Lynne V. Abruzzo

Lymphoid neoplasms associated with concurrent t(14;18) and 8q24/c-MYC translocation generally have a poor prognosis.

We identified 14 B-cell neoplasms with concurrent t(14;18) and chromosome 8q24 or c-MYC translocations shown by conventional cytogenetics or fluorescence in situ hybridization analysis. All cases assessed by conventional cytogenetics had a complex karyotype. There were 10 men and four women, with a median age of 55 years (range, 29–72). None of these patients had a history of follicular lymphoma. The biopsy specimens were obtained from bone marrow, lymph node, and extranodal sites. Morphologically, nine neoplasms had features of Burkitt or atypical Burkitt lymphoma/leukemia and three were diffuse large B-cell lymphoma with high-grade cytologic features. The remaining two cases were plasmablastic myeloma and low-grade B-cell lymphoma, respectively. All cases expressed BCL-2. The proliferation index assessed by using Ki-67 (MIB1) was 5% in the low-grade B-cell lymphoma, 80% in the plasmablastic myeloma, 90–95% in three cases of diffuse large B-cell lymphoma, and ranged from 90 to >99% in most Burkitt and atypical Burkitt neoplasms. The patient with low-grade B-cell lymphoma was treated with rituximab. All other patients received intensive combination chemotherapy. Two of these patients underwent bone marrow transplantation, and one patient received radiation therapy in addition to transplantation. The median follow-up period was 9 months (range, 3–81). In all, 10 patients died with a median survival of 9 months (range, 3–81). We conclude that most B-cell lymphomas with concurrent t(14;18) and 8q24/c-MYC translocations fall within the morphologic spectrum of diffuse large B-cell and Burkitt lymphoma. These neoplasms are high-grade and are associated with a poor prognosis. However, this combination of molecular abnormalities can also rarely occur in other neoplasms, such as the cases of low-grade B-cell lymphoma and plasmablastic myeloma in this study.

De novo deletion 17p13.1 chronic lymphocytic leukemia shows significant clinical heterogeneity: the M. D. Anderson and Mayo Clinic experience

To determine the clinical fate of patients with de novo deletion 17p13.1 (17p-) chronic lymphocytic leukemia (CLL), we retrospectively studied the outcome of 99 treatment-naive 17p- CLL patients from the M. D. Anderson Cancer Center (n = 64) and the Mayo Clinic (n = 35). Among 67 asymptomatic patients followed for progression, 53% developed CLL requiring treatment over 3 years. Patients who had not progressed by 18 months subsequently had stable disease, with 3 of 19 patients progressing after follow-up of up to 70 months. Risk factors for progressive disease were Rai stage of 1 or higher and unmutated immunoglobulin variable region heavy chain (IgVH). The overall survival rate was 65% at 3 years. Rai stage 1 or higher, unmutated IgVH, and 17p- in 25% or more of nuclei were adverse factors for survival. The 3-year survival rates of patients with 1 or fewer, 2, and 3 of these factors were 95%, 74%, and 22%, respectively (P < .001). Response rates to therapy with rituximab (n = 6); purine analogues and rituximab (n = 25); and purine analogues, rituximab, and alemtuzumab (n = 16) combinations were 50%, 72%, and 81%, respectively. Patients with 17p- CLL exhibit clinical heterogeneity, with some patients experiencing an indolent course. Survival can be predicted using clinical and biologic characteristics.

Mutational profile and prognostic significance of TP53 in diffuse large B-cell lymphoma patients treated with R-CHOP: Report from an International DLBCL Rituximab-CHOP Consortium Program Study

TP53 mutation is an independent marker of poor prognosis in patients with diffuse large B-cell lymphoma (DLBCL) treated with cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone (CHOP) therapy. However, its prognostic value in the rituximab immunochemotherapy era remains undefined. In the present study of a large cohort of DLBCL patients treated with rituximab plus CHOP (R-CHOP), we show that those with TP53 mutations had worse overall and progression-free survival compared with those without. Unlike earlier studies of patients treated with CHOP, TP53 mutation has predictive value for R-CHOP-treated patients with either the germinal center B-cell or activated B-cell DLBCL subtypes. Furthermore, we identified the loop-sheet-helix and L3 motifs in the DNA-binding domain to be the most critical structures for maintaining p53 function. In contrast, TP53 deletion and loss of heterozygosity did not confer worse survival. If gene mutation data are not available, immunohistochemical analysis showing > 50% cells expressing p53 protein is a useful surrogate and was able to stratify patients with significantly different prognoses. We conclude that assessment of TP53 mutation status is important for stratifying R-CHOP-treated patients into distinct prognostic subsets and has significant value in the design of future therapeutic strategies.

Identifying differentially expressed genes in cDNA microarray experiments

A major goal of microarray experiments is to determine which genes are differentially expressed between samples. Differential expression has been assessed by taking ratios of expression levels of different samples at a spot on the array and flagging spots (genes) where the magnitude of the fold difference exceeds some threshold. More recent work has attempted to incorporate the fact that the variability of these ratios is not constant. Most methods are variants of Students t-test. These variants standardize the ratios by dividing by an estimate of the standard deviation of that ratio; spots with large standardized values are flagged. Estimating these standard deviations requires replication of the measurements, either within a slide or between slides, or the use of a model describing what the standard deviation should be. Starting from considerations of the kinetics driving microarray hybridization, we derive models for the intensity of a replicated spot, when replication is performed within and between arrays. Replication within slides leads to a beta-binomial model, and replication between slides leads to a gamma-Poisson model. These models predict how the variance of a log ratio changes with the total intensity of the signal at the spot, independent of the identity of the gene. Ratios for genes with a small amount of total signal are highly variable, whereas ratios for genes with a large amount of total signal are fairly stable. Log ratios are scaled by the standard deviations given by these functions, giving model-based versions of Studentization. An example is given.

Differences in gene expression between B-cell chronic lymphocytic leukemia and normal B cells: a meta-analysis of three microarray studies

MOTIVATION A major focus of current cancer research is to identify genes that can be used as markers for prognosis and diagnosis, and as targets for therapy. Microarray technology has been applied extensively for this purpose, even though it has been reported that the agreement between microarray platforms is poor. A critical question is: how can we best combine the measurements of matched genes across microarray platforms to develop diagnostic and prognostic tools related to the underlying biology? RESULTS We introduce a statistical approach within a Bayesian framework to combine the microarray data on matched genes from three investigations of gene expression profiling of B-cell chronic lymphocytic leukemia (CLL) and normal B cells (NBC) using three different microarray platforms, oligonucleotide arrays, cDNA arrays printed on glass slides and cDNA arrays printed on nylon membranes. Using this approach, we identified a number of genes that were consistently differentially expressed between CLL and NBC samples.

Validation of oligonucleotide microarray data using microfluidic low-density arrays: a new statistical method to normalize real-time RT-PCR data

Profiling studies using microarrays to measure messenger RNA (mRNA) expression frequently identify long lists of differentially expressed genes. Differential expression is often validated using real-time reverse transcription PCR (RT-PCR) assays. In conventional real-time RT-PCR assays, expression is normalized to a control, or housekeeping gene. However, no single housekeeping gene can be used for all studies. We used TaqMan Low-Density Arrays, a medium-throughput method for real-time RT-PCR using microfluidics to simultaneously assay the expression of 96 genes in nine samples of chronic lymphocytic leukemia (CLL). We developed a novel statistical method, based on linear mixed-effects models, to analyze the data. This method automatically identifies the genes whose expression does not vary significantly over the samples, allowing them to be used to normalize the remaining genes. We compared the normalized real-time RT-PCR values with results obtained from Affymetrix Hu133A GeneChip oligonucleotide microarrays. We found that real-time RT-PCR using TaqMan Low-Density Arrays yielded reproducible measurements over seven orders of magnitude. Our model identified numerous genes that were expressed at nearly constant levels, including the housekeeping genes PGK1, GAPD, GUSB, TFRC, and 18S rRNA. After normalizing to the geometric mean of the unvarying genes, the correlation between real-time RT-PCR and microarrays was high for genes that were moderately expressed and varied across samples.

Immunohistochemical detection of ZAP-70 in 341 cases of non-Hodgkin and Hodgkin lymphoma

Using immunohistochemical methods, we evaluated zeta-associated protein (ZAP)-70 expression in 341 cases of non-Hodgkin and Hodgkin lymphoma. In B-cell NHL, ZAP-70 was positive in five of six (83%) precursor B-lymphoblastic lymphoma, 11 of 37 (30%) chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), five of 39 (13%) mantle cell lymphoma, one of 12 (8%) Burkitt lymphoma, and one of 12 (8%) nodal marginal zone B-cell lymphoma. In 22 cases of CLL/SLL, seven of nine (78%) with unmutated IgVH genes expressed ZAP-70, compared with one of 13 (8%) with mutated IgVH genes (P=0.0015 Fishers exact test). ZAP-70 expression was not detected in diffuse large B-cell lymphoma (n=26), extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (n=24), follicular lymphoma (n=21), plasma cell myeloma/plasmacytoma (n=10), lymphoplasmacytic lymphoma (n=10), or splenic marginal zone lymphoma (n=6). In T/NK-cell NHL, ZAP-70 was positive in all extranodal natural killer (NK) / T-cell lymphoma, nasal-type (n=6) and enteropathy-type T-cell lymphoma (n=4), four of five (80%) subcutaneous panniculitis-like T-cell lymphoma, six of eight (75%) mycosis fungoides, three of five (60%) precursor T-lymphoblastic lymphoma, 10 of 17 (59%) peripheral T-cell lymphoma, two of four (50%) blastic NK-cell lymphoma, one of three (33%) T-cell prolymphocytic leukemia, 13 of 52 (25%) anaplastic large cell lymphoma, and one of six (17%) angioimmunoblastic T-cell lymphoma. Seven of 12 (58%) cutaneous CD30-positive lymphoproliferative disorders were also ZAP-70-positive. In Hodgkin lymphoma, ZAP-70 was negative in neoplastic cells in all cases tested. ZAP-70 staining in B-cell lymphomas and reactive T cells was predominantly nuclear with variable cytoplasmic staining. By contrast, ZAP-70 staining in T/NK-cell lymphomas was heterogeneous, and a shift from predominantly nuclear to predominantly cytoplasmic staining was observed, particularly in those neoplasms with high-grade morphology. In summary, ZAP-70 is expressed by many lymphoma types, correlates with immunoglobulin heavy-chain variable region gene mutational status in CLL/SLL, and can be detected reliably using immunohistochemical methods.

Hepatosplenic gamma/delta T-Cell Lymphoma in Bone Marrow A Sinusoidal Neoplasm With Blastic Cytologic Features

We report 8 cases of hepatosplenic T-cell lymphoma (HSTCL) involving bone marrow and correlate histologic findings with disease progression. Immunophenotypic analysis demonstrated mature, aberrant gamma/delta T-cell immunophenotypes. Isochromosome 7q was identified in 4 cases; 1 case showed the t(7;14)(q34;q13). Seven of 7 cases tested had monoclonal TCR gamma gene rearrangements. The initial diagnostic bone marrow biopsy specimens were hypercellular with a frequently subtle, predominantly sinusoidal infiltrate of atypical small to medium-sized lymphoid cells. In all cases, aspirate smears at diagnosis and in subsequent specimens contained malignant cells that resembled blasts, some with fine cytoplasmic granules. With progression, the pattern of HSTCL in bone marrow biopsy specimens became increasingly interstitial, and the neoplastic cells became larger. In aspirate smears, the proportion of blasts increased. Seven patients died; 1 was lost to follow-up. Autopsy performed on 1 patient demonstrated malignant cells within vascular channels in all organs sampled, with relatively little tumor formation, resembling intravascular lymphoma at these sites. HSTCL often can be recognized in bone marrow by its unique combination of a sinusoidal pattern in core biopsy specimens and blastic cytology in aspirate smears.

Clonal evolution in patients with chronic lymphocytic leukaemia developing resistance to BTK inhibition

Resistance to the Brutons tyrosine kinase (BTK) inhibitor ibrutinib has been attributed solely to mutations in BTK and related pathway molecules. Using whole-exome and deep-targeted sequencing, we dissect evolution of ibrutinib resistance in serial samples from five chronic lymphocytic leukaemia patients. In two patients, we detect BTK-C481S mutation or multiple PLCG2 mutations. The other three patients exhibit an expansion of clones harbouring del(8p) with additional driver mutations (EP300, MLL2 and EIF2A), with one patient developing trans-differentiation into CD19-negative histiocytic sarcoma. Using droplet-microfluidic technology and growth kinetic analyses, we demonstrate the presence of ibrutinib-resistant subclones and estimate subclone size before treatment initiation. Haploinsufficiency of TRAIL-R, a consequence of del(8p), results in TRAIL insensitivity, which may contribute to ibrutinib resistance. These findings demonstrate that the ibrutinib therapy favours selection and expansion of rare subclones already present before ibrutinib treatment, and provide insight into the heterogeneity of genetic changes associated with ibrutinib resistance.

Expression of Mcl-1 in mantle cell lymphoma is associated with high-grade morphology, a high proliferative state, and p53 overexpression

Mantle cell lymphoma (MCL) is a distinct type of B‐cell non‐Hodgkins lymphoma characterized by the t(11;14)(q13;q32) and cyclin D1 overexpression. Defects in apoptosis may contribute to pathogenesis. This study evaluated the expression of the anti‐apoptotic protein Mcl‐1 in two MCL cell lines and five frozen MCL tumours (four small‐cell, one blastoid/large‐cell) using western blot analysis. Mcl‐1 expression was also assessed in 36 formalin‐fixed, paraffin wax‐embedded MCL tumours (24 small‐cell, 12 blastoid/large‐cell) by immunohistochemistry. Western blot analysis revealed the expected 37 kD protein product in both MCL cell lines and in five frozen tumours, with the blastoid case having the highest expression level. Using a cut‐off of >10% immunolabelled cells for Mcl‐1, it was found that 12 of 36 MCL tumours were positive. Mcl‐1‐positive tumours had a higher frequency of blastoid/large‐cell morphology (8/12 versus 4/24, p = 0.009), p53 overexpression (3/10 versus 1/23, p = 0.04), and higher Ki67 immuno‐labelling (p = 0.002). It is concluded that expression of Mcl‐1 in MCL is heterogeneous. A relatively high level of Mcl‐1 expression correlates with high‐grade morphology, a high proliferative state, and p53 overexpression. Copyright