Douglas M. Dahl

Isolation and Characterization of Circulating Tumor Cells from Patients with Localized and Metastatic Prostate Cancer

Automated imaging of prostate-specific cancer cells from the blood provides a measure of circulating tumor cell half-life after tumor resection. Circling Cancers Out Oftentimes a patient and his or her clinician learn together at the flip of a radiological imaging scan that a solid tumor, previously removed, has returned either at the original site or at new locations to which it has spread. A failure of cancer treatment—but could it have been predicted? Whether it is freely floating tumor DNA or tumor cells, the circulation of cancer-derived material in the blood holds great promise for the early detection and prevention of cancer metastases. The accurate identification, enumeration, and molecular classification of blood-borne cells—although postulated more than 140 years ago—remain the greatest challenge. Now, in a small cohort of individuals with and without prostate cancer, Stott and colleagues have used a silicon microfluidic cell-capture technology that, when coupled to an automated imaging system, enables the detection and enumeration of prostate cancer cells fished out from the blood. These cells express a surface protein that uniquely identifies epithelial cells in the circulation. Once efficiently captured by antibody to this protein, the cells are counterstained with antibodies that are prostate-specific and that indicate cell proliferation, suggesting that these circulating cells are ready to repopulate distant metastatic sites. In their study, tumor cells obtained from the blood of cancer patients were monitored before and after surgery; some circulating cells persisted months after surgery while others rapidly declined shortly thereafter. Whether the persistence or disappearance of lurking cancer cells reflects an intrinsic capacity for reseeding remains to be established, but the system used in the study offers great potential for oncologists to detect cancer-related changes earlier and to monitor responses to drug treatments. In the hope of ultimately applying personalized treatments to cancer patients, based on “real-time” monitoring of the tumor cell genetic makeup, this approach to identifying these circulating cells early on moves us beyond the finality of the films currently offered to patients in the clinic. Rare circulating tumor cells (CTCs) are present in the blood of patients with metastatic epithelial cancers but have been difficult to measure routinely. We report a quantitative automated imaging system for analysis of prostate CTCs, taking advantage of prostate-specific antigen (PSA), a unique prostate tumor–associated marker. The specificity of PSA staining enabled optimization of criteria for baseline image intensity, morphometric measurements, and integration of multiple signals in a three-dimensional microfluidic device. In a pilot analysis, we detected CTCs in prostate cancer patients with localized disease, before surgical tumor removal in 8 of 19 (42%) patients (range, 38 to 222 CTCs per milliliter). For 6 of the 8 patients with preoperative CTCs, a precipitous postoperative decline (<24 hours) suggests a short half-life for CTCs in the blood circulation. Other patients had persistent CTCs for up to 3 months after prostate removal, suggesting early but transient disseminated tumor deposits. In patients with metastatic prostate cancer, CTCs were detected in 23 of 36 (64%) cases (range, 14 to 5000 CTCs per milliliter). In previously untreated patients followed longitudinally, the numbers of CTCs declined after the initiation of effective therapy. The prostate cancer–specific TMPRSS2-ERG fusion was detectable in RNA extracted from CTCs from 9 of 20 (45%) patients with metastatic disease, and dual staining of captured CTCs for PSA and the cell division marker Ki67 indicated a broad range for the proportion of proliferating cells among CTCs. This method for analysis of CTCs will facilitate the application of noninvasive tumor sampling to direct targeted therapies in advanced prostate cancer and warrants the initiation of long-term clinical studies to test the importance of CTCs in invasive localized disease.

RNA-Seq of single prostate CTCs implicates noncanonical Wnt signaling in antiandrogen resistance.

Circulating signals of drug resistance Cancer drugs often lose their effectiveness because tumors acquire genetic changes that confer drug resistance. Ideally, patients would be switched to a different drug before tumor growth resumes, but this requires early knowledge of how resistance arose. Miyamoto et al. have developed a non-invasive method to spot resistance by sequencing RNA transcripts in single circulating tumor cells (CTCs) (see the Perspective by Nanus and Giannakakou). For example, in prostate cancer patients, drug resistance was triggered by activation of the Wnt signaling pathway. But CTCs are rare and fragile, and the technology needs further development before it is used in clinical practice. Science, this issue p. 1351; see also p. 1283 Analysis of circulating tumor cells from prostate cancer patients reveals a mechanism that contributes to treatment failure. [Also see Perspective by Nanus and Giannakakou] Prostate cancer is initially responsive to androgen deprivation, but the effectiveness of androgen receptor (AR) inhibitors in recurrent disease is variable. Biopsy of bone metastases is challenging; hence, sampling circulating tumor cells (CTCs) may reveal drug-resistance mechanisms. We established single-cell RNA-sequencing (RNA-Seq) profiles of 77 intact CTCs isolated from 13 patients (mean six CTCs per patient), by using microfluidic enrichment. Single CTCs from each individual display considerable heterogeneity, including expression of AR gene mutations and splicing variants. Retrospective analysis of CTCs from patients progressing under treatment with an AR inhibitor, compared with untreated cases, indicates activation of noncanonical Wnt signaling (P = 0.0064). Ectopic expression of Wnt5a in prostate cancer cells attenuates the antiproliferative effect of AR inhibition, whereas its suppression in drug-resistant cells restores partial sensitivity, a correlation also evident in an established mouse model. Thus, single-cell analysis of prostate CTCs reveals heterogeneity in signaling pathways that could contribute to treatment failure.

NCCN Guidelines Insights: Prostate Cancer Early Detection, Version 2.2016

The NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for Prostate Cancer Early Detection provide recommendations for prostate cancer screening in healthy men who have elected to participate in an early detection program. The NCCN Guidelines focus on minimizing unnecessary procedures and limiting the detection of indolent disease. These NCCN Guidelines Insights summarize the NCCN Prostate Cancer Early Detection Panels most significant discussions for the 2016 guideline update, which included issues surrounding screening in high-risk populations (ie, African Americans, BRCA1/2 mutation carriers), approaches to refine patient selection for initial and repeat biopsies, and approaches to improve biopsy specificity.

Lymphotropic nanoparticle-enhanced magnetic resonance imaging (LNMRI) identifies occult lymph node metastases in prostate cancer patients prior to salvage radiation therapy ☆

Twenty-six patients with prostate cancer status post-radical prostatectomy who were candidates for salvage radiation therapy (SRT) underwent lymphotropic nanoparticle enhanced MRI (LNMRI) using superparamagnetic nanoparticle ferumoxtran-10. LNMRI was well tolerated, with only two adverse events, both Grade 2. Six (23%) of the 26 patients, previously believed to be node negative, tested lymph node positive by LNMRI. A total of nine positive lymph nodes were identified in these six patients, none of which were enlarged based on size criteria.

Pilot study evaluating use of lymphotrophic nanoparticle-enhanced magnetic resonance imaging for assessing lymph nodes in renal cell cancer.

OBJECTIVE To assess lymphotrophic nanoparticle-enhanced magnetic resonance imaging (LNMRI) in identifying malignant nodal involvement in patients with renal neoplasms. METHODS MRI was performed in 9 patients with renal masses. All patients were imaged on a GE 1.5T system with phased array body coil. Protocols included T2 and T2* weighted imaging before and after administration of ferumoxtran-10 (Combidex) for the evaluation of lymph node (LN) involvement. All 9 patients underwent nephrectomy. Lymph node dissection (LND) was performed in patients with stage 2 renal cell cancer (RCC), or transitional cell cancer (TCC), per routine clinical practice. Data analysis was performed by 2 radiologists, who were blinded to pathologic results. Nodes that lacked contrast uptake were deemed malignant, and those with homogeneous uptake were deemed benign. Quantitative, retrospective analysis was performed on primary tumors by quantifying T2* with a monoexponential fitting algorithm (Osirix). T2* was quantified before, immediately after, and 24 hours after the administration of ferumoxtran-10. RESULTS MRI demonstrated 26 lymph nodes within the 9 patients imaged (24 benign and 2 malignant). Pathologic results allowed comparison in 22 of the 26 lymph nodes and demonstrated high sensitivity (100%) and specificity (95.7%). CONCLUSIONS LNMRI demonstrated high sensitivity (100%) and specificity (95.7%) in patients with renal neoplasms. Although in a relatively small sample size, the results are encouraging and warrant a larger, prospective trial.

Decision analysis using individual patient preferences to determine optimal treatment for localized prostate cancer.

Selecting treatment for clinically localized prostate cancer remains an ongoing challenge. Previous decision analyses focused on a hypothetical patient with average preferences, but preferences differ for clinically similar patients, implying that their optimal therapies may also differ.

Hand-assisted laparoscopic donor nephrectomy minimizes warm ischemia

OBJECTIVES Traditional open donor nephrectomy is associated with good donor outcomes and excellent allograft function. Laparoscopic donor nephrectomy may accomplish these same goals with less morbidity. We report our initial experience with hand-assisted laparoscopic living donor nephrectomy using a commercially available hand-assist device. METHODS Donor and allograft outcomes for the first 30 patients undergoing hand-assisted laparoscopic live donor nephrectomy in our institution were prospectively analyzed. RESULTS Hand-assisted laparoscopic donor nephrectomy was successfully completed in 29 (97%) of 30 donors. Organ dissection was carried out purely laparoscopically. Vessel division and allograft extraction were performed using a hand-assisted technique. The average operative time was 275 minutes (range 193 to 360), with an estimated blood loss of 99 mL (range 50 to 300). Pneumoperitoneum was consistently maintained during the hand-assisted portion of the procedure. The mean warm ischemic time was 72.5 seconds (range 30 to 165). On average, the regular diet was resumed after 2.2 days (range 1 to 3), and patients were discharged home 3.4 days (range 2 to 5) after surgery. Eight minor complications occurred in the donor group. Immediate graft function occurred in all 30 cases. No ureteral complications occurred. The recipient creatinine levels ranged from 0.6 to 2.4 mg/dL at an average follow-up of 11.5 months (range 1 to 23). CONCLUSIONS Laparoscopic donor nephrectomy is technically feasible and can be performed with minimal morbidity. Hand-assisted kidney extraction may help to facilitate immediate allograft function by minimizing the warm ischemic time.

New imaging modalities in bladder cancer.

Carcinoma of the urinary bladder is estimated to account for 61,420 new cases of cancer and 13,060 cancer related deaths in the United States in 2006 [1]. Of these approximately 80% of patients will present with superWcial cancer which can be treated by endoscopic resection with or with out neoadjuvant intravesical chemotherapy. However, despite adequate treatment of superWcial cancer there is a 70% recurrence rate at 3 years and a 10–30% chance of development of invasive cancer [2, 3]. Invasive but organ conWned disease is treated by radical cystectomy while more advanced tumors are treated by palliative chemotherapy or radiation. Hence, accurate pretreatment clinical and radiological staging at presentation is vital for therapeutic decision making and accurate prognosis. Traditionally urine cytology and standard cystoscopy have been eVective in the diagnosis of bladder cancer. However, urine cytology is insensitive for low-grade lesions and does not provide information about the stage of disease. Standard cystoscopy is minimally invasive and lacks speciWcity for Xat malignancies [3, 4]. Therefore non-invasive imaging plays a signiWcant role not only in the diagnosis and staging of bladder cancer but also in post treatment surveillance. The development of newer imaging techniques such as positron emission tomography (PET), lymphotropic nanoparticle enhanced magnetic resonance imaging (LNMRI), CT/ MR cystography, virtual cystoscopy, MR spectroscopy and intra-operative ultrasound has brought imaging to the forefront of bladder cancer evaluation. This article will review these newer imaging modalities and describe speciWc applications of each technique.

Bronchial carcinoid cells contain neural-type intermediate filaments.

Monospecific antibodies and indirect immunofluorescent microscopic examination, combined with immunochemical analysis, were used to examine intermediate filaments in four cases of bronchial carcinoid tumors. The results show that carcinoid cells express intermediate filaments of neural type (neurofilaments) but are negative for intermediate filaments of mesenchymal type (vimentin), epithelial type (keratin), muscle type (desmin), and glial type (glial fibrillary acidic protein). Since the expression of intermediate filaments shows a high degree of tissue specificity, the results suggest either derviation of bronchial carcinoid cells from maternal cells displaying neural characteristics or from cells with the capacity to acquire neural properties on neoplastic growth. It is also suggested that antineurofilament antibodies can be used as a useful aid in differential diagnosis of bronchial carcinoids from other pulmonary tumors.

Long-term Outcomes After Bladder-preserving Tri-modality Therapy for Patients with Muscle-invasive Bladder Cancer: An Updated Analysis of the Massachusetts General Hospital Experience

BACKGROUND Tri-modality therapy (TMT) is a recognized treatment strategy for selected patients with muscle-invasive bladder cancer (MIBC). OBJECTIVE Report long-term outcomes of patients with MIBC treated by TMT. DESIGN, SETTING, AND PARTICIPANTS Four hundred and seventy-five patients with cT2-T4a MIBC were enrolled on protocols or treated as per protocol at the Massachusetts General Hospital between 1986 and 2013. INTERVENTION Patients underwent transurethral resection of bladder tumor followed by concurrent radiation and chemotherapy. Patients with less than a complete response (CR) to chemoradiation or with an invasive recurrence were recommended to undergo salvage radical cystectomy. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Disease-specific survival (DSS) and overall survival (OS) were calculated using the Kaplan-Meier method. RESULTS AND LIMITATIONS Median follow-up for surviving patients was 7.21 yr. Five- and 10-yr DSS rates were 66% and 59%, respectively. Five- and 10-yr OS rates were 57% and 39%, respectively. The risk of salvage cystectomy at 5 yr was 29%. In multivariate analyses, T2 disease (OS hazard ratio [HR]: 0.57, 95% confidence interval [CI]: 0.44-0.75, DSS HR: 0.51, 95% CI: 0.36-0.73), CR to chemoradiation (OS HR: 0.61, 95% CI: 0.46-0.81, DSS HR: 0.49, 95% CI: 0.34-0.71), and presence of tumor-associated carcinoma in situ (OS HR: 1.56, 95% CI: 1.17-2.08, DSS HR: 1.50, 95% CI: 1.03-2.17) were significant predictors for OS and DSS. When evaluating our cohort over treatment eras, rates of CR improved from 66% to 88% and 5-yr DSS improved from 60% to 84% during the eras of 1986-1995 to 2005-2013, while the 5-yr risk of salvage radical cystectomy rate decreased from 42% to 16%. CONCLUSIONS These data demonstrate high rates of CR and bladder preservation in patients receiving TMT, and confirm DSS rates similar to modern cystectomy series. Contemporary results are particularly encouraging, and therefore TMT should be discussed and offered as a treatment option for selected patients. PATIENT SUMMARY Tri-modality therapy is an alternative to radical cystectomy for patients with muscle-invasive bladder cancer, and is associated with comparable long-term survival and high rates of bladder preservation.