Mutation profiling identifies numerous rare drug targets and distinct mutation patterns in different clinical subtypes of breast cancers

The mutation pattern of breast cancer molecular subtypes is incompletely understood. The purpose of this study was to identify mutations in genes that may be targeted with currently available investigational drugs in the three major breast cancer subtypes (ER+/HER2-, HER2+, and Triple Negative). We extracted DNA from fine needle aspirations of 267 stage I-III breast cancers. These tumor specimens typically consisted of >80% neoplastic cells. We examined 28 genes for 163 known cancer-related nucleic acid variations by Sequenom technology. We observed at least one mutation in 38 alleles corresponding to 15 genes in 108 (40%) samples, including PIK3CA (16.1% of all samples), FBXW7 (8%), BRAF (3.0%), EGFR (2.6%), AKT1 and CTNNB1 (1.9% each), KIT and KRAS (1.5% each), and PDGFR-α (1.1%). We also checked for the polymorphism in PHLPP2 that is known to activate AKT and it was found at 13.5% of the patient samples. PIK3CA mutations were more frequent in estrogen receptor-positive cancers compared to triple negative breast cancer (TNBC) (19 vs. 8%, p=0.001). High frequency of PIK3CA mutations (28%) were also found in HER2+ breast tumors. In TNBC, FBXW7 mutations were significantly more frequent compared to ER+ tumors (13 vs. 5%, p=0.037). We performed validation for all mutated alleles with allele-specific PCR or direct sequencing; alleles analyzed by two different sequencing techniques showed 95-100% concordance for mutation status. In conclusion, different breast cancer subtypes harbor different type of mutations and approximately 40 % of tumors contained individually rare mutations in signaling pathways that can be potentially targeted with drugs. Simultaneous testing of many different mutations in a single needle biopsy is feasible and allows the design of prospective clinical trials that could test the functional importance of these mutations in the future.     

原发灶不明转移癌的临床诊断

原发灶不明转移癌(CUP)是目前临床诊疗的一大难题,患者大多预后较差.综合患者的病史与体检、病理学、影像学以及内镜检查结果等相关信息,对部分患者可发现原发灶,但其检出率仍较低.随着分子医学的进步和正电子发射计算机体层摄影(PET-CT)的临床应用,CUP患者原发灶的检出率较前明显提高.     

Risk factors for cancers of unknown primary site: Results from the prospective EPIC cohort

Cancer of unknown primary site (CUP) may be called an “orphan” disease, as it is diagnosed when metastases are detected while the primary tumor typically remains undetected, and because little research has been done on its primary causes. So far, few epidemiological studies, if any, have addressed possible risk factors for CUP. We analyzed data from the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort (N = 476,940). During prospective follow‐up, a total of 651 cases of incident cases of CUP were detected (ICD‐O‐2 code C809). Proportional hazards models were conducted to examine the associations of lifetime history of smoking habits, alcohol consumption, levels of education and anthropometric indices of adiposity with risk of being diagnosed with CUP. Risk of being diagnosed with CUP was strongly related to smoking, with a relative risk of 3.66 [95% C.I., 2.24–5.97] for current, heavy smokers (26+ cigarettes/day) compared to never smokers (adjusted for alcohol consumption, body mass index, waist circumference and level of education) and a relative risk of 5.12 [3.09–8.47] for cases with CUP who died within 12 months. For alcohol consumption and level of education, weaker associations were observed but attenuated and no longer statistically significant after adjusting for smoking and indices of obesity. Finally, risk of CUP was increased by approximately 30 per cent for subjects in the highest versus lowest quartiles of waist circumference. Our analyses provide further documentation, in addition to autopsy studies, that a substantial proportion of cancers of unknown primary site may have their origin in smoking‐related tumors, in particular.     

18F-FDG PET-CT在原发灶不明转移性肿瘤诊断中的应用

目的 探讨18F-FDG PET-CT显像在原发灶不明转移性肿瘤中的应用价值.方法 应用PET-CT技术,对67例原发灶不明转移性肿瘤患者行显像检查,将结果与病理学检查或临床结果对照,并比较原发灶与转移灶标准摄取值(SUV)的相关性和差异.结果 67例患者中,18F-FDG PET-CT发现可疑原发灶39例,经病理或临床诊断证实36例,检出率为53.7%(36/67).新检出远处转移灶13个,淋巴结转移灶17个.原发灶SUV(6.3103±3.1162)与转移灶SUV(10.4586±5.6337)有相关性(r=0.738,P=0.000);原发灶SUV较转移灶SUV低,且差异有统计学意义(t=3.470,P=0.001).结论 18F-FIX;PET-ET对寻找转移性肿瘤的原发灶有重要价值,为寻找转移性肿瘤的原发灶提供了一种新的检查手段.临床上可充分利用功能显像的优势,进一步研究恶性肿瘤的生物学特性.     

Survival in cancer of unknown primary site: population-based analysis by site and histology

BackgroundCancer of unknown primary (CUP) is diagnosed at a metastatic stage, conferring an unfavorable prognosis. The natural history of the disease is poorly understood, which complicates diagnosis, treatment and follow-up. Population-based survival data are lacking regarding location and histology of metastases.Patients and methodsFrom the Swedish Cancer Registry, 18 911 CUP patients were identified between years 1987 and 2008. Survival was analyzed by Kaplan–Meier survival curves and Cox regression.ResultsAdenocarcinoma accounted for 70% of all extranodal cases with a 12-month survival of 17% and the median survival of 3 months. Adenocarcinoma was also the most common histology (33.4%) when metastases were limited to lymph nodes, with a 12-month survival of 41% and median survival of 8 months. For extranodal metastases, the extremes in survival were small intestinal cancer with poor prognosis and mediastinal cancer with favorable prognosis. For nodal metastases, patients affected in the head and neck, axillary and inguinal regions had the best prognosis and those with abdominal and intrapelvic metastases the worst prognosis.ConclusionsThe present data underline the importance of histology and location of metastasis in assisting clinical decision making: hazard ratios differed by a factor of five among extranodal and nodal metastases.     

Genomic scars as biomarkers of homologous recombination deficiency and drug response in breast and ovarian cancers

Poly (ADP-ribose) polymerase (PARP) inhibitors and platinum-based chemotherapies have been found to be particularly effective in tumors that harbor deleterious germline or somatic mutations in the BRCA1 or BRCA2 genes, the products of which contribute to the conservative homologous recombination repair of DNA double-strand breaks. Nonetheless, several setbacks in clinical trial settings have highlighted some of the issues surrounding the investigation of PARP inhibitors, especially the identification of patients who stand to benefit from such drugs. One potential approach to finding this patient subpopulation is to examine the tumor DNA for evidence of a homologous recombination defect. However, although the genomes of many breast and ovarian cancers are replete with aberrations, the presence of numerous factors able to shape the genomic landscape means that only some of the observed DNA abnormalities are the outcome of a cancer cell’s inability to faithfully repair DNA double-strand breaks. Consequently, recently developed methods for comprehensively capturing the diverse ways in which homologous recombination deficiencies may arise beyond BRCA1/2 mutation have used DNA microarray and sequencing data to account for potentially confounding features in the genome. Scores capturing telomeric allelic imbalance, loss of heterozygosity (LOH) and large scale transition score, as well as the total number of coding mutations are measures that summarize the total burden of certain forms of genomic abnormality. By contrast, other studies have comprehensively catalogued different types of mutational pattern and their relative contributions to a given tumor sample. Although at least one study to explore the use of the LOH scar in a prospective clinical trial of a PARP inhibitor in ovarian cancer is under way, limitations that result in a relatively low positive predictive value for these biomarkers remain. Tumors whose genome has undergone one or more events that restore high-fidelity homologous recombination are likely to be misclassified as double-strand break repair-deficient and thereby sensitive to PARP inhibitors and DNA damaging chemotherapies as a result of prior repair deficiency and its genomic scarring. Therefore, we propose that integration of a genomic scar-based biomarker with a marker of resistance in a high genomic scarring burden context may improve the performance of any companion diagnostic for PARP inhibitors.     

MicroRNA expression profiling with a droplet digital PCR assay enables molecular diagnosis and prognosis of cancers of unknown primary

Metastasis is responsible for the majority of cancer‐related deaths. Particularly, challenging is the management of metastatic cancer of unknown primary site (CUP), whose tissue of origin (TOO) remains undetermined even after extensive investigations and whose therapy is rather unspecific and poorly effective. Molecular approaches to identify the most probable TOO of CUPs can overcome some of these issues. In this study, we applied a predetermined set of 89 microRNAs (miRNAs) to infer the TOO of 53 metastatic cancers of unknown or uncertain origin. The miRNA expression was assessed with droplet digital PCR in 159 samples, including primary tumors from 17 tumor classes (reference set) and metastases of known and unknown origin (test set). We combined two different statistical models for class prediction to obtain the most probable TOOs: the nearest shrunken centroids approach of Prediction Analysis of Microarrays (PAMR) and the least absolute shrinkage and selection operator (LASSO) models. The molecular test was successful for all formalin‐fixed paraffin‐embedded samples and provided a TOO identification within 1 week from the biopsy procedure. The most frequently predicted origins were gastrointestinal, pancreas, breast, lung, and bile duct. The assay was applied also to multiple metastases from the same CUP, collected from different metastatic sites: The predictions showed a strong agreement, intrinsically validating our assay. The final CUPs'' TOO prediction was compared with the clinicopathological hypothesis of primary site. Moreover, a panel of 13 miRNAs proved to have prognostic value and be associated with overall survival in CUP patients. Our study demonstrated that miRNA expression profiling in CUP samples could be employed as diagnostic and prognostic test. Our molecular analysis can be performed on request, concomitantly with standard diagnostic workup and in association with genetic profiling, to offer valuable indications about the possible primary site, thereby supporting treatment decisions.     

Supraclavicular lymph node metastases of unknown origin: HPV-typing identifies the primary tumour

Cancers of unknown primary origin (CUP) account for 0.5–10% of all malignancies. CUP patients with metastases have a median survival of approximately 6 months, despite therapy. Identification of the primary tumour site may offer the opportunity of a specific and more efficient treatment. The case of a 45-year-old woman with supraclavicular lymph node metastases of a squamous cell CUP is reported. A staging laparoscopy with multiple biopsies and a loop diathermy excision of the cervix were performed. Human papillomavirus (HPV)-testing in the tissues revealed the tumour cells as metastases of an occult cervical cancer. Primary platin-based chemotherapy combined with paclitaxel leads to a complete apparative remission. Twelve months later, staging positron emission tomography with 2-[¹⁸F]fluoro-2-deoxy-D-glucose in combination with computed tomography identified an isolated left renal lymph node metastasis. The patient received targeted radiation therapy, combined with cisplatin. To date, 19 months after diagnosis, she is doing well without any evidence of disease. The presented case report addresses the difficulties involving the identification of CUP. HPV-DNA is found in over 95% of cervical cancers. As the presented case illustrates, testing for this virus DNA in human tissues can be a useful diagnostic tool in patients with CUP where cervical cancer is the possible primary tumour.     

Chemotherapy in carcinomas of unknown primary site: A high-dose intensity policy

Background: Unknown primary tumors are highly malignant diseases which portend a dire prognosis. We designed a prospective high dose-intensity policy with the aim of improving the results obtained with conventional chemotherapy.Patients and methods: Chemotherapy regimens were determined according to clinical features. In patients younger than 61 years with an ECOG performance status of 0 or 1, poorly differentied adenocarcinoma or poorly differentiated carcinoma, and no evidence of brain or bone marrow involvement (group A), the treatment plan included four sequential high-dose courses with hematopoietic progenitor cell and growth factor support. Peripheral blood progenitor cells were collected by apheresis as the leukocyte counts recovered from the nadir induced by the first cycle of chemotherapy (doxorubicin 75 mg/m2, cyclophosphamide 6000 mg/m2). Patients then received two cycles of etoposide (800 mg/m2) and carboplatin (900 mg/m2) separated by one cycle of doxorubicin (75 mg/m2) and cyclophosphamide (3000 mg/m2). G-CSF (5 µg/kg/d) was given until engraftment. It was planned that cycles would be delivered every three weeks. The remaining patients (group B) received alternative cycles of AC (doxorubicin 50 mg/m2, cyclophosphamide 1000 mg/m2) and EP (etoposide 300 mg/m2, cisplatin 100 mg/m2). Cycles were given at two-week intervals with GM-CSF support (5 µg/kg/d) from day 4 to day 10. Patients without measurable lesions were included, since the major endpoint was survival.Results: Sixty patients entered the study. Twenty patients were assigned to group A and 40 patients to group B. In group A, 5 of 12 patients with measurable lesions (42%; 95% confidence interval (95% CI): 22%–62%) achieved major responses to chemotherapy, including one complete response. The duration of the overall median survival was 11 months. In group B, a major response was observed in 12 (39%; 95% CI: 28%–50%) of 31 patients with measurable lesions, including three complete responses. The overall median survival was 8 months. Hematological toxicities were noteworthy in both groups. Two toxic deaths occurred in group B.Conclusion: Using these doses and schedules of chemotherapy, a high-dose intensity policy does not appear to improve the outcome of patients with carcinoma of unknown primary site. Alternative studies dealing with new drugs are required.     

Cancer of unknown primary site: review of consecutive cases at the National Cancer Center Hospital of Japan

Background Cancer of unknown primary (CUP) is not a rare clinical entity, accounting for 3%–5% of all solid malignancies. Methods We retrospectively reviewed 86 (38 male/48 female) patients with a diagnosis of CUP (exclusive of female patients with adenocarcinoma involving the axillary lymph nodes alone and patients with squamous cell carcinoma of the cervical lymph nodes) who were referred to the National Cancer Center Hospital between April 1996 and October 2002. Results The median interval between the first visit to a local community hospital and referral to our hospital was 1 month (range, 1 to 45 months). The histological diagnosis was adenocarcinoma in 61 patients (71%), poorly differentiated carcinoma in 18 patients (21%), and squamous cell carcinoma in 4 patients (5%). Twenty-three female patients had peritoneal carcinomatosis of adenocarcinoma. Seventy-eight patients (91%) received platinum-containing chemotherapy. Sixty-one of the 86 patients (71%) were categorized as a subgroup of CUP without a specific therapy, and 55 of these 61 patients (90%) received platinum-containing regimens. The median survivals of all 86 patients and the 61 patients in the subgroup without a specific therapy in this series were 13 months and 11 months, respectively. Conclusion In this series, the survival of the patients in the CUP subgroup without a specific therapy did not seem worse than that in previous reports. Empirical chemotherapy with platinum-containing regimens may benefit some CUP patients in a subgroup without a specific chemotherapy.     

Perspectives for targeted therapies in cancer of unknown primary site

Cancer of unknown primary site (CUP) ranks as the fourth most common cause of cancer deaths. Regression of the primary, early development of systemic metastases and resistance to therapy are hallmarks of this heterogeneous clinical entity. Targeted therapy offers promise for improvement of outcome of such patients, but it is currently hindered by lack of known molecular targets on which tumours are dependent for growth. In this review, we present the gene and protein profiling studies done on expression of oncogenes, tumour-suppressor genes and angiogenesis effectors and discuss the therapeutic potential of developed targeted agents. Existing data show occasional overexpression of Ras, BCL2 oncoproteins, absence of active EGFR/c-KIT/PDGFR signalling, uncommon presence of tumour-suppressor gene mutations and highly active angiogenesis in CUP. High-throughput multi-gene, multi-protein platforms offer promise for unravelling the complex molecular pathophysiology of CUP, for identification of targets suitable for modulation and ultimately hope for abrogation of its aggressive natural history.