Patterns of bone marrow involvement with small cell lung cancer

The extent of tumor involvement, necrosis, reticulin, and megakaryocytosis were assessed in 25 patients with small cell lung cancer in their bone marrow at diagnosis. The pattern of marrow involvement was compared with clinical outcome and tolerance of therapy. Any marrow involvement, no matter how minor, is a poor prognostic factor. Neither more extensive involvement, nor greater marrow fibrosis significantly worsened the prognosis.     

Elimination of small cell lung cancer cells in vitro from human bone marrow by a monoclonal antibody

We report here a useful method for elimination of small cell lung cancer cells in vitro from bone marrow. A monoclonal antibody, TFS-2, which mediates complement lysis and recognizes an antigen present on small cell lung cancer cells but not lymphoid cells or bone marrow cells, was used to clear infiltrated bone marrow. The antibody in the presence of complement effectively killed tumor cells, but it was not cytotoxic to bone marrow cells. When mixed populations consisting of tumor cells and bone marrow cells were treated with antibody and complement, the tumor cells were also effectively killed, except when large numbers of bone marrow cells were present, whereas TFS-2 had no significant effect on bone marrow stem cells, as judged by colony-forming unit assays.     

G-banded chromosome analysis of bone marrow and peripheral blood cells from small cell lung cancer (SCLC) patients

Detailed G-banded chromosome analysis was carried out on the bone marrow and/or PHA-stimulated peripheral blood cells from 17 SCLC patients (14 males and 3 females), who were diagnosed cytologically or pathologically or both. Twelve of them had no prior treatment and 16 had a heavy smoking history. High chromosome aberration rates were found in the bone marrow (31% for average structural aberration rate and 63% for numerical aberration rate) and peripheral blood cells (37% for average structural aberration rate and 49% for numerical aberration rate). The smoking index, as a whole, was positively correlated to the structural chromosome aberration rate, indicating that smoking is one of the most important environmental factors in causing chromosome aberrations. But some patients gave a high aberration rate dis-proportional to their smoking index, suggesting that genetically determined susceptibility to smoking or even other factors also play an important role. The structural chromosome aberrations in the bone marrow and peripheral blood cells were mainly clustered on chromosome 3 and chromosomes 1, 9 and 11, respectively. The aberration types were manifold and complicated. No consistent or specific aberration as del 3p14-23 for SCLC was found in this study.     

Examination of bone marrow biopsy specimens and staging of small cell lung cancer

Bone marrow biopsy specimens were evaluated retrospectively in 63 of 88 (72%) patients with small cell lung cancer (SCLC). Significant differences were not found between extensive disease (ED) patients with or without bone marrow metastases in survival nor in nadirs of leucocytes or platelets subsequent to chemotherapy. A panel of antibodies was used to investigate whether immunohistochemical analysis on routinely processed bone marrow biopsy specimens could detect marrow metastases more effectively than conventional microscopy. In histologically proven marrow metastases and in control SCLC sections a combination of an antibody against cytokeratin 8, 18 and 19 (NCL5D3) and an antibody against neurone specific enolase was validated for detection of metastases. In histologically negative marrow biopsy samples, however, this combination did not yield any additional tumour positive cases. Therefore, histological evaluation of a bone marrow biopsy specimen, even when analysed by immunohistochemistry, does not contribute information relevant for staging, therapy evaluation or prognosis in SCLC.     

The value of the bone scan and bone marrow biopsy staging small cell lung cancer

The charts of 112 patients with small cell lung cancer were reviewed in a retrospective fashion in order to define the role of the radionuclide bone scan and bone marrow biopsy in the staging of this disease. Both a radionuclide bone scan and bone marrow biopsy were performed on all patients at the time of diagnosis. Sixty-one percent of patients had a negative bone scan and negative biopsy; 22% had a positive bone scan and negative biopsy; 8% had a negative scan and positive biopsy; and 9% had a positive scan and positive biopsy. In 21 of the 44 patients with osseous involvement, no other focus of distant metastasis was found. The bone scan showed greater than or equal to 3 areas of increased uptake in 15 patients, 2 areas of increased uptake in 13 patients, and 1 area in 7 patients. The number of patients with bone marrow biopsy results positive for tumor in these 3 groups were 5, 3, and 2, respectively. Our study shows a lack of correlation between bone scan and bone marrow biopsy results. The bone scan and bone marrow biopsy identify independent patterns of osseous metastasis. Both procedures should be performed in the evaluation of patients with small cell lung cancer.     

Detection of small cell lung cancer bone marrow involvement by discontinuous gradient sedimentation

Marrow involvement by small cell lung cancer (SCLC) is detected in 10-23% of patients at initial diagnosis by marrow aspirate and biopsy techniques. To improve the detection and potentially monitor marrow involvement by SCLC we have attempted to concentrate malignant cells with clonogenic potential on a discontinuous density gradient (DDG). The bone marrow from 43 patients with SCLC (36 with histologically negative marrow aspirates and biopsies) were separated on a Ficoll-based DDG. Samples were also separated by conventional Ficoll-diatrizoate (FD) (density, 1.077) gradient sedimentation. The cellular interphase from three fractions (F X) corresponding to specific densities 1.050 (F X 1), 1.055 (F X 2), and 1.060 (F X 3) as well as cells separated by Ficoll-diatrizoate (F X FD) centrifugation were isolated and 2.5 X 10(5) cells from each fraction were cultured in 2 ml of 0.3% agar in McCoy's media with 10% fetal calf serum, 2.5 micrograms transferrin, 1 microgram insulin, and 1% penicillin-streptomycin. Colony growth was assessed after 14 days of culture at 37 degrees C and 6% CO2. Tumor colony growth was seen in eight of 36 (22%) patients with histologically negative marrows as well as in four of seven (57%) patients with known involvement. Mean colony growth per 2.5 X 10(5) cells for all 12 patients was 4.3 colonies for F X 1; 8.8 for F X 2; and 2.7 for F X 3. In contrast mean growth from the F X FD was 1.0 colonies. Cells with clonogenic potential could be demonstrated from F X 2 and F X 3 in seven of 12 and eight of 12 patients, respectively; in F X FD four of 12 patients had tumor growth. We conclude that separation of marrow samples by DDG concentrates malignant cells with clonogenic potential at least 8-fold compared to FD separation and that the sensitivity of the clonogenic assay in detecting marrow involvement by SCLC is enhanced by DDG sedimentation.     

Purging of small cell lung cancer cells from human bone marrow using ethiofos (WR-2721) and light-activated merocyanine 540 phototreatment

One limitation of autologous bone marrow transplantation for patients with cancer has been the presence of tumor cells in the bone marrow. Methods to eliminate tumor cells while preserving hematopoietic stem cells have been sought. The present study was performed to analyze the in vitro effectiveness of light-activated merocyanine 540 phototreatment (LAMP) and an aminothiol (ethiofos) as a marrow-purging regimen for small cell lung cancer (SCLC). Two human SCLC cell lines (ATCC HTB-119 and HTB-120) were treated with LAMP and exposed to light for varying periods of time up to 120 min. LAMP reduced SCLC cell proliferation and colony formation in a light exposure-dependent manner; colony formation was not totally inhibited until light exposure of 120 min was used. At this light exposure interval, multipotential hematopoietic progenitors, colony-forming units-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM), were substantially reduced. In an attempt to diminish hematopoietic toxicity, SCLC cells were incubated with ethiofos (formerly WR-2721) for 1 hour before LAMP. SCLC colony formation was eliminated at light exposure intervals (90 min or less) which had no inhibitory effect on CFU-GEMM. Ethiofos did not protect CFU-GEMM from LAMP inhibition at 120 min. Ethiofos alone had no effect on the SCLC or hematopoietic cells. When normal bone marrow was contaminated with 1 or 5% SCLC cells, ethiofos plus 60 min of LAMP eliminated SCLC cells but had no effect on CFU-GEMM. The results suggest that ethiofos sensitized SCLC cells to LAMP; thus ethiofos-enhanced LAMP may be an effective method for removing metastatic SCLC cells from bone marrow used for autologous marrow transplantation after high dose chemotherapy.     

High dose chemotherapy with autologous bone marrow rescue for small cell lung cancer]

The efficacy of high dose chemotherapy with autologous bone marrow rescue (ABMR) was given to 10 patients with small cell lung cancer (SCLC). Four patients received high dose methotrexate plus ABMR followed by chest irradiation. Six patients giving complete response after induction chemotherapy were given intensive chemotherapy plus ABMR only. The over-all survival of these 10 patients was 30.5 months with a range of 6-96 months. Five of them are surviving as of this writing, 3 survived for more than two years and 2 for more than 5 years. Toxic reactions were not severe. Systemic metastasis was the Leading cause of treatment failure. ABMR was effective and safe. When combined with radiotherapy ABMR could be expected to improve the result of intensive chemotherapy for limited SCLC.     

High dose chemotherapy with autologous bone marrow transplantation for limited small cell lung cancer

In 1980 we initiated a pilot study of high dose chemotherapy intensified with autologous bone marrow transplantation (ABMT) for seven patients with limited small cell lung cancer. Six patients were given the ABMT initially while one male patient received the ABMT late to intensify three courses of induction therapy. He further received radiotherapy and underwent a left pneumonectomy and adoptive immunotherapy, because the presence of residual tumor cells, and has maintained disease-free survival for over 117 weeks. Among the first six patients there were two with complete responses, two with partial responses and one who experienced no change, a total response rate of 80%. One patient died of cardiac tamponade at day 7 and could not be evaluated. The median survival time of 41 (range 34-108) weeks was not encouraging. In conclusion: an increased drug dose with ABMT to intensify the chemotherapy provided disappointing results both when the ABMT was used initially and when it was given late, after induction chemotherapy.     

The selective removal of red blood cells from bone marrow cell suspensions.

A method for the selective removal of red blood cells (RBC) from bone marrow cell suspension is described. The technique is based on the use of a dense (D = 1.090) Ficoll gradient, and a low speed centrifugation. Results show that more than 90% of the nucleated cells (NC) could be collected by this technique. RBC contamination in the NC fraction was less than 1 RBC per 8 NC. 1 NC per 1300 or more RBC was found in the pellet. This method may provide a useful step in the standardization of the bone marrow cell suspension for in vitro studies on colony forming cells as well as for any other experiment requiring purified red cell or nucleated cell populations.     

Monoclonal antibodies in the detection of bone marrow metastases in small cell lung cancer.

Using conventional examination (CE) of H&E stained slides from bone marrow aspirates, metastases can be detected in approximately 25% of patients with small cell lung cancer. We investigated a panel of monoclonal antibodies using immunohistochemistry in the diagnosis of bone marrow infiltration from SCLC and compared the results with CE. Seven monoclonal antibodies raised against epithelial antigens (CAM 5.2, MOV 15, NCCST 433, PE 35, LCA1/L38, HMFG 1 AND HMFG 2) were applied on bone marrow sections from three groups of patients (pts): (1) 19 pts in whom SCLC-metastases were detected by CE, (2) 44 pts with SCLC in whom metastases could not be detected by CE, and (3) 20 pts with non-malignant bone marrow diseases. All the antibodies except LCA1/L38 were positive in 60-90% of the slides with infiltrating tumour cells in group 1. No positive tumour cells were detected in group 2. A few plasma cells and megakaryocytes were slightly positive for MOV 15 and NCCST 433, but no other positive cells were detected in group 3. In conclusion, the monoclonal antibodies used in this study may be useful for diagnostic purposes when a suspicious looking infiltration is detected by CE. However, these antibodies could not detect metastatic tumour cells in the bone marrow sections from patients in whom CE did not reveal any tumour cells.     

Immunological evaluation of personalized peptide vaccination in refractory small cell lung cancer

Since the prognosis of small cell lung cancer (SCLC) remains poor, development of new therapeutic approaches, including immunotherapies, would be desirable. In the current study, to evaluate immunological responses in refractory SCLC patients, we conducted a small scale phase II clinical trial of personalized peptide vaccination (PPV), in which vaccine antigens are selected based on pre-existing host immunity. Ten refractory SCLC patients, who had failed to respond to chemo- and/or chemoradiotherapies (median number of regimens, 2.5; median duration, 20.5 months), were enrolled. A maximum of four human leukocyte antigen (HLA)-matched peptides showing higher antigen-specific humoral responses were subcutaneously administered (weekly for six consecutive weeks and then bi-weekly thereafter). PPV was terminated before the 3rd administration in four patients because of rapid disease progression, whereas the remaining six patients completed at least one cycle (six times) of vaccinations. Peptide-specific immunological boosting was observed in all of the six patients at the end of the first cycle of vaccinations, with their survival time of 25, 24.5 (alive), 10 (alive), 9.5, 6.5, and 6 months. Number of previous chemotherapy regimens and frequency of CD3(+) CD26(+) cells in peripheral blood were potentially prognostic in the vaccinated patients (hazard ratio [HR] = 2.540, 95% confidence interval [CI] = 1.188-5.431, P = 0.016; HR = 0.941, 95% CI = 0.878-1.008, P = 0.084; respectively). Based on the feasible immune responses in refractory SCLC patients who received at least one cycle (six times) of vaccinations, PPV could be recommended for a next stage of larger-scale, prospective clinical trials.     

Bone marrow biopsy in the staging of small cell lung cancer

From April 1982 to December 1987, 71 patients with small cell lung cancer entered a randomized clinical trial, and underwent bone marrow biopsy (BMB) as part of staging procedures. We identified 8 patients (11%) with bone marrow metastases, 6 with extensive disease independently of BMB, and 2 with extensive disease on the basis of the BMB only. BMB determined a change in the stage in only 3% (2/71) of the cases. No differences were found in the hematological parameters of the patients with or without bone marrow metastases. The median survival of the patients with bone marrow involvement was the same (41 weeks) as those with extensive disease but without bone marrow involvement. We conclude that unilateral BMB without aspiration detects a substantial proportion of bone marrow metastases in patients with extensive disease. This fact does not worsen the prognosis. A small proportion of patients with apparently limited disease has bone marrow involvement. The technique therefore contributes, to a small extent, to the definition of the clinical stage of the disease. However, bone marrow involvement is an important data of natural history, and therefore new methods to better assess this peculiar site of the disease are needed.     

Effect on prognosis of bone marrow infiltration detected by magnetic resonance imaging in small cell lung cancer

The staging system of limited disease (LD) and extensive disease (ED) is widely used and has been shown to provide useful prognostic information in cases of small cell lung cancer (SCLC). However, accurate examinations are necessary for correct staging. In this report, we evaluated the clinical usefulness of magnetic resonance imaging (MRI) of bone marrow in SCLC. 37 patients with LD by standard staging and 41 with ED were examined with bone marrow MRI. Results of bone marrow MRI did not influence the choice of treatment in patients with LD. For subsequent analysis, patients with LD were divided into two groups: patients in whom bone marrow infiltration was detected with MRI (MRI-positive LD group) and those in whom it was not (MRI-negative LD group). Focal or diffuse metastases to bone marrow were detected with MRI in 46% (36/78) of all patients and 35% (13/37) of LD patients. The response rates to treatment in patients with MRI-positive LD were lower than those in patients with MRI-negative LD (P=0.006). The survival of patients with MRI-positive LD was worse than that of MRI-negative LD (generalised Wilcoxon test: P=0.0157), and closer to that of ED. Multivariate analyses using a Cox model that included the result of bone marrow MRI, performance status, chemotherapy regimen, radiotherapy and serum lactose dehydrogenase (LDH) level showed that the result of bone marrow MRI remained a prognostic factor in SCLC patients with limited disease. Bone marrow examination with MRI is useful for better staging of SCLC. According to our analysis of response rates and survival, MRI-positive LD should be considered a type of ED.     

小细胞肺癌患者骨髓微小转移病灶的检测与临床应用

小细胞肺癌(SCLC)是一种高侵袭性肿瘤,治疗结果不佳,进一步了解其生物学行为,特别是与临床治疗及预后相关的因素以指导l临床治疗是非常重要的.肿瘤微小转移病灶(MRD)的研究是近年来研究的热点,在小细胞肺癌中具有更加重要的意义.本文对小细胞肺癌骨髓微小转移病灶的检测方法及其在临床中的应用和意义进行了综述.     

小细胞肺癌干细胞样细胞的分离鉴定

背景与目的:小细胞肺癌是高度侵袭的恶性肿瘤,患者的5年生存率不足10%,提示小细胞肺癌组织中可能富含较多的肿瘤干细胞.本研究设计从小细胞肺癌组织中原代培养肿瘤细胞并建立细胞系,从早期传代的细胞系中分离鉴定肺癌干细胞.方法:用无血清培养基原代培养肺癌细胞,传代后建立细胞系.应用流式细胞分析及分选技术,从细胞系第3代或第4代的细胞中寻找肺癌干细胞.应用单细胞克隆形成实验、平板集落形成试验及细胞球形成实验,研究肺癌干细胞的生物学特性.结果:应用原代细胞培养技术成功地从1例小细胞肺癌标本中培养出高纯度的原代肺癌细胞,其传代次数可达25代以上.流式细胞学分析显示该细胞系中有5.1%的细胞CD44呈强阳性表达(CD44++),相比于占主群的CD44弱阳性细胞(CD44+),CD44++细胞具有较强的平板集落形成能力,只有它们可以形成完全克隆(holoclone),在超低黏附的96孔培养板中可形成细胞球,而CD44+细胞不能形成完全克隆和细胞球.这些结果提示小细胞肺癌干细胞富集在CD44++细胞群中,而CD44+细胞中没有干细胞.应用CD44和CD90共同标记,可将小细胞肺癌细胞系中的细胞分为4群,分别是CD44+CD90-、CD44+CD90+、CD44++CD90-和CD44++CD90+细胞,其中CD44++CD90+细胞所占比例为1.9%.相比于其他细胞群,CD44++CD90+细胞形成细胞球的能力最强,提示CD90可能也是肺癌的干细胞标记.结论:小细胞肺癌细胞系中存在肿瘤干细胞样细胞,CD44和CD90可能是小细胞肺癌的干细胞标记.     

Elimination of small cell carcinoma of the lung from human bone marrow by monoclonal antibodies and immunomagnetic beads

The majority of patients with small cell carcinoma of the lung (SCCL) have bone marrow involvement detected by monoclonal antibodies (mAb). High dose chemotherapy followed by autologous bone marrow transplantation may improve treatment results for patients with SCCL, but the bone marrow may need to be purged of contaminating tumor cells. This study investigates the reactivity of a panel of mAb with two SCCL cell lines and normal bone marrow and the ability of the mAb and immunomagnetic beads to eliminate the SCCL cells from a mixture of 90% normal bone marrow cells and 10% SCCL cells. The mAb and immunomagnetic beads removed 4 to 5 log of SCCL cells in the model system. The immunomagnetic separation did not significantly adversely affect normal hematopoietic progenitor cells, as determined by bone marrow colony-forming units. The results suggest that the mAb and immunomagnetic beads could safely and effectively separate SCCL cells from the bone marrow for autologous bone marrow transplantation following high dose chemotherapy.     

小细胞肺癌干细胞信号通路的研究进展

小细胞肺癌是具有高度侵袭性的肺肿瘤,其主要临床特征是化疗有效率高但易在短时间内复发转移,这一特点可能与肿瘤干细胞的存在有关。肿瘤干细胞被认为是恶性肿瘤发生发展、耐药、复发及转移的根源。目前多认为肿瘤干细胞与正常干细胞有着相同的信号通路,如Hedgehog、Notch、Wnt等通路。本文就这几条信号通路在小细胞肺癌干细胞中所起的作用以及针对这几条信号通路治疗药物的研究进展和可能的信号通路交互作用等方面进行综述。     

Detection of bone marrow relapse in patients with small cell carcinoma of the lung

From a total of 874 patients with small cell lung cancer (SCC), a series of 104 patients were reviewed to determine if bone marrow relapses are detectable by routine clinical investigations. Autopsy, including microscopical bone marrow examination, was performed in all the 104 patients and bone metastases were disclosed in 36 patients (35%). After retrospective evaluation it was concluded that dose modification, occurrence of leukopenia plus fever, need of blood transfusion, leukopenia, and thrombopenia during treatment all were without predictive value in the detection of bone marrow relapse. Increased concentrations of lactate dehydrogenase and alkaline phosphatase were, however, positively correlated to the finding of bone marrow relapse.