Monosomy 12 and deletion of 13q34 in a case of chronic lymphocytic leukemia with concomitant lung cancer

Background. We described a patient with chronic lymphocytic leukemia (CLL) and lung cancer and unusual chromosomal aberrations. Case report. At the same time with the diagnosis of B-cell CLL, squamocellular lung carcinoma diagnosis was established. Using interphase fluoresecence in situ hybridization technique (FISH) we detected monosomy 12 and deletion of 13q34 occured in the same clone. One month after the beginning of examination the patient died unexpectedly during sleep immediately before we applied a specific treatment for CLL or lung carcinoma. Conclusion. Simultaneous occurrence of monosomy 12 and deletion of 13q34 in a patient with B-cell CLL has been described only once before, but as a part of a complex karyotype. The prognostic significance of these abnormalities remains uncertain

Coexistence of trisomy 12 and del(13)(q14.3) in two patients with chronic lymphocytic leukemia

We describe two patients with diagnosis of chronic lymphocytic leukemia (CLL) in whom interphase fluo­rescence in situ hybridization (FISH) analysis revealed trisomy 12 and del(13)(q14.3) occurring in the same clone. These abnormalities are rarely seen together and the prognostic relevance of their coexistence is still unclear. According to some data, a probable adverse prognosis for this group of patients is suggested. Our patients have been in a stable phase of the disease for more than one year since the given abnormalities were documented in their karyotypes. Further study is necessary to determine the prognostic significance of coexistence of these abnormalities in CLL patients

Brza progresija hronične limfocitne leukemije u Rihterov sindrom kod bolesnika sa kariotipom blizu triploidnog broja hromozom

Introduction. The presence of aneuploidy in patients diagnosed with chronic lymphocytic leukemia (CLL), except trisomy 12, is considered quite uncommon. Hyperdiploidy or near-tetraploidy (occurring in 1–3% of all CLL patients) usually confer a poor prognosis. Case report. We report a patient in a progressive phase of CLL with near–triploid karyotype. The prognosis of the disease was more precisely determined by applying the cytogenetic analysis of the karyotype and was complemented with molecular methods and pathohistological examination. The complex karyotype was accompanied by the TP53, C-MYC, and IGH gene disruptions, the most probable cause of rapid evolution into Richter’s syndrome. Conclusion. The use of comprehensive contemporary diagnostic techniques is highly recommended in patients who are in the progressive phase of CLL, primarily for the adequate choice of management strategy. The presented case confirms that aneuploidy in CLL patients indicates poor prognosis, which is in accordance with previous publications reporting on cases of CLL patients with aneuploidy.Uvod. Prisustvo aneuploidije kod bolesnika sa dijagnozom hronične limfocitne leukemije (HLL), sa izuzetkom trizomije 12, smatra se retkom pojavom. Pojava hiperdiploidnog ili kariotipa blizu tetraploidnog broja hromozoma (koji se javlja kod 1–3% svih bolesnika sa HLL) smatra se lošim prognostičkim parametrom. Prikaz bolesnika. Prikazan je bolesnik u uznapredovaloj fazi HLL sa kariotipom blizu triploidnog broja hromozoma. Prognoza bolesti je preciznije određena citogenetičkom analizom kariotipa bolesnika, i dopunjena molekularnim metodama i patohistološkom analizom. Otkriveno je prisustvo kompleksnog kariotipa udruženog sa poremećajima u genima TP53, C-MYC i IGH, što je najverovatnije bio uzrok brze progresije u Rihterov sindrom. Zaključak. Primena savremenih dijagnostičkih metoda veoma je značajna kod bolesnika u uznapredovaloj fazi HLL, prvenstveno zbog adekvatnog terapijskog pristupa. Prikazani slučaj ukazuje da je prisustvo aneuploidije kod bolesnika sa HLL loš prognostički znak, što je u saglasnosti sa prethodno publikovanim prikazima bolesnika sa HLL i sa aneuploidijom u kariotipu

Signifi cance of molecular diagnosti cs in therapy of chronic lymphocytic leukemia

Chronic lymphocyti c leukemia (CLL) is a malignancy of mature CD5+ B lymphocytes that is characterized by excepti onal clinical and biological heterogeneity. The Rai and Binet staging systems, developed in the late 1970s to early 1980s, are used in clinical practi ce to strati fy CLL pati ents into risk categories and to help guide clinical follow-up opti ons: to treat or to watch and wait. However, in early-stage disease, these systems are unable to predict what pati ents will face the progression to a more aggressive disease. That means, a number of molecular markers with prognosti c and/ or predicti ve impact exist and their assessment is strongly recommended in all pati ents prior to treatment initi ati on. One of the fi rst recognized prognosti c genomic aberrati ons in CLL include those detected by fl uorescence in situ hybridizati on (FISH): del(17p), del(11q), trisomy 12 and del(13q), and the immunoglobulin heavy variable (IGHV) gene somati c hypermutati on (SHM) status. Moreover, the rapid development of genomics techniques greatly expanded the understanding of CLL at the molecular level in the past decade. This resulted in the discovery of many newer prognosti c markers based on chromosomal aberrati ons or gene mutati ons. For instance, next-generati on sequencing (NGS) studies have led to the discovery of recurrently mutated genes in CLL, such as NOTCH1, SF3B1, BIRC3, XPO1, POT1, NFKBIE and EGR2, that are associated with poor clinical outcome. Among all of these biomarkers, the disti ncti on between markers of prognosti c and predicti ve values should be made. Prognosti c markers refer to biomarkers that can provide informati on regarding the pati ent's outcome regardless of treatment. They are o����� en assessed before treatment to help guide decisions on to treat or not. Markers associated with overall survival (OS) or ti me to fi rst treatment (TTFT) represent such examples. On the other hand, predicti ve markers are related to therapeuti c interventi ons with the ability to predict treatment response to a drug. These markers are normally assessed when pati ents receive the parti cular therapy. Some markers can be both prognosti c and predicti ve. The Nati onal Comprehensive Cancer Network guideline recommends testi ng of TP53 geneti c alterati ons, IGHV mutati on status, and several well-established cytogeneti c markers for CLL prognosti cati on. Of these, TP53 mutati ons, IGHV unmutated status, del(17p), and del(11q), as well as complex karyotype (the presence of three or more unrelated clonal chromosomal abnormaliti es in a sample), are associated with poor prognosis. Normal karyotype and trisomy 12 are considered as intermediate prognosti c factors, whereas del(13q) is associated with a favorable prognosis. The higher frequencies of the previously menti oned unfavorable markers (except for IGHV) found in the treated populati on usually imply the clonal evoluti on during disease progression or change in clonal dynamics induced by therapies, especially chemotherapies. Diff erent molecular and genomic techniques are employed for detecti ng molecular biomarkers in CLL. For IGHV mutati on status, the preferred method is Sanger sequencing to detect mutati ons in genomic DNA or cDNA following PCR, and align the resulti ng sequences to the germline IGHV using the IMGT/V-QUEST analyti c tool, where ≥ 98% homology to the germ line is interpreted as unmutated, >2% nonhomology as mutated, and 97.0% to 97.9% is interpreted as borderline. Prognosti cally signifi cant chromosomal abnormaliti es are frequently detected using fl uorescence in situ hybridizati on, array comparati ve genomic hybridizati on or conventi onal karyotyping. Fluorescence in situ hybridizati on, although off ers a high sensiti vity and specifi city, requires prior knowledge of chromosomal lesions for the probe designs and are limited to the chosen panel genes. The technique has limitati ons in detecti ng possible complex cytogeneti c abnormaliti es, as well. On the other hand, karyotyping and array comparati ve genomic hybridizati on provide genome-wide coverage. Despite the fact that array comparati ve genomic hybridizati on does not eff ecti vely detect balanced chromosomal rearrangements, it uncovers more genomic abnormaliti es than karyotyping as the probe-based technology examines the chromosomal structure at a much higher resoluti on. Development in NGS technology in the past two decades, made the technique, especially targeted sequencing of gene panels, much less costly and accessible. Currently, in Serbia, geneti c techniques such as FISH, conventi onal karyotyping, Sanger sequencing and NGS are available for detecti on of CLL biomarkers. Advances in the understanding of CLL pathogenesis have consequently led to the development of several highly eff ecti ve targeted therapies, including Bruton tyrosine kinase (BTK), phosphati dylinositol 3-kinase, and BCL2 apoptosis regulator (BCL2) directed inhibitors. B-cell survival and proliferati on is regulated by the BCR signaling pathway. In normal B cells, BCR is triggered by anti gen ligati on, leading to acti vati on of a cascade of tyrosine kinases, including BTK. BCR signaling is aberrantly acti vated in many B-cell malignancies, including CLL. Ibruti nib has demonstrated high clinical effi cacy acti ng as an irreversible potent inhibitor of Bruton's tyrosine kinase and targets several key components of the BCR pathway. However, despite having 80% to 90% response rate, 10% to 15% of CLL pati ents, who respond initi ally, develop ibruti nib resistance and disease relapse in 2 to 3 years on ibruti nib treatment, mainly because of the acquisiti on of a BTK C481S mutati on. The mutati on prevents the drug from forming a covalent bond with the C481 residue that weakened the drug-BTK binding by 500-fold. As a result, BCR signaling and cell proliferati on were restored in the tumor cells. BTK mutati ons may be found in approximately 70% of CLL pati ents who progressed on ibruti nib treatment. Another resistance mechanism is through acquired acti vati ng mutati ons in PLCG2, which is found in approximately 10% of the cases. Given these evidences, the current Nati onal Comprehensive Cancer Network guideline recommends testi ng for BTK and PLCG2 mutati ons for CLL pati ents receiving ibruti nib who are suspected of having disease progression. NGS has become the opti mal method for detecti ng BTK or PLCG2 mutati ons in the se����� ng of ibruti nib treatment, as multi ple mutati ons in both genes may occur in the same specimen. Currently, approximately 20% of CLL pati ents who progressed on ibruti nib do not have either BTK or PLCG2 mutati ons; thus, with NGS, it is possible to uncover other less common but yet undefi ned drug-resistance mutati ons. In additi on to BTK and PLCG2 mutati ons known to confer ibruti nib resistance, other molecular markers have been associated with an upfront high risk of relapse on ibruti nib treatment. It has been reported that complex karyotype, del(17p)/TP53 mutati on, and del(18p) at baseline before ibruti nib treatment are strongly associated with disease relapse. Other approved targeted agents for CLL treatment include the phosphati dylinositol 3-kinase inhibitors idelalisib and duvelisib and BCL2 inhibitor venetoclax. For venetoclax, a novel BCL2-G101V mutati on was identi fi ed to prevent drug acti vity through drug-protein interacti on. Each pati ent with CLL may have several clinical and molecular markers of confl icti ng prognosti c signifi cance simultaneously, making the precise prognosti cati on challenging. Today is of the greatest importance to apply ultrasensiti ve techniques to reveal molecular relapses a����� er therapy initi ati on and to detect minimal residual disease a����� er pati ents achieve complete responses

Acute promyelocytic leukemia lacking t(15;17): Molecular evidence of atypical PML/RAR-α transcriptional variant by gene sequencing

Uvod. Precizno dijagnostikovanje akutne promijelocitne leukemije (APL), ne samo na osnovu morfoloških i kliničkih parametara, već i na molekularnom nivou, veoma je važno radi primene adekvatne ciljane terapije. Prikaz bolesnika. Prikazali smo bolesnicu, staru 62 godine, sa dijagnozom APL. Primenom standardne citogenetičke analize, kao i primenom fluorescentne in situ hibridizacije (FISH), nije bilo potvrđeno prisustvo t(15;17) kod opisane bolesnice. Primenom metode reverzna transkriptazalančana reakcija polimeraze (RT-PCR), identifikovana su dva atipična promyelotic leukemia/retinoic acid receptor alpha (PML/RAR-α) fuziona transkripta. Oba transkripta su predstavljala izoforme. Duži transkript je zadržao "okvir čitanja" i kodirao je funkcionalan PML/RAR-α aberantni protein, dok je kraći transkript bio van "okvira čitanja". Zaključak. Naša studija ukazuje na potrebu za primenom molekularne metodologije u svakodnevnoj kliničkoj praksi. Precizna karakterizacija PML/RAR-α fuzionih transkipta čini osnovu za identifikovanje retkih bolesnika čije lečenje zahteva dodatni oprez. Prema našim saznanjima, ovo je tek peti slučaj opisanog atipičnog PML/RAR-α transkripta koji u sebi sadrži celokupan PML egzon 7a, a među njima jedini koji se nije mogao detektovati primenom citogenetičke i FISH analize. Svi ovde predstavljeni slučajevi su imali smrtni ishod. Zbog toga, naši rezulatati, zajedno sa sličnim slučajevima opisanim u literaturi, naglašavaju značaj detaljne identifikacije atipičnih PML/RAR-α fuzija, ne samo u svrhu prepoznavanja njihove uloge u procesu leukemogeneze, veći i u smislu procene njihovog uticaja na ishod lečenja.Introduction. The accurate diagnosis of acute promyelocytic leukemia (APL), not only on the morphological and clinical, but also on the molecular level, is very important for application of targeted therapies. Case report. A 62year-old woman presented with APL. By using conventional cytogenetic analysis as well as applying the fluorescence in situ hybridization (FISH) analysis it has not been possible to confirm the presence of t(15;17) in the presented patient. Using reverse transcriptase polymerase chain reaction (RT-PCR) two atypical promyelotic leukemia/retinoic acid receptor alpha (PML/RAR-α) fusion transcripts were identified. Both detected transcripts were isoforms. The larger transcript was in-frame, coding for functional aberrant PML/RAR-α protein, while the shorter transcript was an out-of-frame. Conclusion. Our study highlights the need for the application of molecular methodology in daily clinical practice. Precise characterization of PML/RAR-α fusion transcript creates a basis for identifying rare individual cases that require special caution when treating such patients. To our knowledge this is only the fifth case of atypical PML/RAR-α transcript containing full PML exon 7a, and among them the only one that was cytogenetically cryptic and FISH negative. All of the herein presented cases had lethal outcome. Therefore, our findings with the additional review of the literature, emphasizes the importance of detailed identification of atypical PML/RAR-α fusions, not only for the purpose of knowing their role in leukemogenesis, but also for the assessment of the impact that they can have on the outcome of the treatment

Comparative analysis of International Prognostic Index for chronic lymphocytic leukemia, progression-risk score, and MD Anderson Cancer Center 2011 score: A single center experience

Uvod/Cilj Prognoza hronične limfocitne leukemije (HLL) značajno je unapređena u poslednje vreme. Među nekoliko prognostičkih modela čiji je cilj predviđanje vremena do prve terapije (eng. TTFT) izdvajaju se skor rizika od progresije (eng. PRS) i skor Centra za rak MD Anderson iz 2011. God. (eng. MDACC 2011), dok se internacionalni prognostički indeks za HLL (eng. CLL-IPI), iako primarno ustanovljen za predikciju ukupnog preživljavanja (eng. OS), dobro pokazao i u predikciji TTFT. Cilj ovog rada je da se ispita značaj pomenutih prognostičkih modela u pogledu predviđanja TTFT i OS. Metode Analizirana kohorta je obuhvatila 57 neselektovanih bolesnika sa HLL Univerzitetskog kliničkog centra Srbije sa prosečno agresivnijim profilom bolesti u odnosu na opštu populaciju de novo bolesnika sa HLL. Bolesnici su ocenjivani prema navedenim skorovima uz analizu TTFT i OS. Rezultati Bolesnici sa višim vrednostima CLL-IPI, PRS i MDACC 2011 primili su prvu terapiju značajno ranije u poređenju sa bolesnicima sa nižim vrednostima ovih skorova (p = 0,002, p = 0,019 i p lt 0,001, redom). U multivarijantnoj analizi, MDACC 2011 i CLL-IPI su zadržali prognostički značaj u predikciji TTFT (p = 0,001, odnosno p = 0,018), dok je PRS ovaj značaj izgubio. CLL-IPI je bio jedini značajan prediktor OS u univarijantnoj (p = 0,005) i u multivarijantnoj analizi (p = 0,013). Zaključak CLL-IPI, PRS i naročito MDACC 2011 su dobri prediktori TTFT čak i u kohortama bolesnika sa agresivnijom bolešću, dok je za predikciju OS od ova tri prognostička modela CLL-IPI jedini primenljiv. Ovi rezultati pokazuju da bi prognostičke modele trebalo ispitati na bolesnicima sa HLL u različitim fazama bolesti, kakvi se sreću u realnoj kliničkoj praksi.Introduction/Objective Prognostication of chronic lymphocytic leukemia (CLL) has been substantially improved in recent times. Among several prognostic models (PMs) focused on the prediction of time to first treatment (TTFT), progression-risk score (PRS), and MD Anderson Cancer Center score 2011 (MDACC 2011) are the most relevant, while CLL-International Prognostic Index (CLL-IPI), although originally developed to predict overall survival (OS), is also being used to estimate TTFT. The aim of this study was to investigate CLL-IPI, PRS, and MDACC 2011 prognostic values regarding TTFT and OS. Methods The analyzed cohort included 57 unselected Serbian CLL patients from a single institution, with the basic characteristics reflecting more aggressive disease than in the general de novo CLL population. The eligible patients were assigned investigated PMs, and TTFT and OS analyses were performed. Results Patients with higher risk scores according to CLL-IPI, PRS, and MDACC 2011 underwent treatment significantly earlier than patients with lower risk scores (p = 0.002, p = 0.019, and p lt 0.001, respectively). In multivariate analysis, MDACC 2011 and CLL-IPI retained their significance regarding TTFT (p = 0.001 and p = 0.018, respectively), while PRS did not. CLL-IPI was the only significant predictor of OS both at the univariate (p = 0.005) and multivariate (p = 0.013) levels. Conclusion CLL-IPI, PRS, and particularly MDACC 2011 are able to predict TTFT even in cohorts with more advanced-disease patients, while for prediction of OS, CLL-IPI is the only applicable among the three PMs. These results imply that PMs should be investigated in more diverse CLL populations, as it is in real-life setting

Pattern of trisomy 1q in hematological malignancies: a single institution experience

An extra copy of 1q usually originates from the translocated unbalanced derivative chromosome, isochromosome, or "jumping translocation." We report a pattern of partial trisomies and unbalanced whole-arm translocations of 1q in 10 patients: 5 with myelodysplastic syndrome, 3 with acute myeloid leukemia, and a single patient with acute lymphoblastic leukemia and myeloproliferative syndrome. The trisomy of 1q was registered as the sole karyotype aberration in one patient, while it was accompanied by a limited number of additional chromosomal changes in nine patients. These patients are a subset of a larger group of 92 adults carrying a wide variety of chromosome 1 anomalies within a complex cytogenetic context observed over a period between 1994 and 2006 in a panel of 3,786 hematologic patients at the Institute of Hematology in Belgrade. Conventional cytogenetics was supplemented by fluorescence in situ hybridization with a probe specific for the paracentric region of 1q. Whole-arm 1q translocations involved chromosomes Y, 7, 14, 15, 16, and 19. This study suggests that gain of 1q as the sole cytogenetic abnormality may be sufficiently mutagenic to favor leukemogenesis and hematopoietic tissue degeneration (trilineage myelodysplasia)

COEXISTENCE OF TRISOMY 12 AND DEL(13)(Q14.3) IN TWO PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA

Abstract — We describe two patients with diagnosis of chronic lymphocytic leukemia (CLL) in whom interphase fluorescence in situ hybridization (FISH) analysis revealed trisomy 12 and del(13)(q14.3) occurring in the same clone. These abnormalities are rarely seen together and the prognostic relevance of their coexistence is still unclear. According to some data, a probable adverse prognosis for this group of patients is suggested. Our patients have been in a stable phase of the disease for more than one year since the given abnormalities were documented in their karyotypes. Further study is necessary to determine the prognostic significance of coexistence of these abnormalities in CLL patients

The predictive value of morphological findings in early diagnosis of acute myeloid leukemia with recurrent cytogenetic abnormalities

This study explores cytomorphologic features and their predictive role for early identification of acute myeloid leukemia (AML) with morphological distinctive recurrent cytogenetic abnormalities (RCA): t(15;17), t(8;21) and inv(16)/t(16;16). We retrospectively evaluated 396 de novo AML cases, diagnosed and treated at single institution, between 2013-2017. Specific cytomorphologic features suggesting distinctive AML-RCA were revealed at diagnosis in 62 (15.65%) patients, including AML with t(15;17) in 41 (66.13%), t(8;21) in 13 (20.97%) and inv(16)/t(16;16) in 8 (12.90%). Final diagnoses of AML-RCA according to WHO integrated diagnostic criteria were established in 66 (16.66%) cases, including AML with t(15;17) 40 (60.60%), t(8;21) 17 (25.76%), and inv(16)/t(16;16) 9 (13.64%). Discordance between cytomorphological and other integrated criteria was detected as missed/wrong-call in 0/1 for t(15;17), 6/2 for t(8;21) and 2/1 for inv(16)/t(16;16). The cytomorphological accuracy was 97.56% (40/41) for t(15;17), 57.89% (11/19) for t(8;21) and 70% (7/10) for inv (16)/t(16;16). Positive/negative predictive values of cytomorphological evaluation were: 97.56%/100% for t(15;17); 84.62%/88.68% for t(8;21);87.50%/96.65% for inv(16)/t(16;16). Sensitivity/specificity were: 100%/96.15% for t(15;17);64.10%/95.92% for t(8;21);77.78%/98.25% for inv(16)/t(16;16). We confirmed that morphology is still a highly relevant evaluation method in diagnosing several common AML-RCAs before completing cytogenetic and molecular studies, enabling early detection, particularly of AML with t(15;17)

Association of SLC28A3 Gene Expression and CYP2B6*6 Allele with the Response to Fludarabine Plus Cyclophosphamide in Chronic Lymphocytic Leukemia Patients

Fludarabine plus cyclophosphamide (FC) chemotherapy is the basis of treatment protocols used in management of chronic lymphocytic leukemia (CLL). In some patients, response to therapy may be affected by aberrant function of genes involved in pharmacokinetics and pharmacodynamics of the drugs. The aim of this research was to assess the impact of pharmacogenetic variability, namely expression of SLC28A3 gene and the presence of CYP2B6*6 variant allele, on the FC treatment efficacy. Forty-four CLL patients with functional TP53 gene at the time of FC initiation were enrolled in this study. CYP2B6 genotyping was performed by polymerase chain reaction and direct sequencing. SLC28A3 expression was measured by quantitative reverse-transcriptase polymerase chain reaction. Significantly higher pretreatment levels of SLC28A3 mRNA were detected in patients who failed to respond to FC in comparison to patients who achieved complete and partial response (p = 0.01). SLC28A3 high-expressing cases were almost ten times more likely not to respond to FC than low-expressing cases (OR = 9.8; p = 0.046). However, association of SLC28A3 expression with progression-free survival (PFS) and overall survival (OS) was not observed. CYP2B6*6 allele, detected in 24 patients (54.6%), exerted no association with the attainment of response to FC, as well as with PFS and OS. The results of this study demonstrate that SLC28A3 expression is a significant predictor of FC efficacy in CLL patients with intact TP53. Elevated SLC28A3 mRNA levels are associated with inferior short-term response to FC, suggesting that, if validated on larger cohorts, SLC28A3 expression may become a biomarker useful for pretreatment stratification of patients