The clinically distinct CLL subtypes are characterized by high and low numbers of somatic hypermutations in the variable region of the immunoglobulin genes. CLL is divided into two main subgroups based on the presence or absence of acquired somatic mutations in the immunoglobulin heavy chain gene (IGHV) expressed by leukemic B cells. Patients whose tumor cells express an IGHV gene carrying somatic mutations (M-CLL) have a more indolent disease and longer overall survival than do patients whose tumors express an IGHV gene in the germ line or unmutated configuration (UM-CLL). In the seminal paper by Hamblin et al., the median survival was 117 months for patients with UM-CLL compared with 293 months for patients with M-CLL (Hamblin et al. 1999). IgVH mutation status had prognostic significance even when consideration was restricted to patients with early stage (Binet stage A) CLL: median survival durations were 95 months (UM-CLL) and 293 months (M-CLL). Oscier et al. have confirmed the prognostic significance of IgVH mutation in multivariate analyses and additionally have shown that the presence of unmutated IgVH genes was significantly associated with deletion of chromosome 11q23, absence of deletion of chromosome 13q14, atypical lymphocyte morphology, and more than 30 % CD38 expression (Oscier et al. 2002).

Patients with UM-CLL tended to have high-risk genomic aberrations such as del17p and del11q, whereas favorable aberrations such as del13q were overrepresented in the M-CLL group. Patients with UM-CLL were also more likely to develop additional karyotypic changes (clonal evolution). Krober et al. demonstrated striking differences in the incidence of prognostically important categories between the two groups, with unfavorable cytogenetic abnormalities (17p and 11q deletions) being found almost exclusively in cases with UM-CLL (Krober et al. 2002). Interestingly, even within the UM-CLL group, those cases with high-risk genomic aberrations (as defined by 17p and/or 11q deletion) had significantly inferior survival.

Another prognostic feature, ZAP-70, was identified only a decade ago (Rassenti et al. 2004). ZAP-70 belongs to a family of protein tyrosine kinases closely related to src and is one of the membrane components associated with early cell activation in T lymphocytes and natural killer (NK) lymphocytes (Mustelin and Tasken 2003). Rassenti et al. found that patients with 20–30 % of ZAP-70 positivity were above the threshold and had a shorter life span (Rassenti et al. 2004). They reported that the median time from diagnosis to initial treatment among patients with ZAP-70-positive CLL cells that expressed an unmutated IgVH was 2.8 years, which was similar to the 4.2 years for patients with ZAP-70-positive CLL cells that expressed a mutated IgVH gene. Since their study involved a large pool of 307 patients, they observed discordance between ZAP-70 and IgVH mutational status. Further studies using microarray experiments found that ZAP-70 was differentially expressed in UM-CLL and M-CLL, and its expression has been validated as a powerful predictor of unmutated IgVH status, rapid disease progression, and inferior survival (Orchard et al. 2004).

The most frequently deleted genomic region in CLL occurs at chromosome 13q14 and is associated with the indolent form of the disease. Two microRNA genes, mir-15a and mir-16-1, which are located in the crucial 13q14 region, have been implicated in CLL pathogenesis. CLLs that have a 13q14 deletion as the sole abnormality have a favorable disease course. Detailed deletion and expression analysis showed that miR15 and miR16 are located within a 30-kb region of loss in CLL and that both genes are deleted or downregulated in most CLL cases (approximately 68 %). The presence of both clonal homozygous and heterozygous deletions and the very high frequency of 13q14 loss suggest that these deletions are of pathogenic significance. BCL-2 is a target of miR15/16 and is a critical oncogene in a number of hematological malignancies as well as in solid tumors. It functions by promoting survival and inhibiting cell death. A high-resolution map of 13q14 deletions in 171 CLL samples indicated that this region also contains the DLEU7 gene, which was previously identified as a candidate tumor-suppressor gene located telomeric to miR15/16. Importantly, compared with normal B cells, malignant CLL lymphocytes showed lower expression of DELU7 (Pekarsky and Croce 2014). The 13q14 region of homozygous loss is of particular interest because it contains at least four noncoding genes including the two miR15/16 and Leu-1 and Leu-2 (Calin et al. 2002). Noncoding RNAs have roles in a great variety of processes, including transcription and chromosome structure, RNA processing and modifications, mRNA stability and translation, and protein stability and transport. Therefore, it is possible that the CLL gene(s) on 13q14 acts in a different way compared with classic tumor-suppressor genes.

Deletion of 17p involves the loss of the TP53 tumor-suppressor gene and is found in only 5–7 % of patients with early stage CLL, but the incidence increases to 25–40 % in patients with advanced refractory disease (Fink et al. 2006), and 17p deletion constitutes a poor prognostic factor (Trojani et al. 2010). Interphase fluorescence in situ hybridization (I-FISH) was used to characterize recurrent CLL-related chromosomal abnormalities, and this analysis clearly showed that patients with deletions of 17p13 containing the TP53 locus (del(17p)) had a significantly shorter median overall survival (32 months) than did patients with normal karyotype (111 months) and also compared with patients with other recurrent chromosomal aberrations (79 months for deletion 11q, 114 months for trisomy 12, 133 months for deletion 13q) (Dohner et al. 2000). The highest incidence of TP53 mutation is seen in patients with fludarabine-refractory CLL, and much of the heterogeneity in mutation prevalence is explained by different patient cohorts. The European Research Initiative on CLL (ERIC) has compiled a large set of TP53 mutations from CLL samples and compared this profile with the currently available TP53 mutation databases (Zenz et al. 2010). From four different cohorts of CLL patients, they demonstrated that the TP53 mutation profile is independent of 17p deletion or previous therapy. These findings suggest that the TP53 mutation (detectable after therapy) is selected rather than being caused by, for example, alkylating agents. These findings also support the growing evidence that the clinical consequences of 17p deletion (and TP53 mutation) are very similar to those for the TP53 mutation in the absence of 17p. They found codon 209 frameshift mutation to be a “hot spot” for CLL.

Deletion of 11q involves the loss of the ATM gene, which phosphorylates p53 upon DNA damage. This deletion is found in 10–20 % of CLL patients and confers an impaired clinical outcome, and mutations in the remaining allele confer a worse clinical prognosis. The I-FISH study showed that 11q deletion is the second most common aberration in CLL, occurring in 43 (20 %) of 214 cases as shown by use of the YAC probe for 11q22.3-23.1. Deletions were associated with extensive peripheral, abdominal, and mediastinal lymphadenopathy, and patients with 11q deletions had more rapid disease progression. The prognostic effect on survival strongly depended on the age of the patients. In patients less than 55 years old, median survival time was significantly shorter among patients with deletion, whereas patients older than 55 years had no significant difference compared with such patients with other chromosomal changes. Mutations can be present in the germ line, suggesting that individuals heterozygous for ATM mutations may be predisposed to develop CLL. Recently, loss of distal 11q (which includes the ATM gene) was associated with DNA repair deficiency and increased sensitivity to ionizing radiation (Parikh et al. 2007). BIRC3 mutations were recently identified in CLL and may be important for elucidating the molecular genetics of 11q22-q23 deletion (Rossi et al. 2012a).

Mutations in ATM predispose patients to lymphoid malignancies. Using FISH, a large series (more than 200 B-CLLs) found that deletion of the chromosomal region 11q22-q23 is a recurrent aberration in B-CLL (Dohner et al. 1997). Loss of the region was shown to be a prognostic marker predicting poor survival. The commonly deleted region was defined as a 2–3 mega-base-pair (Mbp) segment containing ATM, RDX, FRDX1, RAB39, CUL5, ACAT1, NPAT, KDELC2, EXPH5, MRE11, H2AX, and BIRC3. The size of the 11q deletion seems to vary between tumors but generally can be classified either as a large 11q deletion (more frequent) or a small 11q deletion (rare). Although the prognostic significance of these differences is unclear at present, the smaller deletions tend to coincide with mutations in the ATM gene, and it has been proposed that haploinsufficiency of a larger number of genes on chromosome 11q may provide a proliferative advantage comparable to biallelic inactivation of ATM (Gardiner et al. 2012). CLL subsets with 11q deletion are associated with an elevation of gene copy number alterations, representing genomic instability. Dohner and coworkers reported a median survival of 79 months in 56 patients with an 11q deletion (Dohner et al. 1997). Catovsky and coworkers found that an 11q deletion was associated with a complete response plus nodular partial response rate of 28 % and that in addition to 17p deletion and unmutated IGHV genes, 11q deletion was also an independent factor predicting shorter progression-free survival and poorer response to treatment (Catovsky 2007). In a study conducted at MD Anderson Cancer Center, CLL with an 11q22 deletion was associated with prolonged survival (97 % and 91 % at 1 and 3 years, respectively) in all patients studied and with high rates of response and relapse-free survival in patients who required therapy. In particular, regimens containing fludarabine-cyclophosphamide-rituximab (FCR) resulted in a response rate of 100 % (complete response, 76 %). In all patients, the relapse-free survival rate was 77 % at 3 years (Tsimberidou et al. 2009).

A recent study that analyzed the ATM coding region in 318 patients with CLL (140 with a chromosome 11q deletion and 178 with no 11q deletion) and 281 controls found that, compared with healthy individuals, constitutional pathogenic ATM mutations were increased in patients with chromosome 11q deletions but not in those without 11q deletions. These results suggest that ATM germ line heterozygosity does not play a role in CLL initiation but rather influences rapid disease progression through ATM loss (Skowronska et al. 2012).

Saiya-Cork and colleagues found that the insulin receptor (INSR) was significantly overexpressed in about 25 % of CLL cases, many of which carried 11q deletion (Saiya-Cork et al. 2011). INSR expression varied across a large cohort of CLLs but was overexpressed in 11q subset. Approximately two thirds of the deletion 11q CLLs showed elevated INSR expression. This receptor was functional, in that insulin was able to induce activation of the AKT and RAS/RAF/extracellular signal-regulated kinase (ERK) pathways and tyrosine phosphorylation of IRS1. These signals provide survival support to the CLLs, which show a reduction in apoptosis in response to insulin.

Trisomy 12 is among the most common aberrations in CLL (10–20 %), but the genes involved in the pathogenesis of CLL with trisomy 12 are unknown (Crossen and Horn 1987). Despite the frequency of trisomy in CLL, little is known about its origin, pathogenic significance, or the mechanism by which it may contribute to malignant transformation or disease progression. Through restriction fragment length polymorphism analysis, the extra chromosome 12 has been shown to derive from duplication of one chromosome 12, with retention of the other homologue rather than from triplication of one homologue (Einhorn et al. 1989). Trisomy 12 can be detected in about one third of CLL patients with clonal chromosome abnormalities and occurs as a single aberration in more than half of them. The presence of trisomy 12 is an adverse prognostic factor and predicts a short treatment-free survival.

At present, the biological significance of this different distribution of +12 cells in CLL is unknown, as is the role (if any) of this trisomy in the pathogenesis of lymphoproliferative disorders. A study of adhesion molecules in B-CLL cells, simultaneously evaluated in the peripheral blood, bone marrow, and lymph nodes, has shown that CD44 and CD54 on CD5+ CD23+ lymphocytes were more dominant in more numbers of cells in the lymph nodes than in the peripheral blood or bone marrow. In some patients with chronic lymphoproliferative diseases other than CLL, polysomy 12 detected in lymph node cells was clearly associated with progressive disease (Callea et al. 1996; Younes et al. 1994).

While not that common, trisomy 19 has also been identified in CLL (Schwaenen et al. 2004). Schwaenen and colleagues investigated 106 B-CLL cases by array comparative genomic hybridization (CGH) and found trisomy 19 as a chromosomal aberration in almost 5 % of cases (Schwaenen et al. 2004). Interestingly, this aberration seemed to be correlated with trisomy 12 and mutated IGHV genes. A similar incidence was also observed in another study of 125 B-CLL cases (Dicker et al. 2006).

The Spanish CLL Genome Consortium performed the first sequencing in CLL. This study by Puente et al. reported the WGS of two cases of CLL without mutations in immunoglobulin genes (IGHV unmutated) and two with mutations in immunoglobulin genes (IGHV mutated) (Puente et al. 2011). Whole-genome sequencing of the tumors using germ line DNA as a baseline identified approximately 1,000 somatic mutations in each of the tumors, consistent with prior estimates of one mutation per Mb for leukemias. From all of the somatic mutations identified in the above four patients, 46 mutations were predicted to alter the protein coding sequence of 45 genes, of which 26 were found to be expressed at the RNA level and of potential biological relevance. Sanger sequencing in a validation cohort of 169 additional CLL patients confirmed that four of these genes (NOTCH1, MYD88, XPO1, and KLHL6) were recurrently mutated.