Functional mapping of epigenetic regulators uncovers coordinated tumor suppression by the HBO1 and MLL1 complexes.

Epigenetic dysregulation is widespread in cancer. However, the specific epigenetic regulators and the processes they control to drive cancer phenotypes are poorly understood. Here, we employed a novel, scalable and high-throughput in vivo method to perform iterative functional screens of over 250 epigenetic regulatory genes within autochthonous oncogenic KRAS-driven lung tumors. We identified multiple novel epigenetic tumor suppressor and tumor dependency genes. We show that a specific HBO1 complex and the MLL1 complex are among the most impactful tumor suppressive epigenetic regulators in lung. The histone modifications generated by the HBO1 complex are frequently absent or reduced in human lung adenocarcinomas. The HBO1 and MLL1 complexes regulate chromatin accessibility of shared genomic regions, lineage fidelity and the expression of canonical tumor suppressor genes. The HBO1 and MLL1 complexes are epistatic during lung tumorigenesis, and their functional correlation is conserved in human cancer cell lines. Together, these results demonstrate the value of quantitative methods to generate a phenotypic roadmap of epigenetic regulatory genes in tumorigenesis in vivo .

Combinatorial in vivo genome editing identifies widespread epistasis during lung tumorigenesis.

Lung adenocarcinoma, the most common subtype of lung cancer, is genomically complex, with tumors containing tens to hundreds of non-synonymous mutations. However, little is understood about how genes interact with each other to enable tumorigenesis in vivo , largely due to a lack of methods for investigating genetic interactions in a high-throughput and multiplexed manner. Here, we employed a novel platform to generate tumors with all pairwise inactivation of ten tumor suppressor genes within an autochthonous mouse model of oncogenic KRAS-driven lung cancer. By quantifying the fitness of tumors with every single and double mutant genotype, we show that most tumor suppressor genetic interactions exhibited negative epistasis, with diminishing returns on tumor fitness. In contrast, Apc inactivation showed positive epistasis with the inactivation of several other genes, including dramatically synergistic effects on tumor fitness in combination with Lkb1 or Nf1 inactivation. This approach has the potential to expand the scope of genetic interactions that may be functionally characterized in vivo , which could lead to a better understanding of how complex tumor genotypes impact each step of carcinogenesis.

Fully accessible fitness landscape of oncogene-negative lung adenocarcinoma.

Cancer genomes are almost invariably complex with genomic alterations cooperating during each step of carcinogenesis. In cancers that lack a single dominant oncogene mutation, cooperation between the inactivation of multiple tumor suppressor genes can drive tumor initiation and growth. Here, we shed light on how the sequential acquisition of genomic alterations generates oncogene-negative lung tumors. We couple tumor barcoding with combinatorial and multiplexed somatic genome editing to characterize the fitness landscapes of three tumor suppressor genes NF1, RASA1, and PTEN, the inactivation of which jointly drives oncogene-negative lung adenocarcinoma initiation and growth. The fitness landscape was surprisingly accessible, with each additional mutation leading to growth advantage. Furthermore, the fitness landscapes remained fully accessible across backgrounds with the inactivation of additional tumor suppressor genes. These results suggest that while predicting cancer evolution will be challenging, acquiring the multiple alterations that drive the growth of oncogene-negative tumors can be facilitated by the lack of constraints on mutational order.

Fully accessible fitness landscape of oncogene-negative lung adenocarcinoma.

Cancer genomes are almost invariably complex with genomic alterations cooperating during each step of carcinogenesis. In cancers that lack a single dominant oncogene mutation, cooperation between the inactivation of multiple tumor suppressor genes can drive tumor initiation and growth. Here, we shed light on how the sequential acquisition of genomic alterations generates oncogene-negative lung tumors. We couple tumor barcoding with combinatorial and multiplexed somatic genome editing to characterize the fitness landscapes of three tumor suppressor genes NF1, RASA1, and PTEN, the inactivation of which jointly drives oncogene-negative lung adenocarcinoma initiation and growth. The fitness landscape was surprisingly accessible, with each additional mutation leading to growth advantage. Furthermore, the fitness landscapes remained fully accessible across backgrounds with additional tumor suppressor mutations. These results suggest that while predicting cancer evolution will be challenging, acquiring the multiple alterations required for the growth of oncogene-negative tumors can be facilitated by the lack of constraints on mutational order.

Combinatorial Inactivation of Tumor Suppressors Efficiently Initiates Lung Adenocarcinoma with Therapeutic Vulnerabilities.

Lung cancer is the leading cause of cancer death worldwide, with lung adenocarcinoma being the most common subtype. Many oncogenes and tumor suppressor genes are altered in this cancer type, and the discovery of oncogene mutations has led to the development of targeted therapies that have improved clinical outcomes. However, a large fraction of lung adenocarcinomas lacks mutations in known oncogenes, and the genesis and treatment of these oncogene-negative tumors remain enigmatic. Here, we perform iterative in vivo functional screens using quantitative autochthonous mouse model systems to uncover the genetic and biochemical changes that enable efficient lung tumor initiation in the absence of oncogene alterations. Generation of hundreds of diverse combinations of tumor suppressor alterations demonstrates that inactivation of suppressors of the RAS and PI3K pathways drives the development of oncogene-negative lung adenocarcinoma. Human genomic data and histology identified RAS/MAPK and PI3K pathway activation as a common feature of an event in oncogene-negative human lung adenocarcinomas. These Onc-negativeRAS/PI3K tumors and related cell lines are vulnerable to pharmacologic inhibition of these signaling axes. These results transform our understanding of this prevalent yet understudied subtype of lung adenocarcinoma. SIGNIFICANCE: To address the large fraction of lung adenocarcinomas lacking mutations in proto-oncogenes for which targeted therapies are unavailable, this work uncovers driver pathways of oncogene-negative lung adenocarcinomas and demonstrates their therapeutic vulnerabilities.

Pathogen diversity drives the evolution of generalist MHC-II alleles in human populations.

Central players of the adaptive immune system are the groups of proteins encoded in the major histocompatibility complex (MHC), which shape the immune response against pathogens and tolerance to self-peptides. The corresponding genomic region is of particular interest, as it harbors more disease associations than any other region in the human genome, including associations with infectious diseases, autoimmune disorders, cancers, and neuropsychiatric diseases. Certain MHC molecules can bind to a much wider range of epitopes than others, but the functional implication of such an elevated epitope-binding repertoire has remained largely unclear. It has been suggested that by recognizing more peptide segments, such promiscuous MHC molecules promote immune response against a broader range of pathogens. If so, the geographical distribution of MHC promiscuity level should be shaped by pathogen diversity. Three lines of evidence support the hypothesis. First, we found that in pathogen-rich geographical regions, humans are more likely to carry highly promiscuous MHC class II DRB1 alleles. Second, the switch between specialist and generalist antigen presentation has occurred repeatedly and in a rapid manner during human evolution. Third, molecular positions that define promiscuity level of MHC class II molecules are especially diverse and are under positive selection in human populations. Taken together, our work indicates that pathogen load maintains generalist adaptive immune recognition, with implications for medical genetics and epidemiology.

The role of sentinel node biopsy in male breast cancer.

BACKGROUND: Sentinel lymph node biopsy (SLNB) is a standard procedure in women with breast cancer. The risk of morbidity related to axillary lymph node dissection (ALND) is similar for men and women with breast cancer and SLNB could minimize this risk. METHODS: Between January 2004 and August 2013, 25 men with primary breast cancer were operated on at the Bács-Kiskun County Teaching Hospital. These were reviewed retrospectively. SLNB was performed following lymphoscintigraphy with intraoperative gamma probe detection and blue dye mapping. RESULTS: SLNB was successful in all 16 male patients (100 %), in whom it was attempted. The SLNs were negative in 4 cases (25 %) and were involved in 12. Intraoperative imprint cytology was positive in 9 of the 12 involved cases (75 %) and resulted immediate completion ALND. In 7 patients, the intraoperative imprint cytology was negative, with 3 false-negative results that resulted in delayed completion ALND. After a median follow-up of 48 months, there was only one axillary recurrence after ALND and none in the SLNB group. CONCLUSIONS: SLNB is successful and accurate in male breast cancer patients too. Although compared to women a larger proportion of men have positive nodes, for men with negative nodes, ALND-related morbidity may be reduced by SLNB. We recommend SLNB in male patients with breast cancer and clinically negative axilla.

International multicenter tool to predict the risk of nonsentinel node metastases in breast cancer.

BACKGROUND: Axillary treatment of breast cancer patients is undergoing a paradigm shift, as completion axillary lymph node dissections (ALNDs) are being questioned in the treatment of patients with tumor-positive sentinel nodes. This study aims to develop a novel multi-institutional predictive tool to calculate patient-specific risk of residual axillary disease after tumor-positive sentinel node biopsy. METHODS: Breast cancer patients with a tumor-positive sentinel node and a completion ALND from five European centers formed the original patient series (N = 1000). Statistically significant variables predicting nonsentinel node involvement were identified in logistic regression analysis. A multivariable predictive model was developed and validated by area under the receiver operating characteristics curve (AUC), first internally in 500 additional patients and then externally in 1068 patients from other centers. All statistical tests were two-sided. RESULTS: Nine tumor- and sentinel node-specific variables were identified as statistically significant factors predicting nonsentinel node involvement in logistic regression analysis. A resulting predictive model applied to the internal validation series resulted in an AUC of 0.714 (95% confidence interval [CI] = 0.665 to 0.763). For the external validation series, the AUC was 0.719 (95% CI = 0.689 to 0.750). The model was well calibrated in the external validation series. CONCLUSIONS: We present a novel, international, multicenter, predictive tool to assess the risk of additional axillary metastases after tumor-positive sentinel node biopsy in breast cancer. The predictive model performed well in internal and external validation but needs to be further studied in each center before application to clinical use.

Marked genetic differences between BRAF and NRAS mutated primary melanomas as revealed by array comparative genomic hybridization.

Somatic mutations of BRAF and NRAS oncogenes are thought to be among the first steps in melanoma initiation, but these mutations alone are insufficient to cause tumor progression. Our group studied the distinct genomic imbalances of primary melanomas harboring different BRAF or NRAS genotypes. We also aimed to highlight regions of change commonly seen together in different melanoma subgroups. Array comparative genomic hybridization was performed to assess copy number changes in 47 primary melanomas. BRAF and NRAS were screened for mutations by melting curve analysis. Reverse transcription PCR and fluorescence in-situ hybridization were performed to confirm the array comparative genomic hybridization results. Pairwise comparisons revealed distinct genomic profiles between melanomas harboring different mutations. Primary melanomas with the BRAF mutation exhibited more frequent losses on 10q23-q26 and gains on chromosome 7 and 1q23-q25 compared with melanomas with the NRAS mutation. Loss on the 11q23-q25 sequence was found mainly in conjunction with the NRAS mutation. Primary melanomas without the BRAF or the NRAS mutation showed frequent alterations in chromosomes 17 and 4. Correlation analysis revealed chromosomal alterations that coexist more often in these tumor subgroups. To find classifiers for BRAF mutation, random forest analysis was used. Fifteen candidates emerged with 87% prediction accuracy. Signaling interactions between the EGF/MAPK-JAK pathways were observed to be extensively altered in melanomas with the BRAF mutation. We found marked differences in the genetic pattern of the BRAF and NRAS mutated melanoma subgroups that might suggest that these mutations contribute to malignant melanoma in conjunction with distinct cooperating oncogenic events.

[Analysis of predictive tools for further axillary involvement in patients with sentinel-lymph-node-positive, small (< or =15 mm) invasive breast cancer]. / A hónalji nyirokcsomók további érintettségére vonatkozó modellek elemzése kisméretu (< / =15 mm) ôrszemnyirokcsomó-áttétes emlôrákokban.

Small breast cancers often require different treatment than larger ones. The frequency and predictability of further nodal involvement was evaluated in patients with positive sentinel lymph nodes and breast cancers < or =15 mm by means of 8 different predictive tools. Of 506 patients with such small tumors 138 with positive sentinel nodes underwent axillary dissection and 39 of these had non-sentinel node involvement too. The Stanford nomogram and the micrometastatic nomogram were the predictive tools identifying a small group of patients with low probability of further axillary involvement that might not require completion axillary lymph node dissection. Our data also suggest that the Tenon score can separate subsets of patients with a low and a higher risk of non-sentinel node metastasis. Predictive tools based on multivariate models can help in omitting completion axillary dissection in patients with low risk of non-sentinel lymph node metastasis based on their small tumor size.

Predicting non-sentinel lymph node status after positive sentinel biopsy in breast cancer: what model performs the best in a Czech population?

Several models have previously been proposed to predict the probability of non-sentinel lymph node (NSLN) metastases after a positive sentinel lymph node (SLN) biopsy in breast cancer. The aim of this study was to assess the accuracy of two previously published nomograms (MSKCC, Stanford) and to develop an alternative model with the best predictive accuracy in a Czech population. In the basic population of 330 SLN-positive patients from the Czech Republic, the accuracy of the MSKCC and the Stanford nomograms was tested by the area under the receiver operating characteristics curve (AUC). A new model (MOU nomogram) was proposed according to the results of multivariate analysis of relevant clinicopathologic variables. The new model was validated in an independent test population from Hungary (383 patients). In the basic population, six of 27 patients with isolated tumor cells (ITC) in the SLN harbored additional NSLN metastases. The AUCs of the MSKCC and Stanford nomograms were 0.68 and 0.66, respectively; for the MOU nomogram it reached 0.76. In the test population, the AUC of the MOU nomogram was similar to that of the basic population (0.74). The presence of only ITC in SLN does not preclude further nodal involvement. Additional variables are beneficial when considering the probability of NSLN metastases. In the basic population, the previously published nomograms (MSKCC and Stanford) showed only limited accuracy. The developed MOU nomogram proved more suitable for the basic population, such as for another independent population from a mid-European country.

[Combined surgery and radiotherapy in the treatment of ductal carcinoma in situ of the breast: preliminary results of the Hungarian multicenter prospective randomised study]. / In situ duktális emlokarcinóma kombinált sebészi- és sugárkezelése: a magyarországi multicentrikus prospektív.

The aim of this work is to report the preliminary results of the Hungarian multicentric randomised DCIS study. Between 2000 and 2007, 278 patients with ductal carcinoma in situ (DCIS) treated by breast-conserving surgery were randomised according to predetermined risk groups. Low/intermediate-risk patients (n=29) were randomised to 50 Gy whole-breast irradiation (WBI) or observation. High-risk cases (n=235) were allocated to receive 50 Gy WBI vs. 50 Gy WBI plus 16 Gy tumour bed boost. Very high-risk patients (patients with involved surgical margins; n=14) were randomised to 50 Gy WBI plus 16 Gy tumour bed boost or reoperation (reexcision plus radiotherapy or mastectomy alone). Immunohistochemistry (IHC) was performed to detect the expression of potential molecular prognostic markers (ER, PR, Her2, p53, Bcl-2 and Ki-67). At a median follow-up of 36 months no recurrence was observed in the low/intermediate- and very high-risk patient groups. In the high-risk group, 4 (1.7%) local recurrences and 1 (0.4%) distant metastasis occurred. No patient died of breast cancer. In the high-risk group of patients, the 3- and 5-year probability of local recurrence was 1.1% and 3.1%, respectively. The positive immunostaining for Her2 (38%), p53 (37%) and Ki-67 (44%) correlated with a high nuclear grade. Significant inverse correlation was found between the expression of ER (77%), PR (67%), Bcl-2 (64%) and grade. Preliminary results suggest that breast-conserving surgery followed by radiotherapy yields an annual local recurrence rate of less than 1% in patients with DCIS. IHC of molecular prognostic markers can assist to gain insight into the biologic heterogeneity of DCIS.

The role of radiotherapy in the conservative treatment of ductal carcinoma in situ of the breast.

Breast-conserving surgery (BCS) followed by radiotherapy (RT) has become the standard of care for the treatment of early-stage (St. I-II) invasive breast carcinoma. However, controversy exists regarding the value of RT in the conservative treatment of ductal carcinoma in situ (DCIS). In this article we review the role of RT in the management of DCIS. Retrospective and prospective trials and meta-analyses published between 1975 and 2007 in the MEDLINE database, and recent issues of relevant journals/handbooks relating to DCIS, BCS and RT were searched for. In retrospective series (10,194 patients) the 10-year rate of local recurrence (LR) with and without RT was reported in the range of 9-28% and 22-54%, respectively. In four large randomised controlled trials (NSABP-B-17, EORTC-10853, UKCCCR, SweDCIS; 4,568 patients) 50 Gy whole-breast RT significantly decreased the 5-year LR rate from 16-22% (annual LR rate: 2.6-5.0%) to 7-10% (annual LR rate: 1.3-1.9%). In a recent meta-analysis of randomised trials the addition of RT to BCS resulted in a 60% risk reduction of both invasive and in situ recurrences. In a multicentre retrospective study, an additional dose of 10 Gy to the tumour bed yielded a further 55% risk reduction compared to RT without boost. To date, no subgroups have been reliably identified that do not benefit from RT after BCS. In the NSABP-B-24 trial, the addition of tamoxifen (TAM) to RT reduced ipsilateral (11.1% vs. 7.7%) and contralateral (4.9% vs. 2.3%) breast events significantly. In contrast, in the UKCCCR study, TAM produced no significant reduction in all breast events. Based on available evidence obtained from retrospective and prospective trials, all patients with DCIS have potential benefit from RT after BCS. Further prospective studies are warranted to identify subgroups of low-risk patients with DCIS for whom RT can be safely omitted. Until long-term results of ongoing studies on outcomes of patients treated with BCS alone (with or without TAM or aromatase inhibitors) are available, RT should be routinely recommended after BCS for all patients except those with contraindication.

[Sentinel lymph node biopsy for in situ carcinoma of the breast. Experience at the Bács-Kiskun County Hospital and review of the literature]. / Orszemnyirokcsomó-biopszia in situ emlorákban. A Bács-Kiskun Megyei Onkormányzat Kórházának tapasztalatai és irodalmi összefoglalás.

UNLABELLED: Sentinel node biopsy (SNB) is controversial for in situ breast cancers. We reviewed our experience with in situ and microinvasive carcinomas and surveyed the literature. METHODS: SNB was performed with intraparenchymal administration of vital dye alone or combined with radiocolloid. The SNs were assessed histologically with haematoxylin eosin staining and cytokeratin immunohistochemistry. RESULTS: Patients with in situ (36) or microinvasive (20) carcinomas underwent SNB: 59 axillary and 1 parasternal, and 39 axillary and 1 parasternal SNs were recovered, respectively. The SNs were positive in 4 patients and 1 patient, respectively: 1 micrometastasis and 3 isolated tumour cells, and 1 micrometastasis in the respective groups. No further axillary nodes were found positive after dissection. Further 21 invasive carcinomas (often with extensive intraductal component) had an in situ carcinoma diagnosis preoperatively: of 39 axillary and 3 parasternal SNs 10 patients had nodal involvement in 13 axillary SNs; 5 patients also had further lymph nodes involved after dissection. CONCLUSIONS: The definitive diagnosis of in situ carcinoma does not warrant SNB. This procedure should be considered if the tumour is to be removed by mastectomy, or if the diagnosis is preoperative and there are associated high-risk factors for the subsequent diagnosis of invasive cancer.

[Response rates following neoadjuvant chemotherapy and breast preserving treatment in patients with locally advanced breast cancer]. / A neoadjuváns, avagy primer szisztémás kemoterápia eredményei es az ezt követo emlomegtartó mutétek aránya a loko- regionálisan elorehaladott emlorákos betegeknél.

INTRODUCTION: The neoadjuvant chemotherapy is increasingly being used in the treatment of patients with locally advanced breast cancer. We describe the hypothesis of the biological behaviour of breast cancer supporting the reason for the existence of this treatment. The improvement of neoadjuvant chemotherapy is being discussed as well as the advantages, disadvantages and problems of the treatment. THE AIM OF EXAMINATION: To study the results of neoadjuvant chemotherapy in patients with locally advanced breast cancer and the proportion of breast preserving surgery after the treatment. METHODS: Sixty seven patients were given neoadjuvant chemotherapy treatment between 01.01.1999 and 12.31.2003. Twenty three patients were stage III A while 35 stage III B and 9 stage III C. 63% of the patients received CEF chemotherapy and 19% were given MMM. 18% were given neoadjuvant Taxotere + Carboplatin and 4% were given Taxotere + Farmorubicin chemotherapy. RESULTS: After neoadjuvant chemotherapy 5 patients had SD (stable disease), 32 patients had MR (minor response) and in 28 cases patients had PR (partial response). Two patients showed pCR (complete pathologic response). Twenty patients (30%) had breast preserving surgery. CONCLUSIONS: On the basis of our own experience neoadjuvant therapy is justified in patients with locally advanced breast cancer as they have bigger chance for breast preserving surgery. If mastectomy and axillary block dissection has to be carried out they are easier to perform. Taxans must be introduced for neoadjuvant treatment in order to improve our results. A longer follow-up is necessary before drawing final conclusions.

Use and limitations of a nomogram predicting the likelihood of non-sentinel node involvement after a positive sentinel node biopsy in breast cancer patients.

After a positive sentinel lymph node (SLN) biopsy, some patients may be considered to have a very low risk of non-SLN involvement and could be candidates for axillary sparing. The aim of this study was to validate the nomogram created at the Memorial Sloan-Kettering Cancer Center (MSKCC) for the prediction of non-SLN involvement in an independent set of 140 patients with both positive SLNs and axillary dissection. The predicted proportions of positive non-SLNs were compared with the observed percentages of non-SLN metastasis. Although the SLN metastasis size and tumor size did influence the risk of non-SLN involvement, the correlation between the predicted and observed proportions was weaker for our patients (R: 0.84) than for the patients assessed at the MSKCC (R: 0.97). Differences were noted in the intraoperative assessment and in the final histology of the SLNs (imprints vs frozen sections and more detailed vs less detailed, respectively), and these could partly explain the lower level of the correlation. The nomogram could not be validated and was found to be of only limited use for the prediction of non-SLN involvement in patients operated on under similar, though not fully identical conditions. We therefore warn against the unvalidated use of this prediction tool.

Axillary sentinel node and tumour-related factors associated with non-sentinel node involvement in breast cancer.

BACKGROUND: After completion of axillary dissection, many breast cancer patients with axillary sentinel nodal involvement are found to have regional disease limited to the sentinel nodes. These patients are exposed to the morbidity of axillary clearance without any expected therapeutic benefit. METHODS: Sentinel node biopsy was performed either with Patent blue dye or with a combined dye, radiocolloid and gamma-probe-guided method involving peritumoral tracer administration. For a series of 150 consecutive patients with involved axillary sentinel nodes and axillary dissection, factors associated with non-sentinel nodal involvement were analysed in a multivariate analysis based on logistic regression with the use of fractional polynomials. RESULTS: The following variables were found to be potentially associated with non-sentinel node metastases: tumour size, sentinel node metastasis size, number of examined sentinel nodes, percentage of involved sentinel nodes (the latter two were found to be significant only when in combination), and extracapsular perinodal spread. CONCLUSIONS: Isolated tumour cells and micrometastases in axillary sentinel nodes carry a low risk of non-sentinel node metastasis. The risk of metastasis to further echelon nodes is higher with macrometastases, especially if there is extracapsular growth and the proportion of involved sentinel nodes is high.

Comparison of vital dye-guided lymphatic mapping and dye plus gamma probe-guided sentinel node biopsy in breast cancer.

The optimal technique for sentinel lymph node biopsy (SLNB) is still debated. SLNB with peritumoral injection of Patent blue dye was performed in 129 clinically T1-T2 and N0 breast cancers in 127 patients (group A); it was later replaced by combined dye and radiocolloid-guided SLNB preceded by lymphoscintigraphy in 72 breast cancer patients (group B). This study compares these two methods. All patients underwent completion axillary dissection. Means of 1.4 and 1.3 SLNs were identified in groups A and B, respectively. The mean number of non-SLNs for the whole series was 14.9 (range 5-42). The first 53 cases of lymphatic mapping (dye only) comprised the institutional learning period during which the identification rate of at least 1 SLN in 30 consecutive attempts reached 90%. The identification rate for the subsequent 76 group A patients was 92%. The accuracy rate of SLNBs for overall axillary nodal status prediction and the false-negative rate for group A patients (after excluding the learning-phase cases) were 93% and 10%, respectively. All 72 group B cases had at least one SLN identified, and only one false-negative case occurred in this group (accuracy and false-negative rates of 99% and 3%, respectively). Both the dye-only and the combined SLNB methods are suitable for SLN identification, but the latter works better and results in higher accuracy, a higher negative predictive value, and a lower false-negative rate. It is therefore the method of choice.