[Annual progress of interventional diagnosis and treatment of tuberculosis in 2023].

A series of studies on the interventional diagnosis and treatment of tuberculosis(TB)were carried out by domestic and foreign researchers in 2023. The combination of minimally invasive interventional procedures with endoscopes, guidance, material acquisition techniques by multiple ways and multichannel and highly accurate laboratory testing techniques is becoming more and more widely practiced clinically, which has played an important role in the accurate diagnosis of problematic TB. Diagnostic procedures for pulmonary TB, tracheobronchial TB, mediastinal lymphatic TB and extrapulmonary TB included conventional flexible bronchoscopy and specific types of bronchoscopy(ultrathin bronchoscopy and endobronchial ultrasound), transbronchial needle aspiration biopsy, endobronchial ultrasound and virtual bronchoscopic navigation system-guided forceps biopsy, thoracoscopic cryobiopsy of pleura, percutaneous biopsy, and so on. The time to diagnosis has been significantly reduced and the diagnostic efficacy has been improved by the clinical specimen detection using either Gene Xpert MTB/RIF, Ultra, loop-mediated isothermal amplification, metagenomic next-generation sequencing, or nanopore sequencing, etc. Interventional therapy was focused on the following diseases: pulmonary TB with massive hemoptysis, tracheobronchial TB, pleural TB and TB-related fistulas. Interventional treatment of tracheobronchial TB mainly included the application of rigid bronchoscopy, bronchoscopic cold and thermal ablation treatment, endoscopic clamp, dilatations of narrow airway with balloon and stent placement, etc. The interventional treatment of pulmonary TB complicated by massive hemoptysis included endovascular embolization, coated stent placement, etc. Interventional treatment of pleural TB involved the application of thoracoscopy, endoscopic forceps, the implantation of stent and other occlusive devices and the closure of fistulas with autologous fat transplantation. In this article, we reviewed the progress of interventional diagnosis and treatment of TB by the search of published literatures from October 2022 to September 2023.

[Application of liver venous deprivation in secondary hepatic resection of primary liver cancer].

Objective: To investigate the efficacy and safety of liver venous deprivation (LVD) before secondary resection of primary liver cancer. Methods: 56 patients with advanced primary liver cancer who were not suitable for primary resection in Liver Surgery Department of Xinxiang Central Hospital from January 2018 to January 2019 were analyzed retrospectively. They were divided into liver vein deprivation group (LVD group: LVD+ PVE, n=26) and portal vein embolization group (PVE group, n=30). The dynamic changes of liver reserve function and future liver remnant volume (FLR-V), R0 resection rate, surgical complications, postoperative recurrence rate and overall survival rate of two groups before and after LVD/PVE were compared. Results: The success rate of puncture and embolization in LVD group and PVE group was 100%. There were no grade Ⅳ complications, and there was no significant difference of grades Ⅰ, Ⅱ and Ⅲ complications between the groups (P=0.808). The FLR-V of LVD group before embolization, 7, 14 and 21 days after embolization was (493.1±25.8), (673.2±56.1), (779.5±81.6) and (853.3±85.2) cm(3), respectively. The FLR-V of PVE group before embolization, 7, 14 and 21 days after embolization were (502.4±20.1), (688.6±43.9), (656.8±73.7) and (563.5±69.1) cm(3), respectively. There was no significant difference in FLR-V between the two groups before and 7 days after embolization (P>0.05). The FLR-V of LVD group was higher than that of PVE group at 14 and 21 days after embolization (P<0.01). The preparation time of LVD group was (20.4±6.3) days, which was shorter than that of PVE group [(31.5±8.8) days, P=0.045]. The rate of secondary hepatectomy was 92.3% (24/26), which was higher than that of PVE group [70.0% (21/30), P=0.036]. The R0 resection rate was 87.5% (21/24), which was higher than that of the PVE group [57.1% (12/21), P=0.022]. However, there were no significant differences in surgical methods, operation time, intraoperative blood loss, Clavien-Dindo complication grade and length of hospital stay between the two groups (P>0.05). After hepatectomy, the median recurrence time and median survival time of LVD group were 12.6 months and 21.3 months, respectively, which were longer than those of PVE group (9.4 months and 13.5 months, respectively, P<0.01). Conclusions: For patients with advanced liver cancer who are not suitable for primary hepatectomy, preoperative LVD can significantly increase FLR-V, improve the resection rate of secondary surgery, shorten the preparation time of two operations, and do not increase surgical complications. Moreover, patients with LVD can improve the R0 resection rate of secondary surgery. The postoperative recurrence time and overall survival rate of patients with LVD are better than those of patients with PVE, and LVD has a good long-term effect.

LncRNA SNHG16 functions as an oncogene by sponging miR-200a-3p in pancreatic cancer.

Since this article has been suspected of research misconduct and the corresponding authors did not respond to our request to prove originality of data and figures, "LncRNA SNHG16 functions as an oncogene by sponging miR-200a-3p in pancreatic cancer, by J.-Q. Guo, Z.-J. Yang, S. Wang, Z.-Z. Wu, L.-L. Yin, D.-C. Wang, published in Eur Rev Med Pharmacol Sci 2020; 24 (4): 1718-1724-DOI: 10.26355/eurrev_202002_20347-PMID: 32141539" has been withdrawn. The Publisher apologizes for any inconvenience this may cause. https://www.europeanreview.org/article/20347.

LncRNA SNHG16 functions as an oncogene by sponging miR-200a-3p in pancreatic cancer.

OBJECTIVE: Recently, the role of long noncoding RNAs (lncRNAs) is vital in tumor progression. Our study aims to identify the role of SNHG16 in the metastasis of pancreatic carcinoma. PATIENTS AND METHODS: Real-Time quantitative Polymerase Chain Reaction (RT-qPCR) was used to measure SNHG16 expression in 56 pancreatic carcinoma patients' tissues. Function assays, including wound healing assay, and transwell assay, were conducted to detect the effect of SNHG16 on the metastasis of pancreatic carcinoma. Besides, the luciferase assay was performed to explore the underlying mechanism. RESULTS: The expression level of SNHG16 was upregulated in pancreatic carcinoma samples compared with adjacent tissues. Moreover, cell migration and cell invasion were repressed via the knockdown of SNHG16, while cell migration and cell invasion were promoted via the overexpression of SNHG16. Moreover, the expression of miR-200a-3p was upregulated via knockdown of SNHG16 while the expression of miR-200a-3p was downregulated via the upregulation of SNHG16 in vitro. Furthermore, it was discovered that SNHG16 acted as a competing endogenous RNA via sponging miR-200a-3p in pancreatic carcinoma. CONCLUSIONS: Our study suggests that SNHG16 acts as an oncogene in pancreatic carcinoma and promotes cell metastasis via sponging miR-200a-3p, which might be a novel therapeutic strategy in pancreatic carcinoma.

Development of a blocking immunoperoxidase monolayer assay for differentiation between pseudorabies virus-infected and vaccinated animalss.

Pseudorabies (PR) outbreaks have devastated many swine farms in several parts of China since late 2011. The outbreak-associated pseudorabies virus (PRV) variant strains exhibited some typical amino acid changes in glycoprotein E (gE), a diagnostic antigen used for discriminating between PRV-infected and vaccinated animals (DIVA). To counteract the potential impact of epitope variations on current serological diagnostics of PRV, we produced monoclonal antibodies (mAbs) against gE protein of one representative PRV variant strain and developed a blocking immunoperoxidase monolayer assay (b-IPMA) for DIVA. The b-IPMA was based on the inhibition of binding between PRV-infected cells and mAb by PRV-specific antibodies present in clinical swine sera and was validated by comparison with a commercial PRV gpI Antibody Test Kit (IDEXX Laboratories, USA). The diagnostic sensitivity, diagnostic specificity and agreement were determined to be 99.25%, 98.18% and 99.02% respectively upon testing 509 serum samples. b-IPMA detected only PRV-specific antibodies and showed no cross- -reactivity with antibodies elicited by gE-deleted vaccine or other common swine pathogens. Thus, b-IPMA has the potential to be used for high-throughput screening of PRV-infected animals in veterinary clinics.

[Correlation between single nucleotide polymorphisms of rs4778137 located in OCA2 gene and clinical response of breast cancer patients receiving neoadjuvant chemotherapy].

Objective: To evaluate the correlation between single nucleotide polymorphisms (SNPs) of rs4778137 located in OCA2 gene and clinical response of breast cancer patients receiving neoadjuvant chemotherapy. Methods: A total of 140 breast cancer patients receiving neoadjuvant chemotherapy were enrolled to detect DNA in blood sample by DNA extraction kit and the rs4778137 polymorphism by sequenom. The relationship between SNPs of rs4778137 and pathologic complete response (pCR) were analyzed. Results: The frequency of CC, GC and GG genetype of rs4778137 was 48.6%, 31.4% and 20.0%,respectively. Thirty cases (21.4%) achieved pCR with CC allele in 9 cases(13.2%),GC allele in 10 cases (22.7%) and GG allele in 11 cases (39.3%),respectively,with a statistically significant difference(P<0.05). When conducting stratified analysis in accordance with the estrogen receptor (ER) status,only in ER negative group pCR was significantly associated with SNPs of rs4778137 (P<0.05). SNPs of Rs4778137, targeted therapy,subtypes,tumor stage were independent predictors of pCR in multivariate logistic regression analysis (P<0.05),and SNPs of rs4778137 was an independent predictors of pCR in ER negative group (P<0.05), but not in ER positive group group (P>0.05). Conclusion: SNPs of rs4778137 was associated with pCR only in ER negative patients receiving neoadjuvant therapy, and breast cancer patients with the GG allele were more likely to achieve pCR.

[Killing effect of icotinib combined with CIK on human lung adenocarcinoma cells in vitro].

Objective: To explore the inhibitory effect of icotinib combined with cytokine induced killer (CIK) on various human lung adenocarcinoma cell lines in vitro. Methods: The inhibitory effect of icotinib alone or icotinib combined with CIK on HCC827 and A549 cells was detected by cell counting kit-8(CCK-8). The apoptosis was detected by flow cytometry via Annexin V/PI staining. The effect of icotinib on CIK phenotype was detected by flow cytometry. Results: The inhibitory rates of HCC827 cells treated with 1.5, 3, 6, 12 µmol/L icotinib were (5.64±0.05)%, (8.62±0.45)%, (14.57±0.65)% and (18.52±0.91)%, respectively. The inhibitory rates of A549 cells were (1.64±0.48)%, (2.09±0.28)%, (3.69±0.45)%, (4.41±0.58)%, respectively. At the same concentration, the inhibitory rate of HCC827 cells with icotinib treatment was significantly higher than that of A549 cells (P<0.05). When the effector/target ratio was 10∶1, 20∶1 or 40∶1, the inhibitory rates of HCC827 cells co-cultured with CIK were (15.17±2.33)%, (42.59±7.18)%, (62.59±8.95)%, respectively, and the inhibitory rates of A549 were(16.99±2.81)%, (46.31±1.89)%, (58.24±4.23)%, respectively. The inhibitory rate of HCC827 cells co-cultured with CIK was not significantly different from that of A549 cells at the same effector/target ratio (P(10∶1)=0.299, P(20∶1)=0.318, P(40∶1)=0.366). When the effector/target ratio of CIK combined with 6 µmol/L icotinib was 10∶1, 20∶1 or 40∶1, the inhibitory rates of HCC827 cells were (37.07±3.50)%, (76.03±6.55)%, (80.34±10.69)%, respectively, and the inhibitory rates of A549 cells were(25.72±1.41)%, (52.76±3.82)%, (62.26±1.94)%, respectively. The inhibitory rates of 6 µmol/L icotinib combined with CIK were significantly higher than those of icotinib group and CIK group alone at the same effector/target ratio (P<0.05), except for the effector/target ratio at 40︰1 on A549 cells (P=0.089). Moreover, all of the combination index (CI) of combined group were <1 (P<0.05). The apoptotic rates of HCC827 and A549 cells induced by icotinib combined with CIK were significantly higher than those of icotinib group and blank control group (P<0.05), especially the proportion of late apoptotic or necrotic cells.Increasing effector/target ratio of CIK contributed to stronger inhibition(P<0.05). The expressional rate of CIK phenotype with or without icotinib treatment was not significantly different from each other(P>0.05). Conclusions: EGFR mutant lung adenocarcinoma cells are more sensitive to icotinib, while the EGFR mutation status has no effect on the killing effect of CIK cells. icotinib combined with CIK has a synergistic effect on the inhibition of tumor growth, and icotinib has no any impact on the phenotype of CIK cells.

The intrapapillary capillary loop (IPCL) changes in superficial esophageal lesions.

The aims of this study were to investigate intrapapillary capillary loops (IPCLs) of superficial esophageal lesions changes in different types classified by the Japan Esophageal Society classification. The calibers, areas, and densities of IPCLs were detected in 34 cases of esophageal lesions using immunohistochemical analysis. Statistically significant differences in calibers, areas, and densities of IPCLs were observed between type A, type B1/B2, and type B3 area (P < 0.001). In conclusion, the results of this observation showed the Japan Esophageal Society classification of IPCL would help endoscopists to diagnose the type and the invasion depth of lesion in esophagus, and decide the treatment strategy.

[Optimization of the method to cultivate NK cells from abandoned white cells].

OBJECTIVE: Using abandoned white cells separated from preparation of blood products to cultivate NK cells in vitro, and to optimize the method of cultivation of allogeneic NK cells for clinical application. METHODS: Abandoned white cells separated from blood production were collected from 15 healthy donors. PBMCs were isolated from the abandoned white cells and cultured for 17 days using culture bottles as previously coated antibodies (group CD3 mAb was coated with CD3 mAb, group CD 16mAb was coated with CD16mAb, and group CD3 mAb+ CD16 mAb was coated with CD3 mAb and CD16 mAb). Flow cytometry was used to determine the ratio of CD3(-)CD56(+) cells, expression of activated cell surface receptors, and secretion of IFN-γ. The anti-tumor cytotoxicity against K562 and Raji cells was determined using LDH cytotoxicity assay and flow cytometry. RESULTS: After expansion for 17 days, the proportions of CD3(-)CD56(+) cells was (15.19±12.22)% in the group CD3 mAb, (83.63±10.63)% in the group CD16 mAb, (49.40±12.64)% in the group CD3 mAb+ CD16 mAb, and it was (16.34±10.51)% before expansion. The total number of NK cells was more than 10(9). The expression ratios of NK cell surface activated receptors NKp30 and NKp46 were significantly increased, while that of the NKG2D was not significantly changed. The NK cells after expansion showed high cytotoxicity activity against K562 cells, reaching up to(76.97±3.16)% when effector-cell-to-target-cell ratio (E∶T ratio) was 40∶1. CONCLUSIONS: NK cells can be obtained from abandoned white cells after cultivation for 17 days, with a purity up to 90% and total cell number of more than 10(9). Their activity was reinforced, the anti-tumor cytotoxicity activity was increased, and may meet the standard of clinical therapeutic application.

Genetic variability of ERCC1 and ERCC2 influences treatment outcomes in gastric cancer.

We performed a study to investigate the role of ERCC1 (rs11615, rs2298881, and rs3212986) and ERCC2 (rs13181, rs238406, and rs1799793) polymorphisms in the prognosis of gastric cancer. A total of 346 patients with gastric cancer were recruited between May 2009 and May 2012. Single nucleotide polymorphism genotyping was performed using the Sequenom MassARRAY platform. The GA+AA genotype of ERCC2 rs1799793 showed significant and favorable response to chemotherapy than the wide-type GG genotype in multivariate analysis (OR = 1.78, 95%CI = 1.13-2.81). In a Cox proportional hazard model, carriers of ERCC2 rs1799793 GA+AA genotype exhibited longer duration of survival than did those with the GG genotype (hazards ratio = 0.57, 95%CI = 0.35-0.92). In conclusion, our study suggests that ERCC2 rs1799793 polymorphic variation could be used as a predictor for the prognosis of gastric cancer.

Comparison of competitive-nested PCR and real-time PCR in detecting BCR-ABL fusion transcripts in chronic myeloid leukemia patients.

Real-time RT-PCR has great advantages for estimating transcript levels in a variety of situations. These include relative rapid assay times (hours), reliability and ease of performing replicate analyses. In contrast, competitive PCR is a very labor-intensive procedure requiring a few days to generate useful data. We compared the same samples from CML patients by both methods. Importantly, we used the Bcr-Abl junction plasmid DNA, which is used as a competitor in the manual competitive PCR assay, to generate a standard curve for the real-time assay. This permitted reporting the real-time data as the number of BCR-ABL transcripts per microg of total RNA, which is the same format used for the competitive PCR assay. In this study, a total of 435 peripheral blood and marrow samples from 285 CML patients were analyzed by RT-PCR; these patients were undergoing therapy by STI-571, interferon, and bone marrow transplantation treatment. Most samples also had assay values for the Philadelphia chromosome (Ph), FISH and Western blotting for the Bcr-Abl oncoprotein. Our findings indicated that the real-time assay was less sensitive than the manual competitive RT-PCR assay (t = 5.118; P < 0.001). Of interest, the transcript levels in cell line mixtures with various ratios of K562/KG-1 (BCR-ABL positive/negative) cells were also significantly higher with the competitive RT-PCR assays than real-time RT-PCR, except for levels of BCR-ABL below 200 transcripts per microg of RNA. In both patient and cell line experiments, dividing the BCR-ABL transcripts by the total ABL transcripts virtually eliminated the difference between real-time BCR-ABL transcript values and quantitative competitive BCR-ABL transcript values, indicating that both BCR-ABL and ABL transcripts were underestimated by the real-time assay. In addition, the increased sensitivity of the nested, competitive RT-PCR was readily apparent in patients with minimal residual disease, which by the real-time were negative in the majority of patients but were positive by nested, competitive RT-PCR in 44.6% (n = 29) of samples analyzed (n = 65). These findings indicate that real-time RT-PCR, when normalized for the total ABL transcripts, can be used to monitor CML patients during therapy, but we suggest that nested, competitive RT-PCR be used to determine BCR-ABL/ABL transcript ratios at low transcript values or especially when real-time analyses are negative.

Expression of bcr-abl mRNA in individual chronic myelogenous leukaemia cells as determined by in situ amplification.

We present the results of a novel method developed for evaluation of in situ amplification, a molecular genetic method at the cellular level. Reverse transcription polymerase chain reaction (RT-PCR) was used to study bcr-abl transcript levels in individual cells from patients with chronic myelogenous leukaemia (CML). After hybridizing a fluorochrome-labelled probe to the cell-bound RT-PCR product, bcr-abl mRNA-positive cells were determined using image analysis. A dilution series of bcr-abl-positive BV173 into normal cells showed a good correlation between expected and actual values. In 25 CML samples, the percentage of in situ PCR-positive cells showed an excellent correlation with cytogenetic results (r = 0.94, P < 0.0001), interphase fluorescence in situ hybridization (FISH) (r = 0.95, P = 0.001) and hypermetaphase FISH (r = 0.81, P < 0.001). The fluorescence intensity was higher in residual CML cells after interferon (IFN) treatment than in newly diagnosed patients (P = 0.004), and was highest in late-stage CML resistant to IFN therapy and lowest in CML blast crisis (P = 0.001). Mean fluorescence values correlated with bcr-abl protein levels, as determined by Western blot analysis (r = 0.62). Laser scanning cytometry allowing automated analysis of large numbers of cells confirmed the results. Thus, fluorescence in situ PCR provides a novel and quantitative approach for monitoring tumour load and bcr-abl transcript levels in CML.

Expression of a truncated first exon BCR sequence in chronic myelogenous leukemia cells blocks cell growth and induces cell death.

We have shown that a deletion mutant form of Bcr [Bcr(64-413)] is a strong inhibitor of the tyrosine kinase of Bcr-Abl in vitro and also inhibits its oncogenic growth effects (Liu et al., Cancer Res., 56: 5120-5124, 1996). To determine the effects of this Bcr-Abl kinase inhibitor on chronic myelogenous leukemia (CML) cells, we cloned BCR(64-413) into a recombinant, replication-defective adenovirus to express useful quantities of Bcr(64-413) in a wide variety of cells in culture. Infection of Cos1 cells with plaque-purified virus at a multiplicity of infection of 20-40 induced high expression of Bcr(64-413) as detected by Western blotting. Infection of hematopoietic cells at modest multiplicities of infection (20-40) required special conditions involving shifting cycling cells to a nongrowing condition involving serum starvation and cell crowding. Under these conditions, both Bcr-Abl-positive and -negative hematopoietic cells can be efficiently infected by adenovirus, as demonstrated by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining of cells infected by beta-galactosidase (beta-GAL) adenovirus. We found that expression of Bcr(64-413) in Bcr-Abl-positive K562 and BV-173 cells, but not Bcr-Abl-negative SMS-SB cells, increased cell-cell clumping and inhibited cell growth. In contrast to the effects of the Bcr(64-413) adenovirus, the beta-GAL adenovirus, despite infecting both types of cells, did not block growth or increase cell-cell clumping of Bcr-Abl-positive and -negative hematopoietic cells. Expression of Bcr(64-413) protein in primary cultures of cells from CML patients with active disease interfered with cell growth, induced apoptosis (as measured by annexin staining), and increased cell-cell clumping, whereas the beta-GAL adenovirus and mock-infected cells lacked these effects. In contrast, normal marrow cells did not exhibit these effects on infection with Bcr(64-413) adenovirus. We conclude from these findings that Bcr(64-413) interferes with the oncogenic effects of Bcr-Abl and therefore has the potential for use in therapy of CML.

Analysis of Philadelphia chromosome-negative BCR-ABL-positive chronic myelogenous leukemia by hypermetaphase fluorescence in situ hybridization.

BACKGROUND: In 5%-10% of patients with of chronic myelogenous leukemia (CML), the Philadelphia chromosome (Ph) is not identified, despite the presence of the associated BCR-ABL molecular abnormality (Ph-negative, BCR-ABL-positive CML) because of sub-microscopic rearrangements. PATIENTS AND METHODS: Six patients with Ph-negative, BCR-ABL-positive CML were investigated. The Ph chromosome detection via fluorescence in situ hybridization after 24-hour mitotic arrest of bone marrow cultures resulting in several hundreds of metaphases (hypermetaphase FISH or HMF) was useful in explaining the nature of the six cases. RESULTS: Four patients had a low frequency of Ph-positive cells by HMF (5.7%, 4.8%, 3.9%, 0.2%), i.e., a typical Ph translocation. However, two cases involved a 9q34 inserted into chromosome 22q11 (74.2% and 92%), without a deletion from chromosome 22 and reciprocal translocation onto 9, i.e., not a typical Ph translocation. The pattern of UBCR gene rearrangement was characterized by the same genomic recombination of 5-BCR and c-ABL, both in the four cases of typical translocation (9;22) and in the two cases of insertion of 9q34 into chromosome 22q11. CONCLUSIONS: The HMF identified two different bases for Ph-negative, BCR-ABL-positive cells in CML-presence of low frequency of cells with typical Ph translocations or presence of cells with ABL insertions into the BCR gene on chromosome 22.

Multilineage involvement of Philadelphia chromosome positive acute lymphoblastic leukemia.

Acute lymphocytic leukemia (ALL) is considered a clonal disease restricted to the lymphoid compartment. The Philadelphia chromosome (Ph) is found in a subset of ALL with poor prognosis. Here we present the largest series of Ph+ ALL analyzed for involvement of the myeloid compartment. For the first time at a single cell level the presence of Ph in lineages other than lymphoid is demonstrated. Granulocytes from nine patients diagnosed with BCR-ABL + ALL (eight Ph+, one Ph-) were purified using two layer density gradient separation. They were further identified by the morphology of DAPI-stained nuclei and studied for the presence of the Ph by fluorescence in situ hybridization (FISH) using a BCR-ABL dual-color probe. Ph was demonstrated in 30 to 93% of granulocytes in all patients. FISH identified major and minor BCR gene breakpoints (M-bcr and m-bcr). In one patient, with CD19+/34+/33-/2-/3-/7-/10- lymphoblasts, involvement of B cells (CD19+), T cells (CD3+), myeloid (CD13+), erythroid (glycophorin A+) cells was found by FISH following fluorescence-activated cell sorting (FACS). The diagnosis of ALL as opposed to lymphoblastic transformation of CML was established based on clinical and laboratory data including Western blot results demonstrating the presence of p190/m-bcr in five of the nine cases studied. Results suggest that Ph+ ALL originates from a pluripotent stem cell.

Evidence for specific immune response against P210 BCR-ABL in long-term remission CML patients treated with interferon.

Interferon-alpha treatment induces complete cytogenetic remission in 25% of Philadelphia chromosome (Ph)-positive chronic myelogenous leukemia (CML) patients. These remissions are durable unlike remissions induced with other therapies and yet residual leukemia is detectable in most of these patients. Total peripheral blood mononuclear cells (PBMCs) from CML patients in long-term remission following interferon treatment exhibited significantly higher proliferative responses (four- to 15-fold over background) than normals directed against P210 BCR-ABL in extracts of transfected monkey fibroblast cells. Surprisingly, similar enhanced levels of specific proliferative responses were observed with extracts from cells expressing Bcr and/or Abl proteins. In contrast, extracts from vector only or v-Mos-expressing cells had background level responses. Control monkey fibroblast cells lacking BCR-ABL expression failed to induce proliferation over background levels. Normal individuals had no significant responses to Bcr/Abl extracts. On the other hand, peripheral blood mononuclear cells from allogeneic bone marrow transplant CML patients had proliferative responses to cell extracts independent of Bcr-Abl. These data indicate that patients in remission due to alpha-interferon treatment have significantly higher levels of specific cellular immunoreactivity against Bcr/Abl sequences than normal controls, which could play a role in maintaining cytogenetic remission in Ph-positive CML patients.

Analysis of the BCR-ABL protein in Philadelphia chromosome-positive adult acute lymphocytic leukemia.

Philadelphia chromosome (Ph)-positive acute lymphocytic leukemia (ALL) constitutes 15-35% of all ALL in adults. Its detection is prognostically significant. The Ph abnormality is usually detected through standard cytogenetic analysis but 20-30% of patients have insufficient metaphases (IM) with such analysis. To detect the BCR-ABL oncoprotein in peripheral blood specimen of patients with ALL at the time of diagnosis and at follow-up, a new sensitive technique of enhanced chemiluminescence Western blot (ECL-WB) analysis was investigated. Among 41 patients with newly diagnosed ALL, nine were Ph positive by cytogenetic studies; they were also BCR-ABL positive according to ECL-WB. Eight had p190 disease, and one had p210 disease. Among the 16 patients with IM, none demonstrated the oncoprotein through ECL-WB or through simultaneous Southern blot (SB) for p210 rearrangement. Follow-up studies were available for seven patients: four had detectable protein and three of them relapsed 4-20 weeks later; three had undetectable protein and one of them (who had low level protein at the time of diagnosis) relapsed 11 weeks later. Although none of the patients with IM at diagnosis had detectable protein according to ECL-WB, this was probably due to the small number of patients studied. One patient with IM studied at follow-up demonstrated the protein by ECL-WB. In summary, we describe a technique that is useful in the detection of p190/p210 ALL at diagnosis. It is less time consuming, and more cost effective than standard chromosome banding techniques. It may also detect the oncoprotein in cases with IM. Although a larger number of patients should be studied to prove its clinical usefulness, this technique may also be of value for monitoring residual disease at follow-up.

Comparison of bcr-abl protein expression and Philadelphia chromosome analyses in chronic myelogenous leukemia patients.

The Philadelphia chromosome (Ph) is found in most chronic myelogenous leukemia (CML) patients. The bcr-abl oncoprotein (P210 or P185), the product of the fused bcr-abl gene produced by the Ph, is known to be the major factor in initiation and maintenance of the leukemic state in these types of leukemias. The authors have devised a Western blot test to detect and quantitate the bcr-abl oncoprotein in blood and bone marrow cells. The authors analyzed 1,155 peripheral blood samples from CML patients, for which there were same day Ph data, to determine if there was a correlation between bcr-abl protein levels and the percentage of Ph. A ratio of bcr-abl protein (P210 or P185) to normal abl protein (P145), the latter is ubiquitously expressed in all blood cells, has been established as a criterion for quantitating bcr-abl levels. The bcr-abl/abl protein ratio from 399 patient who were 100% Ph-positive had a mean of 2.47 (standard error [SE] of +/- 0.05); no false negatives were detected. A decreasing ratio was found in patients with decreasing percentages of Ph. The relationship between the ratio of bcr-abl/abl proteins to the percentage of Ph-positive cells was nearly linear in 392 patients with Ph percentages between 5% to 95% (r = 0.97, P < .001). For patients in remission with no detectable Ph, the bcr-abl/abl ratio had a mean of 0.01 (SE = 0 +/- 0.00). The level of bcr-abl expression in samples from peripheral blood and bone marrow also were compared. The results indicated that the amount of bcr-abl protein within peripheral blood is usually similar to that in marrow. In conclusion, quantitation of the bcr-abl oncoprotein in peripheral blood cells or marrow cells as measured by a Western blot assay provides reliable data for both diagnosis and monitoring patients with pH-chromosome positive leukemia.

A novel BCR-ABL fusion gene (e6a2) in a patient with Philadelphia chromosome-negative chronic myelogenous leukemia.

A novel variant of the chimeric BCR-ABL mRNA transcript was detected in a patient with Philadelphia chromosome-negative (Ph-) chronic myelogenous leukemia (CML) by multiplex reverse-transcription polymerase chain reaction (RT-PCR). Sequence analysis of the fusion region of the amplified cDNA fragment showed an in-frame joining of exon e6 of the BCR gene and exon a2 of the ABL gene, giving rise to an e6a2 BCR-ABL transcript. This finding was confirmed by Southern blot analysis using a specific probe corresponding to intron 6 of the BCR gene, whereas conventional Southern blot for rearrangement of the major breakpoint cluster region (M-bcr) was negative. Western blot studies detected a BCR-ABL protein slightly larger than p185 BCR-ABL. Metaphase fluorescence in situ hybridization showed an insertion of ABL material into the BCR region without reciprocal BCR translocation. The findings in this case show that atypical BCR-ABL transcripts are detectable even in Ph- CML patients without M-bcr-rearrangements. Multiplex PCR using primers that allow for amplification of all known BCR-ABL transcripts is an appropriate method to exclude these rare variants.