Pogostnost monoklonske B-celične limfocitoze pri zdravih osebah Dolenjske : doktorska disertacija

Bajuk, Petra (author), Podgornik, Helena (mentor), Božič, Borut (comentor)

Monoklonska B-celična limfocitoza (MBL) je predstopnja kronične limfatične levkemije (KLL). Pogosteje se pojavlja pri starejših, moških in pri posameznikih, ki imajo v prvem kolenu sorodnika s KLL. Za določitev MBL mora biti v krvi preiskovanca manj kot 5 × 109 limfocitov B/L, pri čemer s pretočno citometrijo (PC) ugotovimo značilen imunofenotip, ki ga lahko spremljata abnormalno razmerje lahkih verig imunoglobulinov κ : λ in preureditev težkih verig IgVH, obenem pa izključimo KLL ali drugi B-celični limfom. Glede na imunofenotip ločimo KLL-značilno MBL, CD5– MBL in atipično MBL. KLL-značilna MBL ima podoben imunofenotip kot KLL in je najpogostejši podtip MBL. Glede na število monoklonskih limfocitov B jo ločimo še na presejalno MBL (sMBL) in klinično MBL (cMBL). CD5– MBL zaznamuje odsotnost izražanja CD5, atipično MBL pa visoko izražanje CD20. S preiskavo fluorescenčna »in situ« hibridizacija (FISH) lahko pri MBL odkrijemo nekatere citogenetske nepravilnosti, najpogostejši sta del(13)(q14) in trisomija 12. Pojavnost bolezni se v splošni populaciji giblje med 3 % in 5 %, vsako leto pa pri približno 2 % bolnikov bolezen napreduje v KLL. Bolezen se tudi do sedemkrat pogosteje pojavlja pri sorodnikih v prvem kolenu bolnikov s KLL. To je še posebno značilno za družine, kjer sta prisotna vsaj dva primera KLL. V Sloveniji podatkov o MBL še nimamo, zato smo se odločili narediti raziskavo o pojavnosti te bolezni in jo primerjati med zdravo splošno populacijo in sorodniki v prvem kolenu bolnikov s KLL. V doktorski disertaciji smo preverjali naslednje 3 hipoteze: 1. da je pojavnost MBL večja pri sorodnikih v prvem kolenu bolnikov s KLL kot tista v splošni populaciji, z uporabljenim načinom določanja je pojavnost bolezni moč odkriti med 5 % in 15 % preiskovane populacije; 2. da je najpogostejši podtip MBL pri sorodnikih bolnikov s KLL klinična oblika KLL-značilne MBL, pojavnost v obeh skupinah pa je večja pri moških; 3. prisotne so ponavljajoče citogenetske spremembe, najpogostejša je del(13q), status IgVH pa je pri večini vzorcev MBL mutiran. Za področje raziskave smo izbrali Dolenjsko, ki predstavlja tipično slovensko pokrajino. S pomočjo Splošne bolnišnice Novo mesto se je za sodelovanje v študiji iz te regije odločilo 54 sorodnikov v prvem kolenu bolnikov s KLL. Za vsakega od njih smo našli 3 zdrave preiskovance iz splošne populacije, ki so se s posameznim sorodnikom ujemali v spolu, starosti in kraju bivanja. Skupaj smo analizirali 216 vzorcev. Povprečna starost preiskovancev je bila 48,4 ± 13,1, mediana 48, starostni razpon pa od 23 do 88 let. Preiskovana moška populacija je bila mlajša (povprečna starost 46,5 ± 13,3, mediana 45,5, starostni razpon od 23 do 88 let) od ženske (povprečna starost 50 ± 12,9, mediana 49, starostni razpon od 25 do 86 let). Pri vseh vzorcih smo naredili popolno krvno sliko in tako izločili neznane hematološke bolezni. Vzorce smo najkasneje 12 ur po odvzemu krvi analizirali s 5-barvno pretočno citometrijo (κ, λ, CD5, CD19, CD20). Posamezno celično populacijo smo prepoznali kot klon MBL, če smo lahko zaznali vsaj 50 dogodkov z enakimi značilnostmi. Imunofenotip klona MBL smo določili po osamitvi populacije pozitivne na CD19. Izračunali smo tudi razmerje κ : λ. Pri vzorcih, kjer smo odkrili klon MBL, smo opravili standardno citogenetsko preiskavo in FISH. Analizo proganih kromosomov smo delali neposredno na gojenih vzorcih krvi. Iz vzorcev venske krvi MBL smo s pozitivno selekcijo izolirali CD19 pozitivne limfocite B. Na teh celicah smo izvedli analizo FISH, ki smo jo ponovili še na celicah po gojenju brez predhodne izolacije. Preverili smo prisotnost za KLL značilnih kromosomskih preureditev. Iz izoliranih celic smo pridobili DNA za določitev mutacijskega statusa IgVH. Med splošno populacijo smo klon MBL odkrili pri 7 od 162 preiskovancev (4,3 %), med vzorci sorodnikov v prvem kolenu bolnikov s KLL pa pri 3 od 54 preiskovancev (5,6 %). Skupaj smo MBL odkrili v desetih vzorcih oz. pri 4,6 % vseh preiskovancev. Pojavnost je bila pogostejša pri sorodnikih v prvem kolenu bolnikov s KLL, kar je bilo glede na ugotovitve objavljenih tujih študij tudi pričakovano. Povprečna starost preiskovane populacije je bila 48 let, zato bi bila lahko pojavnost MBL ob starejši kohorti še večja. Potrdili smo namreč predhodne ugotovitve tujih študij, da se s starostjo pojavnost bolezni povečuje. Pri 6 od 7 preiskovancev iz splošne populacije, ki so bili starejši od 40 let, smo odkrili MBL. Klon MBL smo pogosteje odkrili pri moških. Z imunofenotipizacijo nam je uspelo določiti vse podtipe MBL: 2 primera KLL-značilne MBL (1 primer cMBL in 1 primer sMBL), 2 primera CD5– MBL in 6 primerov atipične MBL. Za vzorec s klinično obliko KLL-značilne MBL je bilo v primerjavi z ostalimi vzorci značilno izrazito povečanje povprečnih vrednosti limfocitov B zaradi povečanega števila monoklonskih celic B. Z raziskavo smo pridobili prvi vpogled o pojavnosti te bolezni v naši državi. Povzamemo lahko, da je pojavnost MBL v Sloveniji s približno 5 % na podobni ravni kot v objavljenih evropskih študijah, še pogosteje pa se lahko pojavlja pri mlajših sorodnikih (≤ 50 let) v prvem kolenu bolnikov s KLL (okvirno 10 %). Razlike v podtipu MBL bi lahko pripisali izbiri kohorte, saj so tuje študije v povprečju preiskovale bistveno starejše populacije. Iz tega lahko sklepamo, da se predvsem pri mlajših odraslih lahko pogosteje pojavi bolezen z imunofenotipskimi značilnostmi atipične MBL. S preiskavo FISH smo odkrili kromosomske nepravilnosti. V vzorcu M82, ki ustreza cMBL, smo pri 91 % limfocitov B našli kromosomsko nepravilnost del(13q). V vzorcu F68 smo s preiskavo FISH zaznali kromosomsko nepravilnost trisomijo 12 le pri 5 % celic, ki smo jo potrdili tudi s standardno citogenetsko analizo proganih kromosomov (Tabela 16). S standardnimi citogenetskimi preiskavami smo določili tudi mitotski indeks (MI), ki je bil pri vzorcih MBL v povprečju višji (4,45 ‰) kot pri vzorcih zdravih preiskovancev (2,24 ‰). Pri dveh vzorcih s klonom MBL smo lahko določili mutacijski status IgVH. Pri vzorcu KLL-značilne MBL, podtip cMBL (M82), smo odkrili mutiran gen IGHV3-64 (94,1 % homologija). Drugi vzorec (M57c) z mutiranim genom IGHV3-15*02 (89,1 % homologija) pa je tudi predstavljal KLL-značilno MBL, in sicer podtip sMBL. Postavili smo presejalno metodo za analizo s 5-barvno pretočno citometrijo in določili njeno občutljivost s postavitvijo meje detekcije klona limfocitov MBL. Za potrditev klona MBL potrebujemo vsaj 50 dogodkov z istimi značilnostmi. Na desetih MBL vzorcih smo izvedli tudi citogenetske analize in prispevali nove podatke na tem področju, kar je do sedaj storila le malokatera študija. Naša preiskovana kohorta je bila mlajša v primerjavi z veliko večino tujih raziskav, zato bi lahko naša odkritja doprinesla k razumevanju pojavnosti in oblike te bolezni pri mlajših odraslih osebah.

• Language: Slovenian
• Keywords: monoklonska B-celična limfocitoza, incidenca, Dolenjska, disertacije
• Work type: Dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: FFA - Faculty of Pharmacy
• Place of publishing: Ljubljana
• Publisher: [P. Bajuk Franko]
• Year: 2018
• Number of pages: XV, 124 f.
• PID: 20.500.12556/RUL-137375
• UDC: 616.155.3(497.434)(043.3)
• COBISS.SI-ID: 294313728
• Publication date in RUL: 15.06.2022

Secondary language

• Language: English
• Title: Prevalence of monoclonal B-cell lymphocytosis in healthy population of Lower Carniola

Abstract

Monoclonal B-cell lymphocytosis (MBL) is a precursor state for chronic lymphocytic leukemia (CLL). The incidence of MBL is higher in older individuals, men and first-degree relatives of patients with CLL. MBL is defined by the presence of more than 5 × 109 limfocytes B/L in blood of individual. Flow cytometry (FC) is used to define disease-specific immunophenotype. Abnormal ratio of light chains κ : λ and IgVH heavy-chain rearrangement can be present. CLL and other B-cell lymphoma need to be excluded to confirm diagnosis of MBL. Based on the immunophenotype MBL can be further subclassified into CLL-like MBL, CD5– MBL and atypical MBL. CLL-like MBL has immunophenotype similar to that of CLL and is the most common type of MBL. CLL-like MBL is further subclassified based on the number of monoclonal lymphocytes B into screening MBL (sMBL) and clinical MBL (cMBL). CD5– MBL is characterized by the absence of CD5 expression, while atypical MBL displays CD5 positivity along with higher expression of CD20. Fluorescence »in situ« hybridization (FISH) is used to find cytogenetic aberrations in MBL, the most common being del(13)(q14) and trisomy 12. Incidence of MBL in general population is 3 % to 5 % with 2 % of patients with cMBL progressing to CLL every year. The incidence of the disease is up to 7 times higher in first-degree relatives of patients with CLL, especially in families with at least 2 cases of CLL. We currently do not have data on MBL incidence in Slovenia, so we decided to make a study of the disease in our country and compare general population to first-degree relatives of patients with CLL. We considered three hypotheses: 1. incidence of MBL is higher in first-degree relatives of patients with CLL than in general population. We expect to find MBL in 5 % to 15 % of researched population with used detection techniques. 2. we expect that the most common finding in first-degree relatives of patients with CLL is CLL-like MBL, while the incidence of MBL in both groups will be higher in men. 3. we expect cytogenetic aberrations with the most common del(13). Mutation status IgVH is mutated in most cases. We focused on Slovenian region of Lower Carniola that represents typical Slovenian mix of rural and urban environment. With the help of Hospital Novo mesto 54 first-degree relatives of patients with CLL decided to participate in our study. For each relative we found 3 healthy subjects in general population who matched sex, age and location. Alltogether we analysed 216 samples. Average age of our subjects was 48,4 ± 13,1, median 48, age range 23 to 88 years. Male population was younger (average age 46,5 ± 13,3, median 45,5, age range 23 to 88 years) than female population (average age 50 ± 12,9, median 49, age range 25 to 86 years). Complete blood count was made on all samples to exclude unknown hematological diseases. All samples were analysed with 5-color flow cytomety (κ, λ, CD5, CD19, CD20) within 12 hours of blood removal. A single population was recognized as a MBL clone when at least 50 events with same characteristics were detected. Immunophenotype of MBL clone was determined after isolation of population positive on CD19. We also calculated κ : λ ratio. All samples with MBL clone were further analysed with conventional cytogenetics and FISH. Chromosome banding was done on stimulated blood samples. CD19 isolated positive lymphocytes B were isolated with positive selection on MBL samples. FISH analysis was performed on both isolated cells and on stimulated cells without prior isolation. Each MBL sample was analyzed for typical CLL chromosomal aberrations, IgVH mutation status was also determined on previously isolated cells. In the general population MBL was found in 7 of 162 samples (4,3 %) and in 3 of 54 samples (5,6 %) of first-degree relatives of patients with CLL. Alltogether we found MBL in 10 samples (4,6 %). Incidence of MBL was higher in first-degree relatives of patients with CLL, which was expected due to the similar discoveries of foreign studies. Average age of our cohort was 48 years, the incidence of MBL could be even higher if older population was analysed. We confirmed findings of other studies that the incidence of MBL increases with age. In general population, 6 of 7 subjects with MBL were older than 40 years. MBL was more common in men. All three categories of MBL were detected – 2 cases of CLL-like MBL (1 case of cMBL and 1 case of sMBL), 2 cases of CD5– MBL and 6 cases of atypical MBL. In comparisson with other MBL cases in one case with cMBL the number of lymphocytes B was much higher due to higher numbers of monoclonal B cells. With this study we gained first insight into incidence of this disease in our country. We can conclude that the incidence of MBL in Slovenia is around 5 %, similar to findings of foreign European studes. The incidence is higher in younger (≤ 50 years) first-degree relatives of patients with CLL (10 %). The differences in discovered MBL types could be linked to the properties of our cohort, which was younger than cohorts in foreign studies. We can also conclude that atypical MBL is more common in younger male adults. FISH analysis was used to determine chromosomal abnormalities. Sample M82 with cMBL featured del(13q) on 91 % of lymphocytes B. We found +12 with FISH on 5 % of cells, which was also confirmed with standard chromosome banding analysis. Conventional cytogenetics was used to determine mitotic index, which was overal higher in MBL samples (4,45 ‰) than in samples of healthy individuals (2,24 ‰). We were able to determine mutation status IgVH in two MBL samples. Sample with cMBL (M82) had mutated gene IGHV3-64 with 94,1 % homology, while sMBL sample (M57c) had mutated gene IGHV3-15*02 with 89,1 % homology. We determined a screening method for 5-color flow cytometry and determing method sensitivity by setting the detection limit for MBL lymphocytes clone. At least 50 events with the same characteristics are needed to confirm MBL clone. Cytogenetic analysis was performed on all 10 MBL samples, which contributed new data for all MBL subtypes. This was done only by few studies to date. Compared to most other studies, our cohort was overall younger. Our findings could therefore contribute to understanding the disease in younger adults.