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Keywords:

  • transferrin receptor;
  • mRNA;
  • acute myeloid leukaemia

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments.
  7. References

Transferrin receptor (TfR, CD71) is an integral membrane glycoprotein that mediates cellular uptake of iron. In most tissues, TfR expression is correlated positively with proliferation and regulated at the post-transcriptional level. The available data regarding the pattern of TfR gene expression in haematological malignancies are very limited. In the present study, we evaluated TfR gene expression at the molecular level in bone marrow (BM) samples of 44 patients with de novo acute myeloid leukaemia (AML) at diagnosis with BM blasts > 85%. TfR mRNA levels were determined by densitometric analysis of quantitative reverse transcription polymerase chain reaction products corresponding to TfR exons 15–17. Each sample was tested in at least two independent experiments. In 13/44 patients, TfR messages were not detected (this is probably an underestimate as some positive results may be attributed to residual normal erythroid cells present in the samples). In 17/44, TfR mRNA levels were low–intermediate, and were high in the remaining patients (14/44). TfR mRNA positivity was significantly associated with older age. No statistically significant correlations were found either with specific French–American–British (FAB) subtypes or attainment of complete remission, incidence of relapse and survival (after adjusting accordingly for age and FAB subtype). The absence of TfR mRNA transcripts in a significant minority of cases suggests that alternative mechanisms of iron uptake may function in AML blast cells.

Iron is essential for cell proliferation. Control of iron acquisition and utilization must be modulated to meet the varied and diverse demands of the cells (Lash & Saleem, 1995; Lieu et al, 2001). The best known mechanism for the uptake of iron in both normal and neoplastic cells entails binding of serum transferrin (the main carrier protein for iron in serum) to a specific transmembrane glycoprotein receptor (transferrin receptor, TfR) (Testa et al, 1993). However, other mechanisms have also been found to operate in several cell types (Qian & Tang, 1995; Richardson & Ponka, 1997; Lieu et al, 2001).

In most tissues, TfR expression is controlled by iron availability at the post-trascriptional level in a manner resembling feedback inhibition: fewer receptors are expressed when iron is abundant and more receptors are expressed when iron is scarce (Chan et al, 1994). However, in erythroid cells, TfR expression is regulated at the transcriptional level during erythroid differentiation and feedback mechanisms related to iron levels do not seem to be important (Chan et al, 1994). Furthermore, TfR expression is related to the proliferative state of the cells as well as the induction of differentiation (Theil, 1990); thus, the number of TfR (CD71) molecules is larger in cells with a high proliferation rate (Kuhn, 1994).

The available data from previous protein studies on TfR (CD71) expression in haematological malignancies indicate that CD71 may be associated with certain features of the neoplastic cells [e.g. T-cell immunophenotype in acute lymphoblastic leukaemia (Koehler et al, 1993) and non-Hodgkin's lymphomas (Das Gupta & Shah, 1990)] but are inconclusive as regards the possible clinical significance of CD71 expression (Medeiros et al, 1988; Koehler et al, 1993; Bradstock et al, 1994). However, there is no evidence about the pattern of TfR mRNA expression and stability in primary malignant cells. In this study, we analysed TfR mRNA expression in 44 patients with de novo acute myeloid leukaemia (AML) at diagnosis (treated on a single protocol) and investigated possible prognostic implications.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments.
  7. References

Patient samples Bone marrow (BM) samples were collected from 44 patients with de novo AML with BM blasts > 85%; clinical and haematological laboratory data at diagnosis before treatment were available in all 44 patients (Table I). Patients were enrolled in two different treatment protocols according to age (> 60 or < 60 years old); within each group, patients were treated uniformly. In both age groups, AML-M3 cases received specific, all-trans retinoic acid-containing, treatment.

Table I.   Clinical-laboratory data and TfR mRNA expression pattern of the AML cases analysed in the present study.
CODE AGE SEX FAB KARYOTYPE REM REL SURVTFR (atoms/μg RNA)
  1. M, male; F, female; FAB, FAB subtype; REM, remission; CR, complete remission; IF, remission induction failure; REL, relapse; SURV, survival (in months); Y, yes; N, no; TfR, TfR mRNA expression; H, high levels; L, low-intermediate levels.

55650FM046, XXCR 40+H: 5·18 × 106
60544MM046, XYIF 1N
65760MM046, XYCR 26+L: 0·45 × 106
55976MM147, XY, +11, i(11q)IF 2H: 1·86 × 106
57352MM145, XY, −12, −21, inv(3)(p13; q27), del(5)(q13; q33), 5q+CR 25N
60456FM146, XX, del(9)(q22q23)CR 37+H: 1·07 × 106
60769MM146, XY, −3, −16, −17, −20, +20q, +mar1, +mar2, +mar3IF 2L: 0·36 × 106
61377FM147, XX, −11, −17, del(3)(q13), del(5)(q13; q33), +i(11q), +der(17) t(5; 11; 17)IF 1L: 0·85 × 106
65661FM146, XXIF 1L: 0·27 × 106
65851FM146, XXCRY9L: 0·38 × 106
66055FM147, XX, +3, +4, −5, der(3) inv(3) (p24q21), del(5) (q22q35), del(11) (p11p15)CRY7L: 0·62 × 106
66422FM146, XXCR 28+N
52935FM246, XXCRY21H: 5·90 × 106
53833FM245, X, -X, t(8; 21)CRY21N
57728FM248, XX, +4, +8, t(8; 21)CR 38+L: 0·14 × 106
58318FM246, XXCRY38+L: 0·65 × 106
58469FM245, XX, −14, −17, +der(14) t(14; 17)(q11; q11)IF 9H: 6·50 × 106
58568MM246, XYIF 10H: 2·80 × 106
60648MM246, XY, t(8; 21)CRY16N
61421MM246, XY, t(8; 21)CRY11N
68073MM245, X, -Y, t(8; 21)CR 25+L: 0·26 × 106
72459FM246, XX, inv(16)CR 11+H: 0·98 × 106
53142FM346, XX, t(15; 17), t(4; 7)(p11; q21), del(14)(q12q23)CR 47+L: 0·11 × 106
53520MM347, XY, t(15; 17), +8CR 44+N
54455MM346, XY, t(15; 17)IF 2L: 0·20 × 106
59856FM346, XX, t(15; 17)CR 48+L: 0·63 × 106
66721MM346, XY, t(15; 17)CR 27+N
67342MM346, XY, t(15; 17)CRY26+H: 4·20 × 106
70578FM346, XX, t(15; 17)CR 19+N
55134FM446, XXCR 9H: 0·91 × 106
58180FM446, XXIF 38+L: 0·40 × 106
58268MM443, XY, −13, −16, −20, 5q-, t(9; 22)(q34; q11), i(14q), +marED 1L: 0·82 × 106
59343FM446, XXCR 39+N
61119MM445, XY, −6, −17, −18, der(17), t(17; 18)CR 36+N
63041MM446, XYCR 35+H: 1·90 × 106
66647MM445, X, -Y, t(8; 21)CR 28+L: 0·49 × 106
67175MM446, XYCR 3H: 4·60 × 106
70266FM446, XXIF 4H: 1·43 × 106
71415MM446, XYCR 12+N
72948MM446, XY, t(8; 21)CR 7+H: 1·80 × 106
56627MM546, XYCR 40+N
64172FM547, XX, −6, 3q+, 3q-, 7p+IF 1L: 0·66 × 106
74644FM546, XXCR 12+L: 0·12 × 106
55350MM643, XY, −7, −16, −17, der(3)t(3;?)(q11;?), 5q+, 12p+IF 5H: 1·78 × 106

RNA extraction and cDNA preparation Total cellular RNA was isolated by the guanidinium isothiocyanate method (Chomczynski & Sacchi, 1987). In vitro reverse transcription of 3 µg of total cellular RNA to cDNA was performed using Moloney Murine Leukaemia Virus reverse transcriptase (Gibco-BRL, Gaithersburg, MD, USA) and random hexamers as primers (Gibco-BRL). After an initial denaturation of 5 min at 65°C, the reaction mixture was incubated at 37°C for 60 min.

As a control for the presence of amplifiable RNA, 5 µl of the reverse transcription (RT) cDNA product was amplified by polymerase chain reaction (PCR) using primers specific for the retinoic acid receptor alpha (RARα) gene (Borrow et al, 1992). Amplification consisted of an initial denaturation step of 5 min at 94°C, followed by 35 cycles of denaturation at 94°C/1min, annealing at 53°C/1 min and extension at 72°C/1 min with a final extension step of 10 min at 72°C.

Both in the RT reaction and in the ensuing amplification reactions, recommended measures to prevent cross-contamination of samples were followed (Kwok & Higuchi, 1989). In addition, for each experiment, a control with no template was used to check for the presence of any contaminant.

PCR Amplification of TfR cDNA sequences Five microlitres of the RT reaction was amplified by PCR in two separate reactions using oligonucleotides specific for exons 5–11 and 15–17 of the human TfR gene (Table II). PCR was carried out in a final volume of 100 µl with 30 pmol of each oligonucleotide primer, 200 µmol/l each of dNTP, 2·5 U Taq polymerase (Gibco-BRL) in PCR buffer (50 mmol/l KCL, 10 mmol/l Tris-HCl pH 8·0, 1·5 mmol/l MgCl2, 0·01% gelatin). Amplification consisted of an initial denaturation step of 5 min at 94°C, followed by 35 cycles of denaturation at 94°C/1min, annealing at 58°C/1 min and extension at 72°C/1 min with a final extension step of 10 min at 72°C.

Table II.   Oligonucleotides used for the analysis of TfR mRNA transcripts in the present study
5 s:5′ CAG TGG CCA ATT TCT TCT CG 3′
11as:5′ CTG CCT CTG GTG TAT CCT CAC A 3′
15 s:5′ CTG ATC CAT GGG CTA AGA ATC 3′
17as:5′ GTC TAC GGA AAG TGC AGG CTT 3′
5 sγ3: 5′ CAG TGG CCA ATT TCT TCT CGT CCT GGT TGT CTA CCC AT 3′
11asγ4: 5′ CTG CCT CTG GTG TAT CCT CAC ATT GAA GTT CTC AGG ATC 3′

Determination of the specific PCR products was carried out by direct sequencing. For the sequencing analysis, PCR specific bands were isolated and purified from 2% low melting point agarose gel and sequenced by the dideoxy-chain termination method (Sanger et al, 1977) using a modified version of T7 DNA polymerase (Sequenase 2·0, US Biochemicals, OH, USA).

Quantification of RNA Amplification was performed as described above for TfR cDNA sequences corresponding to exons 15–17. For each cDNA sample, multiple reaction tubes were prepared and increasing amounts of standard DNA were added to each reaction. A DNA standard specific for the sequences of exons 15–17 of the TfR gene was prepared using RT-PCR with oligonucleotides 15sγ3 and 17asγ4 (Table II). The standard was designed so that the 5′ and 3′ ends were compatible with the primers used to amplify the cDNA sample. However, the PCR product yielded by the standard was different in size from that produced by the cDNA sample under study (Kollia et al, 1996).

Each PCR product (10 µl) was analysed using electrophoresis on 3% low melting agarose gel, stained with ethidium bromide and photographed. An image captured with a Polaroid camera was analysed for optical density with Epson GT-8000 Laser scanner. The optic density of each band was plotted against the concentration of standard DNA.

Statistical evaluation Statistical analysis was carried out by the spss version 10·0 statistical package. The relationship between TfR mRNA expression and age was explored with the One way Analysis of Variance test and the Kruskal–Wallis test, whereas the relationship between TfR mRNA expression and French–American–British (FAB) subtype was explored using Fischer's Exact test (Lee, 1992). The effect of TfR mRNA positivity on remission, relapse and survival status was examined with the use of a Cox's Proportional Hazards Model (Stanton, 1992) so as to take into account the effect of age and FAB subtype. A significance level of 0·05 was set.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments.
  7. References

Detection of TfR mRNA transcripts

Using qualitative RT-PCR, TfR cDNA sequences were detected in 31/44 cases (70·5%). In all these cases, concordant results were obtained using RT-PCR amplification of both ‘targets’ (i.e. cDNA sequences corresponding to exons 5–11 and 15–17, respectively) (Fig 1). The remaining cases (13/44, 29·5%) were negative for TfR mRNA transcripts.

image

Figure 1.  RT-PCR analysis for TfR mRNA expression in AML patients. Lanes 1–5, RT-PCR analysis for exons 5–11; lanes 7–11, RT-PCR analysis for exons 15–17; lane 6, molecular weight marker (φX174/HaeIII); lanes 4 and 10, Samples negative for TfR mRNA expression.

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Quantification of TfR mRNA transcripts

To quantify TfR transcripts in the 31 cases that were found positive on qualitative RT-PCR, competitive RT-PCR was performed. The number of cycles used for the amplification reaction (n = 30) was chosen so that the reaction would be within the exponential phase. The amount of cDNA present in each sample was determined by identification of the concentration of standard DNA at which the ‘target’ (sample) and standard PCR bands were of equal intensity. The equivalence point of sample cDNA and standard DNA was defined by regression analysis (Cross, 1995). Each sample was tested at least twice. TfR mRNA-positive cases were classified into two groups, one with high TfR mRNA expression (850·0 × 106−6500·0 × 106 atoms ‘target’/µg RNA; 14/31 cases) and the other with intermediate–low TfR mRNA expression (110·0 × 106−850·0 × 106 atoms ‘target’/µg RNA; 17/31 cases) (Fig 2, Table I).

image

Figure 2.  Quantitative RT-PCR analysis for TfR transcripts (exons 15–17) in AML patients: (A) High TfR mRNA levels; (B) Intermediate TfR mRNA levels; (C) Low TfR mRNA levels. Upper band: competitor [at concentrations of 10−2 pg/µl (lane 2), 10−3 pg/µl (lane 3), 10−4 pg/µl (lane 4), 10−5 pg/µl (lane 5)]; lower band: target cDNA. Lane 1: molecular weight marker (φX174/HaeIII).

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Statistical analysis

Patients negative for TfR mRNA had a median age of 30 years, whereas patients with intermediate–low and high TfR mRNA levels had a median age of 58 and 53 years, respectively. This difference was statistically significant at the 0·05 level. The relationship between TfR mRNA expression and specific FAB subtypes was examined using Fischer's exact test; no significant association was detected.

In total, 31/44 patients (70·5%) achieved complete remission (CR). The number of patients achieving CR by TfR mRNA expression is given in Table III. The survival time was recorded for all 44 patients, whose condition was monitored for remission and relapse incidence. Survival status by TfR mRNA levels is shown in Table III. It can be seen that the percentage of patients surviving decreases as TfR mRNA levels increase. However, the relationship between TfR mRNA and survival status was not significant at the 0·05 level (Fisher's exact test = 0·67).

Table III.   Association of TfR mRNA expression with attainment of complete remission and survival.
 HighLowNegative
TFR levelCount%Count%Count%
Survival status
 No750·0847·1538·5
 Yes750·0952·9861·5
Complete response
 No535·7741·117·7
 Yes964·31058·91292·3
Total141001710013100

For analysis purposes, patients who were still alive at the completion of the study were considered censored observations in the survival analysis that follows. There were no patients lost to follow-up. Furthermore, no patient died from an irrelevant cause. Patients who fell into negative and low TfR mRNA groups had the highest mean and median survival, followed by patients with high TfR mRNA values. Figure 3 shows the survival functions for each group (‘negative’, ‘low’ and ‘high’), estimated from the Kaplan–Meier method. Negative and low TfR mRNA groups seem to achieve better survival rates than high TfR. However, the log-rank test, which was computed to test the differences in survival distributions of each TfR mRNA group, was not found to be significant at the level of 0·05 [log-rank test value = 4·6 with two degrees of freedom (d.f.)].

image

Figure 3.  Estimated survival functions for each group (‘negative’, ‘low’ and ‘high’) estimated from the Kaplan–Meier method.

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To further examine the relationship between TfR mRNA levels and survival time, a Cox's proportional hazards model was implemented so as to take into account the effect of age and FAB subtype. Data from living patients were regarded as censored. Each of the three variables was entered separately in the model. The effect of age was found to be significant at the level of 0·05 (chi-square = 4·0 with 1 d.f.). TfR mRNA levels were not found to be significant at the level of 0·05 (chi-square = 1·7 with 2 d.f.). FAB subtype was not found to be significant at the level of 0·05 (chi-square = 0·01 with 1 d.f.) (although the rather limited number of cases and the large – for statistical analysis – number of FAB categories has to be considered in the interpretation of the results). After adjusting for the effect of age, TfR mRNA levels and FAB subtype were still not significant at the level of 0·05 (TfR mRNA: chi-square = 1·8 with 1 d.f.; FAB: chi-square = 0·02 with 2 d.f.). Therefore, it was concluded that TfR mRNA levels were not significantly related to survival, whereas age was significantly related to the outcome.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments.
  7. References

CD71 (TfR) has been detected on dividing cells of all haematopoietic cell lines and on malignant myeloid and lymphoid cells (Testa et al, 1993). CD71 is generally expressed more strongly by proliferating cells – albeit with varying levels of expression (10 000–100 000 molecules per cell; Inoue et al, 1993); this could probably be attributed to increased demands for iron (Qian & Tang, 1995). In the largest clinical series so far, CD71 expression was detected using indirect immunofluorescence in only 140/323 (43%) childhood acute lymphoblastic leukaemia (ALL) cases (Koehler et al, 1993). Similar results have also been reported for other malignant lymphoproliferative disorders (Habeshaw et al, 1983; Medeiros et al, 1988; Das Gupta & Shah, 1990) and in AML as well (33/58 cases, 57%; Bradstock et al, 1994). Several explanations may account for the absence of CD71 from many malignant clones, the most obvious being low sensitivity of the applied methods. Alternatively, TfR protein molecules could be unstable or misfolded (Kuznetsov & Nigam, 1998), while TfR mRNA messages might be inefficiently processed or translated.

Evidently, a crucial issue arising from the aforementioned observations concerns the pattern of TfR mRNA expression in malignant haematopoietic cells. To our knowledge, the present analysis of TfR mRNA transcripts in primary AML blasts is the first study to address this issue. Interestingly, 13/44 AML cases included in our study (29·5%) were negative for TfR cDNA sequences. This is certainly an underestimate of the actual incidence, as some weakly positive RT-PCR signals may derive from rare, residual normal myeloid cells (despite the fact that BM samples analysed in the present study had at least 85% infiltration by myeloid blasts; even a modest contamination with erythroid cells may give positive results). On the other hand, the possibility of a false-negative result was effectively ruled out as all samples were tested RT-PCR-positive for the ‘control’ (RARα) transcripts (Lion & Kidd, 1998). Unfortunately, CD71 expression was assessed using flow cytometry only in 6/44 patients of our series, precluding meaningful conclusions on the fate of TfR messages in AML blasts.

Altogether, the above-mentioned observations and our results support the hypothesis that in certain AML cases alternative, TfR-independent, iron carrier-mediated pathways might be involved in the cellular uptake of iron, similar to that already described for various other cell types (intestinal mucosal cells, liver cells and transformed cultured cells) (Seligman et al, 1991; Chan et al, 1992; Qian & Tang, 1995; Richardson & Ponka, 1997; Trinder & Morgan, 1997, Lieu et al, 2001). In this context, a new TfR-like family member (transferrin receptor 2, TfR2) has been cloned recently and proposed to mediate the cellular uptake of iron, via different mechanism(s) and affinity than classic TfR (Kawabata et al, 1999). In particular, expression of TfR2 is not regulated by cellular iron status, probably because its mRNA does not contain iron-responsive elements (Kawabata et al, 2000).

The expression of activation antigens in a malignant population is related to cell proliferation; thus, it is reasonable to speculate that this expression may be correlated with tumour behaviour and treatment outcome. In this context, it has been shown that TfR is overexpressed in adriamycin-resistant K562 human erythroleukaemic cells and HL60 human myeloid cells, irrespective of P-glycoprotein expression (Barabas & Faulk, 1993). However, generally, the pattern and clinical significance of CD71 expression in haematological malignancies has not been established conclusively. In the series of childhood ALL cases mentioned above, CD71 expression appeared to have no prognostic implications (patients were treated on a single protocol) (Koehler et al, 1993). In non-Hodgkin's lymphomas (NHL), some studies have suggested that CD71 is expressed in more aggressive histological subtypes of B-cell NHL, and that it may negatively affect survival (Habeshaw et al, 1983; Medeiros et al, 1988). In contrast, other studies have reached different conclusions (Das Gupta & Shah, 1990). In a series of AML patients reported by the Australian Leukaemia Study Group 1994 (Bradstock et al, 1994), CD71 reactivity was not predictive of survival duration.

In the present study, we sought to evaluate whether TfR mRNA expression in de novo AML might be associated with various patient characteristics and with response to treatment. An absolute prerequisite for reliable molecular analysis at the mRNA level is the homogeneity of cell sample under study. For this reason, our study was confined to the analysis of BM samples from AML cases with BM blasts > 85%. To perform quantitative measurements of TfR mRNA, we developed a quantitative RT-PCR assay and correlated the intensity of expression to various patient variables and the response to treatment. Bearing in mind that the number of cases examined was not very large, the following conclusions could be drawn: (i) TfR mRNA expression was closely related to age, with younger patients more likely to be negative for TfR mRNA transcripts; (ii) TfR mRNA expression was not associated with specific FAB subtypes; and (iii) the prognostic effect of TfR mRNA expression on remission, relapse and overall survival was not found to be significant, after adjusting for age and FAB subtype. In future studies, it would also be useful to investigate possible correlations between TfR mRNA expression and iron status of the patients (this kind of analysis was not feasible in our study because of insufficient data). Nevertheless, it should not be forgotten that in the haematopoietic tissue the level of iron is not a critical determinant of TfR expression.

In conclusion, these data support the notion that high expression of TfR mRNA might simply represent a manifestation of the adverse biological profile of AML in the elderly. In this context, it is not surprising that TfR mRNA expression was not found to be an independent prognostic factor. Finally, our results point to the existence of as yet undefined mechanisms ensuring adequate supply of AML blasts with iron, which operate independently of TfR.

Acknowledgments.

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments.
  7. References

The authors wish to thank Mr Dimitris Boulamatsis, MSc, who performed the statistical analysis of the data.

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  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments.
  7. References
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