Evaluation of chronic lymphocytic leukemia by oligonucleotide-based microarray analysis uncovers novel aberrations not detected by FISH or cytogenetic analysis
- Kathryn A Kolquist1,
- Roger A Schultz2,
- Marilyn L Slovak2, 5,
- Lisa D McDaniel1, 2,
- Theresa C Brown3,
- Raymond R Tubbs4,
- James R Cook4,
- Karl S Theil4,
- Victoria Cawich2,
- Caitlin Valentin2,
- Sara Minier2,
- Nicholas J Neill2,
- Steve Byerly2,
- S Annie Morton2,
- Trilochan Sahoo2, 6,
- Blake C Ballif2 and
- Lisa G Shaffer2Email author
© Kolquist et al; licensee BioMed Central Ltd. 2011
Received: 26 August 2011
Accepted: 16 November 2011
Published: 16 November 2011
Cytogenetic evaluation is a key component of the diagnosis and prognosis of chronic lymphocytic leukemia (CLL). We performed oligonucleotide-based comparative genomic hybridization microarray analysis on 34 samples with CLL and known abnormal karyotypes previously determined by cytogenetics and/or fluorescence in situ hybridization (FISH).
Using a custom designed microarray that targets >1800 genes involved in hematologic disease and other malignancies, we identified additional cryptic aberrations and novel findings in 59% of cases. These included gains and losses of genes associated with cell cycle regulation, apoptosis and susceptibility loci on 3p21.31, 5q35.2q35.3, 10q23.31q23.33, 11q22.3, and 22q11.23.
Our results show that microarray analysis will detect known aberrations, including microscopic and cryptic alterations. In addition, novel genomic changes will be uncovered that may become important prognostic predictors or treatment targets for CLL in the future.
Chronic lymphocytic leukemia (CLL), the most common leukemia diagnosed in adults from Western countries, is characterized by a monoclonal population of mature activated B lymphocytes that usually express CD5+ and CD23+. However, the clinical features, disease course, and outcomes are highly variable. Most patients diagnosed with CLL can survive for many years, but in a subset of patients the course progresses more rapidly and is fatal despite aggressive treatment.
Currently, the diagnosis of CLL is made using histopathology; flow cytometry with a typical pattern of co-expression of CD5, CD23, CD20(dim), and surface Ig(dim); and chromosomal abnormalities detected by fluorescence in situ hybridization (FISH) probes and karyotyping. Conventional cytogenetics with karyotyping requires the use of an immunostimulatory CpG-oligodinucleotide DSP 30 plus IL-2 cocktail to enhance the yield of detectable chromosome aberrations in CLL cells. This cell culturing process is costly, time consuming, and requires the clinical indication of CLL at sample submission. Alternatively, FISH has been used to detect specific prognostic chromosome markers in CLL using a panel of five to six probes. However, locus-specific FISH does not reveal the complete cytogenetic picture . Prognostic markers, determined primarily using FISH, include deletions of 13q, 17p, and 11q and trisomy 12. Most anomalies detected by cytogenetics in CLL are copy number gains and losses; translocations are rarely identified .
Previously, clinicians waited until patients diagnosed with CLL progressed to a specific stage to initiate therapy. Rai et al. [3, 4] and Binet et al.  created staging systems that, until the past decade, were the hallmark for defining disease extent, prognosis, and initiation of treatment of CLL patients. Although these systems were standard of care, they did not predict the disease course for early-stage disease. In the past decade, our understanding of the pathophysiology of CLL has changed significantly with discoveries such as somatic mutations in the immunoglobulin heavy chain variable region (IGHV) genes, which are associated with a good prognosis [6, 7], and lack of IGHV mutations and increased CD38 and ZAP-70 expression, which are associated with poor prognosis. Along with this has come a change in the way CLL has been approached therapeutically .
Microarray-based comparative genomic hybridization (aCGH) on neoplastic specimens has facilitated diagnosis and gene discovery with the ability to perform genome-wide investigations [7–18]. These array studies have shown concordance with the cytogenetic and FISH results. In addition, these studies demonstrated that appropriate microarray design can facilitate the detection of clinically relevant findings that would be missed using FISH panels. Although promising, most such microarray studies of CLL to date have been technically limited by the use of non-targeted BAC arrays [8, 9, 11], CGH-based oligonucleotide arrays that have either been non-targeted [10, 19] or targeted to a relatively small subset of cancer genes or genomic regions (~15) associated with cancer [16, 20], or SNP-based, whole genome (non-targeted) arrays .
In a novel approach as compared to these previous array studies, we developed a 135K-feature oligonucleotide-based microarray specifically targeted to more than 1800 cancer genes and regions and evaluated 34 patients diagnosed with CLL to compare the performance of this array to that of chromosome analysis and FISH. Our results further support microarray analysis as a diagnostic tool to detect cytogenetic abnormalities associated with CLL. In addition to the detection of known chromosomal rearrangements, we identified cryptic and novel DNA alterations using aCGH.
Thirty-four samples validated on an oligonucleotide array designed for detection of aberrations in leukemia and lymphoma*
Concordant array results
Discordant array results
New findings by microarray
LSI D13S319 96% loss, LSI 13q34 normal, LSI ATM normal, CEP 12 normal, LSI p53 normal
13q14.2q14.3(47,613,553-50,628,718) × 1
22q11.23(22,674,846-22,723,991) × 0
LSI D13S319 77% loss, LSI 13q34 normal, LSI ATM normal, CEP 12 normal, LSI p53 normal
13q14.2q14.3(47,123,245-50,443,082) × 1
LSI D13S319 81% loss (34% mono, 47% bi), LSI 13q34 normal, LSI ATM normal, CEP 12 normal, LSI p53 normal
13q13.3q14.3(36,736,548-51,816,512) × 1, 13q14.3(49,315,855-50,237,971) × 0
15q11.2(19,129,891-19,224,501) × 3, 18p11.32q23(123,388-76,100,854) × 3, 22q11.23(22,674,846-22,723,991) × 0
LSI D13S319 42% loss, LSI 13q34 normal, LSI ATM normal, CEP 12 normal, LSI p53 normal
13q14.13q14.3(45,945,097-50,339,992) × 1
LSI D13S319 normal, LSI 13q34 normal, LSI ATM normal, CEP 12 92% trisomy, LSI p53 normal
12p13.33q24.33(60,861-132,267,241) × 3
LSI D13S319 97% loss, LSI 13q34 normal, LSI ATM normal, CEP 12 74% trisomy, LSI p53 normal
12p13.33q24.33(60,861-132,267,241) × 3, 13q14.3(49,074,574-50,628,718) × 1
19p13.3q13.43(220,598-63,782,017) × 3
LSI D13S319 91% loss, LSI 13q34 normal, LSI ATM 89% loss, CEP 12 normal, LSI p53 normal
11q13.4q24.3(72,459,008-130,030,128) × 1, 13q14.11q21.1(43,421,790-53,019,141) × 1
2p25.3p11.2(44,198-89,912,901) × 3
LSI D13S319 normal, LSI 13q34 normal, LSI ATM normal, CEP 12 63% trisomy, LSI p53 normal
12p13.33q24.33(60,861-132,267,241) × 3
LSI D13S319 normal, LSI 13q34 normal, LSI ATM normal, CEP 12 63% trisomy, LSI p53 normal
12p13.33q24.33(60,861-132,267,241) × 3
LSI D13S319 92% loss, LSI 13q34 12% loss, LSI ATM normal, CEP 12 normal, LSI p53 normal
13q14.3(49,407,720-50,523,594) × 0~1
Array did not detect 13q34 deletion seen in 12% of cells by FISH
12q24.12(110,684,027-110,768,579) × 3
LSI D13S319 normal, LSI 13q34 normal, LSI ATM normal, CEP 12 normal, LSI p53 normal
1p31.3(64,811,810-68,404,781) × 3, 1q21.3q23.1(151,852,847-155,280,574) × 3, 2p16.1p15(59,245,962-62,672,016) × 3, 5q35.2q35.3(174,945,789-178,610,160) × 3, 11p15.4p15.3(7,907,684-11,413,676) × 3, 12p13.33p13.31(2,409,808-5,956,328) × 3, 16q24.1q24.2(82,968,178-86,062,471) × 3, 17q22q23.2(53,582,801-57,412,725) × 3, 18q21.32q22.1(57,089,022-60,760,895) × 3
LSI D13S319 45% loss, LSI 13q34 normal, LSI ATM 47%, CEP 12 normal, LSI p53 normal
11q14.1q24.3(79,218,490-128,057,190) × 1, 13q14.11q21.33(43,525,071-71,879,067) × 1
Yp11.31q12(2,706,656-57,735,230) × 0
LSI D13S319 93% loss, LSI 13q34 normal, LSI ATM normal, CEP 12 normal, LSI p53 normal
13q14.3(49,457,877-50,339,992) × 0
LSI D13S319 normal, LSI 13q34 normal, LSI ATM normal, CEP 12 71% trisomy, LSI p53 normal
12p13.33q24.33(60,861-132,267,241) × 3
14q24.1q32.33 (68,329,913-105,393,508) × 1, 22q11.23(22,674,846-22,723,991) × 0
LSI D13S319 89% loss, LSI 13q34 normal, LSI ATM 98% loss, CEP 12 normal, LSI p53 normal
11q14.3q23.2(88,551,231-114,026,260) × 1, 13q14.2q14.3(47,463,489-51,926,538) × 1
2p16.1p14(56,499,065-66,570,230) × 3, 4p16.3p15.1(45,627-29,325,651) × 1, 5q33.2q35.3(152,262,081-180,619,169) × 3, 7q31.32q36.3(123,057,209-158,821,424) × 3
LSI D13S319 40% loss, LSI 13q34 normal, LSI ATM normal, CEP 12 normal, LSI p53 normal
13q14.2q14.3(47,691,117-50,339,992) × 1
1q32.1(203,529,401-204,498,513) × 1, 12p13.33q24.33(60,861-132,267,241) × 2~3
LSI D13S319 92% loss, LSI 13q34 normal, LSI ATM 11% loss, CEP 12 90% trisomy, LSI p53 normal
12p13.33q24.33(60,861-132,267,241) × 3, 13q13.3q21.1(37,240,922-55,410,522) × 1
Array did not detect deletion of 11q22.3 seen in 11% of cells by FISH
5p15.32p15.31(5,388,368-6,879,401) × 1, 10q21.1q21.3(58,227,677-67,149,424) × 1, 10q23.31q23.33(90,215,922-95,066,500) × 1, 11q22.1q22.2(101,471,877-101,736,881) × 1, Yp11.32q12(1-57,735,230) × 0,
LSI D13S319 70% loss, LSI 13q34 normal, LSI ATM 58% loss, CEP 12 normal, LSI p53 normal
11q14.1q25(78,733,283-131,062,293) × 1, 13q14.2q14.3(48,739,670-50,554,228) × 1
LSI D13S319 96% loss, LSI 13q34 normal, LSI ATM 98% loss, CEP 12 normal, LSI p53 normal
11q14.3q23.3 (91,814,326-116,080,874) × 1, 13q14.2q14.3(48,774,702-50,765,417) × 1
2p25.3p14(44,198-66,539,084) × 3, 2p14p11.2(66,729,955-88,771,193) × 2~3, 4q32.3q35.2(166,094,098-191,152,793) × 1, 6q16.3q27(102,816,244-170,736,131) × 1~2, 7p22.1p11.2(6,480,544-55,435,373) × 2~3, 8q23.1q24.3(107,914,570-146,263,042) × 3, 11p15.1p14.3(21,533,469-22,367,835) × 1, 11p14.3(22,398,459-24,320,961) × 3, 13q13.3(37,240,922-38,696,855) × 1, 13q14.11q14.12(43,995,777-5,551,120) × 3, 13q14.3q21.1(50,795,724-52,401,010) × 3, 13q21.1q21.2(55,083,523-58,084,123) × 3, 13q21.2(59,114,933-59,262,005) × 3, 13q21.31q21.32(63,478,713-65,130,349) × 3, 13q21.33q34(67,650,203-114,103,644) × 3, 19p13.3(220,598-546,817) × 1, 21q22.3(42,101,144-46,915,771) × 2~3
LSI D13S319 normal, LSI 13q34 normal, LSI ATM normal, CEP 12 66% trisomy, LSI p53 62% loss
12p13.33q24.33(60,861-132,267,241) × 3, 17p13.3p11.1 (49,128-22,116,415) × 1
22q11.23(22,674,846-22,731,268) × 0~1, Yp11.2q11.23(8,292,949-26,636,748) × 0
LSI D13S319 92% loss, LSI 13q34 normal, LSI ATM normal, CEP 12 normal, LSI p53 normal
13q14.2q14.3(46,444,224-50,554,228) × 1
LSI D13S319 normal, LSI 13q34 normal, LSI ATM normal, CEP 12 35% trisomy, LSI p53 normal
12p13.33q24.33 (60,861-132,267,241) × 3
LSI D13S319 29% loss, LSI 13q34 normal, LSI ATM normal, CEP 12 normal, LSI p53 normal
13q14.3(49,604,393-49,730,034) × 1
LSI D13S319 88% loss, LSI 13q34 normal, LSI ATM normal, CEP 12 normal, LSI p53 normal
13q14.2q14.3(46,444,224-50,554,228) × 1
44~46,X,X,add(1)(p36.1),i(6)(p10),del(8)(p21p23),add(12)(p11.2),- 13, del(14)(q12q32),-15, add(17)(p11.2),+2-6mar[cp11]/46,XX
LSI D13S319 34% loss, LSI p53 49% loss
13q14.2q14.3(47,524,866-50,523,594) × 1, 17p13.3p13.1(49,128-8,581,862) × 1
Array did not detect del(14)(q12q32) seen on karyotype; -15 shows complexity on array
3p21.31(47,218,579-49,437,299) × 1, 5q35.1q35.3(168,342,673-180,619,169) × 3, 6p21.33p12.1(31,649,559-55,535,574) × 1, 7p22.3p15.3(130,978-21,156,763) × 3, 8p23.3p12(177,781-34,695,588) × 1, 8q24.13q24.3(126,709,259-145,344,434) × 3, 15q11.2q13.3(20,372,901-31,359,613) × 3, 15q13.3q15.1(31,388,923-39,994,112) × 1, 15q15.1q21.1(40,049,653-43,331,117) × 3, 15q21.1q22.2(43,385,070-58,614,700) × 1, 15q22.2q26.3(58,646,551-100,217,531) × 3
LSI D13S319 normal, LSI 13q34 normal, LSI ATM normal, CEP 12 77.5% trisomy, LSI p53 normal
12p13.33q24.33(60,861-132,267,241) × 3
7q34(141,693,456-141,719,136) × 1, 14q22.2(53,498,118-53,858,816) × 3
LSI D13S319 67% loss, LSI ATM 93% loss, CEP 12 15% trisomy
11q13.5q23.3(76,383,882-117,091,784) × 1, 12p13.33q24.33(1-132,349,534) × 3, 13q14.2q14.3(47,172,707-50,586,402) × 1~2
LSI D13S319 normal, LSI 13q34 normal, LSI ATM normal, CEP 12 61% trisomy, LSI p53 normal
12p13.33q24.33(60,861-132,267,241) × 3
LSI D13S319 normal, LSI ATM normal, CEP 12 normal, LSI p53 45% loss
17p13.3p11.2(49,128-21,376,245) × 1
Array and FISH did not detect del(13)(q21q34) seen in 3/20 cells by karyotype
LSI D13S319 normal, LSI 13q34 normal, LSI ATM 10.5% loss, CEP 12 85% trisomy, LSI p53 normal
12p13.33q24.33(60,861-132,267,241) × 3
Array did not detect 11q22.3 deletion seen in 10.5% of cells by FISH and in 4/16 cells by karyotype
13q12.2(27,492,830-27,493,700) × 3, 22q11.23(22,674,846-22,731,268) × 0
3p26.3p25.2(88,832-11,601,487) × 1, 3p24.3p21.31(21,821,826-45,382,789) × 1, 3p21.2p14.2(51,631,148-59,828,728) × 1, 3p11.2q21.3(88,616,646-129,821,181) × 1, 3q21.3q25.1(130,955,610-152,751,429) × 1, 4p16.3p14(45,627-39,064,794) × 1, 4p14p12(40,689,654-48,288,082) × 1, 6q14.1q24.3(83,060,798-147,519,394) × 1, 8p23.3p21.3(1-21,077,797) × 1, 8p21.1q12.1(27,859,739-56,426,895) × 1, 8q12.1q13.1(58,060,709-66,596,062) × 1, 8q21.13q22.1(81,697,427-96,822,423) × 1, 8q22.3q24.13(103,345,568-125,046,236) × 1, 8q24.21q24.3(130,132,793-141,102,164) × 1, 13q14.12q34(45,300,002-114,103,644) × 1
10q24.1q25.2(99,015,708-114,057,326) × 1, 11q22.1q23.3(101,560,685-116,463,713) × 1, 16p13.3p13.2(35,819-8,125,327) × 1, 16p13.13p13.12(12,434,601-14,674,345) × 1, 16q21(57,250,542-61,322,202) × 1, 17p13.3p11.2(49,128-21,376,245) × 1, 18q12.3(38,266,239-38,764,068) × 1, 20p13p12.2(517,864-11,841,120) × 1; Array clarified a deletion on 13q to include LAMP1
LSI D13S319 72% loss, LSI 13q34 normal, LSI ATM normal, CEP 12 normal, LSI p53 79.5% loss
13q14.3(49,378,768-50,262,893) × 1, 17p13.3p11.2(49,128-21,247,183) × 1, 20p13p12.1(1,981,763-15,398,390) × 1, 20p11.21(23,271,365-25,666,747) × 1, 20q11.21q11.22(30,436,259-32,563,890) × 1
3p21.31(46,911,912-49,973,212) × 1, 17q21.1q21.31(35,551,063-37,865,072) × 1
4p16.3p14(45,627-38,504,396) × 1, 4p13q22.3(41,631,408-96,524,997) × 1, 15q11.2q15.1(19,129,891-38,918,282) × 1, 15q21.2q22.2(49,131,112-57,212,181) × 1
del(20)(q11.2q13.3) not detected by aCGH
11q22.3q23.2(106,821,962-113,825,965) × 1, 13q12.11(19,508,097-20,625,750) × 1, 17p13.3(49,128-2,779,693) × 1, 17p13.1p11.2(9,888,292-18,868,118) × 1, 17p11.2(19,082,873-20,794,597) × 1, 22q11.23(22,674,846-22,723,991) × 0
LSI D13S319 62.5% loss, LSI 13q34 normal, LSI ATM 31% loss, CEP 12 normal, LSI p53 normal
11q13.4q25(72,590,406-134,425,038) × 1, 13q14.2q14.3(47,588,669-50,414,293) × 1
-Y not detected by aCGH
7p22.3p12.2(130,978-49,815,456) × 3, 8p23.3p12(1-33,376,370) × 1, 22q12.2q13.33(30,406,286-49,519,766) × 3
Common aberrations identified by microarray analysis
Our results confirm prior studies that show the most common abnormalities found in CLL and identifiable by arrays are deletion of 13q14.3, trisomy 12, deletion of 11q22.3 and deletion of 17p13.1.
All trisomy 12-positive cases were detected by microarray analysis. The percentage of abnormal cells detected ranged from 15% to 92% (as determined by FISH). Trisomy 12 was identified by microarray analysis in one additional case in which FISH analysis was normal (case 16); follow-up studies on this case were not possible.
Nine cases had known deletions of 11q22.3/ATM as determined by FISH and/or karyotype. Eight of these were identified by aCGH. The deletions ranged in size from 0.27 Mb to 61.8 Mb. In case 17 with an 11q22.3 deletion not detected by aCGH, the deletion was present in 11% of the interphase cells scored by FISH. Deletions of 11q that include ATM were found by aCGH in two cases that were missed by karyotype: case 31 had a 14.9-Mb deletion at 11q22.1q23.3, and case 33 had a 7-Mb deletion at 11q22.3q23.2. FISH was not performed in either case.
These cases with commonly found abnormalities illustrate the ability and limitations of aCGH to detect a relatively low number of cells with the abnormal clone (~15%) and to detect aberrations missed by conventional chromosome analysis and FISH. The high-density coverage afforded by the array design used in the current study likely contributes to both improved sensitivity in detecting known lower-level mosaic alterations and an improved ability to recognize new alterations. However, lower-level limits of resolution do persist for aCGH due to the nature of the technology. Thus, even with the improvements described herein, this assay should be used only for new diagnoses or relapse and not for monitoring for minimal residual disease.
Microarray analysis can clarify the karyotypes
Karyotype complexity can be delineated by aCGH. For example, case 25 had a reported karyotype of monosomy 15 with two to six markers. Microarray analysis identified a series of gains and losses that included 15q11.2q13.3 × 3, 15q13.3q15.1 × 1, 15q15.1q21.1 × 3, 15q21.1q22.2 × 1, and 15q22.2q26.3 × 3. This result may represent the markers seen by karyotyping, although FISH was not performed to confirm these findings. For some cases in which karyotyping was not performed, we found highly complex genomic changes (e.g., cases 11 and 19).
In several cases, karyotyping showed abnormalities that should have been detected by microarray analysis but were not (Table 1). For example in case 29, karyotyping showed deletion of 13q that was not detected by array or locus-specific FISH, and in case 33 karyotyping showed a deletion at 20q11.2q13.3 in 16 of 20 cells that was not identified by microarray analysis. These results may indicate that the karyotype was not interpreted correctly and that no deletions are present at these loci.
Finally, aCGH clarified the deletion/rearrangement of 17p13.1 identified by prior karyotyping in two cases (cases 31 and 33). In both cases, microarray analysis identified a deletion of 17p. In case 31, the deletion encompasses TP53 that by karyotyping had been interpreted as an add(17p). Case 33 also had an add(17p) by karyotype analysis. Array analysis showed a deletion of 17p, but the deletion does not include TP53. Deletion of 17p is considered an independent prognostic factor with resistance to treatment, shorter treatment-free interval, and shorter overall survival . Thus, aCGH can clarify the chromosome results, and in some cases, the identification of a deletion involving TP53 would change the prognosis for the patient and may be used to alter treatment or patient management. Additional cases in which the array results changed the prognosis are discussed in the following section.
New prognostic information obtained by microarray analysis
In addition to common aberrations, we identified clinically significant or potentially significant gains or losses that were not known prior to submitting the sample for array analysis in the majority of cases (20/34), including trisomy 18 and 19 and deletions of 6q, which are highlighted here.
Trisomy 18 and trisomy 19, each seen in separate cases in our study, are uncommon in CLL. Trisomy 18 generally presents as the sole abnormality or with a karyotype that includes trisomy 12 or trisomy 19 . Trisomy 19 in addition to trisomy 12 has been associated with IGHV gene mutation . In that study of 705 cases of CLL, trisomy 19 was seen in 11 (1.6%) cases, all of which also had trisomy 12; nine had mutated IGHV genes. Those cases that did not have trisomy 19 but had trisomy 12 primarily had unmutated IGHV genes . In our study, trisomy 12 was seen with trisomy 19 in case 6, although the IGHV mutational status is not known. In case 3 with trisomy 18, neither trisomy 12 nor 19 was detected.
Large (~64 Mb) deletions of 6q were detected in two cases, one of which, case 19, was not known prior to array analysis presumably because chromosome analysis was not performed. Cases of CLL with deletions of 6q are characterized by atypical lymphocyte morphology, CD38 positivity, and intermediate incidence of IgVH somatic hypermutation . Cases of CLL with deletion of 6q (specifically at 6q21) are seen in less than 5% of CLL cases, have been shown to require a more demanding treatment regimen, and have been suggested to comprise an intermediate-risk group [14, 15]. Deletion of 6q with or without other abnormalities may also be predictive of shorter survival .
In four cases (cases 7, 11, 15 and 19) for which only limited FISH was performed prior to aCGH analysis, new information was revealed and showed gains of 2p, which encompassed 2p16.1p15, ranging in size from 3.4 Mb to 89.8 Mb that included the REL and BCL11A genes. Gains in the 2p16.1p15 region have been associated with a poor prognosis and have been seen more frequently in cases that have deletion 17p- , although we did not see this association. The ability to perform microarray analysis on residual or archived material provides an opportunity to analyze the cancer genome in an unbiased and comprehensive approach.
Novel aberrations identified by microarray analysis
We found 10 novel changes by microarray analysis that were not identified by karyotype or FISH and that may have clinical significance. For example in case 17, microarray detected additional losses, including a 265-kb loss on 11q that included BIRC3. This gene is part of the inhibitor of apoptosis (IAP) family, which plays a role in apoptosis and the inflammatory process  and, when fused by translocation to MALT1, is associated with MALT-type lymphoma . Interestingly, we have identified a novel translocation of BIRC3 to SETBP1 in a separate case of CLL (unpublished observation). Furthermore, an NF-ĸB inhibitor has recently been shown to achieve apoptosis induction with potential therapeutic value for CLL in cases with reduced expression of BIRC3 . This suggests that BIRC3 status may be an important factor in determining appropriate therapy and prognosis. However, further investigations are warranted. A recent publication reported atypical deletions of 11q in patients with CLL . However, their minimal deletion region, as established by BAC array analysis, did not include BIRC3, as found in case 17 in our study.
We have used a novel approach of targeting over 1800 cancer feature genes while also providing whole genome coverage to identify novel changes and delineate breakpoints of alterations. Using this approach, we have shown that such an array design in CLL will identify cryptic and novel alterations, clarify the karyotype results and refine breakpoints, which may lead to better prognostic precision in CLL, and may influence treatment or patient management. This approach is likely superior to using small, targeted arrays that may miss important novel changes or high-density, whole-genome arrays that have arbitrary coverage and may yield findings that are difficult to interpret in the context of the patient's disease. Arrays also may be useful for cases in which the chromosome analysis and FISH results are discordant with each other, with the pathology, or with disease course. Because the array uses DNA extracted directly from the specimen, aCGH may be useful for cases of tissue culture failure. Finally, microarrays may be helpful when cytogenetics is negative or ambiguous. Because CLL rarely involves balanced translocations, which are not detectable by aCGH, this technology may be particularly useful for these patients, especially in understanding the cancer genomes for the 10–20% of cases representing young patients , who exhibit a significantly reduced life expectancy relative to healthy controls once symptomatic with this disease .
Materials and methods
DNA was extracted from 34 samples collected from patients with either newly diagnosed CLL or recurrent disease. All samples were karyotypically abnormal by conventional cytogenetics, FISH, or both. The samples consisted of 22 peripheral bloods, 11 bone marrow aspirates, and one lymph node. The specimens were de-identified for demographic details but retained data regarding prior chromosome and FISH analyses. The protocol of testing of de-identified, discarded specimens was approved by the Institutional Review Board (IRB) Spokane, and IRB approval was obtained by the source laboratories where required by local regulations.
Genomic DNA was extracted from unenriched blood and bone marrow specimens using the Gentra Puregene Blood kit (Qiagen, Germantown, MD) according to the manufacturer's instructions. Two million cells or 150 μl (if cell counts were unavailable) of blood or bone marrow were used as starting material. Additional cell lysis solution (Gentra Puregene Blood kit) was added to samples with high viscosity to ensure complete cell lysis. Samples were stabilized in cell lysis solution within 24–48 hours when possible to ensure high-quality DNA for use on the microarray.
DNA quality was assessed by measuring DNA concentration, 260/280 and 260/230 readings on a Nanodrop 2000 Spectrophotometer (Thermo Scientific, Waltham, MA). The DNA was also run on a 1% agarose gel with ethidium bromide to determine if degradation was present. To be included in the study, samples had to have minimal degradation with 260/280 values near 1.8 and 260/230 readings greater than 1.35.
Oligonucleotide microarray labeling, hybridization, and analysis
Oligonucleotide-based microarray analysis was performed using a 135K-feature whole-genome microarray (Signature OncoChip™, designed by Signature Genomic Laboratories, Spokane, WA; manufactured by Roche NimbleGen, Madison, WI). This microarray targets 1893 cancer features, including genes with known roles in hematologic malignancies or solid tumors in which deletions or mutations had been previously reported; genes with suspected roles in cancer based on prior expression studies without specific evidence of genomic copy changes; genes with previously speculated roles based solely upon association with a biological pathway or gene family; and genes involved in protein and miRNA coding. The microarray has an average oligonucleotide coverage of one oligo per 0.2–7 kb for targeted cancer features with additional genomic backbone coverage of approximately one oligo per 35 kb. Purified genomic DNA from the diagnostic specimens was labeled with Cyanine dye Cy5, and DNA from a chromosomally normal control was labeled with Cyanine dye Cy3, using a Roche NimbleGen Dual-Color DNA Labeling Kit according to the manufacturer's instructions. Array hybridization and washing were performed as specified by the manufacturer (Roche NimbleGen). Arrays were scanned at 5 microns using an MS 200 Microarray Scanner (Roche NimbleGen) and analyzed using MS 200 1.0 Scanning Software (Roche NimbleGen), NG Packager 1.0 (Signature Genomics) and NimbleScan 2.6 (Roche NimbleGen). Results were then displayed using custom oligonucleotide aCGH analysis software (Oncoglyphix™, Signature Genomics).
We thank Erin Dodge (Signature Genomic Laboratories) for her critical editing and preparation of the manuscript.
- Patel A, Kang SH, Lennon PA, Li YF, Rao PN, Abruzzo L, Shaw C, Chinault AC, Cheung SW: Validation of a targeted DNA microarray for the clinical evaluation of recurrent abnormalities in chronic lymphocytic leukemia. Am J Hematol 2008,83(7):540–546. 10.1002/ajh.21145PubMedView ArticleGoogle Scholar
- Higgins RA, Gunn SR, Robetorye RS: Clinical application of array-based comparative genomic hybridization for the identification of prognostically important genetic alterations in chronic lymphocytic leukemia. Mol Diagn Ther 2008,12(5):271–280.PubMedView ArticleGoogle Scholar
- Rai KR, Sawitsky A, Cronkite EP, Chanana AD, Levy RN, Pasternack BS: Clinical staging of chronic lymphocytic leukemia. Blood 1975,46(2):219–234.PubMedGoogle Scholar
- Rai KR: A critical analysis of staging in CLL. In Chronic lymphocytic leukemia: recent progress, future direction. Edited by: Gale RP, Rai KR. New York: Alan R. Liss; 1987:253.Google Scholar
- Binet JL, Auquier A, Dighiero G, Chastang C, Piguet H, Goasguen J, Vaugier G, Potron G, Colona P, Oberling F, Thomas M, Tchernia G, Jacquillat C, Boivin P, Lesty C, Duault MT, Monconduit M, Belabbes S, Gremy F: A new prognostic classification of chronic lymphocytic leukemia derived from a multivariate survival analysis. Cancer 1981,48(1):198–206. 10.1002/1097-0142(19810701)48:1<198::AID-CNCR2820480131>3.0.CO;2-VPubMedView ArticleGoogle Scholar
- Damle RN, Wasil T, Fais F, Ghiotto F, Valetto A, Allen SL, Buchbinder A, Budman D, Dittmar K, Kolitz J, Lichtman SM, Schulman P, Vinciguerra VP, Rai KR, Ferrarini M, Chiorazzi N: Ig V gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia. Blood 1999,94(6):1840–1847.PubMedGoogle Scholar
- Hamblin TJ, Davis Z, Gardiner A, Oscier DG, Stevenson FK: Unmutated Ig V(H) genes are associated with a more aggressive form of chronic lymphocytic leukemia. Blood 1999,94(6):1848–1854.PubMedGoogle Scholar
- Shanafelt TD: Predicting clinical outcome in CLL: how and why. Hematology Am Soc Hematol Educ Program 2009, 421–429.Google Scholar
- Maciejewski JP, Mufti GJ: Whole genome scanning as a cytogenetic tool in hematologic malignancies. Blood 2008,112(4):965–974. 10.1182/blood-2008-02-130435PubMed CentralPubMedView ArticleGoogle Scholar
- Lichter P: All you need is a Mir-acle: the role of nontranslated RNAs in the suppression of B cell chronic lymphocytic leukemia. Cancer Cell 2010,17(1):3–4. 10.1016/j.ccr.2009.12.029PubMedView ArticleGoogle Scholar
- Klein U, Lia M, Crespo M, Siegel R, Shen Q, Mo T, Ambesi-Impiombato A, Califano A, Migliazza A, Bhagat G, Dalla-Favera R: The DLEU2/miR-15a/16–1 cluster controls B cell proliferation and its deletion leads to chronic lymphocytic leukemia. Cancer Cell 2010,17(1):28–40. 10.1016/j.ccr.2009.11.019PubMedView ArticleGoogle Scholar
- Parker H, Rose-Zerilli MJ, Parker A, Chaplin T, Wade R, Gardiner A, Griffiths M, Collins A, Young BD, Oscier DG, Strefford JC: 13q deletion anatomy and disease progression in patients with chronic lymphocytic leukemia. Leukemia 2011,25(3):489–497. 10.1038/leu.2010.288PubMedView ArticleGoogle Scholar
- Sellmann L, Gesk S, Walter C, Ritgen M, Harder L, Martin-Subero JI, Schroers R, Siemer D, Nuckel H, Dyer MJ, Duhrsen U, Siebert R, Durig J, Kuppers R: Trisomy 19 is associated with trisomy 12 and mutated IGHV genes in B-chronic lymphocytic leukaemia. Br J Haematol 2007,138(2):217–220. 10.1111/j.1365-2141.2007.06636.xPubMedView ArticleGoogle Scholar
- Cuneo A, Rigolin GM, Bigoni R, De Angeli C, Veronese A, Cavazzini F, Bardi A, Roberti MG, Tammiso E, Agostini P, Ciccone M, Della Porta M, Tieghi A, Cavazzini L, Negrini M, Castoldi G: Chronic lymphocytic leukemia with 6q- shows distinct hematological features and intermediate prognosis. Leukemia 2004,18(3):476–483. 10.1038/sj.leu.2403242PubMedView ArticleGoogle Scholar
- Tsimberidou AM, Wen S, O'Brien S, McLaughlin P, Wierda WG, Ferrajoli A, Faderl S, Manning J, Lerner S, Mai CV, Rodriguez AM, Hess M, Do KA, Freireich EJ, Kantarjian HM, Medeiros LJ, Keating MJ: Assessment of chronic lymphocytic leukemia and small lymphocytic lymphoma by absolute lymphocyte counts in 2,126 patients: 20 years of experience at the University of Texas M.D. Anderson Cancer Center. J Clin Oncol 2007,25(29):4648–4656. 10.1200/JCO.2006.09.4508PubMedView ArticleGoogle Scholar
- Forconi F, Rinaldi A, Kwee I, Sozzi E, Raspadori D, Rancoita PM, Scandurra M, Rossi D, Deambrogi C, Capello D, Zucca E, Marconi D, Bomben R, Gattei V, Lauria F, Gaidano G, Bertoni F: Genome-wide DNA analysis identifies recurrent imbalances predicting outcome in chronic lymphocytic leukaemia with 17p deletion. Br J Haematol 2008,143(4):532–536.PubMedGoogle Scholar
- Byrd JC, Stilgenbauer S, Flinn IW: Chronic lymphocytic leukemia. Hematology Am Soc Hematol Educ Program 2004, 163–183.Google Scholar
- Van Dyke DL: +18 or trisomy 18 in lymphoproliferative disorders. [http://AtlasGeneticsOncology.org/Anomalies/Tri18ID2030.html]
- Grubor V, Krasnitz A, Troge JE, Meth JL, Lakshmi B, Kendall JT, Yamrom B, Alex G, Pai D, Navin N, Hufnagel LA, Lee YH, Cook K, Allen SL, Rai KR, Damle RN, Calissano C, Chiorazzi N, Wigler M, Esposito D: Novel genomic alterations and clonal evolution in chronic lymphocytic leukemia revealed by representational oligonucleotide microarray analysis (ROMA). Blood 2009,113(6):1294–1303. 10.1182/blood-2008-05-158865PubMedView ArticleGoogle Scholar
- Sargent R, Jones D, Abruzzo LV, Yao H, Bonderover J, Cisneros M, Wierda WG, Keating MJ, Luthra R: Customized oligonucleotide array-based comparative genomic hybridization as a clinical assay for genomic profiling of chronic lymphocytic leukemia. J Mol Diagn 2009,11(1):25–34. 10.2353/jmoldx.2009.080037PubMed CentralPubMedView ArticleGoogle Scholar
- Hagenkord JM, Monzon FA, Kash SF, Lilleberg S, Xie Q, Kant JA: Array-based karyotyping for prognostic assessment in chronic lymphocytic leukemia: performance comparison of Affymetrix 10K2.0, 250K Nsp, and SNP6.0 arrays. J Mol Diagn 2010,12(2):184–196. 10.2353/jmoldx.2010.090118PubMed CentralPubMedView ArticleGoogle Scholar
- Dahabreh IJ, Giannouli S, Gota V, Voulgarelis M: GSTT1 and GSTM1 polymorphisms and myelodysplastic syndrome risk: a systematic review and meta-analysis. Int J Cancer 2010,126(7):1716–1723.PubMedGoogle Scholar
- Van Dyke DL, Shanafelt TD, Call TG, Zent CS, Smoley SA, Rabe KG, Schwager SM, Sonbert JC, Slager SL, Kay NE: A comprehensive evaluation of the prognostic significance of 13q deletions in patients with B-chronic lymphocytic leukaemia. Br J Haematol 2010,148(4):544–550. 10.1111/j.1365-2141.2009.07982.xPubMed CentralPubMedView ArticleGoogle Scholar
- Deveraux QL, Leo E, Stennicke HR, Welsh K, Salvesen GS, Reed JC: Cleavage of human inhibitor of apoptosis protein XIAP results in fragments with distinct specificities for caspases. EMBO J 1999,18(19):5242–5251. 10.1093/emboj/18.19.5242PubMed CentralPubMedView ArticleGoogle Scholar
- Akagi T, Motegi M, Tamura A, Suzuki R, Hosokawa Y, Suzuki H, Ota H, Nakamura S, Morishima Y, Taniwaki M, Seto M: A novel gene, MALT1 at 18q21, is involved in t(11;18) (q21;q21) found in low-grade B-cell lymphoma of mucosa-associated lymphoid tissue. Oncogene 1999,18(42):5785–5794. 10.1038/sj.onc.1203018PubMedView ArticleGoogle Scholar
- Kanduri M, Tobin G, Aleskog A, Nilsson K, Rosenquist R: The novel NF-[kappa]B inhibitor IMD-0354 induces apoptosis in chronic lymphocytic leukemia. Blood Cancer Journal 2011, 1: e12. 10.1038/bcj.2011.9PubMed CentralPubMedView ArticleGoogle Scholar
- Gunn SR, Hibbard MK, Ismail SH, Lowery-Nordberg M, Mellink CH, Bahler DW, Abruzzo LV, Enriquez EL, Gorre ME, Mohammed MS, Robetorye RS: Atypical 11q deletions identified by array CGH may be missed by FISH panels for prognostic markers in chronic lymphocytic leukemia. Leukemia 2009,23(5):1011–1017. 10.1038/leu.2008.393PubMedView ArticleGoogle Scholar
- Dessen P, Le Minor S: GSTT1 (glutathione S-transferase theta 1). [http://atlasgeneticsoncology.org/Genes/GC_GSTT1.html]
- Yuille M, Condie A, Hudson C, Kote-Jarai Z, Stone E, Eeles R, Matutes E, Catovsky D, Houlston R: Relationship between glutathione S-transferase M1, T1, and P1 polymorphisms and chronic lymphocytic leukemia. Blood 2002,99(11):4216–4218. 10.1182/blood.V99.11.4216PubMedView ArticleGoogle Scholar
- Gunn SR, Bolla AR, Barron LL, Gorre ME, Mohammed MS, Bahler DW, Mellink CH, van Oers MH, Keating MJ, Ferrajoli A, Coombes KR, Abruzzo LV, Robetorye RS: Array CGH analysis of chronic lymphocytic leukemia reveals frequent cryptic monoallelic and biallelic deletions of chromosome 22q11 that include the PRAME gene. Leuk Res 2009,33(9):1276–1281. 10.1016/j.leukres.2008.10.010PubMedView ArticleGoogle Scholar
- Okazaki N, Takahashi N, Kojima S, Masuho Y, Koga H: Protocadherin LKC, a new candidate for a tumor suppressor of colon and liver cancers, its association with contact inhibition of cell proliferation. Carcinogenesis 2002,23(7):1139–1148. 10.1093/carcin/23.7.1139PubMedView ArticleGoogle Scholar
- Georgantas RW, Hildreth R, Morisot S, Alder J, Liu CG, Heimfeld S, Calin GA, Croce CM, Civin CI: CD34+ hematopoietic stem-progenitor cell microRNA expression and function: a circuit diagram of differentiation control. Proc Natl Acad Sci USA 2007,104(8):2750–2755. 10.1073/pnas.0610983104PubMed CentralPubMedView ArticleGoogle Scholar
- Holzelova E, Vonarbourg C, Stolzenberg MC, Arkwright PD, Selz F, Prieur AM, Blanche S, Bartunkova J, Vilmer E, Fischer A, Le Deist F, Rieux-Laucat F: Autoimmune lymphoproliferative syndrome with somatic Fas mutations. N Engl J Med 2004,351(14):1409–1418. 10.1056/NEJMoa040036PubMedView ArticleGoogle Scholar
- Dunphy WG, Kumagai A: The cdc25 protein contains an intrinsic phosphatase activity. Cell 1991,67(1):189–196. 10.1016/0092-8674(91)90582-JPubMedView ArticleGoogle Scholar
- Altekruse SF, Kosary CL, Krapcho M, Neyman N, Aminou R, Waldron W, Ruhl J, Howlader N, Tatalovich Z, Cho H, Mariotto A, Eisner MP, Lewis DR, Cronin K, Chen HS, Feuer EJ, Stinchcomb DG, Edwards BK: SEER Cancer Statistics Review, 1975–2007. [http://seer.cancer.gov/csr/1975_2007/]
- Mauro FR, Foa R, Giannarelli D, Cordone I, Crescenzi S, Pescarmona E, Sala R, Cerretti R, Mandelli F: Clinical characteristics and outcome of young chronic lymphocytic leukemia patients: a single institution study of 204 cases. Blood 1999,94(2):448–454.PubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.