RESUMEN
Myeloproliferative neoplasms are acquired hematological malignancies, mainly affecting the adult and whose morbidity and mortality stems from haemostasis disorders. The most frequently encountered complications include thrombosis, affecting preferentially the arterial territory, but also atypical locations such as splanchnic vein thrombosis. The pathophysiology of these thromboses is complex and involves different actors: blood cells, endothelium and flow conditions. Numerous studies have been conducted to identify risk factors for thrombosis. To date, only two risk factors have been validated through prospective studies (age over 60 years old, history of thrombotic events) and allow classification of patients as "low risk" and "high risk" as the basis for current treatment recommendations. Haemorrhagic manifestations, less frequent than thrombosis, are mainly related to an alteration of primary haemostasis and are therefore manifested by mucocutaneous bleeding. In these patients, platelet dysfunctions and/or acquired Willebrand syndromes can be found. The pathophysiology of thrombosis and platelet dysfunction during myeloproliferative neoplasms remains to date partially unknown. In this review, we offer to focus on physiopathological mechanisms as well as the latest advances in their understanding.
Asunto(s)
Plaquetas/fisiología , Trastornos Mieloproliferativos/sangre , Trastornos Mieloproliferativos/complicaciones , Trombosis/etiología , Plaquetas/patología , Neoplasias Hematológicas/sangre , Neoplasias Hematológicas/complicaciones , Neoplasias Hematológicas/epidemiología , Neoplasias Hematológicas/fisiopatología , Hemorragia/sangre , Hemorragia/epidemiología , Hemorragia/etiología , Humanos , Trastornos Mieloproliferativos/epidemiología , Trastornos Mieloproliferativos/fisiopatología , Factores de Riesgo , Trombosis/sangre , Trombosis/epidemiología , Trombosis/fisiopatologíaRESUMEN
Chromosomal rearrangements of the human MLL/KMT2A gene are associated with infant, pediatric, adult and therapy-induced acute leukemias. Here we present the data obtained from 2345 acute leukemia patients. Genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) were determined and 11 novel TPGs were identified. Thus, a total of 135 different MLL rearrangements have been identified so far, of which 94 TPGs are now characterized at the molecular level. In all, 35 out of these 94 TPGs occur recurrently, but only 9 specific gene fusions account for more than 90% of all illegitimate recombinations of the MLL gene. We observed an age-dependent breakpoint shift with breakpoints localizing within MLL intron 11 associated with acute lymphoblastic leukemia and younger patients, while breakpoints in MLL intron 9 predominate in AML or older patients. The molecular characterization of MLL breakpoints suggests different etiologies in the different age groups and allows the correlation of functional domains of the MLL gene with clinical outcome. This study provides a comprehensive analysis of the MLL recombinome in acute leukemia and demonstrates that the establishment of patient-specific chromosomal fusion sites allows the design of specific PCR primers for minimal residual disease analyses for all patients.
Asunto(s)
N-Metiltransferasa de Histona-Lisina/genética , Leucemia Mieloide Aguda/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Adulto , Niño , Aberraciones Cromosómicas , Rotura Cromosómica , Femenino , Reordenamiento Génico/genética , Humanos , Lactante , Masculino , Proteínas de Fusión Oncogénica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Translocación Genética/genéticaAsunto(s)
Anemia Refractaria/genética , Anemia Sideroblástica/genética , Calreticulina/genética , Mutación/genética , Trombocitosis/genética , Adulto , Anciano , Anemia Refractaria/diagnóstico , Anemia Sideroblástica/diagnóstico , Estudios de Cohortes , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Trombocitosis/diagnóstico , Adulto JovenRESUMEN
Reliable detection of JAK2-V617F is critical for accurate diagnosis of myeloproliferative neoplasms (MPNs); in addition, sensitive mutation-specific assays can be applied to monitor disease response. However, there has been no consistent approach to JAK2-V617F detection, with assays varying markedly in performance, affecting clinical utility. Therefore, we established a network of 12 laboratories from seven countries to systematically evaluate nine different DNA-based quantitative PCR (qPCR) assays, including those in widespread clinical use. Seven quality control rounds involving over 21,500 qPCR reactions were undertaken using centrally distributed cell line dilutions and plasmid controls. The two best-performing assays were tested on normal blood samples (n=100) to evaluate assay specificity, followed by analysis of serial samples from 28 patients transplanted for JAK2-V617F-positive disease. The most sensitive assay, which performed consistently across a range of qPCR platforms, predicted outcome following transplant, with the mutant allele detected a median of 22 weeks (range 6-85 weeks) before relapse. Four of seven patients achieved molecular remission following donor lymphocyte infusion, indicative of a graft vs MPN effect. This study has established a robust, reliable assay for sensitive JAK2-V617F detection, suitable for assessing response in clinical trials, predicting outcome and guiding management of patients undergoing allogeneic transplant.
Asunto(s)
Janus Quinasa 2/genética , Mutación/genética , Trastornos Mieloproliferativos/genética , Recurrencia Local de Neoplasia/diagnóstico , Neoplasia Residual/diagnóstico , Reacción en Cadena en Tiempo Real de la Polimerasa , Adulto , Anciano , Análisis Citogenético , Europa (Continente) , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Trastornos Mieloproliferativos/terapia , Recurrencia Local de Neoplasia/genética , Neoplasia Residual/genética , Pronóstico , ARN Mensajero/genética , Inducción de Remisión , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trasplante de Células Madre , Trasplante Homólogo , Adulto JovenRESUMEN
Chromosomal rearrangements of the human MLL (mixed lineage leukemia) gene are associated with high-risk infant, pediatric, adult and therapy-induced acute leukemias. We used long-distance inverse-polymerase chain reaction to characterize the chromosomal rearrangement of individual acute leukemia patients. We present data of the molecular characterization of 1590 MLL-rearranged biopsy samples obtained from acute leukemia patients. The precise localization of genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) were determined and novel TPGs identified. All patients were classified according to their gender (852 females and 745 males), age at diagnosis (558 infant, 416 pediatric and 616 adult leukemia patients) and other clinical criteria. Combined data of our study and recently published data revealed a total of 121 different MLL rearrangements, of which 79 TPGs are now characterized at the molecular level. However, only seven rearrangements seem to be predominantly associated with illegitimate recombinations of the MLL gene (≈ 90%): AFF1/AF4, MLLT3/AF9, MLLT1/ENL, MLLT10/AF10, ELL, partial tandem duplications (MLL PTDs) and MLLT4/AF6, respectively. The MLL breakpoint distributions for all clinical relevant subtypes (gender, disease type, age at diagnosis, reciprocal, complex and therapy-induced translocations) are presented. Finally, we present the extending network of reciprocal MLL fusions deriving from complex rearrangements.
Asunto(s)
Rotura Cromosómica , Reordenamiento Génico , Leucemia/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas de Fusión Oncogénica/genética , Translocación Genética/genética , Enfermedad Aguda , Adolescente , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Animales , Niño , Preescolar , Femenino , N-Metiltransferasa de Histona-Lisina , Humanos , Lactante , Recién Nacido , Leucemia/clasificación , Masculino , Ratones , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Pronóstico , Adulto JovenRESUMEN
Refractory anaemia with ring sideroblasts (RARS) and marked thrombocytosis (RARS-T) is a provisional entity in the World Health Organisation 2008 classification and has previously been shown to have a high proportion of JAK2(V617F) (Janus Kinase 2) and SF3B1 (Splicing Factor 3B subunit 1) mutations. The purpose of the present study was to analyse the frequency of SF3B1 mutations in a large cohort of 111 patients with RARS-T and 33 patients with RARS and to explore the prognostic impact of SF3B1 mutational status on RARS-T. The frequency of SF3B1 mutations in RARS-T (96/111, 86.5%) and RARS (28/33, 84.8%) was similar. In RARS-T, median survival was better in SF3B1-mutated patients than in SF3B1-non-mutated patients (6.9 and 3.3 years, respectively, P=0.003). RARS can be differentiated from RARS-T by the frequency of JAK2(V617F) (0% vs 48.6%). In RARS-T patients, SF3B1 (P=0.021) and JAK2 mutations (P=0.016) were independent factors for a better prognosis. Altogether, our results confirm that RARS-T is an independent entity that should be recognised by the next World Health Organisation classification. The assessment of SF3B1 mutations is of prognostic interest in RARS-T patients. Younger age, JAK2(V617F) and SF3B1 mutations are the main predicting factors for survival in RARS-T.
Asunto(s)
Anemia Refractaria/genética , Anemia Refractaria/mortalidad , Janus Quinasa 2/genética , Mutación , Fosfoproteínas/genética , Ribonucleoproteína Nuclear Pequeña U2/genética , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Anemia Refractaria/complicaciones , Anemia Refractaria/diagnóstico , Anemia Sideroblástica/complicaciones , Mapeo Cromosómico , Femenino , Humanos , Cariotipo , Masculino , Persona de Mediana Edad , Tasa de Mutación , Pronóstico , Factores de Empalme de ARN , Trombocitosis/complicaciones , Adulto JovenRESUMEN
Myeloproliferative neoplasms are frequently associated with aberrant constitutive tyrosine kinase (TK) activity resulting from chimaeric fusion genes or point mutations such as BCR-ABL1 or JAK2 V617F. We report here the cloning and functional characterization of two novel fusion genes BCR-RET and FGFR1OP-RET in chronic myelomonocytic leukemia (CMML) cases generated by two balanced translocations t(10;22)(q11;q11) and t(6;10)(q27;q11), respectively. The two RET fusion genes leading to the aberrant activation of RET, are able to transform hematopoietic cells and skew the hematopoietic differentiation program towards the monocytic/macrophage lineage. The RET fusion genes seem to constitutively mimic the same signaling pathway as RAS mutations frequently involved in CMML. One patient was treated with Sorafenib, a specific inhibitor of the RET TK function, and demonstrated cytological and clinical remissions.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Leucemia Mielomonocítica Crónica/patología , Monocitos/citología , Proteínas Proto-Oncogénicas c-ret/genética , Secuencia de Bases , Cartilla de ADN , Humanos , Hibridación Fluorescente in Situ , Leucemia Mielomonocítica Crónica/genética , Mutación Puntual , Reacción en Cadena de la Polimerasa/métodos , Translocación GenéticaAsunto(s)
Infecciones por Helicobacter/complicaciones , Hiperglucemia/complicaciones , Neoplasias Gástricas/epidemiología , Neoplasias Gástricas/microbiología , Adulto , Anciano , Estudios de Cohortes , Femenino , Hemoglobina Glucada/metabolismo , Helicobacter pylori , Humanos , Hiperglucemia/sangre , Incidencia , Japón/epidemiología , Masculino , Persona de Mediana Edad , Análisis Multivariante , Estudios Prospectivos , Factores de RiesgoRESUMEN
Imatinib is an effective first-line therapy for chronic myelogenous leukemia (CML) that acts by targeting the tyrosine kinase activity of BCR-ABL. To overcome resistance, second-generation inhibitors of BCR-ABL have been developed. Among these, nilotinib is more potent against BCR-ABL than imatinib, and is effective against many imatinib-resistant BCR-ABL mutants. In this study, an in vitro flow cytometry assay to analyze imatinib- and nilotinib-induced apoptosis in CML cells has been developed. Both the drugs induced significant apoptosis in CD34+ cells from 36 CML bone marrow samples (P<10(-4)), whereas CD34+ cells from BCR-ABL negative samples were unaffected. When the experiments were carried out in the presence of a cocktail of cytokines, nilotinib- but not imatinib-induced apoptosis was inhibited. This differential inhibition was confirmed on K562 cells. A blocking anti-CD117 antibody alleviated the antiapoptotic effect of cytokines against nilotinib. Moreover, using short hairpin RNA against BCR-ABL, we showed that K562 cells were not dependent on BCR-ABL signaling as long as the stem cell factor (SCF) receptor pathway was activated. We conclude that the c-KIT pathway may substitute for BCR-ABL tyrosine kinase to activate survival signals, and that c-KIT must be inhibited besides Bcr-Abl to allow apoptosis of CML cells.
Asunto(s)
Apoptosis/efectos de los fármacos , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Proteínas Proto-Oncogénicas c-kit/metabolismo , Factor de Células Madre/metabolismo , Benzamidas , Citocinas/farmacología , Humanos , Mesilato de Imatinib , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-kit/efectos de los fármacos , Pirimidinas/farmacología , Factor de Células Madre/antagonistas & inhibidores , Células Tumorales CultivadasRESUMEN
The t(8;16)(p11;p13) is a rare translocation involved in de novo and therapy-related myelomonocytic and monocytic acute leukemia. It fuses two genes encoding histone acetyltransferases (HATs), MYST3 located at 8p11 to CREBBP located at 16p13. Variant translocations involve other HAT-encoding genes such as EP300, MYST4, NCOA2 or NCOA3. MYST3-linked acute myeloid leukemias (AMLs) share specific clinical and biological features and a poor prognosis. Because of its rarity, the molecular biology of MYST3-linked AMLs remains poorly understood. We have established the genome and gene expression profiles of a multicentric series of 61 M4/M5 AMLs including 18 MYST3-linked AMLs by using array comparative genome hybridization (aCGH) (n=52) and DNA microarrays (n=44), respectively. We show that M4/5 AMLs have a variety of rare genomic alterations. One alteration, a gain of the MYB locus, was found recurrently and only in the MYST3-linked AMLs (7/18 vs 0/34). MYST3-AMLs have also a specific a gene expression profile, which includes overexpression of MYB, CD4 and HOXA genes. These features, reminiscent of T-cell acute lymphoid leukemia (ALL), suggest the targeting of a common T-myeloid progenitor.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Genes myb/genética , Histona Acetiltransferasas/genética , Leucemia Mielomonocítica Aguda/genética , Antígenos CD4/genética , Hibridación Genómica Comparativa , Regulación Neoplásica de la Expresión Génica , Genoma Humano , Proteínas de Homeodominio/genética , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Proto-Oncogénicas c-myb/genéticaRESUMEN
Episomes with the NUP214-ABL1 fusion gene have been observed in 6% of T-ALL. In this multicentric study we collected 27 cases of NUP214-ABL1-positive T-ALL. Median age was 15 years with male predominance. Outcome was poor in 12 patients. An associated abnormality involving TLX1 or TLX3 was found in all investigated cases. Fluorescent in situ hybridization revealed a heterogeneous pattern of NUP214-ABL1 amplification. Multiple episomes carrying the fusion were detected in 24 patients. Episomes were observed in a significant number of nuclei in 18 cases, but in only 1-5% of nuclei in 6. In addition, intrachromosomal amplification (small hsr) was identified either as the only change or in association with episomes in four cases and two T-ALL cell lines (PEER and ALL-SIL). One case showed insertion of apparently non-amplified NUP214-ABL1 sequences at 14q12. The amplified sequences were analyzed using array-based CGH.These findings confirm that the NUP214-ABL1 gene requires amplification for oncogenicity; it is part of a multistep process of leukemogenesis; and it can be a late event present only in subpopulations. Data also provide in vivo evidence for a model of episome formation, amplification and optional reintegration into the genome. Implications for the use of kinase inhibitors are discussed.
Asunto(s)
Amplificación de Genes , Leucemia-Linfoma de Células T del Adulto/genética , Proteínas de Fusión Oncogénica/genética , Adolescente , Adulto , Línea Celular Tumoral , Niño , Preescolar , Femenino , Proteínas de Homeodominio/genética , Humanos , Leucemia-Linfoma de Células T del Adulto/etiología , Masculino , Persona de Mediana Edad , Plásmidos , Proteínas Proto-Oncogénicas/genética , Factores Sexuales , Resultado del Tratamiento , Adulto JovenRESUMEN
An early appreciation of treatment efficacy could be very useful in acute myeloblastic leukemia (AML), and a prognostic value has been suggested for the morphological assessment of decrease in blasts during induction therapy. More sensitive, multiparametric flow cytometry (FCM) can detect far lower blast counts, allowing for a precise and reliable calculation of blast cell decrease rate (BDR). Such a multiparametric FCM four-colours/single-tube protocol, combining CD11b, CD45-ECD and CD16-PC5, was applied to peripheral blood samples from 130 AML patients, collected daily during induction chemotherapy. Normalized blast cell percentages were used to calculate the relevant decrease slopes. Slope thresholds (<-25, -25 to -15 and >-15), or the time required to reach 90% depletion of the peripheral blast load (<5, 5 or >5 days), was strongly associated with the achievement of complete remission (P<0.0001). Log-rank test and Cox model showed that they also carried high statistical significance (P<0.0001) for disease-free survival. The prognostic value of cytogenetic features, confirmed in this series, was refined by BDR, which allowed to discriminate between good- and poor-risk patients among those with intermediate or normal karyotypes. This simple FCM protocol allows for an accurate prognostic sequential approach adapted to the determination of decrease in peripheral blast cells during induction chemotherapy.
Asunto(s)
Crisis Blástica/patología , Citometría de Flujo/métodos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Adulto , Anciano , Anciano de 80 o más Años , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Recuento de Células , Femenino , Humanos , Inmunofenotipificación , Cariotipificación , Masculino , Persona de Mediana Edad , Pronóstico , Inducción de Remisión , Adulto JovenRESUMEN
Thirty cases of acute myeloid leukaemia (AML) with MYST histone acetyltransferase 3 (MYST3) rearrangement were collected in a retrospective study from 14 centres in France and Belgium. The mean age at diagnosis was 59.4 years and 67% of the patients were females. Most cases (77%) were secondary to solid cancer (57%), haematological malignancy (35%) or both (8%), and appeared 25 months after the primary disease. Clinically, cutaneous localization and disseminated intravascular coagulation were present in 30 and 40% of the cases, respectively. AMLs were myelomonocytic (7%) or monocytic (93%), with erythrophagocytosis (75%) and cytoplasmic vacuoles (75%). Immunophenotype showed no particularity compared with monocytic leukaemia without MYST3 abnormality. Twenty-eight cases carried t(8;16)(p11;p13) with MYST3-CREBBP fusion, one case carried a variant t(8;22)(p11;q13) and one case carried a t(8;19)(p11;q13). Type I (MYST3 exon 16-CREBBP exon 3) was the most frequent MYST3-CREBBP fusion transcript (65%). MYST3 rearrangement was associated with a poor prognosis, as 50% of patients deceased during the first 10 months. All those particular clinical, cytologic, cytogenetic, molecular and prognostic characteristics of AML with MYST3 rearrangement may have allowed an individualization into the World Health Organization classification.
Asunto(s)
Cromosomas Humanos Par 8 , Reordenamiento Génico , Histona Acetiltransferasas/genética , Leucemia Mieloide Aguda/genética , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Bases , Cartilla de ADN , Femenino , Humanos , Inmunofenotipificación , Hibridación Fluorescente in Situ , Cariotipificación , Leucemia Mieloide Aguda/inmunología , Masculino , Persona de Mediana Edad , Pronóstico , Reacción en Cadena de la Polimerasa de Transcriptasa InversaAsunto(s)
Cromosomas Humanos Par 14/genética , Cromosomas Humanos Par 19/genética , Leucemia de Células B/genética , Leucemia Linfocítica Crónica de Células B/genética , Translocación Genética/genética , Adulto , Anciano , Anciano de 80 o más Años , Proteínas del Linfoma 3 de Células B , Femenino , Humanos , Inmunofenotipificación , Leucemia de Células B/clasificación , Leucemia de Células B/patología , Leucemia Linfocítica Crónica de Células B/clasificación , Leucemia Linfocítica Crónica de Células B/patología , Masculino , Persona de Mediana Edad , FN-kappa B/genética , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Recently we identified galectin-3 (gal-3), which is secreted by colonic epithelial cells (CEC), to be a strong activator of colonic lamina propria fibroblasts (CLPF). Modulation of CLPF function may play a role during stricture and fistula formation in inflammatory bowel disease (IBD). Therefore, we investigated further the expression of gal-3 and effects on CLPF. The aim of this study is to perform a direct comparison of gal-3 between tissue from healthy controls and from patients with either Crohn's disease (CD) or ulcerative colitis (UC). CEC, CLPF and intestinal macrophages (IMAC) were isolated from control and IBD colonic tissue. Interleukin-8 secretion as a readout of CLPF activation was quantified by enzyme-linked immunosorbent assay. Gal-3 in cell cultures and tissue samples was evaluated by Western blot, immunofluorescence and immunohistochemistry. CLPF-migration was assayed in the 48-well modified Boyden chamber. Gal-3 expression was found in all segments of the colon. In the terminal ileum, less gal-3 was found compared with the colon. Immunohistochemistry and immunofluorescence revealed a homogenous distribution of gal-3 in CEC and IMAC of control mucosa and UC. However, significantly less gal-3 was found in IMAC from CD patients. In CD fistulae and stenoses, gal-3 expression was reduced significantly and barely detectable. In co-incubation studies lactose reduced significantly the CLPF-stimulatory potential of gal-3, indicating that the C-terminal domain of gal-3 is responsible for CLPF activation. Gal-3 stimulated CLPF migration in CLPF derived from fistulae. In conclusion, gal-3 expression is down-regulated in CD-fistulae and stenoses as well as in IMAC in CD patients. Gal-3 induces migration of CLPF derived from fistulae. Its role for stricture and fistula formation warrants further investigation.
Asunto(s)
Colitis Ulcerosa/inmunología , Enfermedad de Crohn/inmunología , Fibroblastos/inmunología , Galectina 3/inmunología , Adolescente , Adulto , Anciano , Células Cultivadas , Femenino , Fibroblastos/efectos de los fármacos , Galectina 3/antagonistas & inhibidores , Galectina 3/biosíntesis , Galectina 3/genética , Expresión Génica , Humanos , Íleon/inmunología , Fístula Intestinal/inmunología , Mucosa Intestinal/inmunología , Obstrucción Intestinal/inmunología , Intestino Grueso/inmunología , Lactosa/farmacología , Macrófagos/inmunología , Masculino , Persona de Mediana Edad , ARN Mensajero/genéticaRESUMEN
A series of 38 patients with acute myeloblastic leukemia (AML) with 49 or more chromosomes and without structural abnormalities was selected within the Groupe Francophone de Cytogénétique Hématologique (GFCH) to better define their characteristics. The median age of the patients was 65 years, and all FAB subtypes were represented. Although all chromosomes were gained, some seems to prevail: chromosome 8 (68%), 21 (47%), 19 (37%), and 13 and 14 (34% each). Since MLL rearrangement leads patients in a group with an unfavorable prognosis, search for cryptic rearrangements of MLL was performed in 34 patients and showed abnormalities in 5 (15%). When we applied the most frequent definition of complex karyotypes (three or more abnormalities), all patients with high hyperdiploid AML fall in the unfavorable category. Among the 18 patients without MLL rearrangement receiving an induction therapy, 16 (89%) reached CR and 6 (33%) were still alive after a 31-month median follow-up (14-61 months). Although this study was retrospective, these results suggest that high hyperdiploid AML without chromosome rearrangement seems to be a subgroup of uncommon AML (less than 1%), and may be better classified in the intermediate prognostic group.