Detection of micrometastasis in melanoma-draining lymph nodes is important for staging and prognosis. Immunohistochemical staining (IHC) using S-100p-HMB-45–, and MART-1– specfic antibodies is used for detecting metastases in sentinel lymph nodes (SLN). However, improvement in IHC is needed for melanoma micrometastasis detection.
Paraffin-embedded archival tissue (PEAT) specimens were obtained from 42 non-SLN macrometastases, 42 SLN metastases, and 16 tumor-negative SLNs of 100 melanoma patients who underwent SLN biopsy. PEAT specimens were assessed by IHC with high molecular weight-melanoma-associated antigen (HMW-MAA)– specific monoclonal antibodies (mAb) and with S-100p-, HMB-45–, and MART-1– specific antibodies. Quantitative real-time reverse-transcriptase PCR assay was used for HMW-MAA and MART-1 mRNA detection.
Expression frequency and immunostaining intensity were higher for HMW-MAA than MART-1in nodal macrometastases (P < 0.0001and P < 0.0001, respectively) and micrometastases (P < 0.0001and P = 0.004, respectively). All 52 (100%) macrometastases were positive with HMW-MAA – specific mAbs, whereas 43 (83%) were positive with MART-1– specific mAbs. In a comparison analysis, 23 of 23 (100%) micrometastases were HMW-MAA – positive, whereas 21 (91%) and18 (78%) specimens were S-100p – and HMB-45– positive, respectively. Quantitative real-time reverse-transcriptase PCR analysis of 48 nodal metastases showed HMW-MAA mRNA detection in SLNs with metastases.
HMW-MAA is more sensitive and specific than MART-1, S-100p, and HMB-45 for IHC-based detection of SLN micrometastases. SLN PEAT – based detection specificity of melanoma micrometastases can be improved by IHC with HMW-MAA – specific mAbs.
BACKGROUND: To improve median survival of patients with prostate cancer that has metastasized to bone, we need to better understand the early events of the metastatic process in skeleton and develop molecular tools capable of detecting the early tumor cell dissemination into bones (micrometastasis stage). However, the initial phase of tumor cell dissemination into the bone marrow is promptly followed by the migration of tumor cells into bone matrix, which is a crucial step that signals the transformation of micrometastasis to macrometastasis stage and clinically evident metastasis. The migration of cancer cells into bone matrix requires the activation of local bone resorption. Such an event contributes to tumor cell hiding/ escaping from high immunologic surveillance of bone marrow cells. Within bone matrix, tumor cells are establishing plethoric cell-cell interactions with bone marrow-residing cells, ensuring their survival and growth. Recently, RT-PCR detections of tumor marker transcripts, such as PSA and PSMA mRNA performed in RNA extracts of peripheral blood nucleated cells and bone marrow biopsy, have enabled the stratification of patients with clinically localized prostate cancer being of high risk for extraprostatic disease and bone involvement. Therefore, it is conceivable that bisphosphonate blockade of bone resorption can inhibit the migration of tumor cells into bone matrix during the early phase of disease dissemination into bone marrow (micrometastasis stage). Consequently, assessment of the efficacy and efficiency of bisphosphonates to arrest the evolution of bone lesions in this particular clinical setting of patients with clinically localized prostate cancer and positive molecular staging status (high risk for bone involvement) is warranted.
Occult lymph node metastasis (micrometastasis) is a good prognostic indicator in non ‐ small cell lung cancer (NSCLC) and could be used to direct adjuvant chemotherapy in stage I patients. This study was designed to evaluate molecular markers for detection of occult lymph node metastasis in NSCLC, define the best marker or marker combination to distinguish positive from benign lymph nodes, and evaluate these markers in lymph nodes from pathologically node‐negative (pN0) NSCLC patients. Potential markers were identified through literature and database searches and all markers were analyzed by quantitative reverse transcription‐PCR in a primary screen of six NSCLC specimens and 10 benign nodes. Selected markers were further evaluated on 21 primary NSCLC specimens, 21 positive nodes, and 21 benign nodes, and the best individual markers and combinations were identified. A combination of three markers was further validated on an independent set of 32 benign lymph nodes, 38 histologically positive lymph nodes, and 462 lymph nodes from 68 pN0 NSCLC patients. Forty‐two markers were evaluated in the primary screen and eight promising markers were selected for further analysis. A combination of three markers (SFTPB,TACSTD1, and PVA) was identified that provided perfect classification of benign and positive nodes in all sample sets. PVA and SFTPB are particularly powerful in tumors of squamous and adenocarcinoma histologies, respectively, whereas TACSTD1 is a good general marker for NSCLC metastasis. The combination of these genes identified 32 of 462 (7%) lymph nodes from 20 of 68 (29%) patients as potentially positive for occult metastasis. Long‐term follow‐up will determine the clinical relevance of these findings.
HER-2 expression in prostate cancer is associated with a worse prognosis and is suggested to play a role in androgen resistance. We present a study of HER-2 expression in circulating tumor cells and micrometastasis in bone marrow and the effect of androgen blockage or DES in the presence of HER-2 expressing cells.
Patients and Methods:
A multicenter study of men with prostate cancer, treated with surgery, radiotherapy, or observation, and with or without hormone therapy. Mononuclear cells were separated from blood and bone marrow aspirate by differential centrifugation, touch preps were made from bone marrow biopsy samples. Prostate cells were detected using anti-PSA monoclonal antibody and standard immunocytochemistry. Positive samples were processed using Herceptest® to determine HER-2 expression. After 1 year, patients were re-evaluated and the findings of HER-2 expression and PSA change compared with treatment.
Total 199 men participated, and 97 had a second evaluation 1 year later, frequency of HER-2 expression in circulating tumor cells and micrometastasis was 18% and 21%, respectively. There was no significant difference in HER-2 expression in the pretreatment group, after radical surgery or radiotherapy or with biochemical failure. Men with androgen blockade had a significantly higher expression of HER-2 (58%) (P =0.001). Of the 97 men with a second evaluation, 56 were in the observation arm, 27 androgen blockade, and 14 DES. Use of androgen blockade or DES significantly reduced serum PSA levels in comparison with observation (P =0.001). However, there was a significant increase in HER-2 expression in patients with androgen blockade (P =0.05) en comparison with observation or DES treatment. No patient with observation or DES became HER-2 positive, en comparison 4/22 patients initially HER-2 negative became HER-2 positive with androgen blockade.
The results suggest that HER-2 positive cells are resistant to androgen blockade. In an environment lacking androgens, HER-2 positive cells are selected and survive, while HER-2 negative cells are eliminated thus decreasing the serum PSA. The population of HER-2 positive cells proliferate producing androgen-independent disease. DES does not increase HER-2 expression possibly by stimulating beta-estrogen receptors and blocking HER-2 androgen receptor activation.
Androgen blockade; circulating tumor cells; DES; HER-2; micrometastasis; prostate cancer
The monoclonal antibody D2-40 is a specific lymphatic endothelial markers and D2-40 staining have been applicable to evaluate lymphatic invasion in various malignant neoplasms. In the present study, we investigated lymph node micrometastasis determined by immunohistochemistry (IHC) and reverse transcription–polymerase chain reaction (RT–PCR) in all dissected lymph nodes obtained from 80 patients with node-negative gastric cancer, and analysed the relationship between micrometastasis and clinicopathological findings including lymphatic invasion of the resected primary tumour using D2-40 immunohistochemical staining. The incidence of micrometastasis determined by IHC and RT–PCR was 11.3% (nine out of 80) and 31.3% (25 out of 80), respectively. Although haematoxylin–eosin (HE) staining revealed lymphatic invasion in 11.3% (nine out of 80) of patients, D2-40 staining uncovered new invasion in 23.8% (19 out of 80) of patients. In the diagnosis of HE and D2-40 staining, the incidence of micrometastasis was significantly higher in patients with lymphatic invasion than in those without lymphatic invasion (P=0.0150 and P<0.0001, respectively). Micrometastasis correlated more closely with D2-40 than with HE staining. We demonstrated a high incidence of micrometastasis and lymphatic invasion and a correlation between them even in pN0 gastric cancer. When planning less invasive treatment, the presence of such occult cancer cells should be considered.
gastric cancer; lymph node micrometastasis; lymphatic invasion; reverse transcription–polymerase chain reaction; immunohistochemistry
The origin and clinical relevance of circulating cell-free tumor DNA in the blood of cancer patients is still unclear. Here we investigated whether the detection of this DNA is related to bone marrow (BM) micrometastasis and tumor recurrence in breast cancer patients.
BM aspirates of 81 primary breast cancer patients were analyzed for the presence of disseminated tumor cells (DTC) by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3. PCR-based fluorescence microsatellite analysis was performed for detection of loss of heterozygosity (LOH) at 6 polymorphic markers using cell-free serum DNA. The data were correlated with established risk factors, and patients were followed-up over 6-10 years.
LOH was detected in 33.5% of blood samples. The occurrence of LOH at the entire microsatellite marker set correlated with histopathology (P = 0.05) and grading (P = 0.006) of the primary tumor. The genomic region characterized by marker D9S171 was only affected by LOH in patients with increased tumor stages (pT2-4, P < 0.05) and older age (≥ 55 years, P = 0.05). Kaplan-Meier analysis showed that LOH at D3S1255 (P = 0.009) and D9S171 (P = 0.001) were significantly associated with tumor relapse. In BM, DTC were detected in 39.5% of the patients, and this finding correlated with distant metastases (P < 0.05). Patients with DTC-positive BM had higher DNA yields in their blood than patients with DTC-negative BM (P < 0.05). However, no significant correlations were found between the presence of DTC in BM and the detection of marker-specific LOH on blood DNA.
The detection of LOH on cell-free tumor DNA in blood is unrelated to BM micrometastasis and provides independent information on breast cancer progression.
The aim of the present study was to detect the expression of human mammaglobin (hMAM) mRNA in the bone marrow (BM) of patients with breast cancer and determine the relationship between micrometastasis and clinicopathological parameters as well as selected molecular markers and breast cancer prognosis. The expression of hMAM mRNA in the BM of patients with breast cancer was determined by RT-PCR. The expression of ER, PR and Cath-D in cancer tissues was detected by immunohistochemistry. A positive expression rate for hMAM of 38.2% in 102 patients with stage I–III breast cancer was found. The expression of hMAM was higher in patients with T2–3 (>2 cm) tumors than in those with T1 tumors (≤2 cm) (χ2=19.20, P=0.001) and in patients with stage II or III tumors than in patients with stage I tumors (χ2=15.101, P=0.001). The expression of hMAM in the BM of breast cancer patients categorized as grade 1 was lower than that in those of grade 2 or 3 (χ2=8.522, P=0.014), and hMAM expression was related to the pathological type of tumor (χ2=6.892, P=0.032) and the degree of axillary lymph node metastasis (χ2=14.050, P=0.001). The expression of hMAM in BM was much higher in patients with ER(−) or ER(+) tumors than in those with ER(++ or +++) (χ2=11.800, P=0.003), and those with PR (χ2=8.759, P=0.013). hMAM expression in BM was also significantly positively correlated with Cath-D expression (χ2=6.623, P=0.036). However, no correlation was found between hMAM expression and patient age (χ2=1.056, P=0.304). There was a strong correlation between patients with positive expression of hMAM in the BM and the presence of distant metastases (P=0.009). In conclusion, micrometastasis in the BM correlates with certain clinical pathological parameters and several tumor markers. Patients with positive expression of hMAM in the BM have a greater chance of distant metastasis and poor prognosis. The detection of micrometastasis may be one of the most advantageous markers for predicting the prognosis of breast cancer.
breast cancer; bone marrow micrometastasis; human mammaglobin; biological factor; RT-PCR; immunohistochemistry; prognosis
It is not clear if sentinel lymph node (SLN) mapping can improve outcomes in patients with colorectal cancers. The purpose of this study was to determine the prognostic values of ex vivo sentinel lymph node (SLN) mapping and immunohistochemical (IHC) detection of SLN micrometastasis in colorectal cancers.
Colorectal cancer specimens were obtained during radical resections and the SLN was identified by injecting a 1% isosulfan blue solution submucosally and circumferentially around the tumor within 30 min after surgery. The first node to stain blue was defined as the SLN. SLNs negative by hematoxylin and eosin (HE) staining were further examined for micrometastasis using cytokeratin IHC.
A total of 54 patients between 25 and 82 years of age were enrolled, including 32 males and 22 females. More than 70% of patients were T3 or above, about 86% of patients were stage II or III, and approximately 90% of patients had lesions grade II or above. Sentinel lymph nodes were detected in all 54 patients. There were 32 patients in whom no lymph node micrometastasis were detected by HE staining and 22 patients with positive lymph nodes micrometastasis detected by HE staining in non-SLNs. In contrast only 7 SLNs stained positive with HE. Using HE examination as the standard, the sensitivity, non-detection rate, and accuracy rate of SLN micrometastasis detection were 31.8% (7/22), 68.2% (15/22), and 72.2%, respectively. Micrometastasis were identified by ICH in 4 of the 32 patients with HE-negative stained lymph nodes, resulting in an upstaging rate 12.5% (4/32). The 4 patients who were upstaged consisted of 2 stage I patients and 2 stage II patients who were upstaged to stage III. Those without lymph node metastasis by HE staining who were upstaged by IHC detection of micrometastasis had a significantly poorer disease-free survival (p = 0.001) and overall survival (p = 0.004).
Ex vivo localization and immunohistochemical detection of sentinel lymph node micrometastasis in patients with colorectal cancer can upgrade tumor staging, and may become a factor affecting prognosis and guiding treatment.
The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1350200526694475.
Colorectal carcinoma; Sentinel lymph node; Micrometastasis; Prognosis
Nodal micrometastasis and circulating tumor cells (CTC) detected by multimarker quantitative real-time RT-PCR (qRT) may have prognostic importance in patients with colorectal cancer (CRC).
Paraffin-embedded sentinel lymph nodes (SLNs) from 67 patients and blood from 34 of these patients was evaluated in a prospective multicenter trial of SLN mapping in CRC. SLNs were examined by hematoxylin and eosin staining (H&E) and cytokeratin immunohistochemistry (CK-IHC). Both SLNs and blood were examined by a four-marker qRT assay (c-MET, MAGE-A3, GalNAc-T, CK20); qRT results were correlated with disease stage and outcome.
In H&E SLN negative patients that recurred, CK-IHC and qRT detected metastasis in 30% and 60% of patients, respectively. DFS differed significantly by multimarker qRT upstaged SLN (p=0.014). qRT analysis of blood for CTC correlated with overall survival (p=0.040).
Molecular assessment for micrometastasis in SLN and blood specimens may help identify patients at high-risk for recurrent CRC, who could benefit from adjuvant therapy.
RT-PCR; colorectal cancer; micrometastasis; sentinel lymph node
The purpose of this study was to determine the clinical significance of nodal micrometastasis detected by immunohistochemistry in patients that had undergone curative surgery for pancreatic cancer. Between 2005 and 2006, a total of 208 lymph nodes from 48 consecutive patients with pancreatic cancer that had undergone curative resection were immunostained with monoclonal antibody against pan-ck and CK-19. Micrometastasis was defined as metastasis missed by a routine H&E examination but detected during an immunohistochemical evaluation. Relations between immunohistochemical results and clinical and pathologic features and patient survival were examined. Nodal micrometastases were detected in 5 (29.4%) patients of 17 pN0 patients. Nodal micrometastasis was found to be related to tumor relapse (P = 0.043). Twelve patients without overt nodal metastasis and micrometastasis had better prognosis than 5 patients with only nodal micrometastasis (median survival; 35.9 vs 8.6 months, P < 0.001). The Cox proportional hazard model identified nodal micrometastasis as significant prognostic factors. Although the number of patients with micrometastasis was so small and further study would be needed, our study suggests that the lymph node micrometastasis could be the predictor of worse survival and might indicate aggressive tumor biology among patients undergoing curative resection for pancreas cancer.
Pancreas; Adenocarcinoma; Lymph Nodes; Micrometastasis; Prognosis
The best method of pathological evaluation of sentinel lymph node in breast cancer has not been agreed upon. Immunohistochemical (IHC) techniques have shown a greater sensitivity over conventional histology for the detection of micrometastais. The aim of the study was to determine whether IHC for Epithelial Membrane Antigen (EMA) on the sentinel node could be more sensitive than conventional histology for diagnosing micrometastasis in sentinel lymph nodes. Eighty-four clinically node negative breast cancer patients underwent sentinel node biopsy at time of surgery for breast cancer. The node was subjected to conventional histopathology as well as IHC for EMA. The sensitivity of histology viz a viz IHC for EMA for detection of sentinel node metastasis was 88% and the specitficity was 96%. The overall diagnostic accuray of histology viz a viz IHC was 93%. There were 4 patients with micrometastasis (<2.0 mm), which were positive on IHC but negative on histology. Two patients with poorly differentiated breast cancer had a false negative IHC for EMA result as compared to histology. Immunohistochemistry for Epithelial Membrane Antigen can increase the detection rate of micrometastasis in sentinel lymph node. This can have important bearing on deciding the need of adjuvant systemic therapy. A false negative result for EMA may be seen in patients with poorly differential cancer. Therefore the best policy seems to employ both histopathology and IHC for EMA for the comprehensive evaluation of sentinel lymph node in breast cancer.
Sentinel node; Breast cancer; Immunohistochemistry
The aim of this study was to investigate the effects of different sequences of pulmonary artery and vein ligations during lobectomy on blood micrometastasis of non-small cell lung cancer (NSCLC). Cytokeratin 19 (CK19)/adhesion molecule CD44v6 mRNA were used as markers. A total of 30 NSCLC patients undergoing pulmonary lobectomy were randomly divided into pulmonary artery (PA)-first and pulmonary vein (PV)-first groups according to the order of artery or vein ligation (15 cases in each). Fluorescent quantitative-RT-PCR (FQ-RT-PCR) was used to detect the mRNA expression of CK19 and CD44v6 in pulmonary venous blood at the early and late periods during surgery, and ΔCt values were calculated. Meanwhile, the peripheral blood samples from 10 healthy volunteers were selected as the control. ΔCt values of CD44v6 and CK19 of NSCLC groups at the early period during surgery were 7.83±1.70 and 10.76±2.74, while those of the control group were 9.17±1.04 and 12.76±2.36. The expression of CD44v6 and CK19 genes in venous blood of NSCLC groups was significantly higher than that of the control group (P<0.05). In addition, the ΔCt values of CD44v6 and CK19 in the early and late periods during surgery in the PA-first group were 7.92±1.97 vs. 5.67±2.11 (P= 0.008) and 11.21±3.14 vs. 8.60±4.02 (P= 0.05), respectively. The expression of CD44v6 and CK19 in the late period were both significantly higher than those in the early period, while neither the ΔCt value of CD44v6 nor that of CK19 in the early vs. late periods in the PV-first group exhibited statistically significant differences (7.95±1.91 vs. 7.74±2.10 and 10.60±3.15 vs. 10.30±2.98) (P<0.05). Surgical manipulation itself may stimulate the occurrence of blood micrometastasis and the ligation of the PV first during surgery may help prevent blood micrometastasis.
lung cancer; blood; micrometastasis
Local and distant failure rates remain high despite aggressive chemoradiation (CRT) treatment for stage III non-small cell lung cancer (NSCLC). We conducted preclinical studies of docetaxel cytotoxic and radiosensitizing effects on lung cancer cell lines and designed a pilot study to target distant micrometastasis upfront with one-cycle induction chemotherapy, followed by low-dose radiosensitizing docetaxel CRT.
Methods and Materials
Preclinical study was conducted in human lung cancer cell lines NCI 520 and A549. Cells were treated with two concentrations of docetaxel for 3 hours and then irradiated immediately vs. delayed at 24 hours. Clonogenic survival assay was conducted and analyzed for cytotoxic effects vs. radiosensitizing effects of docetaxel. A pilot clinical study was designed based on pre-clinical study findings. Twenty-two patients were enrolled with a median follow-up of 4 years. Induction chemotherapy consisted of 75 mg/m2 docetaxel and 75 mg/m2 cisplatin on day 1, and rh-GCSF 150 mg/m2 on days 2–10. Concurrent CRT started 3–6 weeks later with twice-weekly docetaxel at 10–12 mg/m2 and daily delayed radiation in 1.8 Gy fractions to 64.5 Gy for gross disease.
Preclinical study demonstrated potent cytotoxic effects of docetaxel and subadditive radiosensitizing effects. Delaying radiation resulted in more cancer cell death. The pilot clinical study resulted in a median survival of 32.6 months for the entire cohort, with a 3-year and 5-year survival of 50% and 19%, respectively, and a distant metastasis-free survival rate of 61% for both 3 and 5 years. Patterns of failure analysis revealed 75% chest failures, and 36% all distant failures. Therapy was well tolerated with grade 3 esophagitis observed in 23% of patients.
One-cycle full-dose docetaxel/cisplatin induction chemotherapy with rh-GCSF followed by pulsed low-dose docetaxel chemoradiation is promising in its anti-tumor activity, low rates of distant failure, and low toxicity, suggesting that this regimen deserves further investigation.
Docetaxel; taxotere; chemoradiation; non-small cell lung cancer; radiosensitization
The independent prognostic significance of isolated tumour cells in bone marrow is still a matter of debate. This study evaluated the possible association of bone marrow micrometastases with tumour progression and prognosis in patients affected by gastric cancer. Bone marrow aspirates from both iliac crests were obtained from 114 consecutive patients operated on for gastric cancer. The specimens were stained with monoclonal antibody CAM 5.2 which reacts predominantly with cytokeratin filaments 8 and 19. Among 114 cases analysed, 33 cases (29%) had cytokeratine-positive cells in the bone marrow. There was no significant relationship between the presence of bone marrow micrometastases and site, depth of tumour invasion, lymph node metastases, presence of metastases. Patients with cytokeratine-positive cells had a trend towards a diffuse type histology (P=0.06). Among the 88 curatively resected patients, median survivals were 40 months and 36 months for cytokeratine-negative and cytokeratine-positive subsets respectively (P=0.9). Recurrence of the disease was observed in 39 cases (44.3%); 11 of 24 (45.8%) in the cytokeratine-positive subset and 28 of 64 (43.7%) in the cytokeratine-negative subset. In conclusion in our experience the presence of cytokeratine-positive cells in the bone marrow of curatively resected gastric cancer patients did not affect outcome and its independent prognostic significance remains to be proven before its official acceptance in the TNM classification.
British Journal of Cancer (2002) 86, 1047–1051. DOI: 10.1038/sj/bjc/6600211 www.bjcancer.com
© 2002 Cancer Research UK
gastric cancer; bone marrow micrometastasis; cytokeratin-positive cells; prognosis
The survival of patients with non–small-cell lung cancer (NSCLC), even when resectable, remains poor. Several small studies suggest that occult metastases (OMs) in pleura, bone marrow (BM), or lymph nodes (LNs) are present in early-stage NSCLC and are associated with a poor outcome. We investigated the prevalence of OMs in resectable NSCLC and their relationship with survival.
Patients and Methods
Eligible patients had previously untreated, potentially resectable NSCLC. Saline lavage of the pleural space, performed before and after pulmonary resection, was examined cytologically. Rib BM and all histologically negative LNs (N0) were examined for OM, diagnosed by cytokeratin immunohistochemistry (IHC). Survival probabilities were estimated using the Kaplan-Meier method. The log-rank test and Cox proportional hazards regression model were used to compare survival of groups of patients. P < .05 was considered significant.
From July 1999 to March 2004, 1,047 eligible patients (538 men and 509 women; median age, 67.2 years) were entered onto the study, of whom 50% had adenocarcinoma and 66% had stage I NSCLC. Pleural lavage was cytologically positive in only 29 patients. OMs were identified in 66 (8.0%) of 821 BM specimens and 130 (22.4%) of 580 LN specimens. In univariate and multivariable analyses OMs in LN but not BM were associated with significantly worse disease-free survival (hazard ratio [HR], 1.50; P = .031) and overall survival (HR, 1.58; P = .009).
In early-stage NSCLC, LN OMs detected by IHC identify patients with a worse prognosis. Future clinical trials should test the role of IHC in identifying patients for adjuvant therapy.
Immunohistochemistry (IHC) with mutation-specific antibodies may be an ancillary method of detecting EGFR mutations in lung cancer patients.
EGFR mutation status was analyzed by DNA assays, and compared with IHC results in five non-small-cell lung cancer (NSCLC) cell lines and tumor samples from 78 stage IV NSCLC patients.
IHC correctly identified del 19 in the H1650 and PC9 cell lines, L858R in H1975, and wild-type EGFR in H460 and A549, as well as wild-type EGFR in tumor samples from 22 patients. IHC with the mAb against EGFR with del 19 was highly positive for the protein in all 17 patients with a 15-bp (ELREA) deletion in exon 19, whereas in patients with other deletions, IHC was weakly positive in 3 cases and negative in 9 cases. IHC with the mAb against the L858R mutation showed high positivity for the protein in 25/27 (93%) patients with exon 21 EGFR mutations (all with L858R) but did not identify the L861Q mutation in the remaining two patients.
IHC with mutation-specific mAbs against EGFR is a promising method for detecting EGFR mutations in NSCLC patients. However these mAbs should be validated with additional studies to clarify their possible role in routine clinical practice for screening EGFR mutations in NSCLC patients.
Matrix metalloproteinase-2 (MMP-2) is important in the dissemination and invasion of tumor cells and activates angiogenesis. We present an immunocytochemical study of MMP-2 expression in circulating prostate cells (CPCs), disseminated tumor cells (DTCs), and micrometastasis (mM) in bone marrow of men with prostate cancer. Methods and Patients. Tumor cells were identified with anti-PSA immunocytochemistry. Positive samples underwent processing with anti-MMP-2, its expression was compared with Gleason score, concordance of expression, and metastatic and nonmetastatic disease. Results. 215 men participated, CPCs were detected in 62.7%, DTCs in 62.2%, and mM in 71.4% in nonmetastatic cancer; in metastatic cancer all had CPCs, DTCs, and mM detected. All CPCs and DTCs expressed MMP-2; in mM MMP-2 expression was positively associated with increasing Gleason score. MMP-2 expression in CPCs and DTCs showed concordance. In low grade tumors, mM and surrounding stromal cells were MMP-2 negative, with variable expression in high grade tumors; in metastatic disease, both mM and stromal cells were MMP-2 positive. Conclusions. CPCs and DTCs are different from mM, with inhibition of MMP-2 expression in mM of low grade tumors. With disease progression, MMP-2 expression increases in both mM and surrounding stromal cells, with implications for the use of bisphosphonates or MMP-2 inhibitors.
Aims: To compare the results of breast cancer sections with HercepTest™ immunohistochemistry (IHC) scores ranging from 0 to 3+ with fluorescence in situ hybridisation (FISH) for HER2 amplification. The HER2 digital scoring application of the Micrometastasis Detection System (MDS™) was used, together with manual scoring of FISH and HercepTest, to determine whether this system provides an accurate alternative.
Methods: Paraffin wax embedded sections were stained using HercepTest and analysed by eye and automated quantitative image analysis. FISH was performed using the PathVysion™ fluorescent probe and scored by eye and automated quantitative image analysis using MDS.
Results: Of 114 cases, 26% were amplified by FISH, whereas only 18% scored 3+; 32% of IHC 2+ cases were amplified by FISH, and one showed borderline amplification. Six percent of IHC negative cases (0 or 1+) were amplified by FISH, and one showed borderline amplification. Of IHC 3+ cases, 10% were non-amplified by FISH. Classification discrepancies were seen in 18% of HercepTest cases scored by eye and using the MDS system. MDS was consistent with visual FISH scoring and correctly differentiated most ambiguous visual IHC scores.
Conclusions: FISH provides a more accurate and consistent scoring system for determining HER2 amplification than HercepTest. The MDS system provides a reliable, consistent alternative to visual IHC and FISH scoring. IHC is still a valuable technique to aid in identification of isolated or heterogeneous tumour populations for subsequent FISH analysis, and a combined FISH and HercepTest approach to all breast cancer cases may be the most efficient strategy.
FISH; HER2; breast cancer; Herceptin; immunohistochemistry
In 125 early breast cancer patients who underwent multiple bone marrow aspirates, there was no significant difference in terms of disease-free and overall survival after a median follow-up of 163 months between the patients with or without micrometastasis at the time of primary surgery. However, when the time-dependent evolution of the bone marrow aspirates was taken into account, some evidence for a longer disease-free and overall survival was found for the patients with negative bone marrow
bone marrow; breast cancer; long-term follow-up; micrometastases
Thirty-five patients with prostate cancer were examined for micrometastases to the bone marrow using reverse transcription-polymerase chain reaction (RT-PCR) with primers specific for the prostate-specific antigen (PSA) gene. Of nine patients with bone metastases detectable by bone scan imaging, five patients had PSA mRNA expression in the bone marrow detectable by RT-PCR. Of 26 patients with negative bone scan findings, seven patients had PSA mRNA expression detectable in the bone marrow. RT-PCR could detect micrometastatic prostate cancer cells in the bone marrow that were not detectable by bone scan imaging. Of 16 patients with a serum PSA concentration of 25 ng ml(-1) or greater, only nine (56.3%) had bone metastases detected by bone scans. Of the remaining seven patients, five had micrometastases to the bone marrow detected by RT-PCR. Overall, 14 of 16 patients (87.5%) with a serum PSA concentration of 25 ng ml(-1) or greater had metastatic bone diseases including bone marrow micrometastases. Of 19 patients with a serum PSA concentration of less than 25 ng ml(-1), two (10.5%) had only micrometastatic disease detected by RT-PCR. A significant correlation was observed between the incidence of bone involvement and the serum PSA concentration. This study suggests that RT-PCR will potentially develop into a relevant tool to assess bone involvement including bone marrow micrometastases and establish a precise correlation between serum PSA concentration and metastatic bone disease in patients with prostate cancer.
To detect metastases in the bone marrow of patients with small cell lung cancer, immunofluorescence with a monoclonal antibody detecting a membrane antigen (MOC-1) associated with small cell lung cancer was performed on 53 bone marrow aspirates from 30 patients. In 19 (63%) patients MOC-1 reactive cells were detected. Simultaneous histopathological examination of the bone marrow biopsy specimens detected tumour cells in only six (20%). The method is more sensitive than conventional histochemical staining of bone marrow aspirate and may eventually be able to show additional subgroups, such as patients with limited disease who might benefit from adjuvant radiotherapy or surgery.
Pigment epithelium–derived factor (PEDF) is expressed in human uveal melanoma. In a mouse model with ocular melanoma, PEDF suppresses tumor angiogenesis, tumor size, and number of hepatic micrometastases.
Pigment epithelium–derived factor (PEDF) is known to be an angiogenesis suppressor and to have antitumor effects. This study investigates whether constitutive overexpression of PEDF inhibits the growth and hepatic micrometastasis of ocular melanoma.
Real-time RT-PCR was used to detect endogenous PEDF expression in human uveal melanoma cell lines and mouse melanoma cells. A lentiviral vector containing a mouse PEDF expression sequence was constructed and transduced into mouse melanoma cells in vitro. Transgene expression was assessed by Western blot analysis. Angiogenesis and transendothelial migration assays were performed in constitutively stable PEDF-overexpressing cells and transduced lentiviral vector control cells. The size and microvessel density of the ocular tumor and the number of hepatic micrometastasis were compared between the mice inoculated with PEDF-overexpressing tumor cells and those mice with the control cell line.
Four human uveal melanoma and three mouse melanoma cell lines were found to express PEDF mRNA. Endogenous overexpressing PEDF melanoma cells lost the ability to migrate and form tubes in vitro. In the animal experiment, the size of the ocular melanoma and the number of hepatic micrometastasis were decreased and microvessel density was also reduced in mice inoculated with constitutively overexpressing PEDF melanoma cells.
Lentivirus-mediated gene transfer of PEDF decreased the growth of ocular melanoma and its hepatic micrometastasis in a mouse ocular melanoma model. Dual antitumor/antiangiogenic activities of PEDF suggest that PEDF gene therapy may be considered an approach for the treatment of ocular melanoma.
Special techniques such as serial macroscopic sectioning (SMS) or immunohistochemical staining (IH) improve the detection rate of micrometastases but this detection is of value only if it improves the prediction of recurrence and survival. We first studied the prognosis of 120 patients with a single micrometastasis detected by SMS in a series of 1,680 primary operable breast carcinoma with a median follow-up of 7 years. A significant difference in recurrence (P = 0.005) and in survival (P = 0.0369) was found between node-negative patients and those with one single SMS micrometastasis, but SMS micrometastases were not a predicting factor by multivariate analyses according to the Cox model. We then studied the prognostic significance of patients with a micrometastasis detected by IH in node-negative carcinoma: 37 micrometastases from a series of 89 invasive lobular carcinoma (ILC) and 13 single micrometastases from a series of 129 invasive ductal carcinoma (IDC). In the ILC group, IH micrometastases had no prognostic value (median follow-up: 9.3 years). In the IDC group, IH micrometastases were correlated with recurrences (P = 0.01) and were the most significant predicting factor, but were less correlated with survival (median follow-up: 15.6 years). Three main points emerge from this study: (1) SMS micrometastases have a prognostic significance and macroscopic sectioning is recommended as a routine technique not requiring excessive work. (2) IH micrometastases in infiltrating lobular carcinoma have no prognostic significance. (3) The value of IH is debatable in infiltrating ductal carcinoma, since the technique is of principal use in predicting recurrences. It should therefore be carefully assessed vs other prognostic factors currently under study.
The detection of bone marrow involvement might be of prognostic value and may influence therapeutic decisions in small cell lung cancer. By unilateral bone marrow aspiration and biopsy, evidence of bone marrow metastases is seen in 15-30% of patients with this disease. Since magnetic resonance imaging of the lower body and immunostaining with monoclonal antibodies have recently been shown to be very sensitive detection methods, we investigated the value of these two techniques in detecting bone marrow involvement in 35 consecutive patients with small cell lung cancer. The results were compared to those obtained with conventional cytohistological analysis. In all cases when cytology and/or bone marrow biopsy were positive, monoclonal antibodies immunostaining and magnetic resonance imaging also detected malignant cells. Furthermore, evidence of bone marrow involvement was shown with magnetic resonance imaging and/or immunostaining in 10 of 26 cases (38%) where routine procedures were unable to detect malignant cells. In one of these 26 patients, magnetic resonance imaging and immunostaining provided the only evidence of metastatic disease. These data suggest that the rate of bone marrow metastases is underestimated by routine procedures. Further investigation is needed to determine whether or not these new non-invasive methods have prognostic value or affect therapeutic choices in small cell lung carcinoma.
Pigment epithelium-derived factor (PEDF) is known to be an angiogensis suppressor and have anti-tumor effects. This study investigates whether constitutive overexpression of PEDF inhibits the growth and hepatic micrometastasis of ocular melanoma.
Real time RT-PCR was used to detect endogenous PEDF expression in human uveal melanoma cell lines and mouse melanoma cells. A lentiviral vector containing a mouse PEDF expression sequence was constructed and transduced into mouse melanoma cells in vitro. Transgene expression was assessed by Western blot analysis. Angiogenesis and transendothelial migration assays were performed in constitutively stable PEDF overexpressing cells and transduced lentiviral vector control cells. The size and microvessel density of the ocular tumor, and the number of hepatic micrometastasis were compared between the mice inoculated with PEDF overexpressing tumor cells and those mice with the control cell line.
Four human uveal melanoma and three mouse melanoma cell lines were found to express PEDF mRNA. Endogenous overexpressing PEDF melanoma cells lost the ability to migrate and form tubes in vitro. In the animal experiment, the size of the ocular melanoma and the number of hepatic micrometastasis were decreased, and microvessel density was also reduced in mice inoculated with constitutively overexpressing PEDF melanoma cells.
Lentivirus-mediated gene transfer of PEDF decreased the growth of ocular melanoma and its hepatic micrometastasis in a mouse ocular melanoma model. Dual antitumor/anti-angiogenic activities of PEDF suggest that PEDF gene therapy may be considered as an approach for treatment of ocular melanoma.