- Email: [email protected]
Do random (non-clonal) chromosome abnormalities in bone marrow predict a clone to come?
Do Random (Non-Clonal) Chromosome Abnormalities in Bone Marrow Predict a Clone to Come? Thomas S. McConnell, Marilyn H. Duncan, Kathryn Foucar, and the Southwestern Oncology Group Leukemia Cytogenetics Subcommittee*
ABSTRACT: The biologic significance of clonal karyotypic abnormalities in human neoplasms is becoming better understood, but the significance of rare chromosomal aberrations is uncertain. Useful, yet arbitrary, cytogenetic definitions of a clone have been established and cases with a frequency of chromosome aberrations less than the accepted convention are explained by random loss, karyotypic instability~evolution, or other technical artifact. Are non-clonal chromosomal abnormalities that may predict future clinically significant clones being ignored? A brief case report is presented raising two such issues in the same myelodysplastic patient. This child had manosomy 7 and, later, trisomy 8, as well as increased numerical~structural aberrations seeming to predict relapse. Preliminary data from the Southwestern Oncology group is also presented. Non-clonal data should be included, when appropriate, in the clinical report.
INTRODUCTION A c l o n e is a p o p u l a t i o n of ceils d e r i v e d f r o m a single p r o g e n i t o r cell, but it is not n e c e s s a r i l y h o m o g e n e o u s [1]. A s t e m - l i n e is further defined as the m o s t f r e q u e n t c h r o m o s o m e c o n s t i t u t i o n of a t u m o r cell p o p u l a t i o n at the t i m e that it is o b s e r v e d in a direct p r e p a r a t i o n or f o l l o w i n g s h o r t - t e r m culture. All other lines are t e r m e d sidelines or sub-lines. If there are a n u m b e r of lines that a p p e a r to be related, t h e s e can be referred to together as a clone. S e c k e r - W a l k e r m a i n t a i n s that this is a c o n t r a d i c t i o n of terms, and that cytogeneticists m u s t m e t i c u l o u s l y classify a c h r o m o s o m a l l y d e f i n e d c l o n e u p o n the c h r o m o s o m e a b n o r m a l i t y c o m m o n to all its m e m b e r cells [2]. R o w l e y suggests that the o b s e r v a t i o n of at least two " p s e u d o d i p l o i d " or h y p e r d i p loid cells or three h y p o d i p l o i d cells, e a c h s h o w i n g the s a m e a b n o r m a l i t y , s h o u l d be c o n s i d e r e d as e v i d e n c e of an a b n o r m a l c l o n e [3]. In patients w h o s e cells s h o w
From the Departments of Pathology and Pediatrics, University of New Mexico Medical Center, Albuquerque, New Mexico.
* The Southwestern Oncolagy Group Leukemia Cytogenetics Subcommittee consists of Ellen Magenis (Chair), Oregon Health Sciences University; C. Nanette Clare, University of Texas San Antonio; Sheila Dobin, Scott & White; Jerry P. Lewis, University of California Davis; Thomas S. McConnell, University of New Mexico; Ramesh Babu Vullabhaneni, Henry Ford Hospital; Karl Thiel, Ohio State University. Address reprint requests to: T. S. McConnell, Department of Pathology, The University of New Mexico Medical Center, Albuquerque, NM, 87131. Received August 17, 1990; accepted September 28, 1990.
257 © 1991 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York. NY 10010
Cancer Genet Cytogenet 53:257-263 (1991) 0165-4608/91/$03.50
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T.G. McConnell et al. chromosomal alterations involving different chromosolnes in different (:ells, tbese must be considered as normal and may be due to technical artifact or random initotic errors [3]. In addition, the definition of a clone developed in the Cancer and Leukemia Group B and Southwestern Oncology Group (SWOG) cooperative cytogenetics studies was expanded to include a chromosome abnormality found in one cell from blasts from an unstimulated preparation and also found in one cell from a bone marrow specimen collected on the same or preceeding day. However, the significance of the same abnormality being found singly in specimens collected and processed months apart remains unresolved. Clonal cytogenetic abnormalities in neoplasia are well described [41, but the significance of non-clonal chromosomal aberrations (including random loss) has not been well defined. Early cytogenetic studies indicated that many factors besides malignant or pre-malignant change can cause non-clonal abberations, including viral, radiation, chemical, and other physical insults [5]. In some cases, such aberrations can be transient and are not found again for weeks or months when a new specimen is processed. In other cases, a single abnormal cell may not fit the usual criteria for clonality, but has an aberration that warrants examination of additional cells or specimens. Serial (sequential) chromosome analyses in hematologic disorders may identify clonal changes that reveal biologic progression in the abnormal cells [6]. Because the phenomenon of clonal evolution is well recognized in neoplasia, some non-clonal abnormalities may actually represent the initial (or important secondary) cytogenetic aberration(s) in patients who develop hematologic neoplasms. Every cytogenetic laboratory has at least one case exemplifying this concept. In some cytogenetic laboratories, the observation of a single cell with + 12 or t(1;19) will be described in the report, including a comment indicating its possible importance while cautioning that "a single cell does not a clone make." We present a case that had one, possibly two, important cytogenetic features. Each seemed to precede and predict a major hematologic event: the finding (four times) of a single metaphase with a "clone to come," and the detection (twice) of random "aneuploidy" that seemed to herald relapse, other explanations notwithstanding.
CASE REPORT
The patient presented in 1979 at 12 years of age with fatigue, pallor, mild anemia (hematocrit 32%), and neutropenia (322 x 109/1). Bone marrow examination revealed hypocellularity with mild granulocytic abnormalities including hypogranular cytoplasm and minimal dyserythropoiesis. No toxic or drug exposures were evident by careful history, and an explanation for the pancytopenia was not apparent, although diagnosis of a low-grade myelodysplastic syndrome was considered. The anemia resolved but the neutropenia persisted until age 15~ years when he developed E. coli sepsis. Repeat bone marrow examination at that time showed striking predominance of myeloblasts in a hypocellular marrow. The patient also exhibited gingival hyperplasia, chronic dermatologic problems, and bone pain. Although he was treated with appropriate antibiotics, no antileukemic therapy was given. The first of nine cytogenetic analyses [see Table 1], performed in month 44 since onset of illness, showed 75% (18 of 24) of metaphases with 46,XY and 21% (5 of 24), 45,XY, - 7. He was diagnosed as myelodysplastic syndrome with chronic neutropenia evolving to hypoplastic acute non-lymphoblastic leukemia. The hematologic abnormalities slowly worsened during the subsequent 3 months, with bone marrow blast count rising from 40% to 55%. Anti-leukemic induction chemotherapy was finally initiated in month 47, using cytosine arabinoside, vincristine, and prednisone. Complete remission was documented by day 36 after first
0 1 4
8 12
15
188
20 25 h
30
0 44 46 47 48 51
55 59
62
65
67 72
77
Refractory c y t o p e n i a Refractory c y t o p e n i a w i t h excess blasts Refractory c y t o p e n i a w i t h excess blasts (at autopsy)
Hypocellular ANLL
Refractory cytopenia
Refractory c y t o p e n i a Refractory cytopenia
Neutropenia H y p o c e l l u l a r ANLL Profound hypocell Chemo induction In r e m i s s i o n Refractory cytopenia
Cytologic data (marrow/periph)
1533
2928 1230
100
840
960 986
322 144 210 12 1800 500
Absolute neutrophil count × 109/1
10
8 9
7
6
5
3 4
-1 2
Chromosome accession
ANLL: acute non-lymphoblastic leukemia.
h Third chemo induction.
8 Second chemo induction.
Three cells with -18, -20, csb(2), respectively.
e One cell with + 4.
d Five cells with - 6, - 8, - 12, - 17, - 19, respectively.
c One cell with - 18.
b "Technical artifact," as defined in the text; cells/total examined; % in parentheses.
a Cells/total examined; % in parentheses.
M o n t h from first c h e m o induction
0
23 23
25
25
32
27 50
-24 32
Number cells examined
Sequential and chronologic clinical, hematologic and cytogenetic data
Month from onset
Table 1
28/32 (88%) 46,XY; 2/32 (6%) - 7 ; one cell = + 8 b 14/25 (56%) 46,XY; 10/25 (40%) - 7 ; one cell = + 8 b 3/25 (12%) 46,XY; 21/25 (84%) - 7 ; one cell = + 8 b 2/23 (87%) 46,XY 5/23 (22%) 46,XY; 10/23 (43%) - 7 ; 8/23 (35%) + 8 post mortem sample autolyzed
27/27 (100%) 46,XY 44/50 (88%) 46,XY one cell = + 8 b
18/24 (75%0 46,XY; 5/24 (21%) - 7 10/32 (31%) 46,XY; 22/32 (69%) - 7
Chromosome analysis a
3/23 (13%) f 0
0
0
1/32 (3%) e
0 5/50 (10%) d
1/24 (4%) c 0
% TA b
[',O ¢31
260
w . s . McConnell et a[.
i n d u c t i o n and the peripheral blood picture normalized. Cytogenetic study of bone marrow in m o n t h 48 revealed no abnormal cells. However, the patient d e v e l o p e d d i s s e m i n a t e d candidiasis; anti-fungal therapy {amphotericin-B) was c o m b i n e d with m a i n t e n a n c e c h e m o t h e r a p y of 6-mercaptopurine and methotrexate. Intermittent vincristine and p r e d n i s o n e pulses were also administered. By m o n t h 51, 4 months post-induction, bone marrow c h r o m o s o m e analysis revealed a single metaphase with trisomy 8, and 10% (5 of 50 cells) technical artifact. Bone marrow cellularity was 40% with 1% myeloblasts. Mild to moderate dyspoiesis, especially in the erythroid cell line, was attributed to c h e m o t h e r a p y effects. A single cell with + 8 occurred again in months 59, 62, and 65. At month 65, w h e n 84% (21 of 25) of marrow cells had m o n o s o m y 7, he d e v e l o p e d a second episode of overt acute n o n - l y m p h o b l a s t i c leukemia with 46% blasts in bone marrow. Re-induction therapy was begun, and by day 48 of the second i n d u c t i o n (month 67), complete bone marrow remission was once again achieved. The marrow cells a p p e a r e d normal cytogenetically, but there was 13% (3 of 24 cells) technical artifact. W h i l e on m a i n t e n a n c e c h e m o t h e r a p y he d e v e l o p e d a bacterial lung abscess. Chemotherapy was w i t h e l d during antibiotic treatment. He later d e v e l o p e d non-A, non-B hepatitis. At 72 months, the patient's bone marrow cells again revealed - 7 (in 43% [10 of 23] of cells), with the a p p e a r a n c e of a second clone of + 8 (in 8 of 23 or 35% of cells). Bone marrow cellularity was 40% with 8% blasts on smears; large clusters of i m m a t u r e cells were identified on clot sections. C h e m o t h e r a p y was reinstituted for the third time, but the patient died of E. coli sepsis associated with a large rectal abscess at 77 months. At autopsy, the marrow blast count was estimated at 15%, suggesting response to re-induction therapy. Overall survival was 6½ years from initial presentation with anemia and neutropenia, and 30 months after developing overt acute n o n - l y m p h o b l a s tic leukemia.
CYTOGENETICS S t a n d a r d G-banded cytogenetic analysis from bone marrow samples utilizing Giemsatrypsin m e t h o d o l o g y was attempted ten times and was successful in nine of those. Table 1 shows these serial analyses over a period of 2.8 years. A single cell with + 8 was found four separate times before an overt + 8 clone developed. Twice, the first time 5 months after first complete remission but 12 months before the second relapse, and the second, a few weeks after second complete remission but 10 months before death, cytogenetic abnormalities consisting of non-clonal structural and/or n u m e r i c a l aberrations higher than background (10% [5 of 50] and 13% [3 of 23], respectively) were found. In p r e l i m i n a r y data from the SWOG Leukemia Cytogenetics S u b c o m m i t t e e [7], 211 studies from 275 r e v i e w e d cases had r a n d o m abnormalities. Eighty-two of the 211 studies s h o w e d abnormal non-clonal cells (these i n c l u d e d one or more metaphases with " r a n d o m gain" a n d / o r structural aberrations but not " r a n d o m loss"). Three patients from three different SWOG institutions exemplified the "clone to come" issue. Patient A, in his pre-treatment acute l y m p h o b l a s t i c leukemia study, had one metaphase with 44,XY, - 3, - 3, - 5, - 8,t(9;22)(q34;q11), + 2mar and 27 metaphases, 46,XY. Sixteen days later, the t(9;22) was present clonally (4 of 22 metaphases). Patient B, in the initial study for acute n o n - l y m p h o b l a s t i c leukemia, had a clone of 5 3 , X Y , + 4 , + 6 , + 8 , + 9 , + 1 0 , + 2 1 , + 2 2 and a single cell with 5 3 , X Y , + X , + 4 , + 6 , + 9, + 10, + 21, + 22 (note + X but not + 8 difference). Six months later, in remission, the patient had only 46,XY cells. A relapse study, performed 9 months later, s h o w e d
Are Random Abnormalities Predictive?
261
only an evolved clone, 54,XY,+X,+4,+ 6 , + 8 , + 9 , + 10,+ 21,+ 22, and a final study, done at 10 months, showed 46,XY/54,XY, + X, + 4, + 6, + 8, + 9, + 10, + 21, + 22. Patient C, with 20 cells 46,XX and 1 cell, 46,XX,-12,-18,del(20)(qll),t(9;22) (q34;q11), + der(12)t(12;?),(q21;?), + der(18)t(18;?)(q21;?) in the pre-treatment study, showed clonality for the abnormal cell line at relapse (acute lymphoblastic leukemia) 8 months later. DISCUSSION
The cytogenetic data on the patient presented in detail show a probable clone of + 8, but at a level below significance by cytogenetic convention, present almost 2 years before death. This clone proliferated, but was classifiable as a clone by current criteria only 5 months before death. Bone marrow morphologic examination was performed with each cytogenetic study. On the samples that showed "technical artifact," there was not a recognizable increase in marrow blasts. Although some dyspoietic changes and above-background non-clonal chromosome aberrations were noted in marrow cells, they were not distinguishable from chemotherapy effects. The last evaluation, 5 months before the patient's death, showed an increase in blasts and progressive dyspoiesis suggestive, but not diagnostic, of early leukemic relapse. Background "technical artifact" in the senior author's laboratory is 4% for all analyses and 7% for bone marrow/unstimulated blood analyses, based on minimum counts of 20 cells. Technical artifact is defined as the percent of metaphases with non-clonal (random) loss or gain, and/or structural aberration including chromatid or iso-chromatid break with significant displacement, but not clonal or constitutional structural abnormalities, nor chromosome heteromorphisms. This definition holds only if the metaphase chromosomes are not strung out more than 1.5 high-power fields and appear to be in the same stage of condensation with similar staining patterns. Because this method of metaphase selection is subjective, it should be defined by each laboratory using internal standards (comparison of one laboratory's or technologist's rate of artifact to another is probably not of real value). Each laboratory's rate of artifact can be found by counting the number of cells in which an abnormality is found in a statistically appropriate number of cases, and expressing this as a background incidence of random abnormalities. Each type of procedure (blood, bone marrow, aminiocyte, etc.) should have its own rate calculated periodically for quality control purposes. The meaning of technical artifact relates to the use to which it is put. We use it, in addition to other parameters, to detect technical, reagent, and procedural problems and to evaluate variations in technician skills. Utilized as a finding of exclusion, when no other factor is uncovered, we mention the possibility of increased random chromosome abnormalities over background as a clue to some in vivo problem. In the analyses reported herein used as their own control, there is 1% technical artifact excluding the two samples with excess technical artifact, or 3.8% including all samples. Recent data from the Mayo Clinic suggests that technical artifact in their hands may be as high as 13% in presumably normal bone marrow donor candidates [8]. When confronted with the issue of true artifact versus an important but non-clonal finding, the laboratory alternatives include the following: count more cells, in order to increase the possibility of finding the second or third cell; increase the next sample size so that more cells are available for examination; procure a better sample; vary technique, enabling crisper banding and better spreading; acquire sequential samples in order to repeat the study or investigate the patient's cytogenetic status at different moments in the disease course. Caution dictates continuance of the current definition of clonal. We do not advocate
262
T.S. McConnell et al.
the suggestion that the m i n i m u m criteria for a clone are met w h e n a second cell with the same abnormality is found several months later. A description of non-clonal chromosome abnormalities, especially those involving classical chromosomal abnormalities [9, 10], should become a standard part of the cytogenetics report in neoplastic conditions. The report should also state the numbers of cells examined and describe all normal and abnormal metaphases identified by the procedure. The presence of non-clonal abnormalities in a single cell should always be brought to the attention of the clinician, and those except r a n d o m loss should be identified as possible "clones to come." This approach to complete reporting seems even more important w h e n the same single cell abnormality is found again on a different sample at a later time. In the detailed case presented, the finding of increased technical artifact may have been heralding events to come, or may have reflected effects from other associated microbiologic, physical, or chemical phenomena. When the calculated level of technical artifact reaches twice the baseline (or twice the level found in the patient's own sequential studies as control), this information is worth m e n t i o n i n g to the clinician. Although its m e a n i n g may be unclear at the time, such information may in some cases prove helpful in an i n d i v i d u a l patient's management. Additional study of cases with non-clonal abnormalities may yield more clinically useful predictive data. The SWOG Leukemia Cytogenetics Subcommittee plans to continue its non-clonal chromosome study in adult leukemia. The i m p l e m e n t a t i o n of interphase cytogenetics utilizing molecular probes on large n u m b e r s of cells may help clarify such issues [11]. This molecular technology promises to be helpful in the quantitation of residual neoplastic disease as well as possible "clones to come." In the meantime, complete reporting of all cytogenetic findings to the clinician may help improve patient management.
The authors wish to acknowledge Drs. Michael Grever and Fred Appelbaum. Chairmen of The SWOG Leukemia Committee, and Dr. Cheryl Willman, Chair of the SWOG Leukemia Biology Research Committee. Supported in part by National Institutes of Health grants CA-32102 and CA-12213 supporting the Southwest Oncology Group Leukemia Biology Program.
REFERENCES
1. ISCN (1985): An International System for Human Cytogenetic Nomenclature, Harnden DG, Klinger HP, eds., S. Karger, Basel, pp. 73-76. 2. Secker-Walker LM (1985): The meaning of a clone. Cancer Genet Cytogenet 16:187-8. 3. Rowley JD (1983): Chromosome abnormalities in leukemia and lymphoma. Ann Clin Lab Sci 13:87-94. 4. Yunis JJ (1989): Genes and chromosomes in the pathogenesis and prognosis of human cancers. Adv Pathol 2:147-88. 5. Longacre TA, Foucar K, Crago S, Chen I, Griffith B, Dressier L, McConnell TS, Duncan M, Gribble J (1989): Hematogones: a multiparameter analysis of bone marrow precursor cells. Blood 73:543-52. 6. Miller BA, Weinstein HJ, Nell M, Henkle CT, Dillon PL, Tantravahi R {1985): Sequential development of distinct clonal chromosome abnormalities in a patient with preleukemia. Br J Haematol 59:411-18. 7. McConnell TS (1990): The significance of random chromosome abnormalities in leukemia: preliminary SWOG data (presentation). Southwestern Oncology Group, Leukemia Cytogenetics Subcommittee, Denver, Colorado. 8. Kuffel DB, Schultz CG. Dewald GW (1990): Normal cytogenetic values for bone marrow (poster #42). Annual meeting of the Association of Cytogenetic Technologists, Santa Fe. New Mexico.
Are Random Abnormalities Predictive?
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9. Trent JM, Kaneko Y, Mitelman F (1989): Report of the committee on structural chromosome changes in neoplasia. Cytogenet Cell Genet 51:533-562. 10. Rowley JD (1990): Molecular cytogenetics: Rosetta stone for understanding cancer. Cancer Res 50:3816-25. 11. Nederlof PM, van der Flier S, Raap AK, Tanke HJ, van der Ploeg M, Kornips F, Geraedts JPM (1989): Detection of chromosome aberrations in interphase tumor nuclei by nonradioactive in situ hybridization. Cancer Genet Cytogenet 42:87-98.
ABSTRACT: The biologic significance of clonal karyotypic abnormalities in human neoplasms is becoming better understood, but the significance of rare chromosomal aberrations is uncertain. Useful, yet arbitrary, cytogenetic definitions of a clone have been established and cases with a frequency of chromosome aberrations less than the accepted convention are explained by random loss, karyotypic instability~evolution, or other technical artifact. Are non-clonal chromosomal abnormalities that may predict future clinically significant clones being ignored? A brief case report is presented raising two such issues in the same myelodysplastic patient. This child had manosomy 7 and, later, trisomy 8, as well as increased numerical~structural aberrations seeming to predict relapse. Preliminary data from the Southwestern Oncology group is also presented. Non-clonal data should be included, when appropriate, in the clinical report.
INTRODUCTION A c l o n e is a p o p u l a t i o n of ceils d e r i v e d f r o m a single p r o g e n i t o r cell, but it is not n e c e s s a r i l y h o m o g e n e o u s [1]. A s t e m - l i n e is further defined as the m o s t f r e q u e n t c h r o m o s o m e c o n s t i t u t i o n of a t u m o r cell p o p u l a t i o n at the t i m e that it is o b s e r v e d in a direct p r e p a r a t i o n or f o l l o w i n g s h o r t - t e r m culture. All other lines are t e r m e d sidelines or sub-lines. If there are a n u m b e r of lines that a p p e a r to be related, t h e s e can be referred to together as a clone. S e c k e r - W a l k e r m a i n t a i n s that this is a c o n t r a d i c t i o n of terms, and that cytogeneticists m u s t m e t i c u l o u s l y classify a c h r o m o s o m a l l y d e f i n e d c l o n e u p o n the c h r o m o s o m e a b n o r m a l i t y c o m m o n to all its m e m b e r cells [2]. R o w l e y suggests that the o b s e r v a t i o n of at least two " p s e u d o d i p l o i d " or h y p e r d i p loid cells or three h y p o d i p l o i d cells, e a c h s h o w i n g the s a m e a b n o r m a l i t y , s h o u l d be c o n s i d e r e d as e v i d e n c e of an a b n o r m a l c l o n e [3]. In patients w h o s e cells s h o w
From the Departments of Pathology and Pediatrics, University of New Mexico Medical Center, Albuquerque, New Mexico.
* The Southwestern Oncolagy Group Leukemia Cytogenetics Subcommittee consists of Ellen Magenis (Chair), Oregon Health Sciences University; C. Nanette Clare, University of Texas San Antonio; Sheila Dobin, Scott & White; Jerry P. Lewis, University of California Davis; Thomas S. McConnell, University of New Mexico; Ramesh Babu Vullabhaneni, Henry Ford Hospital; Karl Thiel, Ohio State University. Address reprint requests to: T. S. McConnell, Department of Pathology, The University of New Mexico Medical Center, Albuquerque, NM, 87131. Received August 17, 1990; accepted September 28, 1990.
257 © 1991 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York. NY 10010
Cancer Genet Cytogenet 53:257-263 (1991) 0165-4608/91/$03.50
258
T.G. McConnell et al. chromosomal alterations involving different chromosolnes in different (:ells, tbese must be considered as normal and may be due to technical artifact or random initotic errors [3]. In addition, the definition of a clone developed in the Cancer and Leukemia Group B and Southwestern Oncology Group (SWOG) cooperative cytogenetics studies was expanded to include a chromosome abnormality found in one cell from blasts from an unstimulated preparation and also found in one cell from a bone marrow specimen collected on the same or preceeding day. However, the significance of the same abnormality being found singly in specimens collected and processed months apart remains unresolved. Clonal cytogenetic abnormalities in neoplasia are well described [41, but the significance of non-clonal chromosomal aberrations (including random loss) has not been well defined. Early cytogenetic studies indicated that many factors besides malignant or pre-malignant change can cause non-clonal abberations, including viral, radiation, chemical, and other physical insults [5]. In some cases, such aberrations can be transient and are not found again for weeks or months when a new specimen is processed. In other cases, a single abnormal cell may not fit the usual criteria for clonality, but has an aberration that warrants examination of additional cells or specimens. Serial (sequential) chromosome analyses in hematologic disorders may identify clonal changes that reveal biologic progression in the abnormal cells [6]. Because the phenomenon of clonal evolution is well recognized in neoplasia, some non-clonal abnormalities may actually represent the initial (or important secondary) cytogenetic aberration(s) in patients who develop hematologic neoplasms. Every cytogenetic laboratory has at least one case exemplifying this concept. In some cytogenetic laboratories, the observation of a single cell with + 12 or t(1;19) will be described in the report, including a comment indicating its possible importance while cautioning that "a single cell does not a clone make." We present a case that had one, possibly two, important cytogenetic features. Each seemed to precede and predict a major hematologic event: the finding (four times) of a single metaphase with a "clone to come," and the detection (twice) of random "aneuploidy" that seemed to herald relapse, other explanations notwithstanding.
CASE REPORT
The patient presented in 1979 at 12 years of age with fatigue, pallor, mild anemia (hematocrit 32%), and neutropenia (322 x 109/1). Bone marrow examination revealed hypocellularity with mild granulocytic abnormalities including hypogranular cytoplasm and minimal dyserythropoiesis. No toxic or drug exposures were evident by careful history, and an explanation for the pancytopenia was not apparent, although diagnosis of a low-grade myelodysplastic syndrome was considered. The anemia resolved but the neutropenia persisted until age 15~ years when he developed E. coli sepsis. Repeat bone marrow examination at that time showed striking predominance of myeloblasts in a hypocellular marrow. The patient also exhibited gingival hyperplasia, chronic dermatologic problems, and bone pain. Although he was treated with appropriate antibiotics, no antileukemic therapy was given. The first of nine cytogenetic analyses [see Table 1], performed in month 44 since onset of illness, showed 75% (18 of 24) of metaphases with 46,XY and 21% (5 of 24), 45,XY, - 7. He was diagnosed as myelodysplastic syndrome with chronic neutropenia evolving to hypoplastic acute non-lymphoblastic leukemia. The hematologic abnormalities slowly worsened during the subsequent 3 months, with bone marrow blast count rising from 40% to 55%. Anti-leukemic induction chemotherapy was finally initiated in month 47, using cytosine arabinoside, vincristine, and prednisone. Complete remission was documented by day 36 after first
0 1 4
8 12
15
188
20 25 h
30
0 44 46 47 48 51
55 59
62
65
67 72
77
Refractory c y t o p e n i a Refractory c y t o p e n i a w i t h excess blasts Refractory c y t o p e n i a w i t h excess blasts (at autopsy)
Hypocellular ANLL
Refractory cytopenia
Refractory c y t o p e n i a Refractory cytopenia
Neutropenia H y p o c e l l u l a r ANLL Profound hypocell Chemo induction In r e m i s s i o n Refractory cytopenia
Cytologic data (marrow/periph)
1533
2928 1230
100
840
960 986
322 144 210 12 1800 500
Absolute neutrophil count × 109/1
10
8 9
7
6
5
3 4
-1 2
Chromosome accession
ANLL: acute non-lymphoblastic leukemia.
h Third chemo induction.
8 Second chemo induction.
Three cells with -18, -20, csb(2), respectively.
e One cell with + 4.
d Five cells with - 6, - 8, - 12, - 17, - 19, respectively.
c One cell with - 18.
b "Technical artifact," as defined in the text; cells/total examined; % in parentheses.
a Cells/total examined; % in parentheses.
M o n t h from first c h e m o induction
0
23 23
25
25
32
27 50
-24 32
Number cells examined
Sequential and chronologic clinical, hematologic and cytogenetic data
Month from onset
Table 1
28/32 (88%) 46,XY; 2/32 (6%) - 7 ; one cell = + 8 b 14/25 (56%) 46,XY; 10/25 (40%) - 7 ; one cell = + 8 b 3/25 (12%) 46,XY; 21/25 (84%) - 7 ; one cell = + 8 b 2/23 (87%) 46,XY 5/23 (22%) 46,XY; 10/23 (43%) - 7 ; 8/23 (35%) + 8 post mortem sample autolyzed
27/27 (100%) 46,XY 44/50 (88%) 46,XY one cell = + 8 b
18/24 (75%0 46,XY; 5/24 (21%) - 7 10/32 (31%) 46,XY; 22/32 (69%) - 7
Chromosome analysis a
3/23 (13%) f 0
0
0
1/32 (3%) e
0 5/50 (10%) d
1/24 (4%) c 0
% TA b
[',O ¢31
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i n d u c t i o n and the peripheral blood picture normalized. Cytogenetic study of bone marrow in m o n t h 48 revealed no abnormal cells. However, the patient d e v e l o p e d d i s s e m i n a t e d candidiasis; anti-fungal therapy {amphotericin-B) was c o m b i n e d with m a i n t e n a n c e c h e m o t h e r a p y of 6-mercaptopurine and methotrexate. Intermittent vincristine and p r e d n i s o n e pulses were also administered. By m o n t h 51, 4 months post-induction, bone marrow c h r o m o s o m e analysis revealed a single metaphase with trisomy 8, and 10% (5 of 50 cells) technical artifact. Bone marrow cellularity was 40% with 1% myeloblasts. Mild to moderate dyspoiesis, especially in the erythroid cell line, was attributed to c h e m o t h e r a p y effects. A single cell with + 8 occurred again in months 59, 62, and 65. At month 65, w h e n 84% (21 of 25) of marrow cells had m o n o s o m y 7, he d e v e l o p e d a second episode of overt acute n o n - l y m p h o b l a s t i c leukemia with 46% blasts in bone marrow. Re-induction therapy was begun, and by day 48 of the second i n d u c t i o n (month 67), complete bone marrow remission was once again achieved. The marrow cells a p p e a r e d normal cytogenetically, but there was 13% (3 of 24 cells) technical artifact. W h i l e on m a i n t e n a n c e c h e m o t h e r a p y he d e v e l o p e d a bacterial lung abscess. Chemotherapy was w i t h e l d during antibiotic treatment. He later d e v e l o p e d non-A, non-B hepatitis. At 72 months, the patient's bone marrow cells again revealed - 7 (in 43% [10 of 23] of cells), with the a p p e a r a n c e of a second clone of + 8 (in 8 of 23 or 35% of cells). Bone marrow cellularity was 40% with 8% blasts on smears; large clusters of i m m a t u r e cells were identified on clot sections. C h e m o t h e r a p y was reinstituted for the third time, but the patient died of E. coli sepsis associated with a large rectal abscess at 77 months. At autopsy, the marrow blast count was estimated at 15%, suggesting response to re-induction therapy. Overall survival was 6½ years from initial presentation with anemia and neutropenia, and 30 months after developing overt acute n o n - l y m p h o b l a s tic leukemia.
CYTOGENETICS S t a n d a r d G-banded cytogenetic analysis from bone marrow samples utilizing Giemsatrypsin m e t h o d o l o g y was attempted ten times and was successful in nine of those. Table 1 shows these serial analyses over a period of 2.8 years. A single cell with + 8 was found four separate times before an overt + 8 clone developed. Twice, the first time 5 months after first complete remission but 12 months before the second relapse, and the second, a few weeks after second complete remission but 10 months before death, cytogenetic abnormalities consisting of non-clonal structural and/or n u m e r i c a l aberrations higher than background (10% [5 of 50] and 13% [3 of 23], respectively) were found. In p r e l i m i n a r y data from the SWOG Leukemia Cytogenetics S u b c o m m i t t e e [7], 211 studies from 275 r e v i e w e d cases had r a n d o m abnormalities. Eighty-two of the 211 studies s h o w e d abnormal non-clonal cells (these i n c l u d e d one or more metaphases with " r a n d o m gain" a n d / o r structural aberrations but not " r a n d o m loss"). Three patients from three different SWOG institutions exemplified the "clone to come" issue. Patient A, in his pre-treatment acute l y m p h o b l a s t i c leukemia study, had one metaphase with 44,XY, - 3, - 3, - 5, - 8,t(9;22)(q34;q11), + 2mar and 27 metaphases, 46,XY. Sixteen days later, the t(9;22) was present clonally (4 of 22 metaphases). Patient B, in the initial study for acute n o n - l y m p h o b l a s t i c leukemia, had a clone of 5 3 , X Y , + 4 , + 6 , + 8 , + 9 , + 1 0 , + 2 1 , + 2 2 and a single cell with 5 3 , X Y , + X , + 4 , + 6 , + 9, + 10, + 21, + 22 (note + X but not + 8 difference). Six months later, in remission, the patient had only 46,XY cells. A relapse study, performed 9 months later, s h o w e d
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only an evolved clone, 54,XY,+X,+4,+ 6 , + 8 , + 9 , + 10,+ 21,+ 22, and a final study, done at 10 months, showed 46,XY/54,XY, + X, + 4, + 6, + 8, + 9, + 10, + 21, + 22. Patient C, with 20 cells 46,XX and 1 cell, 46,XX,-12,-18,del(20)(qll),t(9;22) (q34;q11), + der(12)t(12;?),(q21;?), + der(18)t(18;?)(q21;?) in the pre-treatment study, showed clonality for the abnormal cell line at relapse (acute lymphoblastic leukemia) 8 months later. DISCUSSION
The cytogenetic data on the patient presented in detail show a probable clone of + 8, but at a level below significance by cytogenetic convention, present almost 2 years before death. This clone proliferated, but was classifiable as a clone by current criteria only 5 months before death. Bone marrow morphologic examination was performed with each cytogenetic study. On the samples that showed "technical artifact," there was not a recognizable increase in marrow blasts. Although some dyspoietic changes and above-background non-clonal chromosome aberrations were noted in marrow cells, they were not distinguishable from chemotherapy effects. The last evaluation, 5 months before the patient's death, showed an increase in blasts and progressive dyspoiesis suggestive, but not diagnostic, of early leukemic relapse. Background "technical artifact" in the senior author's laboratory is 4% for all analyses and 7% for bone marrow/unstimulated blood analyses, based on minimum counts of 20 cells. Technical artifact is defined as the percent of metaphases with non-clonal (random) loss or gain, and/or structural aberration including chromatid or iso-chromatid break with significant displacement, but not clonal or constitutional structural abnormalities, nor chromosome heteromorphisms. This definition holds only if the metaphase chromosomes are not strung out more than 1.5 high-power fields and appear to be in the same stage of condensation with similar staining patterns. Because this method of metaphase selection is subjective, it should be defined by each laboratory using internal standards (comparison of one laboratory's or technologist's rate of artifact to another is probably not of real value). Each laboratory's rate of artifact can be found by counting the number of cells in which an abnormality is found in a statistically appropriate number of cases, and expressing this as a background incidence of random abnormalities. Each type of procedure (blood, bone marrow, aminiocyte, etc.) should have its own rate calculated periodically for quality control purposes. The meaning of technical artifact relates to the use to which it is put. We use it, in addition to other parameters, to detect technical, reagent, and procedural problems and to evaluate variations in technician skills. Utilized as a finding of exclusion, when no other factor is uncovered, we mention the possibility of increased random chromosome abnormalities over background as a clue to some in vivo problem. In the analyses reported herein used as their own control, there is 1% technical artifact excluding the two samples with excess technical artifact, or 3.8% including all samples. Recent data from the Mayo Clinic suggests that technical artifact in their hands may be as high as 13% in presumably normal bone marrow donor candidates [8]. When confronted with the issue of true artifact versus an important but non-clonal finding, the laboratory alternatives include the following: count more cells, in order to increase the possibility of finding the second or third cell; increase the next sample size so that more cells are available for examination; procure a better sample; vary technique, enabling crisper banding and better spreading; acquire sequential samples in order to repeat the study or investigate the patient's cytogenetic status at different moments in the disease course. Caution dictates continuance of the current definition of clonal. We do not advocate
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the suggestion that the m i n i m u m criteria for a clone are met w h e n a second cell with the same abnormality is found several months later. A description of non-clonal chromosome abnormalities, especially those involving classical chromosomal abnormalities [9, 10], should become a standard part of the cytogenetics report in neoplastic conditions. The report should also state the numbers of cells examined and describe all normal and abnormal metaphases identified by the procedure. The presence of non-clonal abnormalities in a single cell should always be brought to the attention of the clinician, and those except r a n d o m loss should be identified as possible "clones to come." This approach to complete reporting seems even more important w h e n the same single cell abnormality is found again on a different sample at a later time. In the detailed case presented, the finding of increased technical artifact may have been heralding events to come, or may have reflected effects from other associated microbiologic, physical, or chemical phenomena. When the calculated level of technical artifact reaches twice the baseline (or twice the level found in the patient's own sequential studies as control), this information is worth m e n t i o n i n g to the clinician. Although its m e a n i n g may be unclear at the time, such information may in some cases prove helpful in an i n d i v i d u a l patient's management. Additional study of cases with non-clonal abnormalities may yield more clinically useful predictive data. The SWOG Leukemia Cytogenetics Subcommittee plans to continue its non-clonal chromosome study in adult leukemia. The i m p l e m e n t a t i o n of interphase cytogenetics utilizing molecular probes on large n u m b e r s of cells may help clarify such issues [11]. This molecular technology promises to be helpful in the quantitation of residual neoplastic disease as well as possible "clones to come." In the meantime, complete reporting of all cytogenetic findings to the clinician may help improve patient management.
The authors wish to acknowledge Drs. Michael Grever and Fred Appelbaum. Chairmen of The SWOG Leukemia Committee, and Dr. Cheryl Willman, Chair of the SWOG Leukemia Biology Research Committee. Supported in part by National Institutes of Health grants CA-32102 and CA-12213 supporting the Southwest Oncology Group Leukemia Biology Program.
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