Panel sequencing for clinically oriented variant screening and copy number detection in 142 untreated multiple myeloma patients.

We employed a customized Multiple Myeloma (MM)-specific Mutation Panel (M(3)P) to screen a homogenous cohort of 142 untreated MM patients for relevant mutations in a selection of disease-specific genes. M(3)Pv2.0 includes 77 genes selected for being either actionable targets, potentially related to drug-response or part of known key pathways in MM biology. We identified mutations in potentially actionable genes in 49% of patients and provided prognostic evidence of STAT3 mutations. This panel may serve as a practical alternative to more comprehensive sequencing approaches, providing genomic information in a timely and cost-effective manner, thus allowing clinically oriented variant screening in MM.

Hepatitis C virus and lymphoma.

Hepatitis C virus (HCV) is well known for its aetiological role in chronic non-A, non-B viral hepatitis, liver cirrhosis and hepatocellular carcinoma; in addition, the virus has also been implicated in a number of extra-hepatic "autoimmune" disease manifestations. A causative association between HCV and non-Hodgkin lymphoma (NHL) was postulated relatively recently and has been the subject of intense investigation, as well as some debate. On the strength of epidemiological data, emerging biological investigations and clinical observations, HCV appears to be involved in the pathogenesis of at least a proportion of patients with NHL. Morphologically, HCV-associated lymphomas represent a variety of histological subtypes including marginal zone lymphoma (splenic, nodal and extranodal), small lymphocytic lymphoma/chronic lymphocytic leukaemia, lymphoplasmacytic lymphoma and diffuse large B-cell lymphoma. Remarkably, some HCV-associated NHL appears to be highly responsive to antiviral therapy, providing some clinical evidence for this relationship, as well as the prospect for novel therapeutic intervention.

Minimal residual disease detection in childhood precursor-B-cell acute lymphoblastic leukemia: relation to other risk factors. A Children's Oncology Group study.

Minimal residual disease (MRD) can be detected in the marrows of children undergoing chemotherapy either by flow cytometry or polymerase chain reaction. In this study, we used four-color flow cytometry to detect MRD in 1016 children undergoing therapy on Children's Oncology Group therapeutic protocols for precursor-B-cell ALL. Compliance was excellent, with follow-up samples received at the end of induction on nearly 95% of cases; sensitivity of detection at this time point was at least 1/10,000 in more than 90% of cases. Overall, 28.6% of patients had detectable MRD at the end of induction. Patients with M3 marrows at day 8 were much more likely to be MRD positive (MRD+) than those with M2 or M1 marrows. Different genetically defined groups of patients varied in their prevalence of MRD. Specifically, almost all patients with BCR-ABL had high levels of end-of-induction MRD. Only 8.4% of patients with TEL-AML1 were MRD+>0.01% compared with 20.3% of patients with trisomies of chromosomes 4 and 10. Our results show that MRD correlates with conventional measures of slow early response. However, the high frequency of MRD positivity in favorable trisomy patients suggests that the clinical significance of MRD positivity at the end of induction may not be the same in all patient groups.

Rapid detection of leukemia-associated translocation fusion genes using a novel combined RT-PCR and flow cytometric method.

Efficient detection of recurrent translocation-associated fusion genes is of critical importance for the diagnosis, prognosis and post-therapeutic monitoring of many leukemias. Typically, the presence of such translocations is revealed by RT-PCR technique, followed by Southern blot hybridization to ensure specificity of the PCR product. Though widely employed, post-PCR analysis of this type is relatively laborious and time-intensive. As a departure from standard analytic approaches, we have developed a robust novel method combining both high specificity and sensitivity, based on polystyrene bead capture of fluorescently labeled PCR products, with subsequent analysis by flow cytometry. Results from cell line and patient sample evaluations indicate that this method may be easily incorporated into the diagnostic molecular laboratory as a rapid and cost-effective alternative to currently employed techniques.

Detection of t(15;17)(q24;q21), inv(16)/t(16;16)(p13;q22), and t(8;21)(q22;q22) Anomalies in Acute Myeloid Leukemias.

The acute myeloid leukemias (AMLs) are a relatively heterogeneous group of diseases. However, there is growing awareness that the clinical features and subclassification of morphologic leukemia types is often highly correlated with tumor genetics. Furthermore, distinct genetic subgroups of AML are associated with improved therapeutic sensitivity and a more favorable clinical outcome. These observations have prompted suggestions for a revision of the current French-American-British leukemia classification (1), utilizing genetically defined principles (2). Three recurrent chromosomal translocations are identified in approx 25-30% of de novo adult AMLs. These include the t(15;17), associated with acute promyelocytic leukemia ([APL]; AML-M3); the inv(16) and related t(16;16), associated with AML-M4Eo; and the t(8;21), associated most commonly with AML-M2. Each of these abnormalities results in the formation of a chimeric leukemia-specific fusion gene, which is transcribed and expressed as a fusion protein. The widespread genetic deregulation caused by such fusion proteins is thought to interfere with proliferative control and cell differentiation mechanisms, leading to the leukemic state. The presence of these and other fusion gene events can be specifically and sensitively detected by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis.

Detection of t(14; 18)(q32;q21)-Associated BCL-2/J(H) Gene Fusion in Non-Hodgkin Lymphoma.

The identification and study of nonrandom recurrent chromosomal translocations has substantially increased our understanding of the non-Hodgkin lymphomas. Cytogenetic and molecular genetic data now form an integral part of current lymphoma classifications (1) and provide important information for diagnosis, tumor biology, and in some cases prognosis. The t(14;18)(q32;q21) abnormality is the most common translocation detected in B-lineage lymphoma and results in juxtaposition of the BCL-2 gene (18q21) and the JH locus of the immunoglobulin (Ig) heavy chain gene (14q32) (2-5). More specifically, in the North American population, alterations of the BCL-2 gene are detected in approx 75 to 85% of low-grade follicular lymphomas, 20-30% of aggressive large B-cell lymphomas, and rarely in other B-cell tumors (e.g., chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia) (2,6-9). As a consequence of the BCL-2/JH fusion, deregulated overexpression of the antiapoptotic bcl-2 protein occurs owing to constitutive transcriptional activation of the BCL-2 gene by the Ig heavy chain gene enhancer. The unbridled expression of bcl-2 protein in lymphoid tumors confers resistance to programmed cell death (10,11) and is implicated in primary therapeutic failure and a less favorable prognosis (12-14). Although karyotypic detection of lymphoma-associated translocations such as the t(14;18) has proved to be useful in disease diagnosis and subcategorization, molecular genetic approaches including polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) have gained substantial popularity owing to their rapidity, relatively low cost, and increased sensitivity (6,15-22).

Utility of immunohistochemistry in bone marrow evaluation of T-lineage large granular lymphocyte leukemia.

Although T-lineage large granular lymphocyte (LGL) leukemia has been described for over 20 years, many patients with this neoplasm go unrecognized. Chief among the difficulties in diagnosing this entity is that the morphologic features are nonspecific and that it is difficult to distinguish it from reactive processes. The purpose of this study was to examine the histologic and immunophenotypic appearance of T-LGL leukemia in the peripheral blood and bone marrow, and to determine what features may suggest that ancillary studies such as flow cytometric and molecular analysis should be pursued to make a definitive diagnosis. We took a multidisciplinary approach by using morphology, immunoperoxidase staining, flow cytometric analysis, and molecular studies on 9 cases of T-lineage LGL leukemia. Our findings indicate that T-lineage LGL leukemia typically infiltrates the marrow diffusely. Most cases show a hypercellular marrow with an increase in myeloid precursors relative to the mature cells (i.e., an inversion of the myeloid maturation pyramid) and a decreased myeloid:erythroid ratio. Neutropenia without a left shift is usually seen in the peripheral blood. The tumor cells are usually CD3+, CD8+, CD57+, and TIA-1+. Most notably, the number of CD3+ T cells per high-power field is markedly elevated in T-LGL leukemia compared with normal, reactive, and pathologic marrows with neutropenia (mean values, 559 cells/mm(2) v. 7/mm(2), 11/mm(2), and 263/mm(2), respectively, P<.01). Moreover, CD57 staining also shows an increase in positive cells in T-LGL cases in comparison with normal, reactive, and pathologic marrows with neutropenia. Taken together, these findings indicate immunoperoxidase findings may be a useful tool to identify cases that should proceed to molecular or flow cytometric analysis.

Comparison of capillary electrophoresis and polyacrylamide gel electrophoresis for the evaluation of T and B cell clonality by polymerase chain reaction.

Polymerase chain reaction (PCR) technique is widely used in the diagnosis of lymphoma, and PCR amplification products are typically detected by polyacrylamide gel electrophoresis (PAGE). However, the identification of small clonal populations, or the distinction of clonal PCR products in a polyclonal milieu remains difficult, requiring technically demanding alterations to gel analysis. This study describes an alternative approach using a capillary electrophoresis (CE) system to produce an accurately sized electropherogram. A variety of patient samples were examined, including solid tissue, peripheral blood, bone marrow aspirates, and paraffin-embedded tissue. A total of 28 samples were evaluated by PCR for B-cell clonality by detection of immunoglobulin heavy chain gene rearrangement and 29 samples for T-cell clonality by detection of T-cell gamma locus gene rearrangement. Standard 10% PAGE analysis of PCR products was compared with CE. There was a 100% concordance in the assessment of both B-cell and T-cell clonality. Dilution studies with the SUP-B15 cell line showed a detection limit of 0.03% for B-cell clonality and 0.05% for T-cell clonality using CE, versus 0.2% to 1%, respectively for PAGE. Automated, fluorescent analysis of PCR products by CE seems to be at least equally as effective as gel-based analysis for the detection of clonal B-cell and T-cell populations. Moreover. CE offers superior resolution and improved sensitivity, thus representing a significant improvement over traditional gel electrophoretic techniques in these regards.

Blastic mantle cell leukemia: an unusual presentation of blastic mantle cell lymphoma.

Six patients had blood and bone marrow manifestations characterized by the presence of morphologically immature or blastic B-lineage lymphoid cells expressing CD5 antigen. The median patient age was 70 years, and the male-to-female ratio was 5:1. The presence or degree of lymphadenopathy and splenomegaly was variable among this group at staging evaluation, although two patients did not have these features. One patient had an antecedent diagnosis of classical nodal mantle cell lymphoma, without prior morphologic blood or bone marrow involvement. Other patients lacked a history of underlying lymphoproliferative disorders. The median white blood cell count was 120 x 10(9)/L. Most patients had thrombocytopenia, whereas only one patient had neutropenia at presentation. Leukemic peripheral blood cells in these six cases were small to medium in size with fine or granular nuclear chromatin and small or inconspicuous nucleoli. The pattern of marrow involvement was interstitial or diffuse, with cells showing immature nuclear features resembling acute leukemia or blastic lymphoma. All tumors demonstrated a consistent immunophenotype of B-cell lineage, surface immunoglobulin positivity, and CD5 antigen expression. The progenitor cell-associated markers CD34 and TdT were not expressed, and CD23 antigen was either negative (three of four cases) or only weakly present (one of four cases). The presence of a karyotypic t(11;14)(q13;q32) was documented in one tumor, whereas two other cases had BCL-1 gene rearrangements by either polymerase chain reaction or Southern blot analysis. Cyclin D1 mRNA overexpression was noted in three of four cases tested. This patient group was characterized by very poor overall survival (median, 3 months; range, 0.5 to 6 months). The aggregate clinical, pathologic, and genetic data in these unusual cases are consistent with de novo or predominant leukemic presentations of blastic mantle cell lymphoma. Accurate diagnosis in such cases is greatly facilitated by cytogenetic studies or the demonstration of BCL-1/cyclin D1 abnormalities.

Patient and tumor characteristics of colon cancers with microsatellite instability: a population-based study.

Molecular screening for microsatellite instability (MSI) in colon cancers has been proposed to identify individuals with hereditary nonpolyposis colorectal cancer. To date, most reports of MSI in colorectal cancer have been based on studies of clinical case series or high-risk families. We examined the proportion of incident colon cancers in the general population that exhibit MSI by patient and tumor characteristics. We interviewed 201 colon cancer cases ascertained by the New Mexico Tumor Registry in the metropolitan Albuquerque area for demographic information, lifestyle factors, medical history, and family cancer history. Paired normal and tumor tissue specimens were obtained for each case. Three microsatellite markers were used; instability was defined as observed alteration at two or more loci. Overall, 37 of 201 (18%) colon cancers exhibited instability. MSI was more common among cases >70 years (26%) and most common among cases >80 years (38%). MSI was significantly associated with tumors in the proximal colon and with later stage and poor differentiation among cases >70 years. MSI was not associated with a history of polyps. Family history of colorectal cancer was associated with MSI only among cases <50 years. When all factors were analyzed jointly in a regression model, proximal subsite and poor differentiation remained significantly associated with MSI. One patient, whose tumor exhibited MSI, fulfilled the Amsterdam Criteria for hereditary nonpolyposis colorectal cancer. Our study provides a population-based estimate of MSI in colon tumors and a representative estimate of the proportion of colorectal cancer patients in the general population who consent to be interviewed for family cancer history and to have biological samples analyzed.

Molecular diagnosis in pediatric acute leukemias.

This article summarizes the tremendous progress currently achieved in understanding the molecular basis of the pediatric acute leukemias. The article is organized from the perspective of the most frequently encountered pediatric acute leukemia genetic abnormalities in a molecular diagnostics laboratory setting. For each specific entity, the basic molecular biology, putative mechanisms of leukemogenesis, detection methods, and clinical significance are reviewed. Emphasis is placed on discussing the fusion genes generated from common nonrandom chromosomal translocations in B-lineage acute lymphoblastic leukemia (ALL), although brief summaries of T-lineage and myeloid leukemia, as well as the use of the antigen receptor gene rearrangement for residual disease monitoring in acute lympocytic leukemia are also presented. Finally, an overview of emerging technologies of potential importance in the laboratory diagnosis and evaluation of the pediatric acute leukemias is provided.

Molecular analysis and clinical outcome of adult APL patients with the type V PML-RARalpha isoform: results from intergroup protocol 0129.

The type V (for variable) promyelocytic leukemia retinoic acid receptor (PML-RAR)alpha transcript, found in approximately 8% of adult patients with acute promyelocytic leukemia (APL), is defined molecularly by truncation of PML exon 6 and frequent insertion of genetic material from RARalpha intron 2. To more fully characterize the molecular features of PML-RARalpha V-type transcripts and to determine whether V-form APL patients have a distinct clinical presentation or prognosis, we analyzed 18 adult V-form APL patients enrolled on Intergroup protocol 0129 (INT-0129). Truncations in PML exon 6 ranged from 8 to 146 nucleotides, and 3 to 127 extra nucleotides (1 to 42 extra amino acids) were inserted at the PML exon 6/RARalpha exon 3 junction in 13 cases. No distinguishing morphologic, cytogenetic, or immunophenotypic features of V-form blasts were identified. A total of 5 of 7 patients induced with ATRA and 8 of 11 patients who received chemotherapy for induction achieved complete remission (CR). Six patients have relapsed, 4 after chemotherapy induction and 2 after ATRA. Nine patients (50%) are alive, 6 in continuous CR, 2 after salvage therapy for relapsed or refractory disease, and 1 after alternative treatment following early removal from protocol. Although the failure rate for V-form APL patients was high (61%), the low power of the current study to detect clinically significant differences precludes a meaningful comparison of clinical outcomes between the 18 V-form cases and non-V-form adult APL patients enrolled on INT-0129. (Blood. 2000;95:398-403)

Primary non-Hodgkin's lymphoma and malakoplakia of the vagina: a case report.

The vagina is a rare site for both primary non-Hodgkin's lymphoma and malakoplakia. We report a case of concurrent diffuse large B-cell lymphoma and malakoplakia of the vagina in a 67-year-old woman presenting with a vaginal discharge and a vaginal mass. The patient had two biopsy specimens reported as showing malakoplakia only, followed by a third biopsy specimen 10 months later which was diagnosed as diffuse large B-cell lymphoma. Review of the first two biopsy specimens showed areas of histiocytes with Michaelis-Gutman bodies merging with areas of cells with slightly larger nuclei and more amphophilic cytoplasm. Immunohistochemistry for the B-cell marker L-26 (CD20) and polymerase chain reaction analysis of the immunoglobulin heavy chain gene were helpful in retrospectively distinguishing the population of diffuse large B-cell lymphoma from the areas of malakoplakia. The third biopsy specimen showed sheets of large atypical lymphoid cells characteristic of a large cell lymphoma. Malakoplakia has been described in association with a variety of cancers, and this is only the second report of malakoplakia associated with non-Hodgkin's lymphoma. Considering the rarity of these two entities in the vagina, it is unlikely that the association in this case is coincidental, raising the possibilities of an unusual reaction to the presence of lymphoma or a common pathogenesis such as underlying chronic inflammation. Epstein-Barr virus DNA was detected in the second biopsy specimen, suggesting a possible role in the pathogenesis of this lymphoma.

Incidence of second neoplasms in patients with MALT lymphoma: no increase in risk above the background population.

BACKGROUND: Lymphomas of mucosa associated lymphoid tissue (MALT) are a special type of extranodal lymphoma, possibly related to chronic antigenic stimulation. Increased cancer susceptibility may also contribute to the development of MALT lymphoma (MALToma). It has been suggested that patients with MALToma have an increased incidence of other malignancies. PATIENTS AND METHODS: We retrospectively reviewed the histology and clinical records of 147 patients with MALToma, including 51 cases of gastric MALToma. The incidence of any second malignancy was confirmed with a provincial registry. The relative rates of cancer, excluding MALToma, were calculated relative to the background population of the same age group and secular year. RESULTS: A total of 41 tumors occurred in 32 patients (21%), including 22 solid tumors. The incidence of solid tumors in the gastric MALToma group was 15%. Seven patients had two or more second malignancies. Cancer occurred before diagnosis of MALToma in 29 cases, concurrent with MALToma in three, and after MALToma in nine. Follow-up of the surviving patients is short (median 17.6 months). The relative rate from birth of a second malignancy was 0.86 in the whole group (90% confidence interval (CI): 0.62-1.16) and 0.95 (90% CI: 0.55-1.54) in the gastric MALToma group. The rates were roughly the same if skin cancers were excluded. CONCLUSIONS: The incidence of second cancers in this series is similar to previous reports. However, when compared to an age-matched population followed for the same period of time, MALToma patients do not appear to have a statistically significant increased rate of cancers.

Prognostic significance of anaplastic lymphoma kinase (ALK) protein expression in adults with anaplastic large cell lymphoma.

Anaplastic large cell lymphoma (ALCL) is an aggressive lymphoma that is frequently associated with the t(2;5)(p23;q35), resulting in expression of a fusion protein, nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), which can be detected by either monoclonal or polyclonal antibodies to the ALK protein. The clinical features of adults with ALCL are incompletely described, and the prognostic factors that are useful for predicting survival remain unclear. This report describes the clinical and laboratory findings in 70 adults with systemic ALCL who were treated with curative intent. We attempted to identify the clinical and pathological factors of prognostic importance, including the International Prognostic Index (IPI), immunophenotype, and expression of the ALK protein. The median age of the patients was 49 years (range, 15 to 75). There were 26 women and 44 men with a median follow-up of 50 months for living patients. Advanced stage was present in 56% and B symptoms were noted in 70% of the patients. Immunostains showed that 46% of the cases had a T-cell phenotype, 36% a null phenotype, and 18% a B-cell phenotype. The expression of ALK protein was found in 51% of the cases. The IPI factors were evenly distributed between the ALK+ and ALK- groups, except that the ALK+ patients were younger (median age, 30 v 61 years; P <.002). The ALK+ cohort included cases with null (44%), T-cell (42%), and B-cell (14%) phenotypes. All 10 cases with cytogenetic or molecular evidence of a t(2;5) were ALK+. The 5-year overall survival (OS) of the entire cohort was 65%. The 5-year OS of the ALK+ and ALK- cases was 79% and 46%, respectively (P <.0003). Analysis of only the T-cell/null cases (n = 57) showed a 5-year OS of 93% for the ALK+ cases and only 37% for the ALK- cases (P <.00001). Univariate analysis of the clinical features showed that age

Concurrent chromosomal alterations at 3q27, 8q24 and 18q21 in B-cell lymphomas.

Recurrent chromosomal translocations in malignant lymphomas most commonly involve 18q21(bcl-2), 8q24 (c-myc) and 3q27 (bcl-6), with an incidence of 27%, 11% and 6%, respectively. Individual cases concurrently harbouring two of these three rearrangements have been previously reported. This report describes four patients with cytogenetic alterations affecting all three loci, which was confirmed by molecular analysis in one case. Clinically, each patient had aggressive B-cell lymphoma with disseminated disease often involving the central nervous system, poor response to chemotherapy and short survival. Activation of c-myc in association with deregulation of bcl-2, bcl-6 or both confers high-grade disease with a poor prognosis.

Primary follicular lymphoma of the testis in childhood.

BACKGROUND: Follicular lymphoma in childhood is rare. The authors present four unusual primary follicular lymphomas of the testis in children. METHODS: Tumor tissue was evaluated using light microscopy, immunohistochemistry, flow cytometry, and polymerase chain reaction (PCR) for immunoglobulin heavy chain (IgH) and bcl-2 gene rearrangements. Southern blot and immunohistochemical analyses were used to detect bcl-6 gene rearrangements and protein expression, respectively. RESULTS: Four young boys ranging in age from 3 to 10 years were diagnosed with Stage IE follicular large cell lymphoma (Grade 3). A B-cell phenotype was documented in all four cases; monoclonality was confirmed in three cases by demonstration of light chain restriction or clonal IgH gene rearrangement. None of the lymphomas expressed Bcl-2 or p53 protein, and bcl-2 gene rearrangements were not found in the three lymphomas studied. In contrast, Bcl-6 protein was expressed by all three lymphomas studied, and a bcl-6 gene rearrangement was detected in the one case analyzed by Southern blot. All four boys were treated by orchiectomy and combination chemotherapy and are alive with no evidence of disease 18-44 months following their initial diagnoses. CONCLUSIONS: Follicular lymphomas may rarely occur as primary testicular tumors in prepubertal boys and, when localized, appear to be associated with a favorable prognosis. In contrast to follicular lymphoma in adults, pediatric follicular lymphomas of the testis are usually of large cell type (Grade 3) and lack bcl-2 or p53 abnormalities. The identification, in one case, of a bcl-6 gene rearrangement suggests an alternate molecular pathogenesis for pediatric follicular lymphoma.