Interphase Chromosome Profiling: A Method for Conventional Banded Chromosome Analysis Using Interphase Nuclei.
These limitations can lead to false-negative results if abnormal mitotic cells are few, the relevant cells are not mitotically active in sufficient numbers in cell culture, or the cells do not proliferate in the media or mitogen provided. A more reliable and less costly high-resolution cytogenetic method would be a welcome addition to the cytogenetics armamentarium. Here we describe the development and validation of interphase chromosome profiling (ICP), a novel cytogenetic technology to assess the karyotype of any hematologic neoplasia by using interphase cells. This new approach can detect all numerical abnormalities and most balanced and unbalanced structural aberrations, including characterization of "add" and "marker" chromosomes.
MATERIALS AND METHODS
This study was determined to be exempt from institutional review board review by the Mayo Clinic Institutional Review Board, Rochester, Minnesota.
Conventional chromosome analysis routinely uses metaphase cell preparations, since the chromatin is condensed at this stage and individual chromosomes can be readily visualized. However, there is good evidence that chromosomes behave similarly in both interphase and metaphase, and their length in interphase is similar to that in a 600-band karyotype. (1) With whole chromosome multicolor karyotyping methods (eg, multiplex fluorescence in situ hybridization [M-FISH]), spectral karyotyping, combined binary ratio fluorescence in situ hybridization (COBRA-FISH), some chromosome rearrangements such as translocations can be visualized, (2-5) and intrachromosomal changes such as inversions, deletions, and duplications can be identified with a multicolor metaphase banding approach. (6,7) However, interphase visualization of specific chromosome rearrangements (eg, fiber-FISH) has presented greater challenges, (8,9) and until now, no method has been successfully designed for banded karyotype analysis in interphase nuclei.
Building on the observations of Lemke et al, (1) and by assigning FISH probes to target sequences along the length of each chromosome in an equidistant fashion, entire chromosomes can be evaluated in interphase nuclei in normal and disease states (US patents 8,574,836 and 7,943,304). Each chromosome arm includes at least 1 (18p and Yp) and up to 6 (2q, 4q, and 5q) hybridization sites, each assigned to a specific chromosome band. Subtelomeric and paracentromeric sequences are assigned a pure color (aqua, yellow, and red for pter, centromere, and qter, respectively), and interstitial bands are assigned either a pure (far red or green) or a hybrid (fusion) color. Each chromosome is studied individually, with the results compiled into a composite karyotype. This configuration provides the equivalent of a 600-band resolution karyotype (10) and facilitates identification of copy number changes of whole chromosomes and balanced and unbalanced chromosome rearrangements.
Cell Culture and Microscope Slide Preparation
Microscope slides were prepared after a standard 24- to 48-hour cell culture and harvested by using a 0.075M KCl hypotonic solution for 20 minutes at 37[degrees]C without colcemid followed by 3 changes, at room temperature, of freshly prepared 3:1 methanolglacial acetic acid fixative. A direct preparation without cell culture can be used for STAT cases.
Keeping with the equidistant concept, each chromosome arm was assigned a certain number of bands. The overall length of the chromosome arm was the primary deciding factor for the ultimate number of bands on that arm. In general, longer chromosome arms are assigned a greater number of bands (targets). For example, the short arms of chromosomes 6 and 11 have more (3) bands than the short arm of chromosomes 5 and 10. The long arm of chromosome 11 has 5 bands, whereas the long arm of chromosome 9 has 4 bands because although the chromosomes are similar in length, 11q is longer than 9q when the 9q heterochromatic region is excluded.
Individual bacterial artificial chromosome (BAC) clones were selected from a Children's Hospital Oakland Research Institute library for each target on the chromosome. (11) Two BACs were chosen for each target location. Figure 1 provides a cytogenetic ideogram with the targets and the corresponding probes with their fluorescent labels. Table 1 lists the number of bands in each chromosome arm, and Figure 2, A and B, depicts the color scheme of short and long arms. With the exception of the green color, which was assigned to a short arm as well as a long arm band on some chromosomes, bands in each arm received distinct colors. For example, aqua and far red were restricted to the short arm, and yellow and red were restricted to the long arm. In a few instances when a proper BAC was not available in the long arm, the short arm pericentromeric area was chosen. However, the total number of pure colors in any arm was limited to 3. The interstitial hybrid colors were simply distinct combinations of the pure colors belonging to that arm. The following scheme was used in assigning the number of hybrid bands in any arm. First, 2 landmarks were chosen for each arm. The telomere (aqua) and the most proximal band (green) were the landmarks for the short arm. The centromere (yellow) and the telomere (red) were considered the landmarks for the long arm. In order for a chromosome arm to receive a hybrid band, it needs to have more than the 2 landmark bands. Therefore, chromosome arms large enough to require 4 bands received 1 hybrid distal to the green landmark, and the proximal band to the telomere landmark received the remaining pure color (far red) in the short arm. Similarly, for the long arm, the proximal band to the red landmark received hybrid color, and the distal band to the centromere received the remaining pure color (green). For an arm with 5 bands, the 3 interstitial bands were made hybrids. For an arm with 6 bands, the distal band to the telomere was given the remaining pure color (green), and the 3 remaining interstitial bands became hybrids. Finally, when any arm has only the landmarks, the third pure color was eliminated. Since the short arms of chromosomes 18 and Y were small, only a telomere landmark band was assigned. For each probe, we used a 3-step verification process: (1) checking for known genomic variants, (12) (2) end sequencing to match the intended chromosomal location, (11) and (3) confirmation of the expected metaphase band location. (13,14) Only probes that passed these selection criteria were used for further validation.
FISH probes were generated by using standard nick translation protocols from BAC clone DNA. (15) Briefly, 1 to 2 [micro]g nick translation reactions were run for each BAC clone. The average size of the selected BAC clones was approximately 200 kb, and the individual fragments that collectively make up a single probe for each selected target were adjusted to be around 200 base pairs in length to facilitate efficient hybridization. The ratio of dTTP to fluorophore dUTPs was optimized. Individual chromosome hybridizations were done on 4 slides with 6 areas of hybridization on each slide, following standard FISH protocols (16) with minor adjustments. About 20 ng of probe was used for each chromosomal target and the overnight hybridization was done in a 10-mm area under a round coverslip. Posthybridization washing conditions included 2 minutes in 0.4X saline-sodium citrate (SSC)/0.3% NP-40 at 69[degrees]C, followed by 1 minute in 2X SSC/0.1% NP-40 at room temperature; 4',6-diamidino-2-phenylindole (DAPI) counterstaining was omitted. Appropriate filter sets from Semrock (Rochester, NY) were used to detect fluorophores DEAC (aqua), Fluorescein-12 (green), Cyanine555 (yellow), Cyanine647 (far red), and CF594 (red).
Initial scanning to place the cells in the correct plane was done with the filter for Cyanine555. A minimum of 20 interphase cells were analyzed for each chromosome. The usual guidelines of metaphase analysis were followed with minor adjustments to identify an abnormal clone: at least 4 of 20 cells for both structural and numerical abnormalities.
To get familiarized with the banding pattern generated for each chromosome, known normal metaphases were studied. The individual color patterns were studied in metaphase spreads and interphase nuclei, and the combined results of all color combinations were verified to assure concordance with the expected result (Figure 3 and Figure 4, A and B).
In contrast to usual single-target interphase FISH assays, the usual guideline of 20 metaphases is considered adequate and each chromosome is analyzed in 20 nuclei, since the entire chromosome is profiled. To accommodate the maximum number of filter cubes on any fluorescent microscope, DAPI counterstaining was eliminated. This did not create any problem in clearly identifying nuclei, as there was enough autofluorescence of the nuclei. To analyze each chromosome, the following protocol was observed: the guidance provided in Table 1 and in Figure 2, A and B, was followed to make sure all of the bands were present on any chromosome. In interphase nuclei, each chromosome typically occupies a "domain," and one would count the signals within this domain. For example, chromosome 8 has 3 red, green, and yellow signals and 1 aqua signal. Even though there are 3 hybrid bands on this chromosome, by simply counting the total number of bands (both homologs, 2 domains) in each color channel (filter cube), one can determine if the chromosome has the expected number of bands. Any deviation from this signal pattern would indicate a numerical abnormality. A trisomy would produce 9 red, 9 green, 9 yellow, and 3 aqua signals, whereas a monosomy would produce a 3 red, 3 green, 3 yellow, and 1 aqua signal pattern. The number of domains then varies: 2 for normal, 3 for trisomy, and 1 for monosomy.
Signal patterns for a normal chromosome in interphase, a normal chromosome in metaphase, structurally abnormal chromosomes, and a numerical abnormality are all shown in Figure 5, A, B, C through H, and I, respectively. When there is a structural abnormality such as a translocation, whether it be balanced or unbalanced, 3 domains are created (Figure 5, C and D). However, only 1 domain exhibits the expected "normal size and signal pattern" and 2 abnormally patterned domains represent the 2 broken chromosomal segments. The size of the abnormal domains then depends on the translocation breakpoint. Deletions are recognized by the count of signals as well as the smaller size of the domain (Figure 4, A and B; and Figure 5, D, G, and H). Similarly, when there are duplications, depending on the extent of the abnormality, a relatively larger domain would be apparent. A characteristic difference exists between similarly sized duplications depending on whether they represent tandem or not. For tandem duplications, there is no change in the number of domains (Figure 5, E and F), whereas a duplication resulting from an unbalanced translocation will exhibit 3 domains (Figure 5, D).
This form of chromosome rearrangement is easily recognized in ICP as there will be duplication of 1 arm and deletion of the other. So in the abnormal domain for an isochromosome of the long arm, there will be clear absence of the characteristic color band(s) from the short arm, that is, aqua (Figure 5, H). Similarly, for an isochromosome of the short arm, absence of 1 or all red bands would be apparent.
When there is an overlap of 2 signals representing the same band/chromosome location, traditional FISH assays would create an erroneous, deletion signal pattern. In ICP, since the entire chromosome is profiled even when there is an overlap, it is easily recognized by taking into consideration the total number of bands of that color as well as the adjoining signals on that chromosome. This is accomplished by switching the filter cubes during analysis. When 2 telomeres are situated very close to each other, it is possible to assign either of the signals to any of the homologs, and this should not cause misinterpretation as long as the total count for those color sign patterns remained as expected (Figure 5, C). As with traditional FISH, analysis of multiple nuclei will also clarify a misleading pattern in 1 nucleus. Generally, any uncertainty of signal pattern in 1 cell is easily clarified in the analysis of the other 19 cells.
Hybrid/Juxta Color Bands
Depending on the total number of bands, each chromosome arm can have zero, 1, or 3 hybrid bands. When the count matches with the expected number for each color, it is reliable to predict that all expected hybrid patterns exist. Confirmation of the hybrid band is done by simply isolating/focusing a single-color signal under the microscope and switching the filter to observe the other color at the same location. A pure color band, for example a telomere band, will have no other color signal at that same location. Superimposing the pictures taken during or after analysis will clearly identify the hybrid/juxta bands.
Interstitial Deletions and Duplications
While large interstitial deletions and duplications are easily recognized in ICP, small changes either between the adjacent bands or within the band are difficult to identify with the current scheme. This is not dissimilar to the well-known 2-band uncertainty principle in metaphase banding analysis.
Each color band in ICP was given a specific high-resolution G-banded location. When there is a structural abnormality, generally the breakpoint is assigned to the closest distal band to the original remaining band on the chromosome. For example, chromosome 9 has 2 distal bands on the long arm: one at 9q32 and the other at 9q34.3. When the red band at 9q34.3 is separated as a result of a translocation, the breakpoint is assigned to 9q33, the band distal to 9q32.
It is well recognized that 1 of the X chromosomes in females is inactivated, and there could be differences in the physical length of the 2 chromosomes, based on their inactivation status. The active X chromosome could be more diffuse and longer than the inactive chromosome, which is very tight and short and also exhibits a bend during the metaphase stage at the inactivation center.17 In ICP, a similar phenomenon is frequently observed for almost every chromosome. The mechanism for this is not well understood at this point, but in interphase nuclei, it is frequently observed that the homologs differ in overall length. Therefore, while there may be an obvious size difference between the homologs, neither chromosome should be interpreted as abnormal, since the overall difference between the adjacent bands, either due to stretching or condensation, seems to be constant. Chromosome condensation differences are likewise common in metaphase spreads, wherein the homolog positioned at the periphery of the spread is often measurably longer than its partner positioned in the center of the spread.
Since only 1 chromosome pair is analyzed at a time, it is uncommon for a cell to be unanalyzable when using the ICP scoring criteria. As detailed above, even when there is signal overlap, individual chromosomes can be traced following the expected pattern from the short arm telomere through the centromere to the long arm telomere. The only time a cell may have to be considered unanalyzable and therefore rejected would be when most signals are so diffuse that an accurate count is not possible. Diffuse and weak signals could exist in some cells when hybridization conditions are suboptimal, and in these scenarios, these cells could be rejected.
Clinical validation of the ICP method was done in 2 steps. Once the normal chromosome banding pattern was verified and familiarized, 20 bone marrow samples with known cytogenetic results were chosen for blinded analysis (Table 2). Three institutions participated in this study. The institution that developed the methodology received 15 samples from the second institution, and the third institution studied 5 samples in its laboratory.
Next, 2 principal attributes of ICP were tested: improved reliability and sensitivity as compared to conventional chromosome analysis. Interphase nuclei from 39 bone marrow samples were provided by 4 institutions for a blinded ICP analysis (Table 3).
These included 29 samples with no cytogenetic results (cell culture failure) and 10 samples with known cytogenetic results. One participating institution studied 25 samples blindly. To test interlaboratory reproducibility, 5 of these 25 samples were shared with another laboratory.
The first step in the clinical validation used 20 blinded samples with known cytogenetic results, identified by conventional cytogenetics, FISH, or both, and included trisomy, monosomy, deletions, duplications, and balanced and unbalanced rearrangements (Table 2). In almost all cases, there was complete concordance with the conventional cytogenetic or FISH results. In case 5, ICP did not identify the t(1;3) clone.
In case 6, ICP also identified cells with monosomy 7; the originating laboratory was unable to revisit this case. In case 9, ICP did not identify evidence of an abnormality on the short arm of the "add(X)." In case 15, the original testing laboratory was unable to distinguish the small terminal 8p deletion. For several cases with unidentified material in "add" rearrangements, ICP characterized the origin of the additional chromosome material.
The second step in the clinical validation tested the ability of ICP to identify the abnormalities in another set of 35 blinded cases with known cytogenetic results, plus a series of 29 cases for which conventional cytogenetics failed to produce a result (Table 3). Five of the 35 blinded cases were evaluated by using ICP in 2 laboratories, with similar results, which served to demonstrate the interlaboratory reproducibility of the assay.
As with the first series of cases, again there was nearly complete concordance with the conventional cytogenetic or FISH results. In 6 cytogenetically normal cases (cases 35, 4245, and 50) and 1 normal FISH case (case 46), ICP detected clonal abnormalities diagnostic and/or prognostic of various diseases (Tables 2 and 3). In 4 cases with cytogenetic abnormality, ICP failed to detect the abnormal clone. These included 2 cases with inversions (cases 25, 30) and 2 with a very low level (2 cells) abnormality (cases 15, 26). In 1 case (case 5), ICP failed to detect the t(1;3); however, the same (or a similar) translocation, even when present in low level in the cytogenetic preparations in a different case (case 6), was observed by ICP.
ICP identified the origin of the chromosome material in 4 cases (cases 3, 11, 14, 52) with additional material of unknown origin, that is, "add" (Figure 6, A through D; Tables 2 and 3).
In 3 cases, ICP identified the origin of a marker chromosome (cases 53, 54, 55; Figure 6, A through D). One of these cases had 2 identical copies of a marker chromosome derived from chromosome 9, with a potential neocentromere, since these "acentric" markers were very stable (Figure 6, A through D; Table 3). In 1 case (case 57), ICP refined the breakpoints, which were diagnostic for an IGK/CDK6 fusion (Table 3). In 6 cytogenetically abnormal cases (23, 32, 37, 38, 40, 41), ICP identified additional clonal abnormalities (Tables 2 and 3). Example ICP results are depicted in Figure 5, A through I, and Figure 6, A through D.
The ICP method yielded analyzable results in all 29 bone marrow samples for which conventional cytogenetics was attempted but unsuccessful. Ten had an abnormal ICP karyotype (cases 56-65), and 19 had a normal ICP karyotype (cases 66-84). In a separate preliminary study (R.B., unpublished data, 2015), ICP confirmed a normal conventional chromosome analysis result in 9 cases, confirmed a normal myelodysplastic syndrome or myeloma FISH panel result in 4 cases, and combined normal chromosome analysis and myeloma FISH panel results in 7 cases.
The whole study involved sample types of peripheral blood and bone marrow aspirates (Tables 2 and 3) after cell culture without mitogens. There was no difference in the quality of ICP results between the blood and bone marrow cells. The only time a significant difference in the signal quality was observed was when ICP was done on direct preparations, which tended to yield less satisfactory signal patterns (Figure 7, A and B).
Since the clinical management of patients with hematologic malignancies is often influenced by karyotype findings, obtaining this information in a fast, accurate, and failure-proof manner is critical. Even though other focused molecular technologies can be useful, when the working clinical diagnosis is questionable or there is a differential diagnosis, which often is the case in hematologic malignancies, karyotype analysis is the only approach that identifies genome-wide gross chromosome abnormalities. In this study, we have developed a novel molecular method, which we termed interphase chromosome profiling. Using this process, we showed that a molecular karyotype can be obtained from interphase nuclei without any prior clinical information, mitogen stimulation, or other cell culture modifications. The experiments described here indicate that this technique is robust and highly reproducible.
The resolution obtained from the ICP design yields a karyotype roughly equivalent to a 600-band level, in contrast to the usual karyotypes obtained at a 400-or-fewer band level from hematologic samples. This higher resolution allows for confident breakpoint localization and identification of chromosome material residing in marker chromosomes and unbalanced rearrangements that are difficult to characterize.
As demonstrated, results can be obtained in 48 hours by using a brief cell culture, without the use of mitogens or mitotic arresting agents. In the case of STAT situations for acute promyelocytic leukemia with promyelocytic leukemia/ retinoic acid receptor alpha (PML-RARA), a direct interphase cell preparation can be used (Figure 7, A and B), which allows for a next-day ICP karyotype report.
This study demonstrates that ICP is extremely reliable. All 84 samples studied using ICP--including 29 cytogenetically failed cases--had a clinically interpretable karyotype result. This is critical in the workup of hematologic samples. Since the costs and risks associated with a repeated bone marrow aspirate are not trivial, ICP as a first-line or backup method will find utility in patient management from both diagnostic and follow-up treatment samples.
Improved Analytic Sensitivity
Incorrect breakpoint assignment of structural abnormalities has obvious clinical implications. Obtaining the diagnostic chromosome abnormality at the initial workup of patients with hematologic disorders is crucial for disease classification and management. In 7 of the cases in this study, only normal karyotypes were obtained, but 1 or more clonal abnormalities were detected in these by ICP, including 1 with a variant t(15;17;11) characteristic of acute promyelocytic leukemia (case 35). This 3-way translocation was confirmed by using probes flanking the chromosomal breakpoints (data not shown), which neither conventional cytogenetics nor FISH analysis revealed.
Accurate identification of rearrangement breakpoints, and more specifically, determination of the extent of the rearrangement, that is, deletions and duplications, has a prognostic implication. In 1 case with a limited number of metaphase cells and poor morphology, the initial breakpoint assignment of the t(2;7)(p21;q22) failed to recognize a potential CDK6 gene rearrangement at band 7q21, which was identified by ICP.
In general, a more complex karyotype is associated with a less favorable outcome. (18) Historically, conventional cytogenetics can only indicate the presence of a marker but cannot identify the nature or origin of it. In several cases, ICP was able to identify the chromosomal origin of marker chromosomes and "add" material, and it identified other abnormalities. In case 55, ICP established the mechanism of marker formation as a stable acentric chromosome with a neocentromere.
ICP on Metaphase Chromosomes
Even though ICP is designed to analyze interphase chromosomes, it certainly can be used as an adjunct, as demonstrated in this study, in characterizing the marker chromosomes and identifying the material in "add" chromosomes. An additional advantage, provided by the identification of material both in the marker chromosomes as well as the material of unknown origin as usually described in the standard karyotypes as "add," is the possibility of discovering novel fusion genes. The detection of such novel genes was hampered until now by the lack of precise characterization of "material of unknown origin." ICP clears the path for discovering potential fusion genes by identifying targets for further molecular elucidation with methods such as mate-pair sequencing. (19,20) This has clear implications as the pharmaceutical industry develops treatments specifically geared toward fusion gene products.
Some of the small rearrangements involving breakpoints close to telomeres, such as balanced reciprocal translocations exchanging similarly banded material, escape detection by conventional methods. (21) One example of that is t(12;21) in pediatric acute lymphocytic leukemia cases (Figure 5, A through I). As illustrated by our results in case 37, significant rearrangements involving telomere regions can be missed in a complex karyotype. ICP identified the balanced translocation between chromosomes 8 and 22, and this was later confirmed by standard interphase FISH with probes for MYC and IGL (Table 3). Likewise, a small terminal deletion involving the chromosome 8 short arm was identified only by the ICP method (case 15).
Generally, ICP is used when traditional cytogenetics fails to produce any result. However, ICP is a very flexible technique, and based on the clinical situation on hand, it can be used by careful selection of only a few chromosomes, that is, partial ICP. For example, in a follow-up failed cytogenetics on a patient with a proven diagnosis of chronic myelogenous leukemia, chromosomes 8, 17, and 22 can be used for partial ICP analysis to rule out blast crisis. With just these 3 chromosomes, the most common blast crisis-related changes--that is, trisomy 8, isochromosome for the long arm of 17, and additional copies of the Philadelphia chromosome--can be assessed. Similarly, in any failed cytogenetic case with established chromosome changes in the original diagnostic or previous studies, for which no FISH probes are currently available, partial ICP for the selected chromosomes will be very useful in providing the current status.
Confirmation of a Translocation
As opposed to conventional karyotyping, whereby all the chromosomes in a cell are analyzed at the same time, in ICP each chromosome pair is analyzed separately. As a result, in a case where a patient has a clonal abnormality, for example, a balanced translocation between chromosomes 12 and 21, when chromosome 12 is analyzed it will show a displacement of the chromosome segment distal to the breakpoint of the translocation (Figure 5, C). Similarly, there will be a displacement on chromosome 21. This is a simple scenario where there is only 1 balanced translocation and only 2 chromosomes are involved. More complex karyotypes may exhibit more than 1 balanced translocation or a translocation involving multiple chromosomes. Therefore, it may be necessary to confirm that what was presumed from the initial ICP analysis is indeed the case with respect to the partners involved in a translocation. We have confirmed the involvement of the presumed partners in a given translocation by designing probes flanking the breakpoints on the involved chromosomes and analyzing both hybrid chromosomes in the same cell. We performed the confirmatory analyses for all common translocations observed in hematologic malignancies, and a few examples are shown in Figure 8, A through D. In clinical practice, each laboratory would have to do these confirmatory tests only once during the initial validation studies. This can be done by using the existing targeted FISH probes for translocations. Afterwards, in a simple scenario involving only 2 chromosomes, it can be reliably presumed that the 2 chromosomes with displaced chromosome segments actually formed the balanced translocation. Additional confirmatory testing is only needed when there are more than 2 chromosomes involved.
Guidelines for Calling a Clonal Abnormality
Historically, the definition for a clone in the cytogenetics literature requires 2 cells with a structural abnormality and 3 cells with a numerical abnormality, for metaphase analysis. (10) However, for certain neoplasms, the cell with an abnormality may have a selective proliferative advantage in culture, and as such if only 2 cells are detected with an abnormality in a standard 20 metaphase cell analysis, ICP may have a disadvantage in identifying the abnormal clone in a 20 interphase cell analysis. Increasing the number of cells analyzed could alleviate this concern. There were 2 such discordant cases in this study. Based on 5 cases with normal karyotype by standard metaphase and FISH analyses, it appears that when an isolated abnormality is present in 3 or fewer cells on ICP preparations, it may represent a nonclonal change. Therefore, for ICP analysis, a minimum of 4 cells is required to consider a clonal abnormality, either structural or numerical.
Any time an abnormality of any kind was present in 4 or more ICP cells, it was also present in the conventional chromosome analysis. Therefore, it is considered a major" abnormality for the purpose of ICP analysis. Thus, we consider an abnormality present in 3 or fewer cells to be a minor" abnormality for the purpose of ICP analysis. As discussed already, a minor abnormality was not always confirmed in the standard cytogenetic analysis. Therefore, at the present time, the clinical significance of the minor abnormalities detected in the ICP analyses is unclear. Of course, in clinical practice additional analysis may be useful to confirm the presence of a clone. Large prospective studies are needed to clarify the importance of minor ICP abnormalities, and such studies are underway.
Until enough experience is gained from the studies using this technology and the International System for Human Cytogenetic Nomenclature committee issues guidance, we propose to add a prefix icp" to describe the results generated by using the technology described here. We also use cp" routinely, because the karyotype interpretation is assembled from multiple interphase cells. For example, a sample from a male with a balanced translocation between chromosomes 9 and 22 with a break in band 9q34 and 22q11.2 could be described as icp.46,XY,t(9;22)(q34;q11.2)[cp20].
The single case where ICP could not detect the t(1;3) identified in the cytogenetic study, even after extending the analysis to a large number of cells, may represent a laboratory error. Since no specimen remained to reexamine the case, the possibility of specimen mix cannot be excluded. Additionally, ICP clearly detected the same t(1;3) in a different case from the same collaborating laboratory even though the abnormality was present in only 4 cells in the cytogenetics study. Both inversion 11q and inversion 3q were missed by ICP. This is an inherent limitation of the current design of ICP, and therefore alternative methods including standard FISH should be used to rule out inversions. Thus for all practical purposes, inversions are the only cytogenetic abnormality that ICP cannot reliably identify.
ICP is virtually failure-proof and can detect both numerical and structural aberrations including characteriza tion of marker chromosomes and add material. For the workup of hematologic malignancies with failed cytogenetics and with normal" results in plasma cell myeloma cases, ICP for all 24 chromosomes can be a preferred reflex test, since standard FISH panels do not detect all clinically relevant abnormalities. This technology may prove useful for other areas of cytogenetic investigations such as products of conception. Such studies are underway, and the results will be published elsewhere. ICP offers the additional potential to examine hundreds of interphase nuclei for a specific abnormality, similar to conventional FISH studies, as well as evaluation of individual nuclei in prenatal diagnostic studies to evaluate mosaicism. ICP is a very fast method, with a 24hour reporting time (for STAT cases), and provides very reliable analysis on interphase nuclei. It is faster and less costly than chromosomal microarray or DNA sequencing, while also providing chromosomal structural information in addition to copy number.
The authors would like to thank Srikanthi Kopuri, MS, and Yvonne Banol, HSD, for their technical aid; Lisa Plumley, BS, for whole data organization and specimen mailing; and Anna Chockalingam, PhD, for her help with blind studies.
(1.) Lemke J, Claussen J, Michel S, et al. The DNA-based structure of human chromosome 5 in interphase. Am J Hum Genet. 2002;71(5):1051-1059.
(2.) Speicher MR, Ballard SG, Ward DC. Karyotyping human chromosomes by combinatorial multi-fluor FISH. Nat Genet. 1996;12(4):368-375.
(3.) Schrock E, du Manoir S, Veldman T, et al. Multicolor spectral karyotyping of human chromosomes. Science. 1996;273(5274):494-497.
(4.) Zhang FF, Murata-Collins JL, Gaytan P, et al. Twenty-four-color spectral karyotyping reveals chromosome aberrations in cytogenetically normal acute myeloid leukemia. Genes Chromosomes Cancer. 2000;28(3):318-328.
(5.) Barbouti A, Johansson B, Hoglund M, et al. Multicolor COBRA-FISH analysis of chronic myeloid leukemia reveals novel cryptic balanced translocations during disease progression. Genes Chromosomes Cancer. 2002;35(2):127137.
(6.) Chudoba I, Plesch A, Lorch T, Lemke J, Claussen U, Senger G. High resolution multicolor-banding: a new technique for refined FISH analysis of human chromosomes. Cytogenet Cell Genet. 1999;84(3-4):156-160.
(7.) Bint SM, Davies AF, Ogilvie CM. Multicolor banding remains an important adjunct to array CGH and conventional karyotyping. Mol Cytogenet. 2013;6(1): 55.
(8.) Vaandrager J-W, Schuuring E, Raap T, Philippo K, Kleiverda K, Kluin P. Interphase FISH detection of BCL2 rearrangement in follicular lymphoma using breakpoint-flanking probes. Genes Chromosomes Cancer. 2000;27(1):85-94.
(9.) Shimojima K, Okamoto N, Inazu T, Yamamoto T. Tandem configurations of variably duplicated segments of22q11.2 confirmed by fiber-FISH analysis. J Hum Genet. 2011;56(11):810-812.
(10.) Shaffer LG, McGowan-Jordan J, Schmid M, eds. ISCN (2013): An International System for Human Cytogenetic Nomenclature. Basel: S. Karger; 2013.
(11.) FISH mapped clones information. BACPAC Resources Center at Children's Hospital Oakland Research Institute website. https://bacpacresources.org/ pmapped-clones.htm. Accessed October 4, 2016.
(12.) MacDonald JR, Ziman R, Yuen RK, Feuk L, Scherer SW. The database of genomic variants: a curated collection of structural variation in the human genome. Nucleic Acids Res. 2014;42(D1):D986-D992.
(13.) Inazawa J, Ariyama T, Tokino T, Tanigami A, Nakamura Y, Abe T. High resolution ordering of DNA markers by multi-color fluorescent in situ hybridization of prophase chromosomes. Cytogenet Cell Genet. 1994;65(1-2): 130-135.
(14.) Ried T, Baldini A, Rand TC, Ward DC. Simultaneous visualization of seven different DNA probes by in situ hybridization using combinatorial fluorescence and digital imaging microscopy. Proc Natl Acad Sci U S A. 1992;89(4):13881392.
(15.) Maniatis T, Fritsch EF, Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory; 1982.
(16.) Pinkel D, Straume T, Gray JW. Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization. Proc Natl Acad Sci USA. 1986; 83(9):2934-2938.
(17.) Flejter WL, Van Dyke DL, Weiss L. Bends in human mitotic metaphase chromosomes, including a bend marking the X inactivation center. Am J Hum Genet. 1984;36(1):218-226.
(18.) Woyach JA, Lozanski G, Ruppert AS, et al. Outcome of patients with relapsed or refractory chronic lymphocytic leukemia treated with flavopiridol: impact of genetic features. Leukemia. 2012;26(6):1442-1444.
(19.) Boddicker RL, Razidlo GL, Dasari S, et al. Integrated mate-pair and RNA sequencing identifies novel, targetable gene fusions in peripheral T-cell lymphoma. Blood. 2016;128(9):1234-1245.
(20.) Yang R, Chen L, Newman S, et al. Integrated analysis of whole-genome paired-end and mate-pair sequencing data for identifying genomic structural variations in multiple myeloma. Cancer Inform. 2014;13(suppl 2):49-53.
(21.) Nordkamp LO, Mellink C, van derr Schoot E, van den Berg H. Karyotyping, FISH, and PCR in acute lymphoblastic leukemia: competing or complementary diagnostics? J Pediatr Hematol Oncol. 2009;31(12):930-935.
Ramesh Babu, PhD; Daniel L. Van Dyke, PhD; Vaithilingam G. Dev, PhD; Prasad Koduru, PhD; Nagesh Rao, PhD; Navnit S. Mitter, PhD; Mingya Liu, MS; Ernesto Fuentes, BS; Sarah Fuentes, BS; Stephen Papa, BS
Accepted for publication May 15, 2017.
Published as an Early Online Release October 5, 2017.
From the Department of Research and Development, InteGen LLC, Orlando, Florida (Dr Babu, Messrs E. Fuentes and Papa, and Ms S. Fuentes); the Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota (Dr Van Dyke); the Department of Clinical Cytogenetics, Genetics Associates Inc, Nashville, Tennessee (Dr Dev and Ms Liu); the Department of Pathology, UT Southwestern Medical Center, Dallas, Texas (Dr Koduru); the Department of Pathology and Laboratory Medicine, David Geffen UCLA School of Medicine, Los Angeles, California (Dr Rao); and the Department of Clinical Cytogenetics, Dianon Pathology (LabCorp), Shelton, Connecticut (Dr Mitter).
Dr Babu is the founder and chief executive officer of InteGen LLC, the company that developed this technology. Mr E. Fuentes, Ms S. Fuentes, and Mr Papa are also employees of InteGen LLC. InteGen LLC has financial interests in this technology. The other authors have no relevant financial interest in the products or companies described in this article.
Reprints: Ramesh Babu, PhD, Research and Development, InteGen LLC, 8865 Commodity Circle Suite 2, Orlando, FL 32819 (email: firstname.lastname@example.org).
Caption: Figure 1. Illustration of the ideogram at approximately 600-band level showing each color band and its corresponding International System for Human Cytogenetic Nomenclature band designation.
Caption: Figure 2. Interphase chromosome profiling (ICP) color scheme. (A) and (B) illustrate the color patterns, for example, green and aqua in a short arm with 2 bands, and for a short arm with 5 bands, from distal to proximal: aqua, fused aqua/far red, fused far red/green, fused green/aqua, and green.
Caption: Figure 3. This composite karyotype of individual metaphase chromosomes illustrates the interphase chromosome profiling color banding pattern as per the Figure 1 ideogram.
Caption: Figure 4. These interphase nuclei exemplify the appearance of a normal chromosome 5 pair at left (A), and a typical myelodysplastic syndromeassociated 5q deletion at right (B). In the normal nucleus and cartoon below it, 2 chromosomal domains are apparent with the expected interphase chromosome profiling (ICP) banding pattern for chromosome 5. In the abnormal nucleus, 1 domain exhibits a similar normal ICP banding pattern, whereas the second, smaller domain illustrates an interstitial deletion with loss of signals at bands 5q14.1/q 13.3, 5q21.2, and 5q23.3. This example uses chromosome 5 to permit a more direct comparison with the interphase multicolor banding study of Lemke et al,1 which used chromosome paints rather than discrete localized color band fluorescence in situ hybridization signals.
Caption: Figure 5. A, An interphase with normal chromosome 11 pair depicting the interphase chromosome profiling (ICP) color banding pattern. B, A normal metaphase with chromosome 1 depicting the ICP color banding pattern. C, A balanced t(12;21)(p13;q21), with the chromosome 12 color banding pattern at left, and the chromosome 21 color banding pattern at right. The aqua signal, located distal to p13 (12p13.33), is identified by the arrow as being displaced from the circled homolog. The red signal, located distal to q21 (21q22.3), is identified by the arrow as being displaced from the circled homolog. D, An unbalanced der(19)t(1;19)(q23;p13) with chromosome 1 at left and chromosome 19 at right. The duplication distal to 1q23 is circled in the image to the left, and the 2 normal homologs are not circled. The segment distal to 19p13, including the aqua signal (19p13.3), has been deleted. The homolog with the deletion is circled. E, Tandem triplication involving the long arm of chromosome 1. Arrows are pointing to the triplication of band 1q24.3. F, Tandem duplication involving the long arm of chromosome 1. Arrows are pointing to the duplication of band 1q24.3. G, Deletion involving the long arm of chromosome 6. The homolog with the terminal deletion distal to 6q15 is circled. H, A dicentric isochromosome of the chromosome 17 long arm with concomitant loss of the short arm. The isodicentric homolog is circled, and the 2 arrows point to the centromeres. I, Trisomy 8, as seen through each individual color filter and at bottom center showing the composite image.
Caption: Figure 6. A, At the top left is a Y chromosome with additional chromatin (add) attached to the short arm. At the bottom is a chromosome 4 with additional chromatin (add) attached to the distal long arm. An arrow points to the breakpoint on each chromosome. A normal chromosome 2 is placed next to each abnormal chromosome to compare the G-banding pattern on the "add" material, and at right is a chromosome 2 color band ideogram. B, An interphase nucleus showing 2 normal domains of chromosome 2 (unboxed) and 2 additional small abnormal domains (boxed) representing the distal chromosome 2 short arm material from band 2p25.3 to 2p21 (aqua, far red, and hybrid aqua/far red). C, From another subject, an interphase nucleus showing 1 domain of chromosome 9 and 2 small domains (boxes) with distal 9p and 9q arm signals with color bands for 9p24.3, 9p21.2, and 9q34.3, with deletion of the interstitial segment including the centromere. D, A normal chromosome 9 and 2 previously unidentified "marker" chromosomes from the same case shown in (C), with the chromosome 9 color band ideogram for reference.
Caption: Figure 7. This image shows the normal appearance of 3 yellow signals from the chromosome 10 long arm from a direct harvest (A) and harvest after overnight culture (B).
Caption: Figure 8. A, On the chromosome 21 domains, green and red signals flank the translocation breakpoint. The circled red and green signals represent the normal signal pattern for the chromosome 21q22 area. The noncircled aqua and yellow signals represent the normal signal pattern for the chromosome 8q22 area. B, An interphase nucleus showing abnormal signal patterns for the derivative chromosomes 8 and 21 from an acute myelogenous leukemia case with t(8;21)(q22;q22). On the chromosome 8 domains, yellow and aqua signals flank the translocation breakpoint. The green and aqua, and yellow and red combinations represent the derivative chromosomes. The circled red and yellow signals represent the abnormal signal pattern for the chromosome 8q22 area. The circled aqua and green signals represent the abnormal signal pattern for the chromosome 21q22 area. The noncircled aqua and yellow signals represent the normal signal pattern for the chromosome 8q22 area. The noncircled red and green signals represent the normal signal pattern for the chromosome 21q22 area. (C) and (D) represent an acute promyelocytic leukemia-associated t(15;17)(q24;q21) with green and red flanking the translocation breakpoint on chromosome 15, and yellow and aqua flanking the translocation breakpoint on chromosome 17. The circled red and yellow signals represent the abnormal signal pattern for the chromosome 17q24 area. The circled aqua and green signals represent the abnormal signal pattern for the chromosome 15q21 area. The noncircled aqua and yellow signals represent the normal signal pattern for the chromosome 17q24 area. The noncircled red and green signals represent the normal signal pattern for the chromosome 15q21 area.
Table 1. Interphase Chromosome Profiling Probe Design: The Number of Color Bands in the Short and Long Arms of Each Chromosome Chr Chr Chr 1 p5/q5 9 p2/q4 17 p2/q4 2 p4/q6 10 p2/q5 18 p1/q4 3 p4/q5 11 p3/q5 19 p2/q3 4 p2/q6 12 p2/q5 20 p2/q3 5 p2/q6 13 p0/q5 21 p0/q3 6 p3/q5 14 p0/q5 22 p0/q3 7 p2/q5 15 p0/q5 X p3/q5 8 p2/q5 16 p2/q3 Y p1/q2 Abbreviation: Chr, chromosome. Table 2. Twenty Cases With Known Cytogenetic Results Studied by Interphase Chromosome Profiling (ICP) to Test for Concordance of Results Case ST RFR/Diagnosis 1 BM B-cell ALL 2 BM AML-M5 acute leukemia 3 BM Acute leukemia 4 BM MDS, thrombocy- topenia 5 BM MDS, leukocytosis, monocytosis 6 BM MDS/AML 7 BM Suspect CML, leukocytosis 8 BM Myeloproliferative disease 9 BM History of anaplastic large cell lymphoma, leukemic phase 10 BM AML 11 BM Multiple myeloma 12 BM Pediatric B-cell ALL ALL 13 BM Possible leukemia 14 BM Rule out AML 15 BM Pediatric T-cell ALL 16 BM Multiple myeloma 17 BM AML 18 BM AML 19 BM Multiple myeloma 20 BM AML Case ISCN 1 nuc ish(D10Z1x3)[143/200]/ (ETV6x2), (RUNX1x3),(ETV6 con RUNX1x2) [414/500]/ (PBX1x3,TCF3x4)[80/500]/ (D4Z1x3,D10Z1x4,D17Z1x4)[21/200]/ (CDKN2A,D9Z1)x3[24/200]/ (ABL1x3,BCRx4)[73/500]/ (ETV6x3), (RUNX1x5), (ETV6 con RUNX1x3) [70/500]/ (MLLx3)[23/200]/(IGHx4) [33/200] 2 46,XX,t(8;16)(p11.2;p13.3)/46,XX, sl,der(10)t(1;10)q21;p13)/46,XX,sl, der(20)t(1;20)(q21;q13.3)/46,XX,sl, der(21)t(1;21)(q12;p13)/46,XX 3 46,XX,t(8;16)(p11.2;p13.3),add(11) (q25) 4 46,XX,ider(20)(q13.3)del(20)(q11.2 q13.3)/ 47,sl,+20,ider(20)del(20) /47,sdl1,t(X;3) (q22;q27)/ 48,sdl1,del(20)(q11.2q13.3) / 48,sdl1,+ider(20)del(20) 5 46,XY,t(1;3)(p36;q21)/46,XY 6 46,X,-Y,t(1;3)(p36;q21),+mar/ 53,idem, +X,+6,+11,+12,+13,+22,+mar 7 46,XX,t(8;22)(p11.2;q11.2) 8 46,XX,t(8;9)(p11.2;q34)?c 9 47,XY,+add(X)(p22.1),t(2;5)(p23;q35) 10 47,XX,t(6;9)(p23;q34),+13/46,XX 11 47,XY,add(1)(p13),add(3)(p25),add(8)(q22), add(12)(p11.2),add(14)(q32),der(14) t(11;14)(q13;q32), t(15;17;19) (q22;q21 ;p13.1),+18,i(21)(q10)[cp20] 12 46,XX,t(4;11)(q21;q23)/46,XX 13 48,+X,idic(X)(q13)x2,+21/46,XX  14 43-46,XX,-3,add(5)(q11.2),add(6)(p21.3), add(7)(p11.2),-15, del(16)(q12.1), -17,-22, +mar[cp20] 15 46,XY,t(1;14)(p32;q11.2),t(5;17)(q31;q21) /46,XY 16 53,X,-X,t(1;14)(q21;q32),+3,+5,+5,+7, del(8)(q23q24),+9,+11,-13,+15, +15, +19/46,XX 17 47,XX,+4[cp20] 18 46,XY,t(12;18)(p13;q12) 19 57,XY,t(2;8)(p11.2;q24.1),+3,+5,+6,+9, +9,+11,+15,+15,+18,+19,+21/46, XY 20 46,XY,i(17)(q10)/46,XY Case ICP Findings Remarks 1 t(12;21)(p13.32;q22.2),+4, Concordant +9,+10,+10,+11,+14, +17,+22,+22[cp20] 2 t(8;16)(p11.2-21.2;p13.2), Concordant; der(21)t(1;21) of likely 20 cells jumping translocation 3 t(8;16)(p11.2-p21.2;p13.2), Concordant and der(11) t(1;11) identified (q42.11q44;q25) "add" material 4 ider(20)(q13.3) del(20) Concordant (q11.2q13.3)/del(20) (q13.3)  5 46,XY Did not identify the t(1;3) clone 6 XXY,t(1;3)(p36;q21),+6,+11, Concordant; +12, +13,+22 also identified monosomy 7 in 6 cells; no marker identified 7 t(18;22)(p11.2;q11.2],+21 Concordant 8 t(8;9) Concordant 9 XY,+del(X)(p22.1),t(2;5) Concordant for the t(2;5) 10 t(6;9),+13 Concordant 11 t(11;14)t(15;17;19), i(21q), Concordant and dup(X) (q24q28)(17); identified dup(11)(1q3-q25)(17); "add" dup(19) (p12-p13.2)(15), 18 material + 18 12 t(4;11) Concordant 13 dic(X)(q13)x3,-7 Concordant and monosomy 7 in 4 cells 14 dup(1)(p13.3p36,21), Concordant and dup(3)(q25q29), -3,-15,-17, identified -22[cp20] "add" material 15 t(1;14)(p35;q11.2),del(8) t(1;14) seen, (p23.2) t(5:17) not seen, and 8p deletion in eIGHt cells 16 t(1;14),del(8q),+3,+5,+5,+7, Concordant +9,+11,-13, +15,+15, +19[cp7] 17 +4 Concordant 18 t(12;18) Concordant 19 t(2;8),+3,+5,+6,+9,+9,+11, Concordant +15,+15,+18,+19, +21[cp10] 20 i(17q) Concordant Abbreviations: ALL, acute lymphocytic leukemia;AML, acute myelogenous leukemia; BM, bone marrow;CML, chronic myelogenous leukemia; ISCN, International Standard for Cytogenetic Nomenclature;MDS, myelodysplastic syndrome;RFR, reason for referral;ST, specimen type. Table 3. Additional Cases to Compare Conventional Cytogenetics and Fluorescence In Situ Hybridization (FISH) With Interphase Chromosome Profiling (ICP) Findings, and to Illustrate the Utility of ICP When Cell Culture for Metaphase Chromosome Analysis Fails Case ST RFR/Diagnosis 21 BM ALL 22 BM Anemia 23 PB CLL 24 BM MDS 25 BM CLL, r/o MDS 26 PB r/o CLL 27 BM Pancytopenia 28 BM MPN/eosinophilia 29 BM Pancytopenia 30 BM Cytopenia 31 BM Macrocytic anemia 32 BM Follicular lymphoma 33 BM Low-grade lymphoma 34 BM DLBL 35 BM AML 36 BM Follicular lymphoma 37 BM Macrocytic anemia 38 BM CLL 39 PB Developmental delay 40 BM NHL 41 BM MZL 42 BM hx mantle cell lymphoma 43 BM Lymphocytosis 44 BM hx PV 45 BM MPN? 46 BM ALL 47 BM ALL 48 BM ALL 49 BM ALL 50 BM Multiple myeloma 51 BM Multiple myeloma 52 BM Lymphoma, thrombo- cytopenia. Flow: large B-cell lymphoma 53 BM Anemia, renal insufficiency 54 BM Anemia, leucopenia. Flow: MDS 55 BM Anemia, leukopenia, MDS. Flow: MDS 56 PB AML 57 PB Paraproteinemia, leukocytosis 58 PB CLL 59 PB MDS 60 PB CLL 61 PB MDS/MPN 62 PB None 63 BM Thrombocytopenia 64 PB CML 65 BM 66 PB Leukocytosis 67 PB CLL 68 PB NHL 69 PB Polycythemia, leukocytosis 70 PB Leukocytosis 71 PB hx leukopenia 72 PB None 73 PB Leukocytosis 74 BM MDS, leukemia, lymphoma 75 PB None 76 PB Macrocytosis 77 PB Leukopenia 78 PB Nonspecific histologic findings 79 BM Anemia, DIC hemolysis, hx colon/ovarian cancer 80 PB MPN 81 PB Erythrocytosis 82 PB MDS 83 BM 84 PB Anemia Case ISCN 21 46,XY,t(9;22)(q34;q11.2),+17/46,XY 22 46,XX,del(13)(q12q22) 23 47,XX,+12/46,XX 24 46,XY,del(5)(q13q33),del(11)(q23)/46, idem, del(13)(q12q22) 25 45,X,-Y,inv(11)(q21q23)?c/46,XY,inv(11) (q21q23)?c 26 46,XY,i(7)(q10)/46,XY 27 45,XX,t(1;3)(q25;q26),-7/46,XX 28 49,XY,+der(1)t(1;5)(q32;q31),dic(5;17) (q13;q10),+8,+9, t(9;22)(q34;q11.2), +19 29 46,XX,-5,add(11)(q23),+der(11)r(11)(p15q25) trp(11)(q22q25), der(17)t(5;17) (p12;p11.2) /47,idem,+6/46,XX 30 46,XY,inv(3)(q21q26.2)/46,XY 31 46,XX 32 46,XY,t(14;18)(q32;q21)/46,XY 33 45,X,-Y 34 47,XX,der(4)t(1;4)(q21;q35),der(8)t(8;14) (q24;q32)t(14;18)(q32;q21),der(14)t(8;14) (q24;q32),+18,der(18)t(14;18) (q32;q21) x2/48,sl,+ider(8)(q10)t(8;14)t(14;18) /47,sdl1,-X 35 46,XY 36 48,XY,add(1)(p13),der(1)add(1)(p36.1)t(1 ;13)(q42;q12),del(2)(q21),add(3)(p25), der(3) t(2;3)(q21;q27)t (1;2)(p13;q31), del(6) (q25q27),del(9)(p13p22),der(13)t (1;13) (q42;q12), t(14;18)(q32;q21), +2mar/ 46,XY 37 52~54,XX,+5,+7,+9,+11,+15,-16,+22, +1~3mar[cp3]/46,XX 38 45~46,XY,t(2;5)(p23;q11.2),t(6;8)(q13;p21), i(8)(q10),del(12)(p11.2p13),-13,?r(13) (p13q22),-14,-15, add(16)(q22),\-17, add(18)(q23),add(18)(q23),-20,+r, +3mar[cp11]/ 46,XY 39 46,XY,der(9)t(5;9)(p15.3;p12) 40 41~42,-Y,add(X)(p22.1),del(1)(q32), add(9)(p22), add(9)(q22), add(11)(q13), t(11;14)(q13;q32), -12,-13,add(17)(p11.2), add(18)(q21),-20,-21[cp2]/46,XY 41 46,XX,add(6)(q25),t(8;14)(q24.1;q32)/ 46,idem,del(16)(q10)/46,XX 42 46,XX,inv(9)(p11q13) 43 46,XY 44 46,XX 45 46,XY 46 nuc ish(D4Z1,D10Z1)x2,(ABL1,BCR) x2,(5'MLL,3'MLL) x2 (5'MLL con 3'MLLx2),(ETV6,RUNX1) x2 47 46,XX.nuc ish(D4Z1,D10Z1)x2, (ABL1, BCR)x2, (5 'MLL,3 'MLL)x2 (5'MLL con 3'MLLx2), (ETV6, RUNX1x2 48 46,XY.ish t(12;21)(ETV6+,RUNX1+; ETV6+,RUNX1 +).nuc ish(D4Z1,D10Z1) x2,(ABL1,BCR)x2, (5 MLL,3 MLL) x2(5'MLL con 3'MLLx2),(ETV6,RUNX1) x3 (ETV6 con RUNX1x2)[188/200] 49 45,X,-X/46,XX.ish t(12;21)(ETV6+, RUNX1+;ETV6+, RUNX1+).nuc ish (D4Z1,D10Z1)x2,(ABL1,BCR)x2, (5'MLL,3'MLL)x2(5'MLL con 3'MLLx2), (ETV6,RUNX1)x3(ETV6 con RUNX1x2) [197/200] 50 46,XX.nuc ish(FGFR3,IGH)x2, (D9Z1,D15Z4)x2, (CCND1,IGH)x2, (RB1x2),(IGH,MAF)x2,(IGH,MAFB) x2,(TP53x2) 51 46,XY.nuc ish(FGFR3,IGH)x2, (D9Z1,D15Z4)x2, (CCND1 ,IGH)x2, (RB1x2),(IGH,MAF)x2, (IGH,MAFB)x2,(TP53x2) 52 46,X,add(Y)(p11.2),add(4) (q35),del(8)(q24.2), add(9)(p22),del(11) (q14),add(14)(q32), del(20)(p11.2)/45,X, -Y/46,XY[cp7] 53 46,XY,del(16)(q22),add(19)(q13.3),-20, +mar/46,XY 54 45,XY,-5,-20,+mar/45,sl,add(Y)(q12)/ 43~44,sl,add(3)(q21),add(7)(q32), der(15;22)(q10;q10),-17,-18,+22, +1~2mar[cp4]/46,XY 55 47,XX,-9,+2mar/46,XX 56 Failed--No result 57 Failed--No result 58 Failed--No result 59 Failed--No result 60 Failed--No result 61 Failed--No result 62 Failed--No result 63 Failed--No result 64 Failed--No result 65 Failed--No result 66 Failed--No result 67 Failed--No result 68 Failed--No result 69 Failed--No result 70 Failed--No result 71 Failed--No result 72 Failed--No result 73 Failed--No result 74 Failed--No result 75 Failed--No result 76 Failed--No result 77 Failed--No result 78 Failed--No result 79 Failed--No result 80 Failed--No result 81 Failed--No result 82 Failed--No result 83 Failed--No result 84 Failed--No result Case ICP Findings Remarks 21 Same Concordant 22 Same Concordant 23 +12, del(19) (q13.4) Cryptic 19q deletion? / normal  24 Same Concordant 25 Normal Did not identify the inv(11)?c 26 Normal Did not identify an i(7) clone 27 Same Concordant 28 Same Concordant 29 Same Concordant 30 Normal Did not identify the inv(3) 31 Same Concordant 32 -Y,t(14;18) Y loss in 4 of 20 cells 33 Same Concordant 34 Same Concordant 35 t(11;15;17) variant Discordant; FISH confirmed variant PML/ RARA 36 Same Concordant 37 Same, plus a t(8;22) Discordant;IGL/ MYC confirmed by FISH 38 Same, plus del(19p), del(20p) Additional changes identified 39 Same Concordant 40 t(1;3),t(11;14),+3.+5,+6,+7, Abnormal clone clarified by +8,+9,+10,+11,+12,+15, ICP +16,+17,18,+19,+22 41 Same, plus dup(15q),del(17q) Additional changes identified 42 t(11;14), del(18q) Discordant; FISH confirmed CCND1/IGH 43 +12,+15,dup(12q), t(14;18), Discordant; FISH confirmed del(17q),del(19q) +12 and IGH/ BCL2 44 +3,+5,+7,+19, del(16q) Discordant, most consistent with an abnormal plasma cell clone 45 +12 Discordant; FISH confirmed +12 46 Same plus +8 Discordant 47 Same Concordant 48 Same Concordant 49 Same Concordant 50 Same plus -13 Discordant 51 Same Concordant 52 dup(2)(p25.3 to p21)x2 Discordant, add(Y) and add(4) are der(Y)t(Y;2) and der(4)t(2;4) 53 Same plus dup(1) Discordant; (p32.3q24.3) marker identified as shown 54 del(5q), del(20p), del(7q), Discordant; del(17p), +22 markers identified as shown 55 del(9)(p and q) x2 with Discordant; neocentromere markers identified 56 del(7)(q31q33) Abnormal 57 t(2;7)(p11;q21) Abnormal 58 t(2;21)(p23;q22), del(13) Abnormal (q11q14,3), del(20)(p13) 59 t(15;17)(q24;q21) Abnormal 60 del(6)(q13q27) Abnormal 61 XXY, t(3;5)(q21-23;q31), Abnormal del(20)(q13.1q13.3), del(10)(p15) 62 t(2;14)(q35;q24) Abnormal 63 t(4;14)(p15-16;q32) Abnormal 64 t(12;21)(q24;q22) Abnormal 65 -16,dup(16)(p23)x4 Abnormal 66 Normal Normal 67 Normal Normal 68 Normal Normal 69 Normal Normal 70 Normal Normal 71 Normal Normal 72 Normal Normal 73 Normal Normal 74 Normal Normal 75 Normal Normal 76 Normal Normal 77 Normal Normal 78 Normal Normal 79 Normal Normal 80 Normal Normal 81 Normal Normal 82 Normal Normal 83 Normal Normal 84 Normal Normal Abbreviations: ALL, acute lymphocytic leukemia; AML, acute myelogenous leukemia; BM, bone marrow;CLL, chronic lymphocytic leukemia; DIC, disseminated intravascular coagulation;DLBL, diffuse large B-cell lymphoma; hx, history of;ISCN, International Standard for Cytogenetic Nomenclature;MDS, myelodysplastic syndrome;MPN, myeloproliferative neoplasm;MZL, marginal zone lymphoma;NHL, non-Hodgkin lymphoma; PB, peripheral blood; PV, polycythemia vera; RFR, reason for referral; r/o, rule out; ST, specimen type.
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|Author:||Babu, Ramesh; Van Dyke, Daniel L.; Dev, Vaithilingam G.; Koduru, Prasad; Rao, Nagesh; Mitter, Navnit|
|Publication:||Archives of Pathology & Laboratory Medicine|
|Date:||Feb 1, 2018|
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