Residual Disease In Peripheral Blood Research Articles

Reshaping MRD Detection Strategies: Next-Generation Sequencing and High DNA Input Identify Previously Missed Measurable Residual Disease in Peripheral Blood of B-Cell Precursor Acute Lmyphoblastic Leukemia

Background: In acute lymphoblastic leukemia (ALL), measurable residual disease (MRD) is the most important prognostic factor. MRD is traditionally quantified in bone marrow aspirates (BM) using real-time quantitative polymerase chain reaction (RQ-PCR) according to EuroMRD standards. Since the emergence of modern molecular methods, it has been a continuous effort to enable MRD assessment from peripheral blood (PB). The less invasive material collection would allow closer MRD monitoring and solve technical challenges of BM aspiration, such as skewed or non-representative MRD values due to hemodilution and unequal distribution of leukemic cells. While several studies demonstrated comparable MRD values of BM and PB in T-ALL, in B-cell precursor ALL (BCP-ALL) MRD determination from PB is still a challenge. Mean MRD levels in PB are considerably lower than in BM and frequently escape detection. Additionally, paired samples show a highly variable MRD level ratio in both pediatric and adult cases (Kotrova et al, Leukemia 2020). Objective: BCP-ALL cases with known MRD positivity in the BM likely also harbor MRD positive cells in the PB, even when missed by traditional methods. With more sensitive assays, either by increased DNA input or by application of amplicon-based next generation sequencing (NGS), MRD detection from PB may improve in these cases. Methods: We retrospectively selected PB - BM sample pairs of 74 BCP-ALL patients with BM MRD positivity (43 with quantifiable MRD positivity and 31 with positivity below quantitative range) and sufficient leftover DNA from a cohort of patients treated according to GMALL protocols, where conventional RQ-PCR of clonal immunoglobuline heavy chain (IGH) VJ/DJ gene rearrangements failed to detect PB MRD. MRD had been determined by standard RQ-PCR with 1.5 µg DNA in BM and PB, respectively, according to the EuroMRD guidelines (van der Velden et al, Leukemia 2007). PB samples were re-tested for MRD with high DNA input RQ-PCR using 6.6 µg DNA and amplicon-based NGS, each using 6.6 µg DNA, as well as standard input NGS. NGS analyses of corresponding PB and BM samples were performed with the EuroClonality amplicon-based NGS assay (Brüggemann et al, Leukemia 2019). Identification and quantification of leukemia-derived clonotypes was done using ARResT/Interrogate. Results: In total, 74 BCP-ALL patients with pre/c-B-ALL (n=60) and pro-B-ALL (n=14) were included. Patient age ranged from 19 - 71 years with a median of 44.5. In all included samples (56 in first-line therapy and 18 salvage treatment), BM was MRD positive and PB was MRD negative in standard RQ-PCR at a sensitivity of at least 10 -4. To assess the effect of high DNA input alone, RQ-PCR was repeated in PB with high DNA input for 59 patients and 63 IGH VJ/DJ markers. At least one positive signal was detected in 19/59 patients (32%) and 20/63 markers (32%) (FIGURE 1A). NGS was performed for 73 PB samples and 84 markers using standard and high DNA input. With standard DNA input, MRD could be detected for 19/73 patients (26%) and 21/84 markers (25%) (FIGURE 1A). In the high input approach, MRD was detected in 36/73 patients (49%) and 40/84 markers (48%) (FIGURE 1A). A quantifiable MRD value was identified in 17 patients and 19 markers. All markers with quantifiable NGS MRD positivity in PB showed higher MRD values in the corresponding BM sample and MRD values below 10 -4 (FIGURE 1B). No correlation of MRD in paired samples could be demonstrated (Pearson correlation coefficient: 0.09) (FIGURE 1B). Conclusion: In BCP-ALL, MRD detection from PB is challenging with conventional methods. Increased DNA input and amplicon-based NGS allowed detection of MRD in a subset of PB samples that were negative in standard RQ-PCR. With identical DNA input, NGS showed a higher sensitivity than RQ-PCR and best results were achieved when high DNA input and NGS were combined, identifying MRD in nearly half of the PB samples. These results demonstrate that PB can be a valuable tool for early MRD detection in BCP-ALL when adjusting traditional MRD assessment strategies. However, in half of the samples, MRD was still missed by high input NGS. Even with assays improved for sensitivity, PB therefore cannot fully replace BM for MRD assessment in BCP-ALL yet and should rather be considered to complement BM analyses or in cases where BM is not accessible.

Measurable residual disease in peripheral blood in myeloma: dream or reality.

Therapeutic advancements in multiple myeloma have led to increasingly deeper and more durable responses, creating a need for highly sensitive and applicable techniques for measurable residual disease (MRD) assessment. Bone marrow assays can deeply assess for MRD, but it is not conducive to performing frequent and dynamic evaluations, which may be needed for MRD-adapted treatment approaches. Recently, numerous techniques for MRD assessment in peripheral blood have come under investigation, and their integration into routine clinical practice is eagerly anticipated. The identification of circulating tumor cells (CTCs), evaluation of cell-free DNA, and measuring monoclonal protein concentration with mass spectrometry are promising research areas for assessing myeloma in peripheral blood. CTCs assessment and cell-free DNA may carry prognostic significance, but they lack the sensitivity of bone marrow-based techniques. Mass spectrometry has already been implemented in clinical practice in certain centers, but its full potential has yet to be fully realized. This review focuses on recent developments in these fields, emphasizing the potential future roles of these assessments. MRD assessment in peripheral blood is still in the development stage but holds promise for not only complementing bone marrow based evaluations but also potential for improving sensitivity.

Safety and Efficacy of Third Generation CD20 Targeted CAR-T (MB-106) for Treatment of Relapsed/Refractory B-NHL and CLL

Safety and Efficacy of Third Generation CD20 Targeted CAR-T (MB-106) for Treatment of Relapsed/Refractory B-NHL and CLL

Tafasitamab combined with idelalisib or venetoclax in patients with CLL previously treated with a BTK inhibitor

Patients with relapsed/refractory chronic lymphocytic leukemia (R/R CLL) whose treatment failed with a Bruton’s tyrosine kinase inhibitor have poor outcomes. We investigated tafasitamab plus idelalisib (cohort A) or venetoclax (cohort B) in this patient population in a phase II study (NCT02639910). In total, 24 patients were enrolled (cohort A: n = 11, median time on study, 7.4 months; cohort B: n = 13, median time on study, 15.6 months). The most common treatment-emergent adverse event (TEAE) in cohort A was anemia (63.6%) and in cohort B was infusion-related reaction (53.8%). The most common severe TEAE was neutropenia (cohort A: 45.5%; cohort B: 46.2%). The best overall response rate was 90.9% (cohort A) and 76.9% (cohort B). Undetectable minimal residual disease in peripheral blood was achieved in 2/8 patients (cohort A) and 6/7 patients (cohort B). Overall, these results suggest that anti-CD19 antibody-based combinations may be important in the treatment of patients with CLL.

Residual methylation of tumor suppressor gene promoters, RASSF6 and RASSF10, as novel biomarkers for minimal residual disease detection in adult acute lymphoblastic leukemia

Aberrant promoter methylation of RASSF6 and RASSF10 occurs at a high frequency in acute lymphoblastic leukemia (ALL). Because of the complexity of the current minimal residual disease (MRD) detecting-methods, the DNA methylation status of the RASSF6 and RASSF10 genes could potentially become biomarkers for the assessment of MRD levels in ALL patients. The promoter methylation status of RASSF6 and RASSF10 was assessed by using methylation-specific PCR (MSP) in the DNA isolated from 280 peripheral blood (PB) samples taken at the time of diagnosis, day 14, 28, and from the subsequent 30-month follow-ups of 45 adult ALL patients. The relative methylation level obtained during the follow-ups by MSP was compared to the MRD results obtained by the amplification of IG/TCR clonal rearrangements using the allele-specific quantitative-PCR (ASO-PCR) assay. Frequently, RASSF6 was methylated in B-ALL, and RASSF10 was methylated in T-ALL. The applicability and accuracy of the assays were increased when these markers were combined (91.1% and 93.8%, respectively). When a cutoff was defined for the PCR-MRD level, results of the 30months of MRD detection showed a significant correlation between the PCR and MSP techniques (r = 0.848; p < 0.001). Due to the high applicability, the non-invasiveness, and promising prospect of longitudinal assessment, the DNA methylation status of the RASSF6 and RASSF10 genes could be potential biomarkers for the assessment of residual disease in PB of patients with ALL.

Using MALDI-TOF mass spectrometry for tracking of minimal residual disease in peripheral blood from patients with multiple myeloma.

e19525 Background: Minimal residual disease (MRD) negativity after completed therapy is associated with longer progression-free survival (PFS) in patients with multiple myeloma (MM). Current standard of care for MRD testing use flow cytometry and/or next generation sequencing (NGS)-based assays applied on bone marrow (BM) aspirate samples. To develop a strategy for MRD tracking in peripheral blood (PB), we were motivated to evaluate MALDI-TOF head-to-head with established bone marrow-based MRD assays. Methods: We used MALDI-TOF mass spectrometry to detect M-proteins in PB. Our cohort included patients who had serum samples available at 2 timepoints including during active disease and within 60 days of MRD results as determined by flow cytometry of BM aspirates. The cohort enrolled 71 patients (26 females, 45 males) with a median age of 61 years (37-78 years). Twenty-seven patients had high-risk cytogenetics at baseline. Patients were classified at diagnosis as ISS1 (n = 38), ISS2 (n = 18) or ISS3 (n = 6). The flow cytometry based MRD assay was performed using MSKCCs 10-color, single-tube method. MALDI-TOF analysis was performed as described by Mills et al. Samples taken during active disease were used to identify the mass/charge ratio of the M-protein at baseline and in follow-up samples. MALDI-TOF results were compared to flow cytometry bone marrow-based MRD results. Results: The median time between diagnosis and the MRD timepoint was 13.4 months (3.4-91 months). MALDI-TOF in PB and flow cytometry BM-based MRD results were concordant for 44/71 (62%) patients (8+/+, 36 -/- respectively) while 27 were discordant (10 +/-, 17-/+). Fifty-four of 71 patients were in complete response (CR) (45/54 in sCR) at the time of MRD. MALDI-TOF was still positive in 13 of these 54 CR patients. In this cohort, the median PFS since MRD assessment was not reached in the 2 subgroups of double negative patients (n = 31) or in patients with a positive result in at least one technique (n = 23) with a median follow-up of 11.2 months (0-34.6 months). Conclusions: In 44/71 (62%) samples, MALDI-TOF of PB results and flow cytometry BM-based MRD results were concordant. MALDI-TOF of PB may be useful for detecting measurable residual disease and for the monitoring of MM patients during maintenance therapy with the future goal to rule out early recurrent disease.

Comparative Value of the Assessment of Minimal Residual Disease in Peripheral Blood at Days 8 and 15 By Flow Cytometry in Childhood Acute Lymphoblastic Leukemia

IntroductionAcute lymphoblastic leukemia (ALL) is the most frequent cancer in childhood, but treatments' progress now allowsto obtain prolonged remission or curein over 90% of the patients. Consequently, therapeutic de-escalation is now an objective for future treatment protocols, providing that biomarkers allow to reliablyidentifygood responders. Among such indicators, low levels of Minimal Residual Disease (MRD) obtained early after induction chemotherapy stand out as good candidates. The latter can be investigated usingmultiparameterflow cytometry (MFC) or real-time polymerase chain reaction (RT-PCR) for immunoglobulins or T-cell receptors (IG TCR) rearrangements.In this study we report the impact on survival of two early points of peripheral blood (PB) MRD assessment by MFC at days 8 and 15 on a cohort of 125 children with B-ALL enrolled in the French FRALLE trial and compared to molecular MRD in the bone marrow (BM) at day 35.Patients and methods.The study enrolled 67 boys and 58 girls and the duration of the study allowed for a median follow up of 52,1months. Median age at diagnosis was 57 months old (range 18 to 196), 101 children were between 1 to 10 years old and 24 were older than 10.Complete blood counts (CBC) at diagnosis showed a median of 6.7x109/L leucocytes (range 0.47 - 151x109/L) and 33% blasts (range 0 to 97%). One hundred and eight children had less than 50x109/L leucocytes while 17 had higher counts. EGIL classification at diagnosis allowed to classify patients as three B-I, 94 B-II, 27 B-III and 1 B-IV. Cytogenetic analyses were performed for 118 patients who were partitioned as follows: low risk n=47, intermediate risk n=55 and high risk n=16 (Harrisson CJ et al., BJH, 2010). Eighty-three patients were in the low risk group and 42 in the high-risk group as described by the FRALLE protocol. Seven patients of the 64 tested had an IKZF1 deletion. During the duration of the study, 20 patients relapsed and 8 died.Corticosensitivitywas defined by less than 1x109/L PB blasts on day 8 andchemosensitivity by less than 5% BM blasts on day 21 on BM smears.PB MRD was assessed in MFC with a single five or ten colors tube adapted to each patient's leukemia associatedimmunophenotypeon a backbone of CD45, CD19, CD10 and CD38.Statistical analyzes examined factors impacting disease-free survival (DFS) using Log rank test and Kaplan-Meier using theMedcalc® software (Ostend, Belgium). P values <0.05 were considered significant.ResultsNone of diagnosis features had any significant impact on DFS: age (p=0,95), risk group (p=0,17), EGIL classification (p=0,55), cytogenetics (p= 0,87), leucocyte count (p=0,36) nor IKZF1 deletion (p=0,2). Of the 125 patients, 9 were corticoresistant, 79 corticosensitive and 37 not evaluable because of less than 1x109/L leucocyte at diagnosis.Corticosensitivity had no impact on DFS (p=0,11). Conversely,chemosensitivity had a significant positive impact on DFS (p= 0,009).Day 8 PB MRD did not oultlineany significantly different DFS, whether considering detectable vs undetectable MRD (p=0.65) or MRD levels (logwisefrom >10-1 to <10-4, p=0,22).Conversely, PB MFC at day 15 appeared highly discriminant. Considering notdetectablevs detectable MRD, 4 years DFS was 91,6+3% vs. 67,6+9% p=0,0013 (Figure 1). Further refining the thresholds of MRD logwisedid not modify the significance (p=0.004; Figure 2). Indeed, DFS at 48 months was 61+15 % (n=16) for MRD >10-3, 74+11% ( n=18) for MRD <10-3->10-4 and 92+3% ( n=91) for MRD<10-4.Comparison of PB MFC MRD on day 15 with day 35 BM molecular MRD showed concordance in 72% of the cases (83 negative/negative and 7 positive/positive, 48 months DFS 94.6+2.7% and 38+20% respectively). Eight patients were negative in PB but positive in BM (DFS 62.5+17%).Twenty seven where positive in PB but negative in BM (DFS 83.5+7.6%).These differences were statistically highly significant (p <0.0001).ConclusionThis study demonstrates that even in the good prognosis context of childhood ALL, early MRD retains a highly significant prognostic value. It is of importance that this result was obtained not only on day 35 BM but interestingly, even earlier on day 15 PB. This less invasive procedure can easily be applied, especially for children. It should allow to detectgood responders, with MFC MRD levels below 10-4 for whom a de-escalation of chemotherapy could be considered. Conversely, the detection of blasts by MFC in day 15 PB is worrisome. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Clinical significance of minimal residual disease in peripheral blood on day 8 induction in childhood B-precursor acute lymphoblastic leukemia: Report from Children’s Cancer Hospital in Egypt.

e21008 Background: Minimal residual disease (MRD) in childhood acute lymphoblastic leukemia (ALL) during the early stages of therapy is statistically significant indicator of outcome. Aim: To study...

Estimation of molecular upper remission limit for monitoring minimal residual disease in peripheral blood of acute myeloid leukemia patients by WT1 expression

To date, approximately one half of acute myeloid leukaemia (AML) patients do not have a suitable specific molecular marker for monitoring minimal residual disease (MRD). The Wilm's tumour gene (WT1) has been suggested as a possible molecular marker of MRD in AML. The expression of WT1 in peripheral blood (PB) was measured using quantitative real-time reverse transcription-polymerase chain reaction in peripheral leukocytes from 151 patients with AML at diagnosis. WT1 expression was significantly elevated, i.e. up to 3 orders of magnitude in the majority (80%) of AML patients at diagnosis compared to the PB of healthy donors. Sequence samples of the long-term followed-up AML patients treated with chemotherapy and/or allogeneic bone marrow transplantation were analysed for WT1 expression. The results revealed that the hematological relapses were preceded (median, 1.8 months) by an increase in WT1 gene expression. For the practical utility of this gene as a molecular marker of relapse, it was necessary to determine an upper remission limit, crossing which would signal hematological relapse. The upper remission limit was determined in our set of patients to be 0.02 WT1/ABL. The AML patients who consequently relapsed crossed this upper remission limit; however, those in permanent remission did not. Therefore, this upper remission limit could be taken as the border of molecular relapse of AML patients. Moreover, insufficient decline of WT1 expression under the upper remission limit following induction and/or consolidation therapy was associated with markedly high risk of relapse. The results show that our upper remission limit can be taken as the border of molecular relapse of AML patients and WT1 levels following initial therapy as a beneficial prognostic marker.

Minimal residual disease in peripheral blood at day 15 identifies a subgroup of childhood B-cell precursor acute lymphoblastic leukemia with superior prognosis

Most minimal residual disease-directed treatment interventions in current treatment protocols for acute lymphoblastic leukemia are based on bone marrow testing, which is a consequence of previous studies showing the superiority of bone marrow over peripheral blood as an investigational material. Those studies typically did not explore the prognostic impact of peripheral blood involvement and lacked samples from very early time points of induction. In this study, we employed real-time quantitative polymerase chain reaction analysis to examine minimal residual disease in 398 pairs of blood and bone marrow follow-up samples taken from 95 children with B-cell precursor acute lymphoblastic leukemia treated with the ALL IC-BFM 2002 protocol. We confirmed the previously published poor correlation between minimal residual disease in blood and marrow at early treatment time points, with levels in bone marrow being higher than in blood in most samples (median 7.9-fold, range 0.04-8,293-fold). A greater involvement of peripheral blood at diagnosis was associated with a higher white blood cell count at diagnosis (P=0.003) and with enlargement of the spleen (P=0.0004) and liver (P=0.05). At day 15, a level of minimal residual disease in blood lower than 10(-4) was associated with an excellent 5-year relapse-free survival in 78 investigated patients (100% versus 69 ± 7%; P=0.0003). Subgroups defined by the level of minimal residual disease in blood at day 15 (high-risk: ≥ 10(-2), intermediate-risk: <10(-2) and ≥ 10(-4), standard-risk: <10(-4)) partially correlated with bone marrow-based stratification described previously, but the risk groups did not match completely. No other time point analyses were predictive of outcome in peripheral blood, except for a weak association at day 8. Minimal residual disease in peripheral blood at day 15 identified a large group of patients with an excellent prognosis and added prognostic information to the risk stratification based on minimal residual disease at day 33 and week 12.

Increase in Minimal Residual Disease in Peripheral Blood before Clinical Relapse in Dogs with Lymphoma that Achieved Complete Remission after Chemotherapy

We developed previously a minimal residual disease (MRD) monitoring system in dogs with lymphoma by exploring a highly sensitive real-time PCR system. To identify the change in MRD before clinical relapse in dogs with lymphoma that achieved complete remission after chemotherapy. Twenty dogs with multicentric high-grade B-cell lymphoma. MRD levels in peripheral blood mononuclear cells (PBMCs) were measured by real-time PCR amplifying the rearranged immunoglobulin heavy chain gene. MRD measurement and clinical assessment were performed every 2-4 weeks for 28-601 days after completion of chemotherapy. An increase in MRD was defined as an increase by more than 0.5, calculated by log₁₀ [copy number of MRD per 10⁵ PBMCs], based on the uncertainty level observed in a canine lymphoma cell line. During the follow-up period, 15 dogs relapsed in 28-320 days (median, 120 days) after completion of chemotherapy. An increase in MRD was detected 2 weeks or more before relapse in 14 of the 15 dogs, but an increase in MRD before relapse could not be detected in the remaining 1 dog. The time from increased MRD to clinical relapse was 0-63 days (median, 42 days). In contrast, no increase in MRD was detected in 5 dogs that did not experience clinical relapse. An increase in MRD can be detected before clinical relapse in dogs with lymphoma. Application of early reinduction therapy based on an increase in MRD before clinical relapse may improve treatment outcome in canine lymphoma.

Small number of HTLV-1-positive cells frequently remains during complete remission after allogeneic hematopoietic stem cell transplantation that are heterogeneous in origin among cases with adult T-cell leukemia/lymphoma

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) can provide long-term remission for patients with adult T-cell leukemia/lymphoma (ATLL) caused by human retrovirus, human T-lymphocyte virus (HTLV-1). To understand how HTLV-1-positive cells including ATLL cells were suppressed by allo-HSCT, we examined HTLV-1 provirus load and residual ATLL cells in peripheral blood of transplant recipients using PCR-based tests. We found that the copy number of HTLV-1 genome, called provirus, became very small in number after allo-HSCT; however, in most cases, provirus did not disappear even among long-term survivors. Tumor-specific PCR tests demonstrated that most of HTLV-1-positive cells that remained long after transplantation were not primary ATLL cells but donor-derived HTLV-1-positive cells. We also found a case having very low amount of residual disease in peripheral blood even long after transplantation. There was only one recipient in whom we failed to show the presence of HTLV-1 genome and antibody against HTLV-1 even with an extensive search, which strongly suggested the elimination of HTLV-1 after allo-HSCT. These results demonstrated that after allo-HSCT the small amount of residual HTLV-1-positive cells were heterogeneous in origin and that long-term disease control for ATLL could be obtained without the complete elimination of HTLV-1.

MELANOMA: A Multidisciplinary Approach for the General Surgeon

Advances in the understanding of the biology and treatment of melanoma have moved the care of melanoma patients into an increasingly multidisciplinary environment. Surgeons must understand these advances because they will often be responsible for directing the overall care of these patients. Most patients with melanomas more than 1 mm in diameter and no evidence of metastatic disease should be offered SLNB to more accurately stage them and direct decisions about participation in postoperative adjuvant therapy trials. Until the results of the MSLT are known, the effect of SLNB and ELND on outcome remains unknown. SLNs should be analyzed with serial sectioning and immunohistochemistry to avoid missing micro-metastatic disease. Based on the results of the ECOG-1684 trial, the FDA approved IFN-alpha 2b for the adjuvant treatment of melanoma patients with thick primary tumors (> 4 mm) or resected nodal disease. IFN-alpha 2b treatment is expensive and potentially toxic. The data from ECOG-1684 do not support the use of IFN-alpha 2b in patients with node-negative disease. In light of the ECOG-1690 trial results, the role of high-dose IFN-alpha 2b in the management of patients with resected nodal disease is considerably less clear. Any recommendations for treatment with high-dose IFN-alpha 2b should be made only after weighing the costs, side effects, and potential benefits for individual patients. Numerous, less toxic, promising, adjuvant immunotherapeutic strategies have been developed and are being tested in multicenter, prospective, randomized trials. These strategies include GMK, PMCV, and Melacine. If the results of any of these trials show a survival advantage compared with placebo or equivalent survival compared with IFN-alpha 2b, these immunotherapeutic agents will become the adjuvant treatment of choice for patients with resected high-risk melanoma. RT-PCR detection of tyrosinase in SLNs can identify patients with submicroscopic nodal disease who may be at increased risk for recurrence or death from melanoma. An ongoing, prospective, randomized trial will determine whether patients with histologically negative but RT-PCR-positive SLNs will benefit from lymphadenectomy or adjuvant IFN-alpha 2b therapy. RT-PCR can also identify minimal residual disease in peripheral blood and bone marrow from patients with high-risk melanoma, but RT-PCR analysis of peripheral blood and bone marrow is still experimental, and procedural details need to be standardized and prospectively validated in large patient groups before its use can be considered the standard of care.

Monitoring minimal residual disease in peripheral blood in B-lineage acute lymphoblastic leukaemia.

The use of peripheral blood rather than marrow has potential advantages for monitoring minimal residual disease during the treatment of leukaemia. To determine the feasibility of using blood, we used a sensitive polymerase chain reaction method to quantify leukaemia in the blood and marrow in 35 paired samples from 15 children during induction treatment. Leukaemic cells in the blood ranged from 1.1 x 10(-2) to < 9.4 x 10(-7) leukaemic cells/total cells, corresponding to 1.3 x 10(7) to < 2 x 10(3) leukaemic cells/l. In 15 paired samples, leukaemia could be quantified in both tissues and in 20 paired samples, leukaemia was not detected in one or both tissues so that only upper level limits could be set. In the former 15 pairs, the level of leukaemia in peripheral blood was directly proportional to that in marrow but was a mean of 11.7-fold lower. Leukaemia in blood was detected in 10/12 pairs in which the level in marrow was > 10(-4), but in only two of 13 pairs in which the level in marrow was < 10(-5). Patients studied at multiple time-points showed parallel declines in the number of leukaemic cells in both tissues. The results showed that leukaemia could be monitored in peripheral blood during induction therapy, and quantitative considerations based on the results suggest that monitoring of blood during post-induction therapy may be of value in detecting molecular relapse.

Selection of a panel of monoclonal antibodies for monitoring residual disease in peripheral blood and bone marrow of interferon‐treated hairy cell leukaemia patients

A panel of monoclonal antibodies (mAbs) directed against B-cell and hairy cell leukaemia (HCL)-associated antigens was used to identify residual hairy cells in the peripheral blood and/or bone marrow samples from 20 patients with HCL, following treatment with interferon-alpha (IFN-alpha) or interferon-beta (IFN-beta). In all cases, hairy cells retained their characteristic phenotype, e.g. positivity for CD22, CD11c, CD25, CD32, and the HCL-associated trimeric protein (t-GP) recognized by the mAbs HML-1, B-ly7, LF61 and Ber-Act8. The most specific marker for identifying a small percentage of hairy cells in peripheral blood cytospins, was t-GP. In alkaline phosphatase/anti alkaline phosphatase (APAAP) stained preparations, t-GP+ hairy cells (provided with large cytoplasm and hairy surface) could be usually distinguished from t-GP+ normal lymphocytes (small-sized cells with smooth surface). In doubtful cases the percentage of residual hairy cells could exactly be estimated by double immunofluorescence staining for CD22 (B-cell marker) and t-GP. The rationale of the test is based on the finding that the small percentage (about 1%) of t-GP+ lymphocytes circulating in the peripheral blood of normal individuals are T-cells of the CD8 subset and not B-cells. The best markers for identifying residual hairy cells in routine bone marrow biopsies were CD45RA (mAb 4KB5) and CD20 (mAb L26). Immunohistological labelling was superior to morphological examination in picking up scattered hairy cells in bone marrow biopsies showing either severe hypoplasia or exuberant hyperplasia of normal haemopoietic series.