Chronic lymphocytic leukemia (CLL) 

Cytomorphologically, in addition to the proliferation of mature lymphoid cells, B-CLL more frequently shows the phenomenon of "Gumprecht's shadows". They occur but do not prove the diagnosis.

If >15% prolymphocytes are present in blood and/or bone marrow, characterized by a more immature appearance with a larger nucleus and a clearly visible nucleolus, integrated diagnostics are essential.

In the rare transformation of a B-CLL (Richter transformation), blastic cells are found.

Chromosome analysis detects more aberrations than FISH

In the past, chromosome analysis played only a minor role because the necessary cultivation of CLL cells in vitro was hardly possible. However, since cultivation of CLL cells has become reliably possible, more aberrations can be detected with it than with FISH analysis (Dicker et al. 2006). Typical for CLL are genomically unbalanced events (Haferlach et al. 2007). The most common alterations are del(13q), del(11q), +12, +12q, del(6q), and del(17p). Rather rare are trisomy 3 or 3q gains, gains of 2p, 8q and 11q as well as translocations involving the immunoglobulin loci: 2p11 (Ig kappa), 14q32 (IgH), 22q11 (Ig lambda) (Döhner et al. 1999, Döhner et al. 2000, Stilgenbauer et al. 2002, Haferlach et al. 2007, Hallek et al. 2008).

Approximately 20% of CLL patients show a complex aberrant karyotype (≥ 3 aberrations) in chromosome banding analysis (Haferlach et al. 2007 & 2010).  A complex karyotype represents an independent prognostic factor (WHO 2022), although the exact number of aberrations as well as cooperating genetic alterations appear to influence the prognostic effect (Baliakas et al. 2019, Kittai et al. 2021).

A complex karyotype, in addition to its prognostic relevance, is also a therapy-decisive marker (Onkopedia guideline CLL 2023).

FISH can support the differential diagnosis of CLL. The diagnostic panel includes IGH rearrangements for this purpose. Thus, the t(11;14)(q13;q32); IGH::CCND1 is characteristic of MCL and the t(14;18)(q32;q21); IGH::BCL2 is typical of follicular lymphoma. In rare cases, other IGH translocations occur, such as the t(14;19)(q32;q13). Therefore, a partner gene-independent IGH probe complements the panel. If IGH translocations occur in confirmed CLL, this is considered to be prognostically unfavorable, only the t(14;18) is still under discussion regarding its prognostic significance (De Braekeleer et al. 2016).

The markers investigated by prognostic panel are summarized in Table 1. They are important for prognostic assessment under currently approved therapies and/or, in the case of del(11q22) and del(17p13), may also influence the choice of therapy.

According to the Onkopedia guideline and the iwCLL guideline, the determination of the IGHV and TP53 mutation status is indicated in every case; both are of prognostic relevance and significantly determine the choice of therapy. The analysis of further molecular genetic alterations can be useful for risk stratification (see below), above all. Molecular genetics also plays an important role in the identification of resistance mutations (see below).

B cell receptor analysis

For the analysis of IGHV status, the burden of somatic hypermutations in the IGHV (immunoglobulin heavy chain variable region) gene is determined. The IGHV gene is considered "unmutated" if ≤ 2% mutations are detected, which is the case for approximately 40% of all CLL patients. Repeated analysis is usually not necessary, as the mutation status for a specific CLL population does not change during progression. Unmutated IGHV ("U-CLL") is a prognostic risk factor and influences the choice of therapy (Onkopedia guideline CLL 2023), accordingly IGHV status should be assessed at the latest at therapy initiation (iwCLL guideline 2018). Approximately 30% of CLL patients show (quasi)identical amino acid sequences in the CD3 region, this is called stereotypy (Messmer et al. 2004, Tobin et al. 2004). Among the stereotyped B cell receptors, subsets can in turn be identified that can significantly influence clinic and prognosis (Stamatopoulos et al. 2017, WHO 2022). For example, subsets #2 and #8 are established risk factors, and their investigation is therefore recommended by the European Research Inititative on CLL (ERIC) (Agathangelidis et al. 2022).

TP53 changes

Here, a distinction must be made between mutations within the TP53 gene and deletions at the chromosomal level (del(17p) (see above)). Like TP53 deletions, TP53 mutations represent a prognostic risk factor. Due to therapeutic relevance, TP53 aberrations should be determined prior to therapy (iwCLL guideline 2018). The time interval to therapy initiation should be <12 weeks, as TP53 aberrations can also occur only during the course (S3 Guideline 2018). The test should be repeated each time therapy is changed (iwCLL-Guideline 2018).

Next-generation sequencing also allows the detection of subclonal TP53 mutations. In this case, an unfavorable effect on prognosis has also been observed (Rossi et al. 2014, Nadeu et al. 2016, Bomben et al. 2018). Patients with subclonal TP53 mutations are also at risk of clonal expansion with chemotherapy (Landau et al. 2013, Rossi et al. 2014, Malcikova et al. 2015, Nadeu et al. 2016, Bomben et al. 2018).

Combined prognostic panel according to Baliakas et al. 2015 and Hallek et al. 2018 (iwCLL guideline): IGHV, TP53, SF3B1

In addition to the IGHV (see above) and TP53 (see above) genes, the SF3B1 gene is also studied in this panel. SF3B1 mutations correlate with U-CLL, del(11q), and a stereotyped IGHV3-21 rearrangement. The prognosis for SF3B1 mutations is considered intermediate (Oscier et al. 2013, Jeromin et al. 2014, Rossi et al. 2013, Stilgenbauer et al. 2014).

Prognostic panel according to Rossi et al. 2013: SF3B1, TP53, BIRC3, NOTCH1

In addition to SF3B1 (see above) and TP53 (see above), the BIRC3 and NOTCH1 genes are being investigated. The BIRC3 gene may be affected by mutations and correlate with the occurrence of del(11q). BIRC3 aberrations are considered prognostically unfavorable (Rossi et al. 2013). NOTCH1 mutations are clustered in U-CLL and are associated with a +12 and a ~20-fold increased risk of transformation to DLBCL; prognosis appears intermediate. NOTCH1 mutations may also be of predictive relevance (Rossi et al. 2012, Rossi et al. 2013, Stilgenbauer et al. 2014). However, this remains to be confirmed in further independent studies.

Development of resistance under ibrutinib and venetoclax therapy

Treatment with the BTK inhibitor ibrutinib can lead to the development of resistance mutations. Genes known to be affected so far are frequently the BTK gene and less frequently the PLCG2 gene (Woyach et al. 2014). BCL2 mutations may also occur with venetoclax therapy (Blombery et al. 2020, Lucas et al. Blood 2020). Testing for resistance mutations is indicated in case of non-response.