas, where genomic duplications involving BRAF and the poorly characterized gene KIAA1549 create fusion proteins with constitutive B-Raf kinase activity. BRAF V600E point mutations are less common and generally occur in nonpilocytic tumors. The development of BRAF inhibitors as drugs has created an urgent need for robust clinical assays to identify activating lesions in BRAF. KIAA1549-BRAF fusion transcripts have been detected in frozen tissue, however, methods for FFPE tissue have not been reported. We developed a panel of FFPE-compatible quantitative RT-PCR assays for the most common KIAA1549-BRAF fusion transcripts. Application of these assays to a collection of 51 low-grade pediatric gliomas showed 97% sensitivity and 91% specificity compared with fluorescence in situ hybridization or array comparative genomic hybridization. In parallel, we assayed samples for the presence of the BRAF V600E mutation by PCR pyrosequencing. The data further support previous observations that these two alterations of the BRAF, KIAA1549 fusions and V600E point mutations, are associated primarily with pilocytic astrocytomas and nonpilocytic gliomas, respectively. These results show that fusion transcripts and mutations can be detected reliably in standard FFPE specimens and may be useful for incorporation into future studies of pediatric gliomas in basic science or clinical trials.

Florid reactive lymphoid hyperplasia of the lower female genital tract (lymphoma-like lesion): a benign condition that frequently harbors clonal immunoglobulin heavy chain gene rearrangements.

Lymphoma-like lesions (LLL) of the lower female genital tract are florid reactive inflammatory processes that mainly occur in women in their reproductive years. Histologically, they are characterized by a dense lymphoid infiltrate with admixed large cells that is often suspicious for lymphoma. In contrast to lymphoma, however, they are superficial lesions that typically show surface erosion and a mixed lymphoid infiltrate and do not have evidence of a mass, deep invasion, or prominent sclerosis. With the advent of widespread molecular genetic testing, it would seem that LLLs should be polyclonal helping make the correct diagnosis. However, we have found cases with morphologic and immunophenotypic features of LLLs and evidence of clonal rearrangement of the immunoglobulin heavy chain (IGH) gene, potentially leading to misdiagnosis. We examined the clinicopathologic features and outcome of 12 patients with LLL (9 in the cervix and 3 in the endometrium). The patients ranged in age from 18 to 54 (median 37) years and came to medical attention because of squamous dysplasia (8 patients), vaginal bleeding (3), or adnexal mass (1). One patient had an endocervical polyp, but otherwise none had a discrete mass. The specimens contained a dense, polymorphous inflammatory infiltrate, commonly associated with mucosal erosion. Immunohistochemical studies showed a mixture of B and T cells without immunoglobulin light chain restriction. Four cases (all cervical) had a clonal IGH gene rearrangement by PCR. There was no evidence of lymphoma on staging or on follow-up in any patient, including the 4 patients with clonal IGH rearrangement, after a mean follow-up of 3.5 years (range: 4 mo to 13 y). In summary, we describe 12 patients with LLL of the lower female genital tract. Four of the 9 cases (44%) analyzed by PCR had a clonal IGH gene rearrangement. The clinical and pathologic features of these cases suggest that a clonal IGH rearrangement in this setting does not warrant a diagnosis of lymphoma. Careful correlation of clinical, histologic, immunophenotypic, and genetic features is required to avoid misdiagnosis and inappropriate treatment. Routine microscopic findings and detailed clinical information remain paramount in establishing the correct diagnosis.

Characteristics of cutaneous marginal zone lymphomas with marked plasmacytic differentiation and a T cell-rich background.

Primary cutaneous marginal zone lymphoma (MZL) is a common B-cell lymphoma of skin and is characterized by an infiltrate of neoplastic marginal zone B cells typically within the marginal zones of reactive lymphoid follicles and the interfollicular region. However, in our experience, many cases have underemphasized features such as marked plasmacytic differentiation and/or a prominent T-cell component, which may obscure the neoplastic B cells and lead to misdiagnosis. We wanted to draw attention to these features and have studied 15 cases of MZL with marked plasmacytic differentiation, 10 of which had numerous T cells, some with cytologic atypia, and few B cells in the interfollicular region. Plasma cells were monotypic in all cases by in situ hybridization. By polymerase chain reaction, 6 of 8 T cell-rich cases had an IGH gene rearrangement, and none were clonal for T-cell receptor gene. We discuss the terminology, morphologic features, molecular profile, behavior, and differential diagnosis of cutaneous MZL.

Temozolomide treatment for aggressive pituitary tumors: correlation of clinical outcome with O(6)-methylguanine methyltransferase (MGMT) promoter methylation and expression.

CONTEXT: The typically indolent behavior of pituitary tumors is juxtaposed with high rates of tumor cell invasion into adjacent dural structures, and occasional aggressive behavior. Although clinically significant invasion and malignant transformation remain uncommon, there are limited treatment options available for the management of these aggressive tumors. Recently, case reports have described efficacy of temozolomide for the treatment of aggressive pituitary tumors. DESIGN: Seven patients with aggressive pituitary tumors have been treated with temozolomide. We compared O(6)-methylguanine methyltransferase (MGMT) promoter methylation and MGMT expression in 14 surgical specimens from these seven patients and correlated these molecular features with the clinical response to temozolomide. RESULTS: Significant tumor regression was seen in two patients (29%), a 20% reduction in tumor volume with subsequent stable tumor size was noted in one patient, arrest of tumor growth occurred in three patients, and progressive metastatic disease developed during treatment in one patient. The DNA promoter site for MGMT was unmethylated in all 14 adequate specimens, and variable MGMT expression was seen in all 14 cases. There was no correlation between MGMT expression and clinical outcomes. CONCLUSIONS: We conclude that medical therapy with temozolomide can be helpful in the management of life-threatening pituitary tumors that have failed to respond to conventional treatments. The optimal duration of treatment in patients with stabilization or reduction of tumor size has not been established, and long-term follow up studies are needed.

Lymphocyte depleted Hodgkin lymphoma: an evaluation with immunophenotyping and genetic analysis.

Lymphocyte depleted classical Hodgkin lymphoma (LDHL) is a vanishing category of classical Hodgkin lymphoma (CHL); many cases previously placed in this category are now recognised as diffuse large B-cell lymphoma (DLBCL), anaplastic large-cell lymphoma (ALCL), or nodular sclerosis CHL with lymphocyte depletion. In addition, the recent recognition of high grade B-cell lymphomas intermediate between DLBCL and CHL (grey-zone lymphomas) raises the question of whether LDHL exists at all as a category of CHL. We studied eight cases that fulfilled diagnostic criteria of LDHL according to the 2008 WHO Classification. The cases involved lymph nodes (7 cases) and pleura (1 case) from four males and four females (age 30-71 years; median 62 years). All tumors contained numerous Hodgkin-Reed-Sternberg (HRS) cells, fibroblasts and histiocytes and scattered lymphocytes. In three cases the tumors had a more diffuse fibrotic appearance, while in five cases they appeared reticular and anaplastic. Neoplastic cells in all cases expressed CD30, CD15, fascin, weak PAX5 and MUM-1 and lacked CD45, Alk-1, EMA, CD3, CD68, Mart-1 and cytokeratin. Oct.2 and/or Bob-1 were expressed in all cases. Two cases variably expressed CD20 but were CD79a negative. Four cases were positive for EBV. All the four cases with adequate DNA had clonally rearranged IGH genes. The combined morphologic, immunophenotypic and molecular genetic features of this group of cases distinguish LDHL from other disease entities, including grey-zone lymphomas.

Carcinoma and multiple lymphomas in one patient: establishing the diagnoses and analyzing risk factors.

Multiple malignancies may occur in the same patient, and a few reports describe cases with multiple hematologic and non-hematologic neoplasms. We report the case of a patient who showed the sequential occurrence of four different lymphoid neoplasms together with a squamous cell carcinoma of the lung. A 62-year-old man with adenopathy was admitted to the hospital, and lymph node biopsy was positive for low-grade follicular lymphoma. He achieved a partial remission with chemotherapy. Two years later, a PET-CT scan showed a left hilar mass in the lung; biopsy showed a squamous cell carcinoma. Simultaneously, he was diagnosed with diffuse large B cell lymphoma in a neck lymph node; after chemo- and radiotherapy, he achieved a complete response. A restaging PET-CT scan 2 years later revealed a retroperitoneal nodule, and biopsy again showed a low-grade follicular lymphoma, while a biopsy of a cutaneous scalp lesion showed a CD30-positive peripheral T cell lymphoma. After some months, a liver biopsy and a right cervical lymph node biopsy showed a CD30-positive peripheral T cell lymphoma consistent with anaplastic lymphoma kinase-negative anaplastic large cell lymphoma. Flow cytometry and cytogenetic and molecular genetic analysis performed at diagnosis and during the patient's follow-up confirmed the presence of two clonally distinct B cell lymphomas, while the two T cell neoplasms were confirmed to be clonally related. We discuss the relationship between multiple neoplasms occurring in the same patient and the various possible risk factors involved in their development.

CD20+ T-cell lymphoma: clinicopathologic analysis of 9 cases and a review of the literature.

Rare cases of CD20+ T-cell lymphoma (TCL) have been reported, but the clinicopathologic spectrum of this disorder is not known. We identified 9 cases of CD20+ TCL diagnosed at our institution and 26 additional cases through a search of the English language literature. Among current cases, there were 7 men (ages 71 to 81, median 75 y) and 2 women (ages 36 and 37 y). Five patients presented with predominantly nodal disease (localized in 3 and widespread in 2 cases) and 4 patients presented with purely extranodal disease involving the parotid glands, skin, or small intestine. CD20 was uniformly and strongly expressed in 5 cases and dimly expressed or present on a subset of neoplastic cells in 4 cases. The proportion of CD20+ T cells changed over time in 3 cases. Three cases fulfilled diagnostic criteria for clinicopathologically defined subtypes of TCL (2 mycosis fungoides; 1 enteropathy-type TCL), whereas 6 were peripheral TCL unspecified with variable cytomorphology, T-cell immunophenotype, and sites of involvement. In 8 of 9 cases, a clonal T-cell population was identified by molecular genetic analysis. Among 8 cases with clinical follow-up, 5 behaved aggressively with death from disease within 3 years of diagnosis in 4 cases (median survival: 11 mo, range: 1 to 35 mo), and recurrent disease at 10 months in 1 case; 1 patient died of an EBV+ B-cell lymphoma (BCL) 66 months after the original diagnosis; in the remaining 2 cases, patients were alive and undergoing treatment (follow-up: 4 and 18 mo). Historical cases showed similar clinicopathologic variability. CD20+ TCL is rare, and clinically and pathologically heterogeneous. When CD20 expression is present in TCL, it may be dimmer than that of normal B cells, suggesting neoplastic transformation of a normal CD20dim+ T-cell subset. Cases of CD20+ TCL in which the proportion of CD20+ cells changes over time may reflect aberrant expression of CD20, possibly as an activation marker, by neoplastic T cells. CD20+ TCL may cause diagnostic difficulty, particularly in cases that clinically and pathologically mimic BCL. Knowledge of the unusual phenomenon of CD20 expression in TCL, in conjunction with careful morphologic analysis, the use of a panel of antibodies, and molecular genetic studies, is important in avoiding a misdiagnosis of BCL.

PIK3CA mutation in colorectal cancer: relationship with genetic and epigenetic alterations.

Somatic PIK3CA mutations are often present in colorectal cancer. Mutant PIK3CA activates AKT signaling, which up-regulates fatty acid synthase (FASN). Microsatellite instability (MSI) and CpG island methylator phenotype (CIMP) are important molecular classifiers in colorectal cancer. However, the relationship between PIK3CA mutation, MSI and CIMP remains uncertain. Using Pyrosequencing technology, we detected PIK3CA mutations in 91 (15%) of 590 population-based colorectal cancers. To determine CIMP status, we quantified DNA methylation in eight CIMP-specific promoters [CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1] by real-time polymerase chain reaction (MethyLight). PIK3CA mutation was significantly associated with mucinous tumors [P = .0002; odds ratio (OR) = 2.44], KRAS mutation (P < .0001; OR = 2.68), CIMP-high (P = .03; OR = 2.08), phospho-ribosomal protein S6 expression (P = .002; OR = 2.19), and FASN expression (P = .02; OR = 1.85) and inversely with p53 expression (P = .01; OR = 0.54) and beta-catenin (CTNNB1) alteration (P = .004; OR = 0.43). In addition, PIK3CA G-to-A mutations were associated with MGMT loss (P = .001; OR = 3.24) but not with MGMT promoter methylation. In conclusion, PIK3CA mutation is significantly associated with other key molecular events in colorectal cancer, and MGMT loss likely contributes to the development of PIK3CA G>A mutation. In addition, Pyrosequencing is useful in detecting PIK3CA mutation in archival paraffin tumor tissue. PIK3CA mutational data further emphasize heterogeneity of colorectal cancer at the molecular level.