Markus Zuber

Expression of Cytokeratins 17 and 5 Identifies a Group of Breast Carcinomas with Poor Clinical Outcome

While several prognostic factors have been identified in breast carcinoma, the clinical outcome remains hard to predict for individual patients. Better predictive markers are needed to help guide difficult treatment decisions. In a previous study of 78 breast carcinoma specimens, we noted an association between poor clinical outcome and the expression of cytokeratin 17 and/or cytokeratin 5 mRNAs. Here we describe the results of immunohistochemistry studies using monoclonal antibodies against these markers to analyze more than 600 paraffin-embedded breast tumors in tissue microarrays. We found that expression of cytokeratin 17 and/or cytokeratin 5/6 in tumor cells was associated with a poor clinical outcome. Moreover, multivariate analysis showed that in node-negative breast carcinoma, expression of these cytokeratins was a prognostic factor independent of tumor size and tumor grade.

Prognostic Relevance of Gene Amplifications and Coamplifications in Breast Cancer

Multiple different oncogenes have been described previously to be amplified in breast cancer including HER2, EGFR, MYC, CCND1, and MDM2. Gene amplification results in oncogene overexpression but may also serve as an indicator of genomic instability. As such, presence of one or several gene amplifications may have prognostic significance. To assess the prognostic importance of amplifications and coamplifications of HER2, EGFR, MYC, CCND1, and MDM2 in breast cancer, we analyzed a breast cancer tissue microarray containing samples from 2197 cancers with follow-up information. Fluorescence in situ hybridizations revealed amplifications of CCND1 in 20.1%, HER2 in 17.3%, MDM2 in 5.7%, MYC in 5.3%, and EGFR in 0.8% of the tumors. All gene amplifications were significantly associated with high grade. HER2 (P < 0.001) and MYC amplification (P < 0.001) were also linked to shortened survival. In case of HER2, this was independent of grade, pT, and pN categories. MYC amplification was almost 3 times more frequent in medullary cancer (15.9%), than in the histologic subtype with the second highest frequency (ductal; 5.6%; P = 0.0046). HER2 and MYC amplification were associated with estrogen receptor/progesterone receptor negativity (P < 0.001) whereas CCND1 amplification was linked to estrogen receptor/progesterone receptor positivity (P < 0.001). Coamplifications were more prevalent than expected based on the individual frequencies. Coamplifications of one or several other oncogenes occurred in 29.6% of CCND1, 43% of HER2, 55.7% of MDM2, 65% of MYC, and 72.8% of EGFR-amplified cancers. HER2/MYC-coamplified cancers had a worse prognosis than tumors with only one of these amplifications. Furthermore, a gradual decrease of survival was observed with increasing number of amplifications. In conclusion, these data support a major prognostic impact of genomic instability as determined by a broad gene amplification survey in breast cancer.

Morbidity of Sentinel Lymph Node Biopsy (SLN) Alone Versus SLN and Completion Axillary Lymph Node Dissection After Breast Cancer Surgery A Prospective Swiss Multicenter Study on 659 Patients

Objective:To assess the morbidity after sentinel lymph node (SLN) biopsy compared with SLN and completion level I and II axillary lymph node dissection (ALND) in a prospective multicenter study. Summary Background Data:ALND after breast cancer surgery is associated with considerable morbidity. We hypothesized: 1) that the morbidity in patients undergoing SLN biopsy only is significantly lower compared with those after SLN and completion ALND level I and II; and 2) that SLN biopsy can be performed with similar intermediate term morbidity in academic and nonacademic centers. Methods:Patients with early stage breast cancer (pT1 and pT2 ≤ 3 cm, cN0) were included between January 2000 and December 2003 in this prospective Swiss multicenter study. All patients underwent SLN biopsy. In all patients with SLN macrometastases and most patients with SLN micrometastases (43 of 68) or isolated tumor cells (11 of 19), a completion ALND was performed. Postoperative morbidity was assessed based on a standardized protocol. Results:SLN biopsy alone was performed in 449 patients, whereas 210 patients underwent SLN and completion ALND. The median follow-ups were 31.0 and 29.5 months for the SLN and SLN and completion ALND groups, respectively. Intermediate-term follow-up information was available from 635 of 659 patients (96.4%) of enrolled patients. The following results were found in the SLN versus SLN and completion ALND group: presence of lymphedema (3.5% vs. 19.1%, P < 0.0001), impaired shoulder range of motion (3.5% vs. 11.3%, P < 0.0001), shoulder/arm pain (8.1% vs. 21.1%, P < 0.0001), and numbness (10.9% vs. 37.7%, P < 0.0001). No significant differences regarding postoperative morbidity after SLN biopsy were noticed between academic and nonacademic hospitals (P = 0.921). Conclusions:The morbidity after SLN biopsy alone is not negligible but significantly lower compared with level I and II ALND. SLN biopsy can be performed with similar short- and intermediate-term morbidity in academic and nonacademic centers.

High Ep-CAM Expression is Associated with Poor Prognosis in Node-positive Breast Cancer

Previous studies in small series of patients with invasive breast cancer suggested a prognostic value of Ep-CAM overexpression in primary tumor tissue. To corroborate these findings, we performed a retrospective analysis of Ep-CAM expression using a tissue microarray containing tissue specimens from a large patient set. Ep-CAM expression was evaluated by immunohistochemistry in breast cancer tissue from 1715 patients with documented raw survival data. High level Ep-CAM expression (overexpression) was found in 41.7% of tumor samples, low level expression was found in 48.0% and no expression in 10.3% of tumor samples. Ep-CAM expression predicted poor overall survival in this patient cohort (p < 0.0001). Overall survival decreased significantly with increasing Ep-CAM expression. However, in this patient sample Ep-CAM expression was not an independent prognostic marker by multivariate analysis. Subgroup analysis revealed that Ep-CAM expression was a prognostic marker in node-positive (p < 0.0001) but not in node-negative (p= 0.58) breast cancer patients. Intriguingly, Ep-CAM expression was predictive for a dismal prognosis in patients receiving adjuvant cytotoxic (p= 0.03) or hormonal therapy (p < 0.0001) but not in untreated patients (p= 0.41). In summary, this study provides strong evidence that expression of Ep-CAM is a powerful marker of poor prognosis in node-positive invasive breast carcinoma and a potential predictive marker of sensitivity to adjuvant hormonal and/or cytotoxic treatment modalities.

Clinical impact of programmed cell death ligand 1 expression in colorectal cancer.

BACKGROUND Programmed cell death 1 (PD-1) receptor triggering by PD ligand 1 (PD-L1) inhibits T cell activation. PD-L1 expression was detected in different malignancies and associated with poor prognosis. Therapeutic antibodies inhibiting PD-1/PD-L1 interaction have been developed. MATERIALS AND METHODS A tissue microarray (n=1491) including healthy colon mucosa and clinically annotated colorectal cancer (CRC) specimens was stained with two PD-L1 specific antibody preparations. Surgically excised CRC specimens were enzymatically digested and analysed for cluster of differentiation 8 (CD8) and PD-1 expression. RESULTS Strong PD-L1 expression was observed in 37% of mismatch repair (MMR)-proficient and in 29% of MMR-deficient CRC. In MMR-proficient CRC strong PD-L1 expression correlated with infiltration by CD8(+) lymphocytes (P = 0.0001) which did not express PD-1. In univariate analysis, strong PD-L1 expression in MMR-proficient CRC was significantly associated with early T stage, absence of lymph node metastases, lower tumour grade, absence of vascular invasion and significantly improved survival in training (P = 0.0001) and validation (P = 0.03) sets. A similar trend (P = 0.052) was also detectable in multivariate analysis including age, sex, T stage, N stage, tumour grade, vascular invasion, invasive margin and MMR status. Interestingly, programmed death receptor ligand 1 (PDL-1) and interferon (IFN)-γ gene expression, as detected by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) in fresh frozen CRC specimens (n = 42) were found to be significantly associated (r = 0.33, P = 0.03). CONCLUSION PD-L1 expression is paradoxically associated with improved survival in MMR-proficient CRC.

Axillary recurrence rate in breast cancer patients with negative sentinel lymph node (SLN) or SLN micrometastases: prospective analysis of 150 patients after SLN biopsy.

Objective:To assess the axillary recurrence rate in breast cancer patients with negative sentinel lymph node (SLN) or SLN micrometastases (>0.2 mm to ≤2.0 mm) after breast surgery and SLN procedure without formal axillary lymph node dissection (ALND). Summary Background Data:Under controlled study conditions, the SLN procedure proved to be a reliable method for the evaluation of the axillary nodal status in patients with early-stage invasive breast cancer. Axillary dissection of levels I and II can thus be omitted if the SLN is free of macrometastases. The prognostic value and potential therapeutic consequences of SLN micrometastases, however, remain a matter of great debate. We present the follow-up data of our prospective SLN study, particularly focusing on the axillary recurrence rate in patients with negative SLN and SLN micrometastases. Methods:In this prospective study, 236 SLN procedures were performed in 234 patients with early-stage breast cancer between April 1998 and September 2002. The SLN were marked and identified with 99m technetium-labeled colloid and blue dye (Isosulfanblue 1%). The excised SLNs were examined by step sectioning and stained with hematoxylin and eosin and immunohistochemistry (cytokeratin antibodies Lu-5 or CK 22). Only patients with SLN macrometastases received formal ALND of levels I and II, while patients with negative SLN or SLN micrometastases did not undergo further axillary surgery. Results:The SLN identification rate was 95% (224/236). SLN macrometastases were found in 33% (74/224) and micrometastases (>0.2 mm to ≤2 mm) in 12% (27/224) of patients. Adjuvant therapy did not differ between the group of SLN-negative patients and those with SLN micrometastases. After a median follow-up of 42 months (range 12–64 months), 99% (222/224) of evaluable patients were reassessed. While 1 patient with a negative SLN developed axillary recurrence (0.7%, 1/122), all 27 patients with SLN micrometastases were disease-free at the last follow-up control. Conclusions:Axillary recurrences in patients with negative SLN or SLN micrometastases did not occur more frequently after SLN biopsy alone compared with results from the recent literature regarding breast cancer patients undergoing formal ALND. Based on a median follow-up of 42 months—one of the longest so far in the literature—the present investigation does not provide evidence that the presence of SLN micrometastases leads to axillary recurrence or distant disease and supports the theory that formal ALND may be omitted in these patients.

Telomerase as a prognostic marker in breast cancer: high‐throughput tissue microarray analysis of hTERT and hTR

Telomerase activity (TA) has been shown to correlate with poor clinical outcome in various tumour entities, indicating that tumours expressing this enzyme may be more aggressive and that TA may be a useful prognostic marker. For breast cancer, however, TA is a controversial prognostic marker; whereas some studies suggest an association between TA and disease outcome, others do not find this association. This study used tissue microarrays (breast carcinoma prognosis arrays) containing 611 samples (each 0.6 mm in diameter) from the tumour centre of paraffin‐embedded breast carcinomas to analyse the catalytic subunit of telomerase, human telomerase reverse‐transcriptase (hTERT), and the internal RNA component (hTR), which are the core components of the telomerase holoenzyme complex. hTERT protein expression was obtained by immunohistochemistry (human anti‐telomerase antibody Ab‐2, Calbiochem), and hTR RNA was measured by radioactive in situ hybridization. hTERT and hTR expression were determined semi‐quantitatively and graded (scores 1–4). Clinical data, such as histological subtype, pT stage, tumour diameter, pN stage, BRE grade, tumour‐specific survival (in months), patients age and others, were available for statistical analysis. A statistically significant correlation was found between tumour‐specific survival (overall survival) and hTERT expression (p < 0.0001) or hTR expression (p = 0.00110). Tumours with higher scores (scores 3, 4) for hTR and/or hTERT were associated with a worse prognosis. In multivariate analysis, hTERT expression was an independent prognostic factor. Previous studies, focusing on analysis of TA in smaller numbers of fresh‐frozen breast carcinomas by the TRAP assay, gave controversial results with respect to TA as a prognostic marker. Using tissue microarrays from 611 breast carcinomas, this study has demonstrated that increased expression levels of the telomerase core components, hTERT and hTR, are associated with lower overall survival. These findings suggest that TA should be included in future validation studies as a prognostic marker in breast cancer. Copyright

Selective axillary surgery in breast cancer patients based on positron emission tomography with 18F-fluoro-2-deoxy-D-glucose: not yet!

We prospectively evaluated 31 patients with invasive breast cancer. Preoperative positron emission tomography (PET) with 18F-fluoro-2-deoxy-D-glucose (18F-FDG) for detection of axillary lymph node metastases was compared with the histopathologic status of the sentinel lymph node (SLN). Sensitivity of PET imaging was 43%, specificity and negative predictive value were 94 and 67%, respectively. The smallest metastasis detected by PET measured 3 mm in diameter. The results of this study suggest that detection of small axillary lymph node metastases is limited by the currently achievable spatial resolution of PET imaging. Selective axillary surgery in breast cancer patients based on 18F-FDG PET is yet not possible.

Tumor infiltration by FcγRIII (CD16)+ myeloid cells is associated with improved survival in patients with colorectal carcinoma

The prognostic significance of macrophage and natural killer (NK) cell infiltration in colorectal carcinoma (CRC) microenvironment is unclear. We investigated the CRC innate inflammatory infiltrate in over 1,600 CRC using two independent tissue microarrays and immunohistochemistry. Survival time was assessed using the Kaplan–Meier method and Cox proportional hazards regression analysis in a multivariable setting. Spearmans rank correlation tested the association between macrophage and lymphocyte infiltration. The Basel study included over 1,400 CRCs. The level of CD16+ cell infiltration correlated with that of CD3+ and CD8+ lymphocytes but not with NK cell infiltration. Patients with high CD16+ cell infiltration (score 2) survived longer than patients with low (score 1) infiltration (p = 0.008), while no survival difference between patients with score 1 or 2 for CD56+ (p = 0.264) or CD57+ cell (p = 0.583) infiltration was detected. CD16+ infiltrate was associated with improved survival even after adjusting for known prognostic factors including pT, pN, grade, vascular invasion, tumor growth and age [(p = 0.001: HR (95% CI) = 0.71 (0.6–0.9)]. These effects were independent from CD8+ lymphocyte infiltration [(p = 0.036: HR (95% CI) = 0.81 (0.7–0.9)] and presence of metastases [(p = 0.002: HR (95% CI) = 0.43 (0.3–0.7)]. Phenotypic studies identified CD16+ as CD45+CD33+CD11b+CD11c+ but CD64− HLA‐DR‐myeloid cells. Beneficial effects of CD16+ cell infiltration were independently validated by a study carried out at the University of Athens confirming that patients with CD16 score 2 survived longer than patients with score 1 CRCs (p = 0.011). Thus, CD16+ cell infiltration represents a novel favorable prognostic factor in CRC.

Identification of Sentinel Lymph Nodes in Colon Cancer Depends on the Amount of Dye Injected Relative to Tumor Size

Recent studies have shown that the sentinel lymph node (SLN) procedure might improve staging in colon cancer. However, low SLN identification and high false negative rates have also been reported. In a two-institution study, the SLN procedure with isosulfan blue 1% was performed according to a standardized protocol in 31 patients with open resections for colon cancer. Data were collected prospectively. The database was analyzed retrospectively to determine factors contributing to a low identification rate. The SLN identification rate was 87% and the false negative rate was 50%. Successful SLN identification was significantly associated with application of higher volumes of dye relative to the tumor diameter (p = 0.04) and more frequent tumor localization in the sigmoid colon (p = 0.04) as compared to missing SLN identification. The tumor diameter was not significantly different in the two groups. Sentinel lymph node identification in colon cancer depends on the amount of dye injected relative to the tumor size. Application of only 1 ml of dye—the amount generally recommended in the literature—is not sufficient in large tumors.