Alarice Lowe

Whole exome sequencing of circulating tumor cells provides a window into metastatic prostate cancer

Comprehensive analyses of cancer genomes promise to inform prognoses and precise cancer treatments. A major barrier, however, is inaccessibility of metastatic tissue. A potential solution is to characterize circulating tumor cells (CTCs), but this requires overcoming the challenges of isolating rare cells and sequencing low-input material. Here we report an integrated process to isolate, qualify and sequence whole exomes of CTCs with high fidelity using a census-based sequencing strategy. Power calculations suggest that mapping of >99.995% of the standard exome is possible in CTCs. We validated our process in two patients with prostate cancer, including one for whom we sequenced CTCs, a lymph node metastasis and nine cores of the primary tumor. Fifty-one of 73 CTC mutations (70%) were present in matched tissue. Moreover, we identified 10 early trunk and 56 metastatic trunk mutations in the non-CTC tumor samples and found 90% and 73% of these mutations, respectively, in CTC exomes. This study establishes a foundation for CTC genomics in the clinic.

Effect of Ischemic Time, Fixation Time, and Fixative Type on HER2/neu Immunohistochemical and Fluorescence In Situ Hybridization Results in Breast Cancer

Accurate determination of HER2/neu status in breast carcinoma is essential. Alteration of preanalytic variables is known to affect HER2/neu results. American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP) issued guidelines to standardize fixation for increased HER2/neu accuracy. We studied the effects of changing preanalytic variables on HER2/neu immunohistochemical and fluorescence in situ hybridization (FISH) results in a known HER2/neu+ invasive carcinoma. The clinical specimen was processed according to ASCO/CAP guidelines, with remaining tumor stored fresh without any fixatives for 4 days at 4°C and cut into core biopsy-sized pieces. Each was fixed in 10% formalin, 15% formalin, Pen-Fix (Richard-Allan Scientific, Kalamazoo, MI), Bouin solution, Sakura molecular fixative (Sakura Tissue-Tek Xpress, Torrance, CA), or zinc formalin for 0 to 168 hours. Immunohistochemical studies and FISH were performed. Compared with the clinical specimen, the samples showed no tumor degradation or marked difference by immunohistochemical studies, except the 1-hour 10% formalin and Bouin samples, or FISH, except the Bouin-fixed samples. Our study demonstrates that HER2/neu results remain accurate beyond ASCO/CAP-recommended preanalytic variables, with the exception of Bouin solution for FISH analysis.

Translational breast cancer research consortium (TBCRC) 022: A phase II trial of neratinib for patients with human epidermal growth factor receptor 2-positive breast cancer and brain metastases

PURPOSE Evidence-based treatments for metastatic, human epidermal growth factor receptor 2 (HER2)-positive breast cancer in the CNS are limited. Neratinib is an irreversible inhibitor of erbB1, HER2, and erbB4, with promising activity in HER2-positive breast cancer; however, its activity in the CNS is unknown. We evaluated the efficacy of treatment with neratinib in patients with HER2-positive breast cancer brain metastases in a multicenter, phase II open-label trial. PATIENTS AND METHODS Eligible patients were those with HER2-positive brain metastases (≥ 1 cm in longest dimension) who experienced progression in the CNS after one or more line of CNS-directed therapy, such as whole-brain radiotherapy, stereotactic radiosurgery, and/or surgical resection. Patients received neratinib 240 mg orally once per day, and tumors were assessed every two cycles. The primary endpoint was composite CNS objective response rate (ORR), requiring all of the following: ≥ 50% reduction in volumetric sum of target CNS lesions and no progression of non-target lesions, new lesions, escalating corticosteroids, progressive neurologic signs/symptoms, or non-CNS progression--the threshold for success was five of 40 responders. RESULTS Forty patients were enrolled between February 2012 and June 2013; 78% of patients had previous whole-brain radiotherapy. Three women achieved a partial response (CNS objective response rate, 8%; 95% CI, 2% to 22%). The median number of cycles received was two (range, one to seven cycles), with a median progression-free survival of 1.9 months. Five women received six or more cycles. The most common grade ≥ 3 event was diarrhea (occurring in 21% of patients taking prespecified loperamide prophylaxis and 28% of those without prophylaxis). Patients in the study experienced a decreased quality of life over time. CONCLUSION Although neratinib had low activity and did not meet our threshold for success, 12.5% of patients received six or more cycles. Studies combining neratinib with chemotherapy in patients with CNS disease are ongoing.

The Effect of Delay in Fixation, Different Fixatives, and Duration of Fixation in Estrogen and Progesterone Receptor Results in Breast Carcinoma

Accurate determination of estrogen receptor (ER) and progesterone receptor (PR) status in breast carcinoma is essential. Preanalytic variation may contribute to discordant results. Recently, American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) made recommendations to normalize fixation for breast biomarkers. To evaluate this, a 4-cm invasive lobular carcinoma was processed according to ASCO/CAP guidelines. The remainder was stored fresh at 4°C for 4 days and cut into biopsy-sized pieces. Each was fixed in 10% formalin, Pen-Fix (Richard-Allan Scientific, Kalamazoo, MI), Bouin solution, Sakura Molecular Fixative (Sakura Tissue-Tek Xpress, Torrance, CA), zinc formalin, or 15% formaldehyde for times ranging between 1 and 168 hours. Immunohistochemical studies for ER and PR were performed and interpreted. After 4 days at 4°C, all samples showed no degradation or ER/PR staining differences, except 2 Bouin-fixed samples, in comparison with the patients sample processed according to ASCO/CAP guidelines. In our study, the preanalytic variables of fixative type, fixation time, and 4 days of ischemic time did not affect immunohistochemical accuracy for ER/PR.

Comparison of fluorescent in situ hybridization HER-2 / neu results on core needle biopsy and excisional biopsy in primary breast cancer

HER-2/neu status is critical for the therapy for breast carcinoma. Fluorescent in situ hybridization for gene amplification and immunohistochemical stains for protein expression are widely used methods to detect HER-2/neu status. Multiple studies have shown fluorescent in situ hybridization and immunohistochemical stain results to have high concordance rates. To our knowledge, a comparison between fluorescent in situ hybridization results for core needle biopsy and the subsequent excisional biopsy specimens has not yet been studied. We retrospectively evaluated the fluorescence in situ hybridization and immunohistochemical results in both the breast core needle and the excisional biopsy of 125 patients with invasive breast carcinoma from 2002 to 2005. There was complete concordance with respect to both immunohistochemical and fluorescence in situ hybridization results for core needle biopsy and excisional biopsy specimens in 87% of the patients evaluated. Comparison of fluorescent in situ hybridization results of the 129 core needle biopsies to the 131 excisional biopsies of all 125 patients showed a concordance rate of 92%. The immunohistochemical stain results of the same core needle and excisional biopsies showed a concordance rate of 98%. Comparison of the immunohistochemical stain results with the fluorescent in situ hybridization results for all 260 cases examined showed 95% concordance. On the basis of our study, we observed that repeating HER-2/neu testing by immunohistochemical stain and/or fluorescent in situ hybridization methods on excisional biopsy is not unreasonable, in particular in cases of intratumoral heterogeneity, indeterminate/borderline HER-2/neu results and after neoadjuvant chemotherapy.

Predictive value of circulating tumor cells (CTCs) in newly-diagnosed and recurrent ovarian cancer patients

OBJECTIVE To determine whether circulating tumor cells (CTCs), as detected and enumerated by the Veridex CellSearch system, could predict for clinical outcomes in women with newly diagnosed or recurrent epithelial ovarian cancer. METHODS Serial measurements of CTC s and paired serum CA125 were collected in a series of 78 women with newly diagnosed or recurrent ovarian cancer seen at our institution over a period of 15 months. Clinical data were abstracted from patient medical records. CTCs were detected and enumerated by the CellSearch protocol, and patients were divided into CTC negative (<2 CTCs) or positive (≥2 CTCs) groups. CTC groups were correlated with clinical characteristics and outcomes. A longitudinal analysis of CTC change and CA125 trends was also performed. RESULTS At least one CTC was isolated from the peripheral blood of over 80% of the women participating in this study, with a range from 0 to 8. No correlations were observed between CTC numbers and clinical characteristics or outcomes. Although both serum CA125 and CTC number exhibited an overall significant decreasing trend over time, there was no correlation observed between change in CTCs and CA125. CONCLUSION Using the FDA-approved CellSearch system, CTCs can be isolated from women with newly diagnosed or recurrent ovarian cancer. However, CTC numbers do not significantly correlate with clinical characteristics or patient outcomes. Future studies should focus on phenotypic characterization of CTCs and whether different isolation protocols yield a higher number of CTCs or add prognostic value.

Benign metastasizing pleomorphic adenoma presenting as a solitary kidney mass: imaging features.

Benign metastasizing pleomorphic adenoma is a rare condition that occurs in patients with a prior history of pleomorphic adenoma of the salivary glands. Metastases to the kidney are extremely rare, and, to the best of our knowledge, their imaging appearance on multiple cross-sectional imaging modalities has not been described. We present a solitary metastasis to the kidney in a 40-year-old woman. Computed tomography and magnetic resonance imaging demonstrated a 2.4 cm, well-marginated, enhancing mass that protruded into the renal sinus fat. Findings were indistinguishable from a primary renal malignancy. Prior history is crucial in suggesting the correct diagnosis.

Young investigator challenge: Application of cytologic techniques to circulating tumor cell specimens: Detecting activation of the oncogenic transcription factor STAT3

The circulating tumor cell (CTC) field is rapidly advancing with the advent of continuously improving technologies for enriching these rare neoplastic cells from blood. CTC enumeration provides prognostic information, and CTC characterization has the potential to provide more useful information for the clinical decision‐making process in this era of personalized medicine and targeted therapeutics. Proof‐of‐principle studies have shown that CTC samples can be characterized with a variety of techniques in the research laboratory environment. The goal of the current study was to validate routine cytologic techniques and immunohistochemical markers in CTC samples in a clinical cytology laboratory, using inducible phosphorylated signal transducer and activator of transcription 3 (pSTAT3) as a clinically important example and Ki‐67 as a positive control.

Circulating Tumor Cells

Circulating tumor cells (CTCs) are rare tumor cells found in the blood of patients with cancer that can be reliably detected by CTC technologies to provide prognostic, predictive, and diagnostic information. CTC sampling reflects intratumoral and intertumoral heterogeneity better than targeted biopsy. CTC samples are minimally invasive and amenable to repeated sampling, allowing real-time evaluation of tumor in response to therapy-related pressures and possibly early detection. Cytology is the most natural arena for integration of CTC testing. CTC technology may also be deployed to enhance and facilitate the practice of cytology and surgical pathology.

Molecular Testing of Nodules with a Suspicious or Malignant Cytologic Diagnosis in the Setting of Non-Invasive Follicular Thyroid Neoplasm with Papillary-Like Nuclear Features (NIFTP)

Non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) is an indolent thyroid tumor characterized by frequent RAS mutations and an absence of the BRAF V600E mutation commonly seen in classical papillary thyroid carcinoma (cPTC). The ability to differentiate potential NIFTP/follicular variant of papillary thyroid carcinoma (FVPTC) from cPTC at the time of fine-needle aspiration (FNA) can facilitate conservative management of NIFTP. The aim of the current study was to investigate how molecular testing may add to cytologic assessment in the pre-operative differentiation of potential NIFTP/FVPTC and cPTC. We had previously evaluated cytologists’ ability to prospectively distinguish potential NIFTP/FVPTC from cPTC in a cohort of 56 consecutive FNAs diagnosed as malignant or suspicious for malignancy. We utilized this cohort to perform molecular analysis. Detected molecular abnormalities were stratified into two groups: (1) those supporting malignancy and (2) those supporting a diagnosis of potential NIFTP/FVPTC. The cytologists’ characterization of cases and the detected molecular alterations were correlated with the final histologic diagnoses. Molecular testing was performed in 52 (93%) of the 56 cases. For the 37 cases cytologists favored to be cPTC, 31 (84%) had a molecular result that supported malignancy (28 BRAF V600E mutations, 2 NTRK1 fusions, 1 AGK-BRAF fusion). For the 8 cases that were favored to be NIFTP/FVPTC by cytologists, 7 (88%) had a molecular result that supported conservative management (1 NRAS mutation, 6 wild-type result). Seven cases were designated as cytomorphologically indeterminate for NIFTP/FVPTC or cPTC, of which 6 (86%) had a molecular result that would have aided in the pre-operative assessment of potential NIFTP/FVPTC or cPTC/malignancy. These included 3 BRAF V600E mutations in nodules that were cPTC on resection, an HRAS mutation, and a wild-type result in the 2 nodules that were NIFTP, and a TERT promoter mutation along with an NRAS mutation in a poorly differentiated thyroid carcinoma. For nodules with an FNA diagnosis of suspicious for malignancy or malignant, cytologists can differentiate most cases of potential NIFTP/FVPTC from cPTC. However, molecular testing may be valuable for a subset of cases, especially those that are indeterminate for potential NIFTP/FVPTC versus cPTC based on cytologic features alone.