Annette Staebler

Sentinel-lymph-node biopsy in patients with breast cancer before and after neoadjuvant chemotherapy (SENTINA): a prospective, multicentre cohort study.

BACKGROUND The optimum timing of sentinel-lymph-node biopsy for breast cancer patients treated with neoadjuvant chemotherapy is uncertain. The SENTINA (SENTinel NeoAdjuvant) study was designed to evaluate a specific algorithm for timing of a standardised sentinel-lymph-node biopsy procedure in patients who undergo neoadjuvant chemotherapy. METHODS SENTINA is a four-arm, prospective, multicentre cohort study undertaken at 103 institutions in Germany and Austria. Women with breast cancer who were scheduled for neoadjuvant chemotherapy were enrolled into the study. Patients with clinically node-negative disease (cN0) underwent sentinel-lymph-node biopsy before neoadjuvant chemotherapy (arm A). If the sentinel node was positive (pN1), a second sentinel-lymph-node biopsy procedure was done after neoadjuvant chemotherapy (arm B). Women with clinically node-positive disease (cN+) received neoadjuvant chemotherapy. Those who converted to clinically node-negative disease after chemotherapy (ycN0; arm C) were treated with sentinel-lymph-node biopsy and axillary dissection. Only patients whose clinical nodal status remained positive (ycN1) underwent axillary dissection without sentinel-lymph-node biopsy (arm D). The primary endpoint was accuracy (false-negative rate) of sentinel-lymph-node biopsy after neoadjuvant chemotherapy for patients who converted from cN1 to ycN0 disease during neoadjuvant chemotherapy (arm C). Secondary endpoints included comparison of the detection rate of sentinel-lymph-node biopsy before and after neoadjuvant chemotherapy, and also the false-negative rate and detection rate of sentinel-lymph-node biopsy after removal of the sentinel lymph node. Analyses were done according to treatment received (per protocol). FINDINGS Of 1737 patients who received treatment, 1022 women underwent sentinel-lymph-node biopsy before neoadjuvant chemotherapy (arms A and B), with a detection rate of 99.1% (95% CI 98.3-99.6; 1013 of 1022). In patients who converted after neoadjuvant chemotherapy from cN+ to ycN0 (arm C), the detection rate was 80.1% (95% CI 76.6-83.2; 474 of 592) and false-negative rate was 14.2% (95% CI 9.9-19.4; 32 of 226). The false-negative rate was 24.3% (17 of 70) for women who had one node removed and 18.5% (10 of 54) for those who had two sentinel nodes removed (arm C). In patients who had a second sentinel-lymph-node biopsy procedure after neoadjuvant chemotherapy (arm B), the detection rate was 60.8% (95% CI 55.6-65.9; 219 of 360) and the false-negative rate was 51.6% (95% CI 38.7-64.2; 33 of 64). INTERPRETATION Sentinel-lymph-node biopsy is a reliable diagnostic method before neoadjuvant chemotherapy. After systemic treatment or early sentinel-lymph-node biopsy, the procedure has a lower detection rate and a higher false-negative rate compared with sentinel-lymph-node biopsy done before neoadjuvant chemotherapy. These limitations should be considered if biopsy is planned after neoadjuvant chemotherapy. FUNDING Brustkrebs Deutschland, German Society for Senology, German Breast Group.

Distinction of endocervical and endometrial adenocarcinomas: Immunohistochemical p16 expression correlated with human papillomavirus (HPV) DNA detection

Determining the origin of uterine adenocarcinomas can be difficult in biopsy and curettage specimens because the morphologic spectrum of endocervical and endometrial adenocarcinomas overlaps. In hysterectomy specimens, the primary site is often equivocal for tumors that involve the lower uterine segment and endocervix and lack identifiable precursor lesions. Most endocervical adenocarcinomas (ECAs) contain high-risk human papillomavirus (HPV) DNA, whereas endometrial adenocarcinomas (EMAs) rarely do. p16 is an inhibitor of cyclin-dependent kinases, and overexpression of p16 has been observed in cervical intraepithelial lesions and invasive carcinomas associated with high-risk HPV types. We evaluated the utility of immunohistochemistry for p16 in the distinction of ECAs and EMAs. p16 expression was assessed in 24 unequivocal EMAs and 19 unequivocal ECAs and correlated with HPV DNA detection by in situ hybridization and polymerase chain reaction. These assays were then used to assist in the classification of four lower uterine segment/upper endocervical adenocarcinomas (LUS/EC-A) of equivocal origin. p16 expression was moderate-strong and diffuse in 18 ECAs (median 90% of tumor cells positive, range 90%-100%), and weak and diffuse in one. Fourteen of these were positive for HPV DNA, whereas 5 lacked detectable HPV DNA by in situ hybridization; one of these 5 was positive by polymerase chain reaction. In contrast, EMAs displayed weaker staining with patchy distribution (median 30% of tumor cells positive, range 5%-70%) and none contained HPV DNA by in situ hybridization. Two LUS/EC-As, which were positive for HPV, exhibited strong, diffuse p16 expression, consistent with endocervical origin of the tumors. The remaining 2 LUS/EC-As showed patchy p16 staining and did not contain detectable HPV DNA, consistent with the endometrial origin of the tumors. The p16 expression pattern can distinguish ECAs from EMAs. Compared with HPV DNA detection by in situ hybridization, p16 immunohistochemistry appears to be a more sensitive and easier to perform method for distinguishing ECAs from EMAs, can be used to assist in the classification of LUS/EC-As of equivocal origin, and should be evaluated for its utility in the prospective classification of uterine adenocarcinomas in curettage specimens prior to hysterectomy.

SOX2 gene amplification and protein overexpression are associated with better outcome in squamous cell lung cancer

The transcription factor SOX2 (3q26.3–q27) is a key regulator of foregut development and an embryonic stem cell factor cooperating during induction of pluripotency in terminally differentiated somatic cells. Recently, we found SOX2 to be amplified in a subset of squamous cell lung and esophageal cancers. The aim of this study was to explore the prognostic role of SOX2 in a large series of squamous cell carcinomas and adenocarcinomas of the lung. A total of 891 samples from two independent population-based cohorts were assessed by fluorescence in situ hybridization and immunohistochemistry. Furthermore, we assessed for associations between SOX2 amplification/upregulation and clinicopathological features. Similar results were found in the two cohorts. Within squamous cell carcinoma cases, 8% high-level as well as 68 and 65% low-level SOX2 amplifications occurred in the two cohorts, respectively. In adenocarcinomas, no high-level amplification was found and low-level amplification occurred in 6% of the two cohorts. Within squamous cell carcinomas of one cohort, SOX2 amplification was associated with lower tumor grade, while higher levels of SOX2 expression were related to younger age, smaller tumor size, and lower probability of angiolymphatic invasion and metastasis. High SOX2 expression levels proved to be a marker for prolonged overall survival among patients with squamous cell carcinomas. In conclusion, SOX2 amplification and upregulation are frequent events in squamous cell carcinomas of the lung and are associated with indicators of favorable prognosis.

Expression of the embryonic stem cell marker SOX2 in early-stage breast carcinoma

BackgroundThe SRY-related HMG-box family of transcription factors member SOX2 has been mainly studied in embryonic stem cells as well as early foregut and neural development. More recently, SOX2 was shown to participate in reprogramming of adult somatic cells to a pluripotent stem cell state and implicated in tumorigenesis in various organs. In breast cancer, SOX2 expression was reported as a feature of basal-like tumors. In this study, we assessed SOX2 expression in 95 primary tumors of postmenopausal breast cancer patients.MethodsSamples from 95 patients diagnosed and treated at the University of Tuebingen Institute of Pathology and Womens Hospital were analyzed by immunohistochemistry for SOX2 expression in the primary tumor samples and in corresponding lymph node metastasis, where present. Furthermore, SOX2 amplification status was assessed by FISH in representative samples. In addition, eighteen fresh frozen samples were analyzed for SOX2, NANOG and OCT4 gene expression by real-time PCR.ResultsSOX2 expression was detected in 28% of invasive breast carcinoma as well as in 44% of ductal carcinoma in situ (DCIS) lesions. A score of SOX2 expression (score 0 to 3) was defined in order to distinguish SOX2 negative (score 0) from SOX2 positive samples (score 1-3) and among latter the subgroup of SOX2 high expressors (score 3 > 50% positive cells). Overall, the incidence of SOX2 expression (score 1-3) was higher than previously reported in a cohort of lymph node negative patients (28% versus 16.7%). SOX2 expression was detected across different breast cancer subtypes and did not correlate with tumor grading. However, high SOX2 expression (score 3) was associated with larger tumor size (p = 0.047) and positive lymph node status (0.018). Corresponding metastatic lymph nodes showed higher SOX2 expression and were significantly more often SOX2 positive than primary tumors (p = 0.0432).ConclusionsIn this report, we show that the embryonic stem cell factor SOX2 is expressed in a variety of early stage postmenopausal breast carcinomas and metastatic lymph nodes. Our data suggest that SOX2 plays an early role in breast carcinogenesis and high expression may promote metastatic potential. Further studies are needed to explore whether SOX2 can predict metastatic potential at an early tumor stage.

Hormone receptor immunohistochemistry and human papillomavirus in situ hybridization are useful for distinguishing endocervical and endometrial adenocarcinomas

Determining the origin of uterine adenocarcinomas can be difficult in biopsy and curettage specimens because the morphologic spectrum of endocervical and endometrial adenocarcinomas overlaps. In addition, in hysterectomy specimens the primary site is often equivocal for tumors that involve predominantly the lower uterine segment and endocervix and lack identifiable precursor lesions. We assessed the value of immunohistochemistry for estrogen and progesterone receptors and in situ hybridization for human papillomavirus DNA detection in making this clinically relevant distinction. We evaluated a set of 48 adenocarcinomas of unequivocal origin (24 endocervical carcinomas and 24 endometrial endometrioid carcinomas without cervical extension) and then tested seven lower uterine segment/endocervical carcinomas of equivocal origin to determine whether patterns established in the initial set would permit definitive assignment of primary site for the equivocal set. Only one (4.2%) of 24 endocervical carcinomas was positive for both estrogen receptor and progesterone receptor, whereas 18 (75%) of 24 endometrial carcinomas were positive for estrogen receptor and 23 (95.8%) of 24 endometrial carcinomas were positive for progesterone receptor (p <0.001, &khgr;2 test). Human papillomavirus DNA was detected in 16 (66.7%) of 24 endocervical carcinomas and in none of 24 endometrial carcinomas (p <0.001, &khgr;2 test). Of the seven tumors of equivocal origin, five could be definitively classified as either endocervical or endometrial in origin based on their demonstration of a characteristic profile with these assays (either estrogen receptor/progesterone receptor-negative/human papillomavirus-positive, consistent with endocervical origin or estrogen receptor/progesterone receptor-positive/human papillomavirus-negative, consistent with endometrial origin). We conclude that hormone receptor immunohistochemistry and human papillomavirus in situ hybridization are useful for distinguishing endocervical and endometrial adenocarcinomas. The clinical utility of these techniques should be evaluated in studies that include curettage and biopsy specimens.

SOX2 amplification is a common event in squamous cell carcinomas of different organ sites

Acquired chromosomal aberrations, including gene copy number alterations, are involved in the development and progression of human malignancies. SOX2, a transcription factor-coding gene located at 3q26.33, is known to be recurrently and specifically amplified in squamous cell carcinomas of the lung, the esophagus, and the oral cavity. In these organs, the SOX2 protein plays an important role in tumorigenesis and tumor survival. The aim of this study was to determine whether SOX2 amplification is also found in squamous cell carcinomas in other organs commonly affected by this tumor entity. In addition, we examined a large spectrum of lung cancer entities with neuroendocrine differentiation (ie, small cell cancers, large cell cancers, typical and atypical carcinoids) for SOX2 and TTF1 copy number gains to reveal potential molecular ties to squamous cell carcinomas or adenocarcinomas of the lung. Applying fluorescence in situ hybridization, we assessed squamous cell carcinomas of the cervix uteri (n = 47), the skin (n = 57), and the penis (n = 53) for SOX2 copy number alterations and detected amplifications in 28%, 28%, and 32% of tumors, respectively. Furthermore, we performed immunohistochemical SOX2 staining and found that SOX2 amplification is significantly associated with overexpression of the corresponding protein in squamous cell carcinomas (P < .001). Of the lung cancer entities with neuroendocrine differentiation, only small cell cancers and large cell cancers exhibited SOX2 or TTF1 amplifications at significant frequencies, indicating that at least a subset of these might be dedifferentiated forms of squamous cell carcinomas or adenocarcinomas of the lung. We conclude that SOX2 amplification and consequent SOX2 protein overexpression may represent important mechanisms of tumor initiation and progression in a considerable subset of squamous cell carcinomas.

ERalpha-status of disseminated tumour cells in bone marrow of primary breast cancer patients

IntroductionIsolated disseminated tumour cells (DTC) are regarded as surrogate markers for minimal residual disease in breast cancer. Characterisation of these cells could help understand the known limitations of adjuvant therapy. Of particular interest is their oestrogen-receptor (ER) status because endocrine adjuvant therapy remains a cornerstone of breast cancer treatment.MethodsBone marrow (BM) aspirates from 254 patients with primary breast cancer were included in this study. A double immunofluorescence staining procedure was established for the identification of cytokeratin (CK) positive/Erα-positive cells. ERα status of the primary tumour was assessed immunohistochemically using the same antibody against ERα.ResultsIn 107 of 254 (42%) breast cancer patients, CK-positive cells could be detected in the BM. More than one DTC in the BM was observed in 38 of the 107 patients. The number of detected cells ranged between 1 and 55 cells per 2 × 106 mononuclear cells. DTCs demonstrated ERα positivity in 12% of the patients. The ERα expression was heterogeneous in 10 of the 38 (26%) patients with more than one DTC. The concordance rate of ERα status between primary tumour and DTC was 28%. Only 12 of 88 patients with ERα-positive tumours also had ERα-positive DTCs.ConclusionsPrimary tumours and DTCs displayed a concordant ERα status in only 28% of cases. Most of the DTCs were ERα negative despite the presence of an ERα-positive primary tumour. These findings further underline the distinct nature of DTCs and may explain the failure rates seen in conventional endocrine adjuvant therapy.

SOX2 Expression Associates with Stem Cell State in Human Ovarian Carcinoma

The SRY-related HMG-box family of transcription factors member SOX2 regulates stemness and pluripotency in embryonic stem cells and plays important roles during early embryogenesis. More recently, SOX2 expression was documented in several tumor types including ovarian carcinoma, suggesting an involvement of SOX2 in regulation of cancer stem cells (CSC). Intriguingly, however, studies exploring the predictive value of SOX2 protein expression with respect to histopathologic and clinical parameters report contradictory results in individual tumors, indicating that SOX2 may play tumor-specific roles. In this report, we analyze the functional relevance of SOX2 expression in human ovarian carcinoma. We report that in human serous ovarian carcinoma (SOC) cells, SOX2 expression increases the expression of CSC markers, the potential to form tumor spheres, and the in vivo tumor-initiating capacity, while leaving cellular proliferation unaltered. Moreover, SOX2-expressing cells display enhanced apoptosis resistance in response to conventional chemotherapies and TRAIL. Hence, our data show that SOX2 associates with stem cell state in ovarian carcinoma and induction of SOX2 imposes CSC properties on SOC cells. We propose the existence of SOX2-expressing ovarian CSCs as a mechanism of tumor aggressiveness and therapy resistance in human SOC.

Measurement of tumour size with mammography, sonography and magnetic resonance imaging as compared to histological tumour size in primary breast cancer

BackgroundTumour size in breast cancer influences therapeutic decisions. The purpose of this study was to evaluate sizing of primary breast cancer using mammography, sonography and magnetic resonance imaging (MRI) and thereby establish which imaging method most accurately corresponds with the size of the histological result.MethodsData from 121 patients with primary breast cancer were analysed in a retrospective study. The results were divided into the groups “ductal carcinoma in situ (DCIS)”, invasive ductal carcinoma (IDC) + ductal carcinoma in situ (DCIS)”, “invasive ductal carcinoma (IDC)”, “invasive lobular carcinoma (ILC)” and “other tumours” (tubular, medullary, mucinous and papillary breast cancer). The largest tumour diameter was chosen as the sizing reference in each case. Bland-Altman analysis was used to determine to what extent the imaging tumour size correlated with the histopathological tumour sizes.ResultsTumour size was found to be significantly underestimated with sonography, especially for the tumour groups IDC + DCIS, IDC and ILC. The greatest difference between sonographic sizing and actual histological tumour size was found with invasive lobular breast cancer. There was no significant difference between mammographic and histological sizing. MRI overestimated non-significantly the tumour size and is superior to the other imaging techniques in sizing of IDC + DCIS and ILC.ConclusionsThe histological subtype should be included in imaging interpretation for planning surgery in order to estimate the histological tumour size as accurately as possible.

Detection of the BRAF V600E mutation in serous ovarian tumors: a comparative analysis of immunohistochemistry with a mutation-specific monoclonal antibody and allele-specific PCR

Mutations of components of the mitogen-activated protein kinase pathway, mainly BRAF, are common in serous ovarian borderline tumors, whereas high-grade serous ovarian carcinomas rarely show this feature. With the advent of specific kinase inhibitors active against BRAF-mutated cancers, rapid and sensitive detection of the BRAF V600E, by far the most common mutation of this gene, is of great practical relevance. Currently, BRAF mutations are detected by DNA-based techniques. Recently, a monoclonal antibody (VE1) specific for the BRAF V600E protein suitable for archival tissues has been described. In this study, we compared detection of the V600E mutation in serous ovarian tumors by VE1 immunostaining and by allele-specific polymerase chain reaction. All 141 cases of high-grade serous ovarian cancer showed negative or rarely weak, diffuse background VE1 immunostaining, and BRAF wild type was confirmed by molecular analysis in all tested cases. In contrast, 1 (14%) of 7 low-grade serous carcinomas and 22 (71%) of 31 serous borderline tumors revealed moderate to strong VE1 positivity. Immunostaining was clearly evaluable in all cases with sufficient tumor cells, and only rare cases with narrow cytoplasm were difficult to interpret. The V600E mutation was confirmed by allele-specific polymerase chain reaction and sequencing in all VE1-positive cases. Two VE1-positive cases with low epithelial cell content required repeat microdissection to confirm the presence of the mutation. Immunohistochemistry with the VE1 antibody is a specific and sensitive tool for detection of the BRAF V600E mutation in serous ovarian tumors and may provide a practical screening test, especially in tumor samples with low epithelial content.