Abstract Liquid biopsy using ultra-low-pass whole-genome sequencing (ULP-WGS, ∼0.25x coverage) is a promising tool to detect circulating tumor DNA (ctDNA) for cancer management, and the use of the native Oxford Nanopore (ONT) sequencing platform adds DNA methylation to the set of detectable features. Here, we test the performance of methylation-based cell-type deconvolution in ULP-WGS samples from diverse epithelial malignancies and investigate several new computational strategies using our CelFiE-ISH deconvolution framework. We find that incorporating larger numbers of markers restricted to the epithelial cell lineage can reduce the cancer fraction limit of detection down to 1.7-3.1%, matching or exceeding the 3% floor of established copy-number alteration (CNA) benchmarks. Our study provides a useful strategy for analysis of ULP-WGS ONT data and indicates that marker selection remains a key challenge for analyzing methylation-based cancer datasets.

The analysis of cell-free DNA (cfDNA) in plasma provides information on pathological processes in the body. Blood cfDNA is in the form of nucleosomes, which maintain their tissue- and cancer-specific epigenetic state. We developed a single-molecule multiparametric assay to comprehensively profile the epigenetics of plasma-isolated nucleosomes (EPINUC), DNA methylation and cancer-specific protein biomarkers. Our system allows for high-resolution detection of six active and repressive histone modifications and their ratios and combinatorial patterns on millions of individual nucleosomes by single-molecule imaging. In addition, our system provides sensitive and quantitative data on plasma proteins, including detection of non-secreted tumor-specific proteins, such as mutant p53. EPINUC analysis of a cohort of 63 colorectal cancer, 10 pancreatic cancer and 33 healthy plasma samples detected cancer with high accuracy and sensitivity, even at early stages. Finally, combining EPINUC with direct single-molecule DNA sequencing revealed the tissue of origin of colorectal, pancreatic, lung and breast tumors. EPINUC provides multilayered information of potential clinical relevance from limited (<1 ml) liquid biopsy material.

Cancer inflicts damage to surrounding normal tissues, which can culminate in fatal organ failure. Here, we demonstrate that cell death in organs affected by cancer can be detected by tissue-specific methylation patterns of circulating cell-free DNA (cfDNA). We detected elevated levels of hepatocyte-derived cfDNA in the plasma of patients with liver metastases originating from different primary tumors, compared with cancer patients without liver metastases. In addition, patients with localized pancreatic or colon cancer showed elevated hepatocyte cfDNA, suggesting liver damage inflicted by micrometastatic disease, by primary pancreatic tumor pressing the bile duct, or by a systemic response to the primary tumor. We also identified elevated neuron-, oligodendrocyte-, and astrocyte-derived cfDNA in a subpopulation of patients with brain metastases compared with cancer patients without brain metastasis. Cell type–specific cfDNA methylation markers enable the identification of collateral tissue damage in cancer, revealing the presence of metastases in specific locations and potentially assisting in early cancer detection.

Small cell lung cancer (SCLC) is an aggressive malignancy with exceptionally poor prognosis and limited therapeutic advances in the past few decades. Although SCLCs are treated as a single disease entity in clinic, emerging data support subtypes of SCLC driven by expression of distinct transcription regulators, which engender unique therapeutic vulnerabilities. However, the translational potential of these observations is limited by access to tumor biopsies. Here, we apply chromatin immunoprecipitation of cell-free nucleosomes carrying active chromatin modifications followed by sequencing (cfChIP-seq) to 286 plasma samples from patients with advanced SCLC, non-SCLC cancers, and healthy adults. In addition to providing reliable estimates of SCLC circulating free DNA (cfDNA) tumor fraction, cfChIP-seq recovers the unique epigenetic states of SCLC tissue and cells of origin, and importantly tumor gene expression. Comparison of cfChIP-seq signals to matched tumor transcriptomes shows genome-wide concordance presenting a direct link between gene expression in the tumor and plasma cell-free nucleosomes. We devise a classifier that discriminates between SCLC lineage-defining transcription factor subtypes based on cfChIP-seq assay. This work sets the stage to non-invasively profile SCLC transcriptomes using plasma cfDNA histone modifications.

Cell-free DNA (cfDNA) in human plasma provides access to molecular information about the pathological processes in the organs or tumors from which it originates. These DNA fragments are derived from fragmented chromatin in dying cells and retain some of the cell-of-origin histone modifications. In this study, we applied chromatin immunoprecipitation of cell-free nucleosomes carrying active chromatin modifications followed by sequencing (cfChIP-seq) to 268 human samples. In healthy donors, we identified bone marrow megakaryocytes, but not erythroblasts, as major contributors to the cfDNA pool. In patients with a range of liver diseases, we showed that we can identify pathology-related changes in hepatocyte transcriptional programs. In patients with metastatic colorectal carcinoma, we detected clinically relevant and patient-specific information, including transcriptionally active human epidermal growth factor receptor 2 (HER2) amplifications. Altogether, cfChIP-seq, using low sequencing depth, provides systemic and genome-wide information and can inform diagnosis and facilitate interrogation of physiological and pathological processes using blood samples.

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ERBB2 amplification is a prognostic marker for aggressive tumors and a predictive marker for prolonged survival following treatment with HER2 inhibitors. We attempt to sub-group HER2+ tumors based on amplicon structures and co-amplified genes. We examined five HER2+ cell lines, three HER2+ xenographs and 57 HER2+ tumor tissues. ERBB2 amplification was analyzed using digital droplet PCR and low coverage whole genome sequencing. In some HER2+ tumors PPM1D, that encodes WIP1, is co-amplified. Cell lines were treated with HER2 and WIP1 inhibitors. We find that inverted duplication is the amplicon structure in the majority of HER2+ tumors. In patients suffering from an early stage disease the ERBB2 amplicon is composed of a single segment while in patients suffering from advanced cancer the amplicon is composed of several different segments. We find robust WIP1 inhibition in some HER2+ PPM1D amplified cell lines. Sub-grouping HER2+ tumors using low coverage whole genome sequencing identifies inverted duplications as the main amplicon structure and based on the number of segments, differentiates between local and advanced tumors. In addition, we found that we could determine if a tumor is a recurrent tumor or second primary tumor and identify co-amplified oncogenes that may serve as targets for therapy.

Hereditary cancer comprises more than 10% of all breast cancer cases. Identification of germinal mutations enables the initiation of a preventive program that can include early detection or preventive treatment and may also have a major impact on cancer therapy. Several recurrent mutations were identified in the BRCA1/2 genes in Jewish populations however, in other ethnic groups in Israel, no recurrent mutations were identified to date. Our group established panel sequencing in cancer patients to identify recurrent, founder, and new mutations in the heterogeneous and diverse populations in Israel, We evaluated five breast cancer patients of Arab descent diagnosed with cancer before the age of 50 years and identified the previously described TP53 mutation, c.541C>T, R181C (rs587782596), in two women from unrelated Arab families. The two probands were diagnosed with breast cancer at a young age (27 and 34 years) and had significant family history spanning a wide range of tumors (breast cancer (BC), papillary thyroid cancer, glioblastoma multiform (GBM), colon cancer and leukemia). The R181C variant is expected to disrupt p53 at the ASPP2 binding domain but not the DNA binding domain and is defined by Clinvar as likely pathogenic and in HGMD as disease mutation. We further tested 85 unrelated Arab cancer patients and father of a BC carrier patient for TP53 c.541C>T using a real time polymerase chain reaction (RT-PCR) approach and identified four additional carriers, two with BC one with lung cancer, and the father of a BC carrier patient, diagnosed with GBM. Another carrier suffering from BC was identified using a Myriad panel, suggesting a recurrent mutation in this population with a frequency of 5/42 (11.9%) of our selected BC patients. We suggest testing Arab women with a breast cancer at a young age, Arab patients with multiple malignancies, or with suggestive family history for TP53 c.541C>T.

Bevacizumab improves survival when added to chemotherapy in metastatic colorectal cancer (mCRC). We assessed the safety and efficacy of bevacizumab in mCRC patients ≥70 years old (YO) vs. those <70 YO. mCRC patients treated from 2005–2012 who received chemotherapy (physician’s choice) plus bevacizumab were included. The primary end point was safety; secondary objectives were progression-free survival (PFS) and overall survival (OS). Data was collected retrospectively. Three-hundred eight patients (92 ≥70 YO, 216 <70 YO) with 20.5 month median follow-up were included. Of the patients, 1.9 % died due to bevacizumab-related adverse effects; all were <70 YO. Grades 3–5 adverse events of interest for bevacizumab in patients ≥70 YO included hypertension (37.0 %), venous thromboembolism (6.5 %), wound-healing complications (5.4 %), bleeding (7.6 %), fistula (4.3 %), arterial thromboembolism (3.3 %), congestive heart failure (2.2 %), and proteinuria (grades 1–2 only, 14.1 %). Treatment was stopped due to adverse effects in 6.0 % of older patients. Older patients had significantly more ischemic heart disease and hypertension at baseline, and were treated less with FOLFOX and more with 5FU/LV monotherapy; nevertheless, OS and PFS were similar in younger and older patients. Compared to younger patients, in older patients, rates of proteinuria (all grades 1–2) were significantly higher (14.1 vs. 5.6 %, p=0.012) and rates of treatment-related hypertension (grades 3–5) were marginally higher (37 vs. 25.9 %, p=0.053); rates of other adverse events were similar in the two groups. In our patient population, bevacizumab was safe and effective in older as well as younger patients.

e16100 Background: , Sequential hormonal deprivation therapy incorporating old inexpensive pharmaceuticals may still have a role in the treatment of castrate resistant metastatic prostate carcinoma(CRPC). Abiraterone Acetate(AA), a CYP 17 inhibitor, initially approved by the FDA for CRPC in the post chemotherapy setting after showing improvement in OS, PFS, PSA and clinical response attained approval in the chemotherapy naïve patient essentially resulting in the majority of patients receiving this agent earlier in their disease course. Diethylstilbestrol(DES) is a nonsteroidal estrogen, first used for CRPC in 1941 . The effectiveness of orchiectomy, LHRH agonists, and the cardiovascular toxicity associated with DES has relegated it to a minor role in the treatment of CRPC. One month supply AA costs $6,285 compared to one month DES $114. Methods: Patient records at a single large academic center were reviewed if they had been treated for metastatic CRPC first with DES and at time of progression later had received in their disease course treatment with AA. We report a series of 18 patients with Mean Age 70(62-83), Gleason 7(4-10), all received LHRH treatment with 8/18 receiving second line bicalutamide to which 3/8 responded. Results: Clinical response to DES occurred in 9/18 patients for Mean 8.5 months (2-12). At time of initiation of AA, Median PSA 104(1.07-1,000), 11/18 had prior docetaxol, all had bone involvement. PSA decline greater 30% occurred 8/18. Clinical benefit with decrease in pain was achieved in 8/18 patients. Conclusions: There is potential utility for a strategy of sequential testosterone ablative therapies to achieve the desired postponement toxic interventions, chemotherapy. Patients responded to AA after progression on DES, showing absence of cross- resistence. Our patient cohort suggests that there may still be a role for old and inexpensive agents including DES and similar to the incremental improvement in outcome seen in the treatment paradigm of other metastatic diseases such as breast and colon cancer where new therapeutics are given sequentially without discarding older more cheaper therapies may be a model to explore for castrate resistant metastatic prostate patients.

Bevacizumab treatment is associated with an increased risk of hypertension (HTN), a potential marker for effectiveness. We aimed to assess whether grades 2-3 HTN during bevacizumab treatment was associated with increased overall survival (OS) or progression-free survival (PFS). One hundred and eighty-one patients with metastatic colorectal cancer (CRC), who were treated in our Department from January 2009-February 2011 were included. Bevacizumab was administered jointly with standard first- or second-line chemotherapy protocols. Blood pressure was measured before each treatment. HTN was graded using common toxicity criteria. There were 181 CRC patients. Grades 2-3 HTN developed in 81 patients (44.75 %) but not in 100 patients (55.25 %); no patient developed grades 4-5 HTN. Median follow-up was 15.2 months. HTN was associated with better OS in HTN-positive versus HTN-negative patients (median not reached vs. 36.8 months, p = 0.029) and better PFS (29.9 vs. 17.2 months, p = 0.024, respectively). Bevacizumab-related HTN may represent a biomarker for clinical benefit in metastatic colorectal cancer patients.

retrospectively involved personal and family antecedents of cancer, clinical and anatomic-pathological variables as well as data concerning the treatments received and the tumor evolution were collected.Results: The mean age was 31years .These patients represent 20 % of all the rectum cancers diagnosed during the same period.Two patients had haemorrhagic recto-colitis antecedent,4 cases had family recto-colic polyposes and one case of Burkitt lymphoma, with an8 months (1month to 24months); Clinical symptomatology was dominated by rectorrhagias (70%), rectal syndrome (48.6%) and 12 patients had visited at the stage of occlusion (16%),cancer detection at a tardive stage (stages C et D in more than 75% of the cases), the tumor was seated in lower part of the rectum (59%),the predominance of the undifferentiated adenocarcinomas and mucous colloids (15.3%) are unfavorable prognoses that affect the treatment and the survival without recidivation neoadjuvant radiotherapy was carried out in 31 patients.The operativeness rate was 69%; forty patients had a curative surgical resection (R0), chemotherapy was proposed to 25 patients postoperatively.Among the 51patients undergone curative resection, 18 patients had recidivation.Recidivations were local-regional (12 cases) or metastatic (6 cases) and survival at 3 years was very low (5%).Conclusion: Rectum cancer in the young patient is not as rare as we may think active detection in patients at risk, the setting of the diagnosis when the treatment would be useful, exercise surgery according to carcinologic rules and the introduction of neo-adjuvant radiochemotherapy in daily practice are means of the improvement of the prognosis and to offer these young patients the best survival.This action requires a multidisciplinary approach of the problem.

e11541 Background: Chemotherapy improves breast cancer outcome, but may impact fertility. Post chemotherapy fertility rates range between 10–90% among studies. Fertility post chemotherapy is most often assessed by rate of menstruation resumption- an indirect method of evaluating childbearing potential. It is noteworthy that future pregnancy is a woman's first concern not menses preservation. Variability in fertility rates along with limited data on post chemotherapy pregnancies led us to undertake this single institute retrospective study evaluating fertility and pregnancy post chemotherapy in ≤ 38 y old breast cancer patients. Methods: We reviewed medical records of 222 consecutive stage II-IIIB breast cancer patients diagnosed, treated and followed at Sharette Institute of Hadassah-Hebrew University Medical Center from 1990–2004. Inclusion criteria included age ≤ 38 years, ≥3 cycles of standard metothrexate or adriamycine based chemotherapy, metastasis-free 12 months post chemotherapy initiation or one year following GnRH analog withdrawal. Patients diagnosed with infertility prior to breast cancer diagnosis (data present for part of patients), bilateral oophorectomy or patients surviving ≤3 years from diagnosis were excluded. Patient pregnancy preference was not recorded. Fertility was defined as resumption of recurrent menses or pregnancy anytime during follow up. In case of recurrence, date of recurrence diagnosis was assigned as date of last follow up. Results: Cohort included 65 patients. Mean age 32.5±4 ys (20.3–38.5) Almost all patients (95.4%), 38 ys or younger, preserve menses following chemotherapy, and 33.9% become pregnant. Most of patients who did not conceive post chemotherapy (66.1%), were under a familial status not promoting pregnancy; 44% of all women who did not give birth post chemotherapy had at least 3 offspring at the time of diagnosis and 83% out of all women who did not have any offspring by the end of follow up were single. Conclusions: This data suggest that pregnancy potential may be even higher than our actual finding, since pregnancy is most probably not attempted by multiparous or most single patients, whom cultural constrains affect their decisions. It seems prudent to offer the subgroup of unmarried women fertility preservation. No significant financial relationships to disclose.

Abstract Abstract #6148 Background Counseling young breast cancer patients regarding the chances of conception following chemotherapy is a subject of great importance. Previous studies have reported a 50-70% rate of amenorrhea following standard breast cancer chemotherapy and very low rates of pregnancy. However a uniform definition of infertility is lacking and as a result many studies reported results observed in pre-menopausal women as a whole without specific analysis and separation according to age. In this retrospective, single institution study, we examined resumption of menses and pregnancy rates in young breast cancer patients who had received chemotherapy. Methods We identified 193 breast cancer patients aged 35 years or younger diagnosed at a single institution between 1996 and 2004. All patients who were treated with adjuvant chemotherapy and had completed at least 1 year of follow-up without evidence of recurrence were included in the study. Results Forty seven patients met the eligibility criteria. The mean time of follow-up was 69 months (range, 12-124 mos.). The mean age at diagnosis was 30 years (range, 19-34 yrs). Chemotherapy consisted of Adriamycin based protocols in 29 cases and CMF (Cyclophosphamide, Methotrexate, 5-Fluorouracil) in 18 cases. GnRH agonists were administered to 28 (61%) of patients for at least two years. All patients had regular menses prior to the initiation of chemotherapy. Three patients continued to receive regular injections with a GnRH agonist beyond two years, one patient underwent oophorectomy, two patients developed amenorrhea whilst 35 patients (35/37 95%) resumed menses subsequent to chemotherapy. Information regarding menstruation was lacking for six patients. Sixteen patients (16/37 43%) gave birth to at least one child after menses resumed at a mean age of 35 years (range, 28-41yrs). Discussion Standard chemotherapy protocols in breast cancer patients under the age of 35 do not preclude fertility preservation and pregnancy. However, not all young breast cancer survivors wish to become pregnant following treatment and therefore the actual rate of fertility is in fact higher than those recorded. Further prospective studies which incorporate documentation regarding patient's desire for future pregnancies need to be undertaken in order to better advise young patients with respect to their individual chances of conceiving following chemotherapy. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6148.