Detection of minimal residual disease (MRD) after bone marrow transplantation (BMT) by multi-parameter flow cytometry (MPFC)

Multi-parameter flow cytometry (MPFC) was used to detect minimal residual disease (MRD) following bone marrow transplantation (BMT) in 21 patients. Bone marrow (BM) was analyzed pre-transplant and 3–4 months post-BMT while the patients were in clinical and morphological remission. MRD was detected by identifying cells with aberrant antigen expression and/or leukemia-associated phenotype (LAP) using MPFC. Prior to BMT, 8 out of 21 patients exhibited normal antigen expression based on normal BM samples while 13 BM aspirates had abnormal MPFC. Pre-BMT MPFC was abnormal in all 10 patients who were not in complete remission (CR) (>5% blasts in BM) as well as 3 patients acute lymphoblastic leukemia (ALL) who were in CR. In BM from ALL patients, an abnormal uniform B cell population was observed however antigen expression patterns varied greatly between patients. BM from acute myeloblastic leukemia (AML) patients showed an abnormal distribution of CD34+ cells. In addition, a correlation was observed between pre-BMT cytogenetics and MPFC. Only 2 out of 8 (25%) patients with normal MPFC pre-autologous bone marrow transplantation (ABMT) relapsed (AML), while 6 out of 13 (46%) patients with abnormal pre-BMT MPFC relapsed including 2 out of 3 patients who were transplanted in clinical CR. Pre-BMT MPFC may thus be an effective tool for detection of MRD by detection of a pre-transplant MPFC abnormality.     

An immunomagnetic epithelial tumor cell enrichment model for minimal residual disease detection of cytokeratin 8+ malignancies

Immunocytochemical detection of isolated tumor cells in peripheral blood and bone marrow is currently the most established method for monitoring early dissemination in epithelial cancer. In this study we used an immunomagnetic selection technique to develop an enrichment model for disseminated tumor cells in blood. Buffy coat cells spiked with varying numbers of BT-474 carcinoma cells were permeabilized and fixed, following which carcinoma cells were magnetically labelled with an anti-cytokeratin 8 mAb. Labelled cells were enriched by the use of magnetic columns. The eluted cytokeratin 8+ tumor cells were detected by flow cytometry and immunocytochemistry. Spiked samples were split and processed freshly in the immunomagnetic enrichment assay, as well as cryopreserved and processed in the assay after thawing. Enumeration of BT-474 cells demonstrated a detection limit of one BT-474 cell in 1.0 x 10(7) leukocytes in both fresh and cryopreserved-thawed samples. The pair wise comparison showed a significantly higher recovery of spiked BT-474 cells from freshly processed samples than from cryopreserved and thawed samples (57% vs 21%). Viability tests suggested that this outcome might be due to a greater susceptibility of BT-474 cells than buffy coat cells to the used cryopreservation and thawing technique. Altogether our findings show that the performance of the immunomagnetic enrichment assay on fresh samples is satisfactory with a recovery rate of almost 60% and a sensitivity of 10(-7). However, performance of the assay on cryopreserved and thawed cells needs to be improved.     

Multidrug resistance (MDR) genes in haematological malignancies

The emergence of drug resistant cells is one of the main obstacles for successful chemotherapeutic treatment of haematological malignancies. Most patients initially respond to chemotherapy at the time of first clinical admission, but often relapse and become refractory to further treatment not only to the drugs used in the first treatment but also to a variety of other drugs. Laboratory investigations have now provided a cellular basis for this clinical observation of multidrug resistance (MDR). Expression of a glycoprotein (referred to as P-glycoprotein) in the membrane of cells made resistantin vitro to naturally occurring anticancer agents like anthracyclines, Vinca alkaloids and epipodophyllotoxins, has been shown to be responsible for the so-called classical MDR phenotype. P-glycoprotein functions as an ATP-dependent, unidirectional drug efflux pump with a broad substrate specificity, that effectively maintains the intracellular cytotoxic drug concentrations under a non-cytotoxic threshold value. Extensive clinical studies have shown that P-glycoprotein is expressed on virtually all types of haematological malignancies, including acute and chronic leukaemias, multiple myelomas and malignant lymphomas. Since in model systems for P-glycoprotein-mediated MDR, drug resistance may be circumvented by the addition of non-cytotoxic agents that can inhibit the outward drug pump, clinical trials have been initiated to determine if such an approach will be feasible in a clinical situation. Preliminary results suggest that some haematological malignancies, among which are acute myelocytic leukaemia, multiple myeloma and non-Hodgkin's lymphoma, might benefit from the simultaneous administration of cytotoxic drugs and P-glycoprotein inhibitors. However, randomised clinical trials are needed to evaluate the use of such resistance modifiers in the clinic.Abbreviations ALL acute lymphocytic leukaemia - AML acute myelocytic leukaemia - BM bone marrow - CAT chloramphenicol acetyltransferase - CLL chronic lymphocytic leukaemia - CML chronic myelocytic leukaemia - CR complete remission - HCL hairy cell leukaemia - MDR multidrug resistance - MDS myelodysplastic syndrome - MM multiple myeloma - MoAb monoclonal antibody - NHL non-Hodgkin's lymphoma - PB peripheral blood - PCR polymerase chain reaction - PLL prolymphocytic leukaemia - RMA resistance modifying agent - VAD vincristine, doxorubicin, dexamethasone     

Cancer Special Issue: Early detection and minimal residual disease

Beryne Odeny discusses PLOS Medicine’s Special Issue on early cancer detection and minimal residual disease.

PLOS Medicine’s editorial team, together with guest editors, Chris Abbosh, Sarah-Jane Dawson and Charles Swanton, are delighted to disseminate several high-quality translational research and clinical studies on advances in early cancer detection. In 2020 alone, there were upward of 19 million new cancer cases and 10 million cancer deaths, worldwide [1]. Cancer kills more people than HIV/AIDS, tuberculosis and malaria combined and should be a top health priority, regardless of region or country [2]. Early detection of cancer and identification of minimal residual disease (MRD), post-treatment, are key to timely treatment and cure. This issue features robust studies that bring cutting edge, and potentially scalable, innovations that have the potential to inform research, policy, and clinical cancer management.Three studies in this issue center on innovations for detection of MRD. Yaqi Wang and colleagues found that combining circulating tumor DNA (ctDNA) and Magnetic Resonance Imaging (MRI) improved prediction of response to neoadjuvant chemoradiotherapy in patients with locally advanced rectal cancer (LARC) before surgery [3]. This combined model also improved stratification of patients at high risk of recurrence, and clearly has important clinical implications for management of LARC as it could potentially inform guidelines on patient selection for non-operative management and targeted treatment strategies for those with highly recurrent diseases. Jeanne Tie and colleagues confirmed the prognostic utility of post-surgery and post-chemotherapy ctDNA in determining the risk of relapse among patients with colorectal cancer with liver metastases (CRCLM) [4]. They demonstrated the function of serial ctDNA measurement as an early marker of treatment of efficacy. This is a noteworthy advance that requires further study around optimized integration of ctDNA analyses in adjuvant chemotherapy for resectable CRCLM. Pradeep S. Chauhan and colleagues applied next-generation sequencing (NGS) for urine tumor DNA (utDNA) detection to assess MRD in patients with muscle-invasive bladder cancer who received neoadjuvant chemotherapy [5]. They found that MRD detection prior to radical cystectomy correlated with pathologic response and may be used to identify candidates for bladder sparing treatment. Urine tumor DNA also offers the ability to determine tumor mutational burden and can therefore facilitate personalized immunotherapy.Two studies in this issue focused on early cancer detection. Jeffrey J. Szymanski and colleagues investigated the use of plasma cell-free DNA (cfDNA) ultra-low-pass whole genome sequencing (ULP-WSG) to distinguish the malignant peripheral nerve sheath tumor (MPNST) from its benign precursor lesion–plexiform neurofibroma–in patients with Neurofibromatosis type1(NF1) [6]. This provides a strong evidence base for use of plasma cfDNA in liquid biopsy to distinguish early between benign and malignant tumors of this hereditary cancer. This is proof of concept that cfDNA can be leveraged as a biomarker for monitoring treatment response in patients with MPNST. Brian D Nicholson and colleagues demonstrated that risk scores based on combinations of risk factors and routine blood tests can be used to stratify patients with unexpected weight loss based on their risk of cancer [7]. They found that these combined risk scores showed superior clinical utility–compared to the symptoms-only model–to discriminate between patients with and without cancer. In this, they clearly demonstrate innovation in the use of routine clinical tools at scale. This type of model could potentially be scaled-up in under-resourced settings.With growing global interest in cancer diagnostics and treatment, these robust assays and tools are a welcome addition to the early cancer detection armamentarium, prior to and post-treatment. Further innovation around low-cost technologies and tools for early detection that can be rapidly tested and scaled up will further galvanize, the universal commitment to defeat cancer in both high and low resource settings.     

A new method for high speed, sensitive detection of minimal residual disease

Investigations of rare cell types in peripheral blood samples, such as tumor, fetal, and endothelial cells, represent an emerging field with several potentially valuable medical applications. Peripheral blood is a particularly attractive body fluid for the detection of rare cells as its collection is minimally invasive and can be repeated throughout the course of the disease. Because the number of rare cells in mononuclear cells can be very low (1 in 10 million), a large number of cells must be quickly screened, which places demanding requirements on the screening technology. While enrichment technology has shown promise in managing metastatic disease, enrichment can cause distortions of cell morphology that limit pathological identification, and the enrichment targeting adds additional constraints that can affect sensitivity. Here, we describe a new approach for detecting rare leukemia cells that does not require prior enrichment. We have developed an immunocytochemical assay for identification of leukemia cells spiked in peripheral blood samples, and a high-speed scanning instrument with high numerical aperture and wide field of view to efficiently locate these cells in large sample sizes. A multiplex immunoassay with four biomarkers was used to uniquely identify the rare cells from leukocytes and labeling artifacts. The cytometer preserves the cell morphology and accurately locates labeled rare cells for subsequent high resolution imaging. The sensitivity and specificity of the approach show promise for detection of a low number of leukemia cells in blood (1 in 10 million nucleated cells). The method enables rapid location of rare circulating cells (25 M cells/min), no specific enrichment step, and excellent imaging of cellular morphology with multiple immunofluorescent markers. The cell imaging is comparable to other imaging approaches such as laser scan cytometry and image flow cytometry, but the cell analysis rate is many orders of magnitude faster making this approach practical for detection of rare cells.     

Treatment outcomes for childhood acute lymphoblastic leukemia in low-middle income country before minimal residual disease risk stratification

BackgroundOutcome of childhood acute lymphoblastic leukemia (ALL) in low- and middle-income countries is lagging in many aspects including diagnosis, risk stratification, access to treatment and supportive care.Objectiveto report the outcome of childhood ALL at Ain Shams University Children’s Hospitals with the use of risk-based protocols before the implementation of minimal residual disease technology and to evaluate the use of double delayed intensification (DDI) in standard risk patients.MethodsTwo hundred and twenty patients with ALL diagnosed between January 2005 and December 2014 were included in the study. Patients were treated according to a modified CCG 1991 and 1961 for standard and high risk respectively. Patients were stratified into three risk groups: standard risk (SR), high-risk standard arm (HR-SA), and high-risk augmented arm (HR-AA).ResultsAmong the whole cohort, the 10-year event-free survival (EFS) and overall survival (OS) were 78.1% and 84.3% respectively. Patients with Pre-B immunophenotype (IPT) had significantly better outcome than T-cell IPT (EFS 82.0% versus 58.6%, p < 0.001; OS 86.9% versus 69%, p = 0.003 for Pre-B and T-cell respectively). Among the SR group, patients treated with single delayed intensification (SDI) had comparable EFS and OS rates when compared to patients treated with DDI with EFS 82.4% versus 87.5%, p = 0.825 and OS 88.2% versus 93.5%, p = 0.638 for SDI and DDI groups, respectively.ConclusionThe use of risk-based protocol with simple laboratory techniques resulted in acceptable survival outcome in resource limited settings. The use of double delayed intensification showed no survival advantage in patients with standard risk.     

Genes of multidrug resistance in haematological malignancies

Since the early 1970s, multiple drug resistance has been known to exist in cancer cells and is thought to be attributable to a membrane-bound, energy-dependent pump protein (P-glycoprotein [P-gp]) capable of extruding various related and unrelated chemotherapeutic drugs. The development of refractory disease in haematological malignancies is frequently associated with the expression of one or several multidrug resistance (MDR) genes. MDR1, multidrug resistance-associated protein (MRP) and lung-resistance protein (LRP) have been identified as important adverse prognostic factors. Recently it has become possible to reverse clinical MDR by blocking P-gp-mediated drug efflux. The potential relevance of these reversal agents of MDR as well as the potential new approaches to treat the refractory disease are discussed in this article. In addition, an array of different molecules and mechanisms by which resistant cells can escape the cytotoxic effect of anticancer drugs has now been identified. These molecules and mechanisms include apoptosis-related proteins and drug inactivation enzymes. Resistance to chemotherapy is believed to cause treatment failure in more than 50% patients. Clearly, if drug resistance could be overcome, the impact on survival would be highly significant. This review focuses on molecular mechanism of drug resistance in haematological malignancies with emphasis on molecules involved in MDR. In addition, it brings the survey of methods involved in determination of MDR, in particular P-gp/MDR1, MRP and LRP.     

Detection and significance of minimal residual disease in colorectal cancer

Colorectal cancer (CRC) is one of the most common causes of cancer death in the developed world. Although the primary treatment for CRC is surgical, disease relapse due to minimal residual disease (MRD) following apparently curative surgery occurs in up to fifty percent of patients. Most patients who develop overt metastases beyond the regional lymph nodes eventually die of the disease. At present adjuvant chemotherapy is used to improve survival in patients with metastases to regional lymph nodes demonstrated by routine histopathology with no other evidence of spread. The ability to identify metastatic disease at an earlier stage could be of considerable benefit in directing adjuvant therapy to patients at high risk of relapse who are not identified by current methods. Several techniques have been developed for the detection of MRD, including immunohistochemical and molecular methods, however their role in clinical practise is not yet established. The purpose of this paper is to review these techniques and their potential clinical use in the management of CRC.     

Use of nanocolonies to detect minimal residual disease in patients with leukemia t(8;12)

Molecular Biology - A complete diagnostic procedure was developed to detect single molecules of the AML1-ETO mRNA in whole blood and bone marrow samples of leukemia t(8;21)(q22;q22) patients. The...     

Hormonal changes in patients with haematological malignancies

Levels of immunologically reactive insulin (IRI), growth hormone (GH), thyroxine, cortisol and ACTH in sera of patients with various haematological malignancies were determined. IRI and GH levels were increased in 80% of the patients regardless of the type of disease. IRI was more elevated in relapse than in remission. Cytostatic treatment returned IRI serum levels to normal. Thyroxine, ACTH and cortisol in serum were higher in only two percent of the patient with haematological malignancies.     

Detection of minimal residual disease in acute leukemia by flow cytometry.

Patients with acute leukemia in clinical remission may still have up to 10(10) residual malignant cells (the upper limit of detection by standard morphologic techniques). Sensitive techniques to detect minimal residual disease (MRD) may allow better estimates of the leukemia burden and help the selection of appropriate therapeutic strategies. Flow cytometry and polymerase chain reaction have emerged as the most promising methods for detecting submicrospopic levels of leukemia. Flowcytometric detection of MRD is based on the identification of immunophenotypic combinations expressed on leukemic cells but not on normal hematopoietic cells. It affords the detection of one leukemic cell among 10,000 normal bone marrow cells, and can be currently applied to at least two thirds of all patients with acute leukemia. Prospective studies in large series of patients have demonstrated a strong correlation between MRD levels during clinical remission and treatment outcome. Therefore, MRD assays can be reliably used to assess early response to treatment and predict relapse. In this review, we discuss methodologic aspects and clinical results of flowcytometric detection of MRD in patients with acute leukemia.     

The role of CCN family genes in haematological malignancies

Haematological malignancies, although a broad range of specific disease types, continue to show considerable overlap in classification, and patients are treated using similar chemotherapy regimes. In this review we look at the role of the CCN family of matricellular proteins and indicate their role in nine haematological malignancies including both myeloid and lymphoid neoplasms. The potential for further haematological neoplasms with CCN family associations is argued by summarising the demonstrated role of CCN family genes in the differentiation of haematopoietic stem cells (HSC) and mesenchymal stem cells. The expanding field of knowledge encompassing CCN family genes and cancers of the HSC-lineage highlights the importance of extracellular matrix-interactions in both normal physiology and tumorigenesis of the blood, bone marrow and lymph nodes.     

Epigenetic inactivation of the hsa-miR-203 in haematological malignancies

miR-203 is a tumour suppressor microRNA (miRNA). We studied the methylation of hsa-miR-203 in 150 samples including acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL), chronic myeloid leukaemia (CML), chronic lymphocytic leukaemia (CLL) and non-Hodgkin's lymphoma (NHL) by methylation-specific PCR, and miRNA expression by stem-loop RT-qPCR. hsa-miR-203 promoter was unmethylated in normal controls but homozygously methylated in two AML and four lymphoma cell lines, in which 5-Aza-2'-deoxycytidine treatment led to promoter demethylation and miR-203 re-expression. Restoration of miR-203 expression in lymphoma cells inhibited cellular proliferation and increased cell death, suggesting an inherent tumour suppressor activity. In primary samples, hsa-miR-203 methylation was absent in CML but detected in 5.0% ALL, 10.0% AML, 42.0% CLL and 38.8% of NHL (including six [60.0%] natural killer-cell, nine [40.9%] B-cell and four [23.5%] T cell NHL). Moreover, hsa-miR-203 methylation was associated with hypermethylation of hsa-miR-34a, -124a and -196b in NHL but not CLL. In CLL, hsa-miR-203 methylation was associated with a higher presenting Hb level (P = 0.033). The projected 10 year overall survival of the CLL patients was 58.2%, which was impacted by Rai stage and high-risk karyotypes but not hsa-miR-203 methylation. hsa-miR-203 was more frequently methylated in lymphoid than myeloid malignancies (P = 0.002). In conclusion, miR-203, a tumour suppressor gene, was hypermethylated in a tumour-specific manner with gene silencing. hsa-miR-203 was more frequently hypermethylated in lymphoid than myeloid malignancies. In NHL, hsa-miR-203 methylation was associated with concomitant methylation of other tumour suppressor miRNAs. The frequent hsa-miR-203 methylation in lymphoid malignancies suggested a pathogenetic role of hsa-miR-203 methylation.     

Immunodiagnosis of childhood malignancies

Immunodiagnosis utilizing immunohistochemical techniques is currently the most commonly utilized and readily available method of ancillary diagnosis in pediatric oncopathology. The methodology comprises relatively simple steps, based on straightforward biologic concepts, and the reagents used are generally well characterized and widely used. The principle of cancer immunodiagnosis is based on the determination of neoplastic lineage using detection of proteins typical of cell differentiation pathways. Methodology sensitivity varies and has become greater with each new generation of tests, but technical drawbacks should be considered to avoid excessive background or nonspecific results. Automated instrumentation offers a degree of accuracy and reproducibility not easily attainable by manual methods.