Establishing optimal quantitative-polymerase chain reaction assays for routine diagnosis and tracking of minimal residual disease in JAK2-V617F-associated myeloproliferative neoplasms: a joint European LeukemiaNet/MPN&MPNr-EuroNet (COST action BM0902) study.

Reliable detection of JAK2-V617F is critical for accurate diagnosis of myeloproliferative neoplasms (MPNs); in addition, sensitive mutation-specific assays can be applied to monitor disease response. However, there has been no consistent approach to JAK2-V617F detection, with assays varying markedly in performance, affecting clinical utility. Therefore, we established a network of 12 laboratories from seven countries to systematically evaluate nine different DNA-based quantitative PCR (qPCR) assays, including those in widespread clinical use. Seven quality control rounds involving over 21,500 qPCR reactions were undertaken using centrally distributed cell line dilutions and plasmid controls. The two best-performing assays were tested on normal blood samples (n=100) to evaluate assay specificity, followed by analysis of serial samples from 28 patients transplanted for JAK2-V617F-positive disease. The most sensitive assay, which performed consistently across a range of qPCR platforms, predicted outcome following transplant, with the mutant allele detected a median of 22 weeks (range 6-85 weeks) before relapse. Four of seven patients achieved molecular remission following donor lymphocyte infusion, indicative of a graft vs MPN effect. This study has established a robust, reliable assay for sensitive JAK2-V617F detection, suitable for assessing response in clinical trials, predicting outcome and guiding management of patients undergoing allogeneic transplant.

Routine EGFR and KRAS Mutation analysis using COLD-PCR in non-small cell lung cancer.

Aims: Epidermal growth factor receptor (EGFR) antagonists are particularly active in non-small cell lung cancer (NSCLC) patients with tumours bearing mutations in the EFGR gene. EGFR mutation prevalence is very low in squamous histology. Response rates using these drugs in patients with KRAS mutations are low, so available KRAS mutation information may aid treatment selection in the second-line setting. Since 2009, patients presenting to this hospital with non-squamous histology have been routinely screened for mutations in both the EGFR and KRAS genes, with results used to inform treatment. We present an analysis of 215 consecutive patients for whom EGFR mutation analysis was informative. Methodology: EGFR and KRAS mutations were identified using a COLD-PCR technique confirmed with sequencing, which makes no prior assumption about location of specific mutations. Results were correlated with clinical and demographic data from hospital records, where available. Results: The prevalence of patients with EGFR mutations was 14% and for KRAS mutations it was 27%. Despite the conventional understanding that EGFR and KRAS mutations are mutually exclusive, we identified two dual mutations. Of 29 patients identified with mutated EGFR, there were 3/8/8/10 mutations in exons 18/19/20/21 respectively. Exon 20 mutations were identified in a proportion exceeding many other series because of the unbiased mutation analysis used, and clinical benefit was seen in some of these. Of 23 different EGFR mutations identified, 11 have not previously been described in the literature. Conclusions: The high prevalence of EGFR, KRAS or both mutations (40%) in this non-squamous population tested in clinical practice supports a policy of routine screening for these mutations in NSCLC.

Development of standardized approaches to reporting of minimal residual disease data using a reporting software package designed within the European LeukemiaNet.

Quantitative PCR (qPCR) for detection of fusion transcripts and overexpressed genes is a promising tool for following minimal residual disease (MRD) in patients with hematological malignancies. Its widespread clinical use has to some extent been hampered by differences in data analysis and presentation that complicate multicenter clinical trials. To address these issues, we designed a highly flexible MRD-reporting software program, in which data from various qPCR platforms can be imported, processed, and presented in a uniform manner to generate intuitively understandable reports. The software was tested in a two-step quality control (QC) study; the first step involved eight centers, whose previous experience with the software ranged from none to extensive. The participants received cDNA from consecutive samples from a BCR-ABL+ chronic myeloid leukemia (CML) patient and an acute myeloid leukemia (AML) patient with both CBFß-MYH11 and WT1 target genes, they conducted qPCR on their respective hardware platforms and generated a series of reports with pre-defined features. In step two, five centers used the software to report BCR-ABL+ MRD in a harmonized manner, applying their recently obtained CML international scale conversion factors. The QC study demonstrated that this MRD-reporting software is suitable for efficient handling of qPCR data, generation of MRD reports and harmonization of MRD data.

Elevated levels of WT1 transcripts in bone marrow harvests are associated with a high relapse risk in patients autografted for acute myeloid leukaemia.

Relapse postautograft in acute myeloid leukaemia (AML), may in part arise from leukaemia cells present in the bone marrow (BM) inoculum, and the level of minimal residual disease (MRD) in BM harvests used for autografting may therefore be clinically important. We have used the WT1 transcript as a marker of MRD, which was quantitated by RQ-PCR, in the BM harvests of 24 patients receiving an ABMT for AML. ABL was used as a control gene with WT1 level being normalised to 10(5) copies of ABL per sample. Median WT1 level was 651 copies (range=113-32 700) for the 13 patients with relapse-free survival (RFS) of less than 5 years, and 174 (range=0-1900) for patients with RFS of over 5 years postautograft (P<0.04). The RFS was 10.5 months for patients with WT1 level of >2000 copies (n=5), and has not yet been reached for patients with WT1 level<2000 (n=21), at a median follow-up of 92 months (P<0.05). We show that elevated levels of MRD in BM harvests are associated with a higher relapse risk in patients autografted for AML.

Monitoring minimal residual disease and predicting relapse in APL by quantitating PML-RARalpha transcripts with a sensitive competitive RT-PCR method.

Qualitative RT-PCR methods used for monitoring minimal residual disease (MRD) in APL patients fail to predict relapse in up to 25% of patients in remission. We report here the development and evaluation of a highly sensitive (10(-5) and 10(-6) with one round and two rounds of PCR, respectively) competitive RT-PCR method to quantitate the PML-RARalpha fusion transcripts. PML-RARalpha transcript's levels were normalised to 10(5) copies of ABL transcript. Serial BM and PB samples from 16 patients with APL and t(15;17) were examined. Presentation samples from three patients (three BM, one PB) showed levels in the range of 0.7 x 10(6)-3.5 x 10(6) and 1.2 x 10(5) molecules in BM and PB samples respectively. Serial quantitation of MRD in both BM and PB samples showed significantly lower levels of PML-RARalpha transcripts in remission, although the majority of samples remain positive for the PML-RARalpha transcripts even those in long-term remission (up to 94 months). Levels of PML-RARalpha in remission samples were up to 2 x 10(2) and up to 5.2 x 10(1) molecules in BM and PB respectively. BM and PB samples taken from two patients 2-4 months before relapse showed significantly higher levels of PML-RARalpha transcripts (1.2 x 10(4) molecules in BM; 3.5 x 102, 1.2 x 10(2) and 1.2 x 10(3) in PB). The same samples, when tested with a standard qualitative RT-PCR for the amplification of PML-RARalpha (with a sensitivity of 10(-4)) produced negative results. This indicates that the qualitative methods would not have predicted relapse in these patients. Our data show that quantitating PML-RARalpha transcripts with a sensitive method may provide a superior approach for monitoring MRD in APL and identifying patients at high risk of relapse.

Molecular quantitation of minimal residual disease in acute myeloid leukemia with t(8;21) can identify patients in durable remission and predict clinical relapse.

One of the most common translocations in acute myeloid leukemia (AML) is the t(8;21), which produces the fusion gene AML1-MTG8. We have developed a sensitive competitive reverse transcriptase-polymerase chain reaction (RT-PCR) assay for AML1-MTG8 transcripts, coupled with a competitive RT-PCR for the ABL transcript as a control to accurately estimate the level of amplifiable RNA. We have shown that AML1-MTG8 and ABL transcripts have equal degradation rates. Thus, this method is useful for multicenter studies. We studied 25 patients with t(8;21) AML by means of serial analysis done on bone marrow (BM) and peripheral blood (PB) samples from 21 patients. Our analysis showed that, in general, a successful induction chemotherapy produces a reduction of 2 to 3 log in the level of AML1-MTG8, followed by a further 2 to 3 log after consolidation/intensification chemotherapy. Levels up to 1 x 10(3) and 1 x 10(2) molecules/microg of RNA in BM and PB, respectively, were compatible with durable remission. On the other hand, 5 patients with levels of 0.71 x 10(5) to 2.27 x 10(5) molecules/microg of RNA in BM and 2.27 x 10(3) to 2.27 x 10(4) molecules/microg of RNA in PB had hematologic relapse within 3 to 6 months. Our data indicate that serial quantitation of AML1-MTG8 transcripts is useful in identifying patients at high risk of relapse and may offer an opportunity for clinical intervention to prevent hematologic relapse. This approach was applied successfully in a patient who had an allogeneic BM transplantation. We also suggest that PB may be used an alternative to BM for quantitating AML1-MTG8 transcripts.

RT-PCR method with increased sensitivity shows persistence of PML-RARA fusion transcripts in patients in long-term remission of APL.

RT-PCR methods have been developed, to date, by various groups to amplify the PML-RARA fusion gene produced by the t(15;17) in APL patients. However, these methods lack the necessary sensitivity to detect minimal residual disease (MRD) below the level of 1 leukaemic cell in 10(4) cells. Patients who test positive by these methods after treatment are likely to relapse. However, up to 25% of patients who test negative after treatment relapse within a short period. We have developed a 'hot-start' RT-PCR method for the amplification of PML-RARA with increased sensitivity at the level of two leukaemic cells in 10(6) cells. Using this method we were able to detect MRD in seven out of 15 patients tested in remission. Of the 11 patients in medium to long-term remission, five patients tested positive. None of these 11 patients tested positive with the standard RT-PCR. These results show that some patients in remission of APL continue to express PML-RARA even in long-term remission, when they can be considered clinically 'cured' of their disease.

Molecular monitoring of minimal residual disease in acute myeloblastic leukemia with t(8;21) by RT-PCR.

The t(8;21) is one of the most common translocations in acute myeloid leukaemia (AML) occurring in approximately 20% of adult and 40% of paediatric AML-M2. This translocation fuses the AML1 gene on chromosome 21q to the MTG8 (ETO) gene on chromosome 8q to produce the fusion gene AML1-MTG8. Transcripts for the AML1-MTG8 fusion gene have been detected in the majority of patients in remission by qualitative RT-PCR methods. Thus for such patients these methods are unsuitable for monitoring minimal residual disease (MRD). Furthermore, the diverse form of transcripts for this fusion gene was found in patients at different phases of their disease, which rules out the usefulness of the expression of any particular set of transcripts as a marker for monitoring MRD in those patients. On the other hand a quantitative RT-PCR method we developed, was able to assess the effectiveness of treatment and predict relapse up to four months before the onset of haematological relapse. This method should distinguish patients in stable remission from those at high risk of relapse and therefore identify patients who would require additional or new treatment such as BMT.

Detection and quantitation of the CBFbeta/MYH11 transcripts associated with the inv(16) in presentation and follow-up samples from patients with AML.

We have developed a competitor-based RT-PCR technique which will detect and quantitate the CBFbeta/MYH11 transcripts associated with inv(16)(q22;p13) and have used it to study presentation and follow-up samples of acute myeloid leukaemia (AML). The levels of the leukaemia-specific transcripts are expressed as a ratio to a ubiquitously expressed mRNA species (Abl) which controls for RNA degradation. This technique has been applied to 75 consecutive patients presenting with either de novo AML or tMDS; 6/75 patients analysed were positive for the inv(16), all were confirmed by conventional cytogenetics. The inv(16) has a strong association with M4Eo, but we found only 2/6-positive patients to have this diagnosis (two patients with M2, one patient M1 and one patient had MDS). At presentation the levels of CBFbeta/MYH11 transcripts were 0.1-10/Abl transcript (mean 3.3/Abl transcript). Seventeen follow-up samples were available on 5/6 of these patients, and on two further patients in whom stored material was available. Following the first cycle of chemotherapy the level of transcripts was at least 10(-2) lower (0.1-10 x 10(-2)/abl transcript) than their presentation sample. Subsequent samples on these patients when in remission gave transcript levels in the range (1.0 x 10(-4) - 2 x 10(-3)/abl transcript), and three long-term follow-up samples were negative. We have developed a quantitative test which opens the possibility of predicting relapse by detecting changes in the numbers of leukaemia-specific transcripts.

Expression of AML1/MTG8 transcripts in clonogenic cells grown from bone marrow of patients in remission of acute myeloid leukaemia with t(8;21).

Patients in long-term remission of acute myeloid leukaemia (AML) M2 with t(8;21) after chemotherapy, with or without bone marrow transplantation, are known to retain residual cells which express AML1/MTG8 transcripts in bone marrow, detectable by RT-PCR. In order to determine whether these residual cells are clonogenic, we have grown remission bone marrow samples in standard semi-solid culture and picked individual CFU-GM and BFU-E colonies which were then analysed for the expression of AML1/MTG8 transcripts using a rapid specific RT-PCR technique. Nine patients were tested in remission, six between 1 and 83 months post chemotherapy, one 103 months post autologous bone marrow transplant and one 41 months post allogeneic bone marrow transplant. One of these patients also had quantitation of AML1/MTG8 transcripts on five occasions after recovery from each course of chemotherapy and at the end of treatment. There was a significant correlation between the percentage of positive colonies and the level of AML1/MTG8 transcripts. Between two and 80 CFU-GM and between two and 21 BFU-E colonies were analysed from each patient sample: 0-23% CFU-GM and 0-17% BFU-E colonies were found to express AML1/MTG8 transcripts suggesting that these residual cells are clonogenic in vitro and that the cell of origin is a multipotent myeloid progenitor.

Monitoring of minimal residual disease by quantitative reverse transcriptase-polymerase chain reaction for AML1-MTG8 transcripts in AML-M2 with t(8; 21).

We have developed a quantitative reverse transcriptase-polymerase chain reaction method for the quantitation of AML1-MTG8 transcripts in patients with AML-M2 and t(8;21) in different phases of the disease. Using this method, we have tested sequential samples from 13 patients to monitor minimal residual disease and were able to show a significant increase in AML1-MTG8 transcripts level in two patients 2 and 4 months before clinical relapse. In five patients tested at presentation and then sequentially at remission, we detected a marked decrease in the level of AML1-MTG8 transcripts as the treatment progressed. Patients in long-term remission of their disease had a level of up to 1 x 10(3) AML1-MTG8 molecules/microgram RNA. Two patients tested 2 and 4 months before hematologic relapse showed a level of 0.71 x 10(5) molecules/microgram RNA and this level increased further during relapse to 0.71 x 10(7) and 2.27 x 10(5) molecules/microgram RNA, respectively. Our results show that quantitation of AML1-MTG8 transcripts by competitive polymerase chain reaction is valuable in predicting early relapse in AML with t(8;21). Identification of at-risk patients may allow treatment to be modified to include additional or alternative therapy such as bone marrow transplantation.

Expression of diverse AML1/MTG8 transcripts is a consistent feature in acute myeloid leukemia with t(8;21) irrespective of disease phase.

The (8;21) chromosomal translocation occurs in 20% of adult patients with AML M2. This translocation interrupts two genes, AML1 on chromosome 21q and MTG8 (ETO) on 8q to form a chimeric gene AML1/MTG8 on the der(8) chromosome. Recent reports have shown the presence of diverse forms of transcript for this chimeric gene. Three alternative out-of-frame transcripts have been previously demonstrated (types II, III, IV) all of which have a stop codon 3' of the runt box encoding a truncated runt polypeptide. We have characterized a novel transcript (V) which is in-frame and has a stop codon 3' to the runt box. We have examined transcript diversity in 10 AML patients with t(8;21) in remission of their disease following chemotherapy or bone marrow transplantation. Specific transcripts detected at presentation in six patients were similarly expressed during remission and at relapse in two patients; thus expression of transcript diversity was unaffected by the disease phase. Alternative transcripts were unhelpful as a marker of remission quality or predictor of relapse. The significance of these diverse transcripts in leukemogenesis remains unknown.

Detection of CBFB/MYH11 transcripts in patients with inversion and other abnormalities of chromosome 16 at presentation and remission.

The pericentric inversion of chromosome 16 [inv(16)(p13q22)] and t(16;16)(p13;q22) are chromosomal rearrangements frequently associated with AML FAB type M4Eo resulting in the production of a fusion gene CBFB/MYH11. We studied 17 patients with a chromosome 16 abnormality (eight M4Eo, two M1, one M2, three M4 without abnormal eosinophils, three MDS) for the presence of CBFB/MYH11 transcripts using an RT-PCR technique. 10 AML patients with inv(16) tested RT-PCR positive (eight at presentation, one in remission, one in remission and relapse). Three of these patients were originally reported by cytogenetic analysis to have del(16q22) but the positive RT-PCR results prompted a cytogenetic re-examination, resulting in the correction of the reports to inv(16). We show that although inv(16) is closely associated with AML M4Eo, it can also be detected in cases of AML M4 without abnormal eosinophils. Three cases of MDS with inv(16) were also RT-PCR positive. Four patients with other chromosome 16 abnormalities were RT-PCR negative. Four AML patients with inv(16) were studied in remission. All were RT-PCR positive, including one patient in remission for 108 months and one 22 months post allogeneic bone marrow transplant. In the latter two remission patients, RT-PCR evaluation was positive in bone marrow (BM) but not in peripheral blood, suggesting that BM may be the more informative. We conclude that this technique is valuable in the accurate molecular classification of AML, particularly as treatment options may be influenced by such information. Though RT-PCR is highly sensitive in detecting CBFB/MYH11 fusion transcripts during remission, monitoring of minimal residual disease in patients with inv(16) remains to be established.

Persistence of RAR alpha-PML fusion mRNA detected by reverse transcriptase polymerase chain reaction in patients in long-term remission of acute promyelocytic leukaemia.

Acute promyelocytic leukaemia (APL) is characterized by t(15;17), which results in the formation of two chimaeric genes, PML-RAR alpha and RAR alpha-PML. PML-RAR alpha transcripts have been detected in all cases of APL whilst those of RAR alpha-PML have been detected in only about 67% of cases. We have used reverse transcriptase polymerase chain reaction (RT-PCR) to detect both fusion transcripts serially in 18 patients in remission of APL after chemotherapy and bone marrow transplantation. All patients were negative for PML-RAR alpha, whereas in six patients (remission 3-9 years) RAR alpha-PML was consistently detected. Only one patient at remission showed the 5' breakpoint RAR alpha-PML, with the rest showing the 3' breakpoint 144 bp RAR alpha-PML. The level of sensitivity for detecting RAR alpha-PML was some 10-fold higher than that for PML-RAR alpha. Serial negative tests for PML-RAR alpha have been correlated with durable remissions, suggesting possible eradication of residual leukaemia in APL. Our results, however, show persistence of t(15;17) cells expressing RAR alpha-PML fusion mRNA in patients in long-term remission of APL. They indicate that patients considered clinically 'cured' of APL still have molecular evidence of minimal residual disease and also provide further insight into the biology of acute myeloid leukaemia.

Detection of t(8;21) by reverse transcriptase polymerase chain reaction in patients in remission of acute myeloid leukaemia type M2 after chemotherapy or bone marrow transplantation.

Seven patients with acute myeloid leukaemia (AML) type M2 and t(8;21), treated with intensive chemotherapy followed in two cases by allogeneic or autologous bone marrow transplant (BMT), were tested for the presence of transcripts of the characteristic chimaeric gene AML1/ETO by reverse transcriptase polymerase chain reaction (RT-PCR) at serial intervals during remission. Six patients, including both BMT patients, demonstrated persistence of t(8;21), one being consistently negative in peripheral blood but bone marrow positive. Bone marrow may, therefore, be more reliable than peripheral blood for detecting residual t(8;21) cells. Our results show persistence of t(8;21) cells in AML-M2 patients following chemotherapy or BMT, some of whom were in long-term remission.

The diagnosis of delayed post-operative endophthalmitis by polymerase chain reaction of bacterial DNA in vitreous samples.

Delayed post-operative endophthalmitis is a complication of modern cataract extraction and posterior chamber lens implantation. Propionibacterium acnes has been isolated in a few such cases but the majority are culture-negative, compounding surgical and medical management decisions. A method of detecting bacterial, and specifically P. acnes, DNA by the polymerase chain reaction (PCR) directed at 16S rDNA is reported. Nested PCR with universal eubacterial primers complimentary to regions of 16S rDNA conserved sequences detected 50 fg of bacterial DNA spike in normal vitreous. Nested PCR with P. acnes primers detected 10 fg of DNA. Vitreous samples from 29 patients undergoing vitrectomy for reasons unrelated to infection and 23 samples from 19 patients with delayed post-operative endophthalmitis were analysed. Four (14%) of 29 normal individuals and 17 (74%) of 23 delayed cases gave positive results with universal eubacterial primers. None of 29 and eight of 23 samples gave positive results with P. acnes primers. The 14% positive rate with universal primers in non-infected cases may limit their use in delayed post-operative endophthalmitis. PCR detection of bacterial DNA with specific primers from vitreous samples may prove a useful means of diagnosing delayed post-operative endophthalmitis and facilitating management decisions when conventional bacterial culture is negative.

Detection of melanocytes from uveal melanoma in peripheral blood using the polymerase chain reaction.

PURPOSE: Uveal melanoma is the most common intraocular malignancy in adults and can cause loss of vision in the affected eye and death from metastasis, usually to the liver. The techniques currently used to detect cellular dissemination from the tumor are inadequate, and lack the sensitivity required for the detection of low levels of melanocytes in the peripheral blood of patients. The detection of circulating melanocytes is important as an early indication of the possibility of metastasis. METHODS: The viability of reverse transcription/polymerase chain reaction amplification of the tyrosinase gene to detect circulating melanocytes was examined as a first sign of dissemination from uveal melanoma. RESULTS: It was shown that it is possible to detect as few as ten circulating melanocytes in 5 ml of blood. Blood-borne dissemination was also detected in three of six patients with uveal melanoma examined. Two of these patients had clinically confirmed widespread metastases. A positive result was also recorded in one patient in whom there was no other evidence for tumor dissemination. Overt metastatic disease developed in this patient 9 months after blood collection. CONCLUSIONS: The success of this technique has important implications for the detection of circulating tumor cells from uveal melanoma, as an early indication of dissemination. This may be important when considering the administration of adjuvant therapy.