The dynamic range of circulating tumor DNA in metastatic breast cancer.

INTRODUCTION: The management of metastatic breast cancer needs improvement. As clinical evaluation is not very accurate in determining the progression of disease, the analysis of circulating tumor DNA (ctDNA) has evolved to a promising noninvasive marker of disease evolution. Indeed, ctDNA was reported to represent a highly sensitive biomarker of metastatic cancer disease directly reflecting tumor burden and dynamics. However, at present little is known about the dynamic range of ctDNA in patients with metastatic breast cancer. METHODS: In this study, 74 plasma DNA samples from 58 patients with metastasized breast cancer were analyzed with a microfluidic device to determine the plasma DNA size distribution and copy number changes in the plasma were identified by whole-genome sequencing (plasma-Seq). Furthermore, in an index patient we conducted whole-genome, exome, or targeted deep sequencing of the primary tumor, metastases, and circulating tumor cells (CTCs). Deep sequencing was done to accurately determine the allele fraction (AFs) of mutated DNA fragments. RESULTS: Although all patients had metastatic disease, plasma analyses demonstrated highly variable AFs of mutant fragments. We analyzed an index patient with more than 100,000 CTCs in detail. We first conducted whole-genome, exome, or targeted deep sequencing of four different regions from the primary tumor and three metastatic lymph node regions, which enabled us to establish the phylogenetic relationships of these lesions, which were consistent with a genetically homogeneous cancer. Subsequent analyses of 551 CTCs confirmed the genetically homogeneous cancer in three serial blood analyses. However, the AFs of ctDNA were only 2% to 3% in each analysis, neither reflecting the tumor burden nor the dynamics of this progressive disease. These results together with high-resolution plasma DNA fragment sizing suggested that differences in phagocytosis and DNA degradation mechanisms likely explain the variable occurrence of mutated DNA fragments in the blood of patients with cancer. CONCLUSIONS: The dynamic range of ctDNA varies substantially in patients with metastatic breast cancer. This has important implications for the use of ctDNA as a predictive and prognostic biomarker.

Establishment of tumor-specific copy number alterations from plasma DNA of patients with cancer.

With the increasing number of available predictive biomarkers, clinical management of cancer is becoming increasingly reliant on the accurate serial monitoring of tumor genotypes. We tested whether tumor-specific copy number changes can be inferred from the peripheral blood of patients with cancer. To this end, we determined the plasma DNA size distribution and the fraction of mutated plasma DNA fragments with deep sequencing and an ultrasensitive mutation-detection method, i.e., the Beads, Emulsion, Amplification, and Magnetics (BEAMing) assay. When analyzing the plasma DNA of 32 patients with Stage IV colorectal carcinoma, we found that a subset of the patients (34.4%) had a biphasic size distribution of plasma DNA fragments that was associated with increased circulating tumor cell numbers and elevated concentration of mutated plasma DNA fragments. In these cases, we were able to establish genome-wide tumor-specific copy number alterations directly from plasma DNA. Thus, we could analyze the current copy number status of the tumor genome, which was in some cases many years after diagnosis of the primary tumor. An unexpected finding was that not all patients with progressive metastatic disease appear to release tumor DNA into the circulation in measurable quantities. When we analyzed plasma DNA from 35 patients with metastatic breast cancer, we made similar observations suggesting that our approach may be applicable to a variety of tumor entities. This is the first description of such a biphasic distribution in a surprisingly high proportion of cancer patients which may have important implications for tumor diagnosis and monitoring.

Multiplex genetic cancer testing identifies pathogenic mutations in TP53 and CDH1 in a patient with bilateral breast and endometrial adenocarcinoma.

BACKGROUND: Germline genetic testing for familial cancer syndromes is usually performed serially for the most likely genetic causes. In recent years the way genetic testing carried out has changed, as next generation sequencing now allows the simultaneous testing of multiple susceptibility genes at low costs. CASE PRESENTATION: Here, we present a female with bilateral breast cancer and endometrial adenocarcinoma. After simultaneous sequencing of 150 genes (890 kb) associated with hereditary cancer we identified pathogenic mutations in two high-penetrance genes, i.e. TP53 and CDH1 that would most likely not have been elucidated by serial screening of candidate genes. CONCLUSION: As the two mutated genes are located on different chromosomes and cause different cancer syndromes these findings had a tremendous impact not only on genetic counseling of the index patient and her family but also on subsequent surveillance strategies.

Adjuvant endocrine therapy plus zoledronic acid in premenopausal women with early-stage breast cancer: 62-month follow-up from the ABCSG-12 randomised trial.

BACKGROUND: Analysis of the Austrian Breast and Colorectal Cancer Study Group trial-12 (ABCSG-12) at 48 months' follow-up showed that addition of zoledronic acid to adjuvant endocrine therapy significantly improved disease-free survival. We have now assessed long-term clinical efficacy including disease-free survival and disease outcomes in patients receiving anastrozole or tamoxifen with or without zoledronic acid. METHODS: ABSCG-12 is a randomised, controlled, open-label, two-by-two factorial, multicentre trial in 1803 premenopausal women with endocrine-receptor-positive early-stage (stage I-II) breast cancer receiving goserelin (3.6 mg every 28 days), comparing the efficacy and safety of anastrozole (1 mg per day) or tamoxifen (20 mg per day) with or without zoledronic acid (4 mg every 6 months) for 3 years. Randomisation (1:1:1:1 ratio) was computerised and based on the Pocock and Simon minimisation method to balance the four treatment arms across eight prognostic variables (age, neoadjuvant chemotherapy, pathological tumour stage; lymph-node involvement, type of surgery or locoregional therapy, complete axillary dissection, intraoperative radiation therapy, and geographical region). Treatment allocation was not masked. The primary endpoint was disease-free survival (defined as disease recurrence or death) and analysis was by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00295646; follow-up is ongoing. FINDINGS: At a median follow-up of 62 months (range 0-114.4 months), more than 2 years after treatment completion, 186 disease-free survival events had been reported (53 events in 450 patients on tamoxifen alone, 57 in 453 patients on anastrozole alone, 36 in 450 patients on tamoxifen plus zoledronic acid, and 40 in 450 patients on anastrozole plus zoledronic acid). Zoledronic acid reduced risk of disease-free survival events overall (HR 0.68, 95% CI 0.51-0.91; p=0.009), although the difference was not significant in the tamoxifen (HR 0.67, 95% CI 0.44-1.03; p=0.067) and anastrozole arms (HR 0.68, 95% CI 0.45-1.02; p=0.061) assessed separately. Zoledronic acid did not significantly affect risk of death (30 deaths with zoledronic acid vs 43 deaths without; HR 0.67, 95% CI 0.41-1.07; p=0.09). There was no difference in disease-free survival between patients on tamoxifen alone versus anastrozole alone (HR 1.08, 95% CI 0.81-1.44; p=0.591), but overall survival was worse with anastrozole than with tamoxifen (46 vs 27 deaths; HR 1.75, 95% CI 1.08-2.83; p=0.02). Treatments were generally well tolerated, with no reports of renal failure or osteonecrosis of the jaw. Bone pain was reported in 601 patients (33%; 349 patients on zoledronic acid vs 252 not on the drug), fatigue in 361 (20%; 192 vs 169), headache in 280 (16%; 147 vs 133), and arthralgia in 266 (15%; 145 vs 121). INTERPRETATION: Addition of zoledronic acid improved disease-free survival in the patients taking anastrozole or tamoxifen. There was no difference in disease-free survival between patients receiving anastrozole and tamoxifen overall, but those on anastrozole alone had inferior overall survival. These data show persistent benefits with zoledronic acid and support its addition to adjuvant endocrine therapy in premenopausal patients with early-stage breast cancer. FUNDING: AstraZeneca; Novartis.

Androgen receptor expression in breast cancer patients tested for BRCA1 and BRCA2 mutations.

AIM: To assess the expression of receptors for androgen (AR), oestrogen (ER) and progesterone (PR) as well as human epidermal growth factor receptor type 2 (Her-2/neu) status of breast carcinomas in breast cancer susceptibility gene (BRCA) BRCA1/2 mutation carriers and BRCA1/2 negative tested women. METHODS: One hundred and thirty-five breast cancers in women tested for BRCA1/2 mutations. Screening for BRCA1 and BRCA2 mutations was performed by direct sequencing of all BRCA1 and BRCA2 exons as well as the surrounding intronic sequences. Additionally, BRCA genes were analysed with multiplex ligation-dependent probe amplification. Consecutive paraffin sections were examined immunohistochemically for AR, ER, PR and Her-2/neu. RESULTS: Of the 135 tumours, 43 (32%) were BRCA1-related, 18 (13%) were BRCA2-related and 74 (55%) were BRCA1/2-negative. Seventy-two per cent of the BRCA1-related, 22% of the BRCA2-related and 12% of the BRCA1/2-negative tumours were triple (ER, PR, Her2neu)-negative. Eighty-four per cent of BRCA1 mutated cancers were high-grade (G3) tumours. ARs were expressed in 30% (13 of 43) of BRCA1-related, in 78% (14 of 18) in BRCA2-related tumours and in 76% (56 of 74) in BRCA1/2 negative tumours. Twenty-one per cent of ER-negative BRCA1-related tumours expressed androgen receptors. CONCLUSION: Approximately one in five BRCA1 mutated breast cancers negative for ER and PR express androgen receptors. Modulation of AR might open a new avenue for treating these high-risk cancers.

Bilateral ovarian well-differentiated Sertoli-Leydig cell tumors with heterologous elements associated with unilateral serous cystadenoma--a case report.

Bilateral Sertoli-Leydig cell tumors (SLCTs) of the ovary, especially in association with a cystadenoma, are exceedingly rare. Some SLCTs, usually of poor differentiation, show heterologous elements. We present a case of a 61-year-old woman with bilateral well-differentiated SLCTs in which the Sertoli-Leydig cell component showed leiomyogenic (heterologous) differentiation. Furthermore, on the left side it also was associated with a serous cystadenoma.

[Genes beyond BRCA1 and BRCA2 for hereditary breast cancer]. / Vererbbarer brustkrebs jenseits von BRCA1- und BRCA2-genmutationen.

BACKGROUND: germline mutations in the tumor suppressor genes BRCA1 and BRCA2 are identified in less than 50% of hereditary breast cancer cases. Besides BRCA1/2 further high-risk breast cancer genes are known; however they account only for a small fraction of inherited breast cancer cases. Most of them are involved in rare cancer predisposition syndromes. Moderate and low-risk breast cancer genes confer modest cancer risk up to 10% and may be more relevant due to polygenic inheritance. The majority of hereditary breast cancer cases are still caused by unknown genes. CLINICAL IMPLICATIONS: genetic testing of other known genes is not yet routinely performed in families tested negative for BRCA1/2-mutations, but can be recommended in special patients. In case of a calculated high-risk situation, participation in an early-detection screening program should be recommended. CONCLUSIONS: genetic susceptibility to breast cancer is heterogeneous and conferred by a large number of identified and yet undetected genes.

[BRCA1- and BRCA2 mutations: Clinical management of patients with hereditary breast and ovarian cancer]. / BRCA1- und BRCA2-Genmutationen: Klinische Konsequenzen beim vererbbaren Brust- und Eierstockkrebs.

BACKGROUND: In total, 5-10% of all breast cancer cases are related to gen mutations. In most cases a mutation in the BRCA1-gen and BRCA2-gen is responsible for insufficient repair of DNA damages that cause breast and ovarian cancer. CLINICAL MANAGEMENT: In patients carrying BRCA1-mutation the risk for developing breast and ovarian cancer is 87% and 40% as well as 47% and 20% for those carrying a BRCA2-mutation. Women at hereditary risk should be informed about existing recommendations for surveillance. Primary prevention of breast and ovarian cancer including prophylactic surgery (bilateral salpingoophorectomy and bilateral mastectomy) should be explained to mutation carriers. The issue of oral antihormonal therapy for prevention of breast cancer should be addressed. Psycho-social support should be offered to mutation carriers. CONCLUSIONS: The clinical management of BRCA1 and BRCA2-mutation carriers is very challenging and should be done in centres specialized in this issue.

Lymphoscintigraphy for Sentinel Lymph Node Biopsy in Breast Cancer: Do We Need a Delayed Image?

BACKGROUND: Sentinel lymph node biopsy has become a standard of care in the treatment of patients with early breast cancer, but clinical guidelines continue to be vague on details of the procedure. We were interested in the results of our 2-day protocol, which includes delayed lymphoscintigraphy at 18 h. METHODS: We reviewed the results of preoperative lymphoscintigrams in patients undergoing surgery for breast cancer. Lymphoscintigraphy was performed 2 h after periareolar injection of 4 × 37 MBq 99mTc nanocolloid (early lymphoscintigraphy) and 18 h following injection (delayed lymphoscintigraphy). The early results were compared with the late results. RESULTS: A total of 238 lymphoscintigraphies were performed in 232 patients (6 bilateral). At 2 h, ≥1 sentinel nodes were visualized in 154/238 (65%) cases; in 84 (35%), no sentinel node was visualized. Delayed lymphoscintigraphy visualized a sentinel node in 40 of 76 (53%) cases with no visualization at 2 h and failed to show a sentinel node in 36 (47%) of these cases (in 8 cases, no delayed lymphoscintigram was obtained). CONCLUSIONS: Delayed lymphoscintigraphy was useful in about 50% of the breast cancer patients in whom immediate scintigraphy failed to demonstrate a sentinel lymph node.

Publisher Correction: Inference of transcription factor binding from cell-free DNA enables tumor subtype prediction and early detection.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

How accurate is frozen section histology of pelvic lymph nodes in patients with endometrial cancer?

OBJECTIVE: Recent prospective data support the trend towards systematic retroperitoneal lymphadenectomy in patients with high-risk endometrial cancer. Because para-aortic node involvement in the absence of pelvic node involvement is uncommon, a reliable finding of negative pelvic lymph nodes (PLN) at intraoperative frozen section examination might allow omitting para-aortic dissection. We analyzed the diagnostic accuracy of frozen section examination of PLN in patients with endometrial cancer. METHODS: We reviewed 131 patients with endometrial cancer who underwent surgery including systematic pelvic lymphadenectomy (n=101) or pelvic and para-aortic lymphadenectomy (n=27). Intraoperative frozen section examination of PLN was performed in 72 (55%) patients. Results of frozen section examination were compared with those of final histopathology and the diagnostic accuracy of frozen section examination of PLN was calculated. One pathologist measured the diameters of PLN metastases retrospectively. RESULTS: A total of 1063 and 2666 PLN were analyzed by frozen section examination and by final histopathology, respectively. PLN metastases were found in 7 cases (10%) at frozen section examination, and in 17 cases (24%) at final histopathology (false negative rate, 59%). No false positive cases were noted. The mean diameter of all PLN metastases at final histopathology was 4.3 mm, as compared to 9.0 mm for the metastases detected at frozen section analyses. The mean diameter of PLN metastases missed at frozen section examination was 2.0 mm. CONCLUSION: In this review at a single institution, intraoperative frozen section histology missed nearly two of three endometrial cancer patients with positive nodes. These results do not support tailoring the extent of lymphadenectomy according to the results of frozen section examination.

Inference of transcription factor binding from cell-free DNA enables tumor subtype prediction and early detection.

Deregulation of transcription factors (TFs) is an important driver of tumorigenesis, but non-invasive assays for assessing transcription factor activity are lacking. Here we develop and validate a minimally invasive method for assessing TF activity based on cell-free DNA sequencing and nucleosome footprint analysis. We analyze whole genome sequencing data for >1,000 cell-free DNA samples from cancer patients and healthy controls using a bioinformatics pipeline developed by us that infers accessibility of TF binding sites from cell-free DNA fragmentation patterns. We observe patient-specific as well as tumor-specific patterns, including accurate prediction of tumor subtypes in prostate cancer, with important clinical implications for the management of patients. Furthermore, we show that cell-free DNA TF profiling is capable of detection of early-stage colorectal carcinomas. Our approach for mapping tumor-specific transcription factor binding in vivo based on blood samples makes a key part of the noncoding genome amenable to clinical analysis.

Neonatal outcome and two-year follow-up after expectant management of second trimester rupture of membranes.

OBJECTIVE: To assess neonatal outcome and 2-year follow-up of pregnancies complicated by second trimester preterm premature rupture of membranes (PPROM). METHODS: A retrospective review of obstetric and neonatal records for 87 pregnancies (56 singletons, 6 twins, 1 triplet) with PPROM between 14+0 and 24+6 weeks of gestation. Patients received antibiotics and steroids for fetal lung maturity once they reached 24 weeks of gestation. Placentas were examined histopathologically. Surviving infants were followed-up at 2 years of age. RESULTS: Median latency from PPROM to delivery was 4 days. Survival rate of 56 singletons was 45% (25/56); and 13 died in hospital. Survival rate of infants discharged from hospital was 23% (12/56). Chorioamnionitis was seen histologically in 42% (5/12) of surviving infants compared with 92% (12/13) of those that died in hospital. Of the 12 surviving infants, 50% had a normal neurological and developmental outcome at 2 years of age. CONCLUSION: Gestational age, birth weight, and histologic chorioamnionitis have prognostic importance in pregnancies complicated by PPROM. Surviving infants have a 50% chance of achieving an adequate health status at 2 years of age.

Neonatal outcome of second twins depending on presentation and mode of delivery.

Our objective was to examine the neonatal outcome of second twins depending on presentation and mode of delivery. Using a database we analyzed the short-term neonatal outcome in twin pregnancies offered a trial of labor with special emphasis on the second twin depending on presentation and mode of delivery. Neonatal outcome was evaluated by Apgar scores, umbilical cord blood pH values, and perinatal or neonatal morbidity and mortality. Overall, in 219 (78%) of 281 pregnancies successful vaginal birth (VB) of both twins (VB-VB) was possible, 48 (17%) women had to be delivered by cesarean section (CS) of both twins (CS-CS), and in 14 (5%) women the second twin had to be delivered by CS after VB of the first twin (VB-CS). Successful VB was most common for vertex-vertex (V/V; n=171, 82%) and vertex-nonvertex (n=48, 75%) presentation (V/NV). Twins delivered by VB-CS had the lowest values for pHart (p=.006) and pHven (p=.010). pHart less than or equal to 7.00 values occurred only in second twins delivered VB-VB or VB-CS. Lower Apgar scores of the second twin occurred more frequently in the VB-CS and in the VB-VB than in the CS-CS groups (ps<.05). Lower levels of pHart (p=.002) and frequency of pHart less than or equal to 7.00 occurred more often in nonvertex second twins than in vertex second twins (p<.022). The high CS rate in V/NV presentation and the significantly worse perinatal short-term outcome of NV second twins after VB of the first twin underline that randomized studies are necessary to evaluate the best delivery mode for V/NV twins.

Inferring expressed genes by whole-genome sequencing of plasma DNA.

The analysis of cell-free DNA (cfDNA) in plasma represents a rapidly advancing field in medicine. cfDNA consists predominantly of nucleosome-protected DNA shed into the bloodstream by cells undergoing apoptosis. We performed whole-genome sequencing of plasma DNA and identified two discrete regions at transcription start sites (TSSs) where nucleosome occupancy results in different read depth coverage patterns for expressed and silent genes. By employing machine learning for gene classification, we found that the plasma DNA read depth patterns from healthy donors reflected the expression signature of hematopoietic cells. In patients with cancer having metastatic disease, we were able to classify expressed cancer driver genes in regions with somatic copy number gains with high accuracy. We were able to determine the expressed isoform of genes with several TSSs, as confirmed by RNA-seq analysis of the matching primary tumor. Our analyses provide functional information about cells releasing their DNA into the circulation.

[Guideline for the prevention and early detection of breast and ovarian cancer in high risk patients, particularly in women from HBOC (hereditary breast and ovarian cancer) families]. / Leitlinie zur Prävention und Früherkennung von Brust- und Eierstockkrebs bei Hochrisikopatientinnen, insbesondere bei Frauen aus HBOC (Hereditary Breast and Ovarian Cancer) Familien.

The Austrian guideline for prevention and early detection of breast and ovarian cancer in high risk patients--particularly in women from hereditary breast and ovarian cancer families--were established with particular consideration of the most recent position paper of the European Society of Breast Cancer Specialists (EUSOMA) by the authors mentioned above. The guideline is aimed at facilitating and standardizing the care and early detection strategies in women with an elevated life time risk for breast and ovarian cancer.

Assessment of risk factors for survival of neonates born after second-trimester PPROM.

OBJECTIVE: Assess fetal risk factors which impact survival of infants delivered after second-trimester PPROM. STUDY DESIGN: Clinical records of 87 patients, who all had second-trimester rupture of membranes between 14+0 and 24+6 weeks of gestation treated January 1998 to July 2005 were reviewed regarding perinatal outcome. This study is based on 25 surviving infants. RESULTS: 13 of these 25 infants died in the hospital. Survivors had a higher birth weight (p=0.008) and higher Apgar scores after 5 min (p=0.005) than those infants dying. No differences in UA pH, the need of catecholamines and no association between histological verified chorioamnionitis and early onset sepsis were seen between survivors and nonsurvivors. CONCLUSION: Higher gestational age at birth, higher birth weight, the absence of histologically verified chorioamnionitis and 5 min Apgar scores of >or= than 6 have positive prognostic value for survival of neonates delivered preterm after second-trimester PPROM.

Is intraoperative frozen section analysis of pelvic lymph nodes accurate after neoadjuvant chemotherapy in patients with cervical cancer?

OBJECTIVE: Intraoperative frozen section examination of pelvic lymph nodes is frequently used in patients with cervical cancer, some of whom have received neoadjuvant chemotherapy (NACT). However, NACT can cause necrosis, fibrosis, or keratinization of tumor deposits in extirpated lymph nodes, and it is unclear whether intraoperative frozen section analysis of extirpated nodes is accurate after NACT. We analyzed the accuracy of frozen section examination of pelvic lymph nodes in patients after NACT for cervical cancer. METHODS: We reviewed 134 patients with invasive cervical cancer who underwent surgery including systematic pelvic lymphadenectomy with intraoperative frozen section examination of pelvic lymph nodes. Results of frozen section examination were related to definitive histology and compared between patient groups of NACT and primary surgery. RESULTS: A total of 1670 pelvic lymph nodes were evaluated intraoperatively by frozen section examination, and 6689 pelvic lymph nodes were analyzed by final histopathology. Overall frozen section analysis had nine false negative results among 53 patients with positive lymph nodes (false negative rate, 16.9%). After NACT, there were two false negative diagnoses in twelve patients with node metastases (false negative rate, 16.7%). No false positive cases were noted. The sensitivity and negative predictive value of frozen section examination were 83% and 82%, respectively, in patients after NACT, and 83% and 91% at primary surgery. CONCLUSION: NACT does not appear to compromise the diagnostic accuracy of intraoperative frozen section examination of pelvic lymph nodes in patients with cervical cancer.