Increased serum interleukin-8 in patients with early and metastatic breast cancer correlates with early dissemination and survival.

PURPOSE: The prognostic significance of serum interleukin (IL)-8 was evaluated in patients with metastatic breast cancer. The predictive value of serum IL-8 for the presence of occult metastatic tumor cells in bone marrow aspirates was evaluated in patients with operable and metastatic breast cancer. EXPERIMENTAL DESIGN: Serum IL-8 was measured in healthy controls, patients with operable breast cancer, and patients with untreated, progressive metastatic breast cancer. In 69 patients with either operable or advanced breast cancer, occult cytokeratin-positive cells were counted in bone marrow aspirates. RESULTS: Serum IL-8 levels are increased in 67% (52 of 77) of patients with advanced breast cancer. Overall, these levels are significantly higher in patients with breast cancer compared with healthy volunteers (P < 0.001). The IL-8 levels increase significantly in patients with more advanced disease. An elevated serum IL-8 is related to an accelerated clinical course, a higher tumor load, and the presence of liver or lymph node involvement. A multivariate analysis indicates that serum IL-8 is an independent significant factor for postrelapse survival. There was a significant difference between serum IL-8 levels in patients with or without occult cytokeratin-positive bone marrow cells (P < 0.04). Serum IL-8 levels also showed an association with the number of these cells (P < 0.01). CONCLUSIONS: Serum IL-8 is increased in patients with breast cancer and has an independent prognostic significance for postrelapse survival. The observations on the relationship between occult cytokeratin-positive bone marrow cells corroborate the concept of IL-8 acting as a contributor to the process of tumor cell dissemination. Similarly, the relationship between serum IL-8 and nodal stage at presentation deserves further study. These results further expand the concept that inflammation and inflammatory cytokines are critical components of tumor progression.     

Immunocytological detection of micrometastatic cells: Comparative evaluation of findings in the peritoneal cavity and the bone marrow of gastric,colorectal and pancreatic cancer patients

The prognosis of digestive cancers is poor mainly due to intraperitoneal relapse by cells which may have already been seeded at the time of surgery. Using immunocytology we investigated the peritoneal cavity and, as a comparison, the bone marrow of 147 patients with gastric, colorectal and pancreatic cancer for micrometastatic cells. Cytological samples from peritoneal cavity lavages and bone marrow aspirates were analyzed using monoclonal antibodies (MAbs) against tumor-associated antigens (TAA) (CEA, CA-19-9, 17-1-A, C-54-0, Ra96) and compared to a MAb staining cytokeratins (KL-I). Patients with benign diseases served as controls. Intraperitoneal micrometastatic cells were detected in 27% of colorectal, 43% of gastric and 58% of pancreatic cancer patients. In the bone marrow, the corresponding data were 29% for colorectal, 25% for gastric and 58% for pancreatic cancer patients. Combined evaluation of both compartments increased the detection rate significantly (colorectal cancer: 40%, gastric cancer: 52%, pancreatic cancer: 72%). No unwarranted reactions were found in the control group. Combining 3 antibodies (CA-19-9, Ra96, C-54-0) enabled good detection for peritoneal cavity samples. In the bone marrow, the use of 2 antibodies (KL-I and CA-19-9) detected 94% of all positive samples, whereas KL-I and CA-19-9 stained approx. 70% of all positive samples in each case. The occurence of stained cells in the peritoneal cavity correlated with classical prognostic factors (TNM classification). © 1994 Wiley-Liss, Inc.     

Epithelial cells in bone marrow of breast cancer patients at time of primary surgery: clinical outcome during long-term follow-up.

PURPOSE: To evaluate the detection of epithelial cells in bone marrow of breast cancer patients as an indicator of metastatic disease. PATIENTS AND METHODS: Between 1989 and 1994, bone marrow biopsies were performed on 393 breast cancer patients during primary surgery. Specimens were stained immunocytochemically for epithelial cells expressing cytokeratins or the epithelial membrane antigen. The long-term outcomes of these patients were analyzed in this study. RESULTS: In 166 of 393 patients, epithelial cells were found in bone marrow (BM) aspirates. These patients were designated BM+. The rate of tumor recurrence or cancer-related death was significantly higher in BM+ patients than in BM- patients. Multivariate analysis using the Cox regression model revealed BM status as a prognostic parameter independent of tumor size and axillary lymph node status. However, tumor size and axillary lymph node status were clearly superior prognostic parameters. CONCLUSION: Disseminated epithelial cells in BM are associated with poor clinical outcome in breast cancer patients. However, the presence of these cells is not a sufficient parameter to predict growing metastases in the majority of patients, suggesting that epithelial cells in the BM of breast cancer patients at the time of surgery have limited metastatic potential. The role of these cells needs to be further evaluated.     

Micrometastases in bone marrow of patients with suspected pancreatic and ampullary cancer

AIMS: This prospective study aimed to evaluate the detection of micrometastases in bone marrow of patients with suspected pancreatic and ampullary cancer and to determine their predictive value on overall survival. METHODS: Between December 1997 and December 1998, 35 patients (19 male, 42-77 years) with suspected pancreatic and ampullary cancer underwent diagnostic laparoscopy as a final staging procedure before exploration. Bone marrow was aspirated from the iliac crest at the beginning of laparoscopy. Mononuclear cells were isolated and stained using the specific monoclonal antibody CAM 5.2. RESULTS: Cytokeratin-positive cells were detected in 12/35 (34%) of all patients. In the 31 patients with a final diagnosis of carcinoma, a positive staining was found in 10/31 (32%) of the bone marrow aspirates. After a median follow-up of 17 months (2-24), 15/31 (48%) patients had died: 7/10 (70%) with and 8/21 (38%) without micrometastases (* P<0.04). All four patients who turned out to have chronic pancreatitis were alive without malignancy. In two of these four patients, distinct cytokeratin-positive cells were seen. CONCLUSIONS: Micrometastases in bone marrow of patients with the final diagnosis pancreatic or ampullary carcinoma seem to predict a significantly shorter survival. However, clinical use of cytokeratin markers cannot be recommended at present, because false-positive staining was found.     

Rare expression of epithelial cell adhesion molecule on residual micrometastatic breast cancer cells after adjuvant chemotherapy.

PURPOSE: Over the past 5 years, several clinical studies on a total of approximately 2500 patients have shown that the immunocytochemical detection of occult metastatic tumor cells in bone marrow (BM) at primary surgery provides important prognostic information in breast cancer (e.g., Ref 13 ). Here, we evaluated whether these cells can survive first-line chemotherapy and express epithelial cell adhesion molecule (Ep-CAM), recently suggested as promising target for immunotherapeutic interventions in breast cancer. Experimental Design: A total of 62 patients with node-negative and -positive breast cancer but without distant metastases (Tumor-Node-Metastasis stage M(0)) was treated with two or more courses of various forms of adjuvant chemotherapy (e.g., cyclophosphamide-methotrexate-5-fluorouracil, anthracyclines). After chemotherapy, BM was aspirated from the upper iliac crest and analyzed for the presence of tumor cells. A first cohort of 34 BM aspirates was enriched for tumor cells by Ficoll density gradient centrifugation, and 2-4 x 10(6) mononuclear cells were analyzed per patient. The tumor cells were detected by anticytokeratin monoclonal antibody (Mab) A45-B/B3 and double labeled with Mab 3B10 against an Ep-CAM-epitope. The subsequent 27 BM aspirates were specifically enriched for Ep-CAM(+) cells using magnetic beads coupled to Mab 3B10, and tumor cells were identified by Fab fragments of Mab A45-B/B3 directly conjugated with alkaline phosphatase. RESULTS: After chemotherapy, 10 of 35 (28.6%) Ficoll-enriched BM samples contained cytokeratin-positive tumor cells. In total, 26 cytokeratin-positive cells were detected, but none of these cells coexpressed Ep-CAM. Even within the second cohort of 27 Ep-CAM-enriched BM samples, only 2 specimens (7.4%) harbored cytokeratin-positive cells costaining with the Ep-CAM antibody. CONCLUSION: Our results indicate that disseminated breast cancer cells in BM can survive first-line adjuvant chemotherapy. Ep-CAM expression is, however, restricted to a subset of these cells, which may limit the broad applicability of Ep-CAM as target for second-line adjuvant therapy in breast cancer.     

Analysis of and prognostic information from disseminated tumour cells in bone marrow in primary breast cancer: a prospective observational study

ABSTRACT: BACKGROUND: Disseminated tumour cells (DTCs) in the bone marrow of patients with breast cancer have been identified as an independent predictor of poor prognosis in patients with non-metastatic disease. This prospective study aimed to evaluate the presence and prognostic value of DTCs in the bone marrow of female patients with primary breast cancer. METHODS: Between 1999 and 2003, bone marrow aspirates were obtained from patients at the time of surgery for primary invasive breast cancer. DTCs in bone marrow were identified using monoclonal antibodies against cytokeratins for detection of epithelial cells. The detection of DTCs was related to clinical follow-up with distant disease-free survival (DDFS) and breast cancer-specific survival as endpoints. Bone marrow aspirates from adult healthy bone marrow donors were analysed separately. RESULTS: DTCs were analysed in 401 patients, and cytokeratin-positive cells were found in 152 of these (38%). An immunofluorescence (IF) staining procedure was used in 327 patients, and immunocytochemistry (IC) was performed in 74 patients. The IF-based method resulted in 40% DTC-positive cases, whereas 30% were positive using IC (p = 0.11). The presence of DTCs in bone marrow was not significantly related to patient or tumour characteristics. The presence of DTCs was not a prognostic factor for DDFS (IF: hazards ratio [HR], 2.2; 95% confidence interval [CI], 0.63--2.2; p = 0.60; IC: HR, 0.84; 95% CI, 0.09--8.1; p = 0.88). Significant prognostic factors were lymph node metastases, oestrogen receptor positivity, Nottingham histological grade, and tumour size using Cox univariate analysis. The analyses were positive for epithelial cells in bone marrow from adult healthy donors in 19 (25%) samples. CONCLUSIONS: The detection of DTCs in bone marrow in primary breast cancer was previously shown to be a predictor of poor prognosis. We were not able to confirm these results in a prospective cohort including unselected patients before the standard procedure was established. Future studies with a standardised patient protocol and improved technique for isolating and detecting DTCs may reveal the clinical applications of DTC detection in patients with micrometastases in the bone marrow.     

贲门癌的显微自体荧光研究

目的:探讨贲门癌显微自体荧光图象的特征表现。方法:采用激光扫描共聚焦显微镜,以氩离子(Ar+)激光(EX=488nm)和氦氖激光(He-Ne)(EX=543nm)为激发光的双通道法,对12例贲门癌手术标本包括贲门癌组织与同体胃体组织进行自体荧光检测,并进行显微图象分析和自体荧光强度的比较。结果:癌细胞的高度增生和浸润使组织原有结构被破坏。贲门癌组织癌细胞浸润区域的平均自体荧光强度绿光为62·03,红光为18·50,而正常胃体组织平均荧光强度为:绿光168·06,红光139·79。与正常组织各层分别比较,癌组织的自体荧光强度均明显减弱,差异有统计学意义,P值均<0·01。结论:贲门癌与正常胃体组织的显微自体荧光无论在形态、颜色和分布上,还是在荧光强度上都存在巨大的差异。     

The presence of bone marrow cytokeratin-immunoreactive cells does not predict outcome in gastric cancer patients

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.     

Isolation of disseminated neuroblastoma cells from bone marrow aspirates for pretreatment risk assessment by array comparative genomic hybridization

In neuroblastoma, tumor biopsies are used for prognostic evaluation and risk assessment by molecular genetic analyses such as fluorescence in situ hybridization (FISH) and array comparative genomic hybridization (array CGH). Analysis of primary tumors by array CGH can be hampered by the lack of sufficient tumor cells due to small biopsy size or availability of invaded bone marrow only. Given the importance of accurate assessment of genetic alterations in the diagnostic work-up of patients with neuroblastoma, we evaluated the possibility to analyze bone marrow metastases in patients with disseminated disease. Disseminated neuroblastoma cells were isolated from bone marrow aspirates by using either laser capture microdissection (LCM) or magnetic activated cell sorting (MACS). The array CGH profiles of these isolated metastases were compared to array CGH profiles and/or FISH data of the corresponding primary tumor. Here, we show that the major recurrent DNA copy number alterations detected in primary neuroblastoma tumors (i.e., 1p, 3p and 11q deletion, 17q gain and MYCN amplification) can be detected, with high sensitivity and specificity, in the disseminated neuroblastoma cells isolated from the bone marrow aspirates, using an array platform with high coverage for these regions. Moreover, we demonstrate that for archived material, for example, for retrospective studies, LCM is the method of choice, while for fresh bone marrow aspirates, acquired at the time of diagnosis, MACS is superior.     

Detection of cytokeratin-positive cells in the bone marrow of breast cancer patients undergoing adjuvant therapy

Summary The presence of cytokeratin-positive cells in the bone marrow of breast cancer patients has been proven to be an independent prognostic factor. Their fate in primary breast cancer patients undergoing adjuvant therapy is of particular interest. We investigated the bone marrow status of 112 patients undergoing postoperative adjuvant treatment before and after therapy. A total of 373 patients with histologically confirmed primary breast cancer underwent bone marrow aspiration at the time of primary surgery. All patients were informed of their bone marrow status and offered repeat aspiration after 12 months. All patients were then treated with adjuvant chemotherapy, endocrine therapy or both based on current treatment recommendations. About 112 patients returned for a second bone marrow aspiration after a mean interval of 12 months following the initiation of adjuvant treatment. In 93 of 112 patients (83%) disseminated tumor cells had been found in the bone marrow before initiation of systemic chemo/endocrine therapy. At the time of follow-up sampling, after surgery and completion of adjuvant chemotherapy, the positivity rate dropped to 24%. Positive bone marrow status during follow-up was only associated with grading (p=0.020). Adjuvant treatment regimens are not able to completely eliminate cytokeratin-positive cells from the bone marrow. Prospective studies need to evaluate, whether these cells could become targets for additional adjuvant therapy.     

Detection of tumor cells in bone marrow of patients with primary breast cancer: a prognostic factor for distant metastasis.

PURPOSE: At the time of primary surgery, approximately 90% of all patients with breast cancer are free of metastases, but in the next 5 years almost 50% of them will relapse. We evaluated the significance of the presence of tumor cells in bone marrow of patients with primary breast cancer to investigate their predictive value for relapse. PATIENTS AND METHODS: Two hundred sixty patients with primary breast cancer were examined for tumor cells in bone marrow aspirates taken from six sites of the skeleton. After density centrifugation, cells in interphase were smeared and stained. For the immunocytologic reaction, we used a new monoclonal antibody (2E11) that was reactive with the core protein of the tumor-associated glycoprotein TAG12. TAG12 is secreted by nearly all human breast carcinomas. RESULTS: A significant correlation was found between tumor-cell detection and tumor stage (P < .0001), nodal status (P < .0001), and tumor grading (P = .002). A good relation to progesterone receptor (PR; P = .008) was found, but there was no correlation to estrogen receptor (ER) and menopausal status. Follow-up examinations showed distant metastases in 26 of 211 patients (15%). Twenty-two relapses occurred among the 81 patients with 2E11-positive cells in bone marrow, but only four occurred among the 130 patients without tumor-cell detection. CONCLUSIONS: This study suggests that tumor-cell detection in bone marrow of patients with primary breast carcinoma is a good predictor for all distant relapses (P < .0005, Cox multiple regression analysis) and provides additional information in regard to other prognostic factors. The highest predicting value for distant metastasis results from the combination of nodal status, negative PR, and tumor-cell presence in bone marrow.     

Prognostic impact of extracellular matrix metalloprotease inducer

BACKGROUND:

Extracellular matrix metalloprotease inducer (EMMPRIN) induces matrix metalloproteinase (MMP) expression, tumor‐stroma cell interaction, and invasion/angiogenesis. The objectives of the current study were to find the first evidence of a prognostic impact of total and relative EMMPRIN expression in colorectal cancer cells and to analyze EMMPRIN in bone marrow‐disseminated tumor cells and normal cells from 2 different gastrointestinal cancer entities.

METHODS:

Tumors and normal tissues from 40 patients with colorectal cancer who were followed prospectively (median follow‐up, 31 months) were analyzed for EMMPRIN by immunohistochemistry. Bone marrow from 51 patients (13 patients with gastric cancer and 38 patients with colorectal cancer) with evidence of disseminated tumor cells was screened for EMMPRIN in tumor cells and normal cells (cytokeratin 18/EMMPRIN double immunocytochemistry).

RESULTS:

A significant correlation between poor disease‐specific survival (P = .037; Kaplan‐Meier method; Mantel‐Cox log‐rank tests) and an increased ratio of EMMPRIN in tumor cells versus corresponding normal epithelial cells were observed. Furthermore, the relative increase of EMMPRIN was associated with a trend toward poor overall and recurrence‐free survival. High relative EMMPRIN expression was associated significantly with positive metastasis status (M1) (P = .001) and with a trend towards advanced pathologic tumor classification. Sixteen percent of disseminated tumor cells in bone marrow samples from patients with colorectal cancer and 48.5% of disseminated tumor cells in bone marrow samples from patients with gastric cancer stained positive for EMMPRIN, and EMMPRIN on micrometastatic cells was associated significantly with parameters of tumor progression (M status, noncurative resectability). A minority of normal bone marrow cells were stained for EMMPRIN, suggesting their suitability for molecular targeting.

CONCLUSIONS:

To the authors' knowledge, this study was the first to indicate that increased relative EMMPRIN protein in tumor‐specific cells compared with normal cells predicts poor disease‐specific survival in patients with colorectal cancer and that EMMPRIN in primary and bone marrow‐disseminated tumor cells is associated with clinical markers of tumor progression in patients with colorectal/gastric cancer. Cancer 2009. © 2009 American Cancer Society.     

Clinical significance of micrometastasis in bone marrow of patients with gastric cancer and its relation to angiogenesis

Background. Detection of micrometastasis is an important problem of clinical significance for a better understanding and control of tumor progression, which will improve patients' survival time. Methods. To identify micrometastases in bone marrow, an immunocytochemical assay for epithelial cytokeratin protein was performed in 106 patients with primary gastric cancer. Also, in 40 of the 106 patients, vascular endothelial growth factor (VEGF) expression and intratumoral vessel density were examined by an immunohistochemical staining method. Results. Of the 106 patients, 22 (20.8%) presented with cytokeratin-positive cells in bone marrow at the time of primary surgery. The positive findings were related to depth of invasion, peritoneal dissemination, and liver metastasis. Patients with cytokeratin-positivity in bone marrow had a higher VEGF positive rate (73%; 8/11) than did cytokeratin-negative patients (48%; 14/29). Intratumoral vessel density in VEGF-positive patients was 26.9 ± 10.3, which was significantly higher than that in VEGF-negative patients (13.2 ± 8.7, P < 0.05). Thus, the presence of cytokeratin-positive cells in bone marrow was closely related to angiogenesis in the primary tumor. Conclusions. Cytokeratin staining can be useful for identifying patients at high risk for metastasis. Prophylactic lymph node dissection, adjuvant chemotherapy, and antiangiogenic treatment may be necessary for patients with micrometastasis. Received for publication on Jan. 27, 1999; accepted on Feb. 26, 1999     

Immunocytochemical detection of breast cancer cells in marrow and peripheral blood of patients undergoing high dose chemotherapy with autologous stem cell support

Summary Detection of small numbers of breast cancer cells is important in staging the disease and can be helpful in assessing the efficacy of purging regimens prior to autologous stem cell infusion. Immunohistochemical methods are potentially useful and broadly applicable for this purpose since they are simple to perform, sensitive, and may be quite specific. We have used a combination of four monoclonal antibodies [260F9, 520C9, 317G5 (Baxter Corp); BrE-3 (Dr. R. Ceriani)] against tumor cell surface glycoproteins in a sensitive immunocytochemical assay to identify breast tumor cells in bone marrow and peripheral blood. Immunostained cytospin preparations were fixed prior to staining to preserve cytological details of immunopositive cells. After immunostaining, slides were counterstained with hematoxylin to confirm the identity of labeled cells. In cytocentrifuge experiments in which small numbers of CAMA human breast tumor cells were added to bone marrow mononuclear cells, a linear relationship between the number of tumor cells added and the number of tumor cells detected was obtained over a broad range of tumor cell concentrations. The probability of detecting tumor cells was dependent on the number of cytocentrifuge slides examined. When ten slides (5 million cells) were examined, the probability of detecting tumor at a concentration of 4 tumor cells per million bone marrow mononuclear cells was 98%. In clinical specimens, tumor cells were detected in marrow aspirates from 73 of 240 (30%) patients undergoing autologous transplantation, including 70 (37%) of 190 patients with clinical stage IV disease, 0 of 7 patients with clinical stage III disease, and 3 of 43 (7%) patients with clinical stage II disease. Seventy-three of 657 peripheral blood specimens from 26 of 155 patients (17%) contained breast cancer cells with counts ranging from 1 to 97 tumor cells per million leukocytes. Tumor cells were most frequently found in the blood of patients with stage IV disease [21 of 107 (20%)] but were also found in a substantial number [5 of 44 (11%)] of patients with stage II disease. Positive selection of CD34-positive hematopoietic progenitor cells as well as negative purging methods such as incubation with 4-hydroxyperoxy-cyclophosphamide (4-HC) were evaluated with respect to tumor cell depletion. Selection of CD34-positive progenitor cells from bone marrow or peripheral blood resulted in log reduction of 1 to > 4 tumor cells reinfused at autologous transplantation. A lesser log reduction (up to 1) was demonstrated following 4-HC purging. We conclude that properly performed and controlled immunocytochemical staining of bone marrow and peripheral blood cytospins is a sensitive and simple way to detect and quantitate breast cancer cells in hematopoietic specimens harvested for autotransplantation and that CD34-positive progenitor cell selection results in significant reduction in the number of breast cancer cells reinfused with marrow or peripheral blood stem cells.     

Detection of isolated tumour cells in the blood and bone marrow of patients with gastric cancer by combined sorting, isolation and determination of MAGE-1, -2 mRNA expression

The detection of isolated (circulating or disseminated) tumour cells (ITC) in patients with cancer requires very sensitive methods, as such cells are very rare. In the present study, the method that combines the negative isolation of CD45- leukocytes from the blood and bone marrow of patients with gastric cancer by flow cytometry, followed by the positive isolation of single cytokeratin-positive (CK+) cells by a Laser Capture Microdissection System for the determination of MAGE-1, -2 mRNA expression was used to detect ITC. This study shows that this method is highly sensitive as it allows to determine beta-actin-mRNA expression in a single CK+ cell. Using > or =5 CK+ cells as a cut-off level, the MAGE-1 mRNA expression was detected in 100% of CK+ cells in the peripheral blood and in 75% of bone marrow samples of patients with gastric cancer. The MAGE-2 mRNA expression was observed in 40 and 58% of samples, respectively. Furthermore, an analysis of primary tumours and locoreginal lymph nodes with respect to the mRNA expression of the two genes showed that MAGE-1 mRNA expression was detected in 88% of the primary tumours and in 67% of the lymph node samples, whereas the MAGE-2 mRNA expression was observed in 72 and 67% of the cases, respectively. Thus, the method described here allows the precise and sensitive determination of tumour-associated gene expression in single ITC present in the blood and bone marrow of patients with gastric cancer.     

Implications of occult metastatic cells for systemic cancer treatment in patients with breast or gastrointestinal cancer.

The early and clinically occult spread of viable tumour cells to the organism is becoming acknowledged as a hallmark in cancer progression, since abundant clinical and experimental data suggest that these cells are precursors of subsequent distant relapse. Using monoclonal antibodies against epithelial cytokeratins or tumour-associated cell membrane glycoproteins, individual carcinoma cells can be detected in cytological bone marrow preparations at frequencies of 10(-5) to 10(-6). Prospective clinical studies have shown that the presence of such immunostained cells in bone marrow is prognostically relevant with regard to relapse-free and overall survival, even in malignancies that do not preferentially metastasise to bone. As current treatment strategies have resulted in a substantial improvement of cancer mortality rates, it is noteworthy to consider the intriguing options of immunocytochemical screening of bone marrow aspirates for occult metastatic cells. Besides improved tumour staging, such screening offers opportunities for guiding patient stratification for adjuvant therapy trials, monitoring response to adjuvant therapies (which, at present, can only be assessed retrospectively after an extended period of clinical follow-up), and specifically targeting tumour-biological therapies against disseminated tumour cells. The present review summarises the current data on the clinical significance of occult metastatic cancer cells in bone marrow.     

Optical molecular imaging detects changes in extracellular pH with the development of head and neck cancer

Noninvasive localized measurement of extracellular pH in cancer tissues can have a significant impact on the management of cancer. Despite its significance, there are limited approaches for rapid and noninvasive measurement of local pH in a clinical environment. In this study, we demonstrate the potential of noninvasive topical delivery of Alexa‐647 labeled pHLIP (pH responsive peptide conjugated with Alexa Fluor^® 647) to image changes in extracellular pH associated with head and neck squamous cell carcinoma using widefield and high resolution imaging. We report a series of preclinical analyses to evaluate the optical contrast achieved after topical delivery of Alexa‐647 labeled pHLIP in intact fresh human tissue specimens using widefield and high‐resolution fluorescence imaging. Using topical delivery, Alexa‐647 labeled pHLIP can be rapidly delivered throughout the epithelium of intact tissues with a depth exceeding 700 µm. Following labeling with Alexa‐647 labeled pHLIP, the mean fluorescent contrast increased four to eight fold higher in clinically abnormal tissues as compared to paired clinically normal biopsies. Furthermore, the imaging approach showed significant differences in fluorescence contrast between the cancer and the normal biopsies across diverse patients and different anatomical sites (unpaired comparison). The fluorescence contrast differences between clinically abnormal and normal tissues were in agreement with the pathologic evaluation. Topical application of fluorescently labeled pHLIP can detect and differentiate normal from cancerous tissues using both widefield and high resolution imaging. This technology will provide an effective tool to assess tumor margins during surgery and improve detection and prognosis of head and neck cancer.     

Preclinical evaluation of near‐infrared (NIR) fluorescently labeled cetuximab as a potential tool for fluorescence‐guided surgery

The high rate of recurrence in patients with pancreatic ductal adenocarcinoma (PDAC) could be reduced by supporting the surgeons in discriminating healthy from diseased tissues with intraoperative fluorescence‐guidance. Here, we studied the suitability of Cetuximab, a therapeutic monoclonal antibody targeting the human epidermal growth factor receptor (EGFR), near‐infrared (NIR) fluorescently labeled as a new tool for fluorescence‐guided surgery. Distribution and binding of systemically injected Cetuximab Alexa Fluor 647 conjugate (Cetux‐Alexa‐647) and the co‐injected control human IgG Alexa Fluor 750 conjugate (hIgG‐Alexa‐750) was studied over 48 h by NIR fluorescence imaging in mice bearing human orthotopic AsPC‐1 and MIA PaCa‐2 PDAC tumors. Cetux‐Alexa‐647, but not the control hIgG‐Alexa‐750 fluorescence, was specifically detected in vivo in both primary pancreatic tumors with maximum fluorescence intensities at 24 h, and in metastases of AsPC‐1 tumors as small as 1 mm. Lifetime analysis and NIR fluorescence microscopy of tumor sections confirmed the binding specificity of Cetux‐Alexa‐647 to PDAC cells. Comparable results were obtained with Cetuximab conjugated to Alexa Fluor 750 dye (Cetux‐Alexa‐750). Fluorescence‐guided dissection, performed 24 h after injection of Cetuximab conjugated to IRDye 800CW (Cetux‐800CW), enabled a real‐time delineation of AsPC‐1 tumor margins, and small metastases. Odyssey scans revealed that only the vital part of the tumor, but not the necrotic part was stained with Cetux‐800CW. NIR fluorescently labeled Cetuximab may be a promising tool that can be applied for fluorescence‐guided surgery to visualize tumor margins and metastatic sites in order to allow a precise surgical resection.