CD133 expression is associated with less DNA repair, better response to chemotherapy and survival in ER-positive/HER2-negative breast cancer.

Purpose: CD133, a cancer stem cells (CSC) marker, has been reported to be associated with treatment resistance and worse survival in triple-negative breast cancer (BC). However, the clinical relevance of CD133 expression in ER-positive/HER2-negative (ER+/HER2-) BC, the most abundant subtype, remains unknown. Methods: The BC cohorts from the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC, n = 1904) and The Cancer Genome Atlas (TCGA, n = 1065) were used to obtain biological variables and gene expression data. Results: Epithelial cells were the exclusive source of CD133 gene expression in a bulk BC. CD133-high ER+/HER2- BC was associated with CD24, NOTCH1, DLL1, and ALDH1A1 gene expressions, as well as with WNT/ß-Catenin, Hedgehog, and Notchsignaling pathways, all characteristic for CSC. Consistent with a CSC phenotype, CD133-low BC was enriched with gene sets related to cell proliferation, such as G2M Checkpoint, MYC Targets V1, E2F Targets, and Ki67 gene expression. CD133-low BC was also linked with enrichment of genes related to DNA repair, such as BRCA1, E2F1, E2F4, CDK1/2. On the other hand, CD133-high tumors had proinflammatory microenvironment, higher activity of immune cells, and higher expression of genes related to inflammation and immune response. Finally, CD133-high tumors had better pathological complete response after neoadjuvant chemotherapy in GSE25066 cohort and better disease-free survival and overall survival in both TCGA and METABRIC cohorts. Conclusion: CD133-high ER+/HER2- BC was associated with CSC phenotype such as less cell proliferation and DNA repair, but also with enhanced inflammation, better response to neoadjuvant chemotherapy and better prognosis.

Clinical relevance of stem cell surface markers CD133, CD24, and CD44 in colorectal cancer.

Colon cancer stem cells (CSC) identified by cell surface markers CD133, CD24, and CD44, have been shown to be involved with tumor formation, chemotherapy resistance, and the progression of metastatic disease. Using an in silico translational approach, we hypothesize that a combination of these CSC markers has prognostic value in a large cohort of patients with colorectal cancer. Clinicopathologic and RNA expression data from a total of 594 colorectal cancer (CRC) patients from TCGA were analyzed. The expression of CD133, CD24, and CD44 was individually defined as "high" or "low" based on the median expression. Disease specific survival (DSS) and overall survival (OS) were not associated with tumors that are CD133-high or CD44-high alone. Patients with CD24-high tumors have significantly better DSS (P<0.001) and OS (P = 0.043). CD24-high, CD44-high and CD133-high tumors were associated with significantly greater EGFR, KRAS and Ki67 expression (all P<0.001). CD133, CD24 and CD44-high tumors were independently enriched for conventional stemness-related signaling pathways such as Wnt/ß-catenin and Hedgehog signaling pathways. There was no survival difference linked to CD133-high/CD44-low patients, but CD44-high/CD24-low patients have worse DSS (P = 0.005) compared with CD44-low/CD24-high patients. CD133-high/CD24-low tumors show significant negative enrichment of MYC targets, E2F targets, G2M checkpoint and mitotic spindle gene sets, suggesting less cell proliferation in these tumors. Patients with CD133-high/CD24-low tumors have worse DSS (P = 0.004) and OS (P = 0.044), and are more likely to have early and late recurrences. In conclusion, we demonstrated that CD133-high/CD24-low tumors may predict colorectal cancer prognosis.

Intratumoral Adipocyte-High Breast Cancer Enrich for Metastatic and Inflammation-Related Pathways but Associated with Less Cancer Cell Proliferation.

Cancer-associated adipocytes are known to cause inflammation, leading to cancer progression and metastasis. The clinicopathological and transcriptomic data from 2256 patients with breast cancer were obtained based on three cohorts: The Cancer Genome Atlas (TCGA), GSE25066, and a study by Yau et al. For the current study, we defined the adipocyte, which is calculated by utilizing a computational algorithm, xCell, as "intratumoral adipocyte". These intratumoral adipocytes appropriately reflected mature adipocytes in a bulk tumor. The amount of intratumoral adipocytes demonstrated no relationship with survival. Intratumoral adipocyte-high tumors significantly enriched for metastasis and inflammation-related gene sets and are associated with a favorable tumor immune microenvironment, especially in the ER+/HER2- subtype. On the other hand, intratumoral adipocyte-low tumors significantly enriched for cell cycle and cell proliferation-related gene sets. Correspondingly, intratumoral adipocyte-low tumors are associated with advanced pathological grades and inversely correlated with MKI67 expression. In conclusion, a high amount of intratumoral adipocytes in breast cancer was associated with inflammation, metastatic pathways, cancer stemness, and favorable tumor immune microenvironment. However, a low amount of adipocytes was associated with a highly proliferative tumor in ER-positive breast cancer. This cancer biology may explain the reason why patient survival did not differ by the amount of adipocytes.

The Impact of Immunofunctional Phenotyping on the Malfunction of the Cancer Immunity Cycle in Breast Cancer.

The cancer-immunity cycle (CIC) is a series of self-sustaining stepwise events to fight cancer growth by the immune system. We hypothesized that immunofunctional phenotyping that represent the malfunction of the CIC is clinically relevant in breast cancer (BC). Total of 2979 BC cases; 1075 from TCGA cohort, 1904 from METABRIC cohort were analyzed. The immunofunctional phenotype was classified as follows: hot T-cell infiltrated (HTI), high immune cytolytic activity (CYT), Cold T-cell infiltrated (CTI), high frequency of CD8+ T cells and low CYT, and non-inflamed, low frequency of CD8+ T cells and low CYT. The analysis of tumor immune microenvironment in the immunofunctional phenotype revealed that not only immunostimulatory factors, but also immunosuppressive factors were significantly elevated and immunosuppressive cells were significantly decreased in HTI. Patients in HTI were significantly associated with better survival in whole cohort and patients in CTI were significantly associated with worse survival in triple negative. Furthers, HTI was inversely related to estrogen responsive signaling. We demonstrated that immunofunctional phenotype not only indicated the degree of anti-cancer immune dysfunction, but also served as a prognostic biomarker and HTI was inversely related to estrogen response.

Incidence and predictors of incisional hernia after cytoreductive surgery and hyperthermic intraperitoneal chemotherapy.

Background and objectives: The incidence of incisional hernia (IH) after cytoreductive surgery plus hyperthermic intraperitoneal chemotherapy (CRS/HIPEC) is largely unknown. Methods: We conducted a retrospective study to identify patients who underwent CRS/HIPEC from 2001 to 2016. Patients were followed postoperatively for a minimum of two years. The primary outcome was the occurrence of an IH identified either on CT scan or physical examination. Univariate and multivariable logistic regression models were used to test associations with IH. Results: We identified 155 patients who underwent CRS/HIPEC; 26 patients (17%) were diagnosed with an IH at a median time of 245 days (Interquartile range [IQR] 175 - 331 days). On multivariable analysis, older age [50-64 vs. 18-49 years: hazard ratio (HR) = 0.08; 95% confidence interval (CI), 0.01 to 0.64)], female gender (HR = 0.09; 95% CI, 0.01 to 0.75), and increased BMI (>30 vs. <25; HR = 0.03; 95% CI, 0.01 to 0.37) were significant independent predictors of IH. Conclusions: The incidence of IH in this high-risk patient population treated with CRS/HIPEC is similar to that after other abdominal cancer operations. Nevertheless, the occurrence of IH is an important patient outcome, so alternative closure techniques for reducing IH should be studied in this patient population. Synopsis In a single-institutional study, the incidence of incisional hernia was 17% after cytoreductive surgery plus hyperthermic intraperitoneal chemotherapy. Independent risk factors of incisional hernia were older age, female gender and obesity.

The use of indocyanine green angiography in postmastectomy reconstruction: Do outcomes improve over time?

BACKGROUND: Indocyanine green angiography (ICGA) reduces ischemic complications by assessing mastectomy flap perfusion intraoperatively. However, outcomes of ICGA can be surgeon-dependent due to its relative novelty. We aimed to determine whether patient outcomes improved with the adoption of ICGA over time. METHODS: We conducted a single-institution retrospective study of mastectomy patients between March 2012 (date of ICGA introduction) and October 2016. We included patients who underwent immediate expander-based reconstruction with intraoperative ICGA, followed by second-stage permanent implant placement. Patients were chronologically sorted into 3 groups, of 45 patients each, based on the date of ICGA. Complications and reconstruction wait times (time between initial expander placement and subsequent final reconstruction) amongst the 3 groups were evaluated. Using the Cochran-Armitage test for trend, we tested the change in median adjusted expander fill volumes (expander fill volume in milliliter per gram of breast removed) over time. RESULTS: We identified 135 patients. Rates of ischemic complications significantly decreased (Group 1, 36%; Group 2, 22%; Group 3, 11%; p = 0.03), despite significantly increasing median adjusted expander fill volumes (Group 1, 0.46 mL/g; Group 2, 0.63 mL/g; Group 3, 0.76 mL/g; p = 0.003) over time. The rates of unexpected returns to the operating room across the 3 groups were not significantly different. The median reconstruction wait time was significantly reduced in the later groups (Group 1, 146 days; Group 2, 122 days; Group 3, 87 days; p = 0.01). CONCLUSIONS: Outcomes for mastectomy with immediate expander-based reconstruction were found to improve with increasing case volume after implementation of ICGA.

Evaluation of a protocol for early detection of delayed brain hemorrhage in head injured patients on warfarin.

PURPOSE: In 2007, Essentia Health St. Mary's Medical Center (SMMC), a Level II trauma center in northeastern Minnesota, implemented a protocol for patients who presented with blunt head trauma and were receiving warfarin for anticoagulation. The purpose of this study was to determine the incidence and risk factors of early delayed, warfarin-associated intracranial hemorrhage (ICH). METHODS: Adult patients with signs and symptoms of head injury on warfarin who were admitted by protocol to SMMC between March 2007 and June 2015 were included. Patients were observed for neurologic change and received a follow-up head CT scan within 24 h after an initial negative scan. RESULTS: Among the 232 episodes of care studied, there were 204 patients. The average age was 71; 51% of patients were female. Most patients presented with Glasgow Coma Scale score of 15 and had signs of head trauma. The majority of patients (63%) had a therapeutic International Normalized Ratio (INR) for their indicated condition, but 19% of patients had a supratherapeutic INR and 19% had a subtherapeutic INR. The incidence of early delayed ICH was 1.7%; none of these cases required operative intervention or were fatal. CONCLUSIONS: For patients who were anticoagulated with warfarin and had sustained minor traumatic brain injury, implementation of our protocol showed low incidence of early delayed ICH in the first 24 h. We believe withholding warfarin for several days and careful follow-up regarding its resumption is warranted, especially in the setting of supratherapeutic INR.

Current survival and treatment trends for surgically resected intrahepatic cholangiocarcinoma in the United States.

BACKGROUND: Intrahepatic cholangiocarcinoma (ICC) is a rare and aggressive disease with an increasing incidence in the United States, and there is no level 1 evidence to help guide treatment decisions. We sought to determine national trends in surgical and medical management of patients with resected ICC, and more specifically, the role of lymphadenectomy (LAD) and utilization of chemotherapy. METHODS: An augmented version of the National Cancer Institute's Surveillance, Epidemiology, and End Results (SEER) cancer database registry was used to identify all surgically resected ICC patients from 2000 to 2014. We evaluated the incidence and adequacy of LAD, and receipt of chemotherapy over time. Next, multivariable logistic regressions were performed to determine the predictors of LAD and receipt of chemotherapy. Overall survival (OS) was evaluated using Kaplan-Meier and Cox proportional hazard models. RESULTS: We identified 1,263 patients who underwent resection for ICC. Lymph nodes (LNs) were removed in 49% of patients, however, only 10% of patients received adequate LAD by the American Joint Committee on Cancer (AJCC) criteria (≥6 nodes). LN metastases were found in 29% of patients who underwent nodal evaluation. Chemotherapy was administered to 40% of patients, was utilized more frequently over time (P<0.05), and was associated with improved survival in node positive patients (P<0.05). Patients who did not have LNs evaluated were significantly less likely to receive chemotherapy than those who did. Lastly, OS for the entire cohort improved over time (P<0.05). CONCLUSIONS: After analyzing the treatment and outcomes of resectable ICC, we concluded: (I) LN evaluation at the time of surgical resection remains inadequate; (II) utilization of chemotherapy has increased over time; (III) the lack of LAD likely results in under-staging and underutilization of chemotherapy; and (IV) despite less than ideal surgical and medical therapy median OS continues to improve.

Tumor grade and progesterone receptor status predict 21-gene recurrence score in early stage invasive breast carcinoma.

PURPOSE: The 21-gene recurrence score (RS) assay is increasingly utilized to predict the risk of recurrence in early stage estrogen receptor (ER)-positive breast cancer. We hypothesize that tumor grade and progesterone receptor (PR) status predict RS categorization. METHODS: We identified women between the ages of 18 and 74 years with stage I or II, ER-positive, invasive carcinoma of the breast from the Surveillance Epidemiology End-Results database from 2010 to 2013. Multivariable logistic regression was performed to determine factors associated with high-risk RS. RESULTS: We identified 42,530 patients that met inclusion criteria. Multivariable logistic regression demonstrated that grade I tumors [OR (odds ratio) 0.33, 95% CI (confidence interval) 0.31-0.37] and PR positive (PR+) status (OR 0.16, 95% CI 0.15-0.17) were significantly less likely to be associated with high-risk RS. Of patients with grade I PR+ tumors, 1% was in the high-risk group by the traditional cutoffs and 4% was in the high-risk group by the TAILORx cutoffs. The percentage of patients with high-risk RS remained low for grade I PR+ tumors regardless of age, race, tumor size, and lymph node status. CONCLUSIONS: We found that grade I PR+ tumors are associated a < 5% probability of having high-risk RS regardless of other patient demographic or pathologic factors. This suggests that the histologic factors of grade and PR status should be taken into consideration before ordering the 21-gene recurrence score assay.

Distribution of 21-Gene Recurrence Scores Among Breast Cancer Histologic Subtypes.

CONTEXT: - The 21-gene recurrence score (RS) provides a probability of distant recurrence for estrogen receptor-positive, human epidermal growth factor receptor 2 (HER2)-negative breast cancers. The utility of RS for rarer histologic subtypes of breast cancer is uncertain. OBJECTIVE: - To determine the distribution of RS among various histologic subtypes using a population database. DESIGN: - Women between the ages of 18 and 75 with estrogen receptor-positive, HER2-negative breast cancer and known RS results were identified using the Surveillance, Epidemiology, and End Results database. Recurrence scores were categorized into risk groups using both traditional and Trial Assigning Individualized Options for Treatment cutoffs. Multivariable logistic regression was used to determine factors associated with high-risk RS. RESULTS: - We identified 45 618 patients with stage I to III, estrogen receptor-positive, HER2-negative breast cancer who had RS available. Overall, 3087 (7%) and 6337 (14%) of cancers were classified as high risk based on traditional and Trial Assigning Individualized Options for Treatment RS cutoffs, respectively. The proportion of high-risk RS ranged from 1% (tubular, 2 of 225) to 68% (medullary, 13 of 19) and 4% (tubular, 10 of 225) to 79% (medullary, 15 of 19) for traditional and Trial Assigning Individualized Options for Treatment cutoffs, respectively. Based on multivariable logistic regression (excluding medullary), subtypes other than invasive ductal carcinoma and papillary carcinoma were significantly associated with lower RS. The strongest predictors of a high-risk RS were higher tumor grade and negative progesterone receptor status. CONCLUSIONS: - We identified distinct distributions of RS among different histologic subtypes of breast cancer. Excluding medullary carcinoma, histologic subtypes other than invasive ductal carcinoma and papillary carcinoma all predict lower RS.

CD133-targeted oncolytic adenovirus demonstrates anti-tumor effect in colorectal cancer.

Oncolytic Adenoviruses (OAds) are one of the most promising anti-cancer agents that can induce cancer specific cell death. Recently, we generated infectivity-selective OAd, and the resultant OAd tumor-specific binding shows strong efficacy and mitigates toxicity. In this study, we applied this strategy based on adenovirus library screening system for generation of CD133-targeted OAd, and examined their oncolytic activity against colorectal cancer (CRC) in vitro and in vivo. CD133 (Prominin-1) is an important cell surface marker of cancer stem (like) cells (CSCs) in various cancers, including CRC. Elimination of CSCs has a high likelihood to improve CRC treatment because CSCs population in the tumor contributes to recurrence, metastases, chemotherapy resistance, and poor survival. The OAd with CD133-targeting motif (AdML-TYML) selectively infected CD133+ cultured cells and lysed them efficiently. Treatment with AdML-TYML prior to tumor inoculation inhibited the establishment of tumor of CD133+ CRC cell lines in nude mice. AdML-TYML also showed strong antitumor effect after intratumoral injections in already established CD133+ CRC subcutaneous xenografts. Our results indicate that CD133-targeted OAd selectively infected CD133+ CRC, and exhibited anti-tumorigenicity and therapeutic effect in established tumors. This novel infectivity selective virus could be a potent tool for the prevention of metastases and relapses in CRC.

Impact of the 21-gene recurrence score on outcome in patients with invasive lobular carcinoma of the breast.

PURPOSE: Invasive lobular carcinoma (ILC) of the breast has unique clinicopathologic characteristics, compared to invasive ductal carcinoma. The role of the 21-gene Recurrence Score (RS) has not been clearly defined for ILC. We sought to determine the prognostic value of RS and the impact of adjuvant chemotherapy on long-term survival in patients with ILC. METHODS: Utilizing the Surveillance, Epidemiology and End Results database from 2004 to 2013, we identified records of women aged 18-74 years, diagnosed with estrogen receptor (ER)-positive ILC (stage I to III) with RS available. We categorized patients into risk groups based on the traditional RS cutoffs and into those of the Trial Assigning Individualized Options for Treatment (TAILORx). Five-year breast cancer-specific survival (BCSS) was analyzed using the Kaplan-Meier method and Cox proportional hazards models. RESULTS: Of the 7316 women included, 21% were in the low-risk; 71%, intermediate-risk; and 8%, high-risk groups as per TAILORx RS cutoffs. The 5-year BCSS was 99% in the low-risk, 99% in the intermediate-risk, and 96% in the high-risk groups. A high-risk RS as per TAILORx cutoff was independently associated with increased mortality (hazard ratio [HR] of death 2.37, 95% confidence interval [CI] 1.14-4.95) when compared to a low-risk RS. In both the high-risk and intermediate-risk groups, adjuvant chemotherapy was not significantly associated with the HR of death (high-risk, HR 1.14, 95% CI 0.55-2.38; intermediate-risk, HR 1.08, 95% CI 0.62-1.87). CONCLUSION: For patients with ER-positive ILC, 8% were in the high-risk and 72% were in the intermediate-risk groups as per the TAILORx RS cutoffs. In the high-risk group, the RS predicted a lower 5-year BCSS. Adjuvant chemotherapy did not seem to confer a survival benefit for either the intermediate- or the high-risk cohorts.

Relative effectiveness of adjuvant chemotherapy for invasive lobular compared with invasive ductal carcinoma of the breast.

BACKGROUND: Invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC) have distinct clinical, pathologic, and genomic characteristics. The objective of the current study was to compare the relative impact of adjuvant chemotherapy on the survival of patients with ILC versus those with IDC. METHODS: Women with estrogen receptor (ER)-positive, human epidermal growth factor receptor 1 (HER2) -negative, stage I/II IDC and ILC who received endocrine therapy were identified from the 2000 to 2014 California Cancer Registry. Patient, tumor, and treatment characteristics were collected. Ten-year overall survival (OS) was estimated using the Kaplan-Meier method and Cox proportional-hazards modeling. RESULTS: In total, 32,997 women with IDC and 4638 with ILC were identified. The receipt of chemotherapy significantly decreased during the study for both subtypes. For patients with IDC, the 10-year OS rate was 95% among those who received endocrine therapy alone versus 93% (P < .01) among those who received endocrine therapy plus chemotherapy. For patients with ILC, the 10-year OS rate was 94% among those who received endocrine therapy alone versus 92% (P < .01) among those who received endocrine therapy plus chemotherapy. After adjusting for patient and treatment factors, adjuvant chemotherapy was significantly associated with a decreased 10-year hazard of death for patients with IDC (hazard ratio, 0.83; 95% confidence interval, 0.74-0.92). In contrast, adjuvant chemotherapy was not independently associated with the adjusted 10-year hazard of death for patients with ILC (hazard ratio, 1.14; 95% confidence interval, 0.90-1.46). CONCLUSIONS: Adjuvant chemotherapy was not associated with improved OS for patients with ER-positive, HER2-negative, stage I/II ILC. Avoidance of ineffective chemotherapy will markedly reduce the adverse effects and economic burden of breast cancer treatment for a large proportion of patients with breast cancer. Cancer 2017;123:3015-21. © 2017 American Cancer Society.

Showing the Way: Oncolytic Adenoviruses as Chaperones of Immunostimulatory Adjuncts.

Oncolytic adenoviruses (OAds) are increasingly recognized as vectors for immunotherapy in the treatment of various solid tumors. The myriads of advantages of using adenovirus include targeted specificity upon infection and selective replication, which lead to localized viral burst, exponential spread of OAds, and antitumor effect. OAds can also induce a strong immune reaction due to the massive release of tumor antigens upon cytolysis and the presence of viral antigens. This review will highlight recent advances in adenoviral vectors expressing immunostimulatory effectors, such as GM-CSF (granulocyte macrophage colony-stimulating factor), interferon-α, interleukin-12, and CD40L. We will also discuss the combination of OAds with other immunotherapeutic strategies and describe the current understanding of how adenoviral vectors interact with the immune system to eliminate cancer cells.

Plac8 links oncogenic mutations to regulation of autophagy and is critical to pancreatic cancer progression.

Mutations in p53 and RAS potently cooperate in oncogenic transformation, and correspondingly, these genetic alterations frequently coexist in pancreatic ductal adenocarcinoma (PDA) and other human cancers. Previously, we identified a set of genes synergistically activated by combined RAS and p53 mutations as frequent downstream mediators of tumorigenesis. Here, we show that the synergistically activated gene Plac8 is critical for pancreatic cancer growth. Silencing of Plac8 in cell lines suppresses tumor formation by blocking autophagy, a process essential for maintaining metabolic homeostasis in PDA, and genetic inactivation in an engineered mouse model inhibits PDA progression. We show that Plac8 is a critical regulator of the autophagic machinery, localizing to the lysosomal compartment and facilitating lysosome-autophagosome fusion. Plac8 thus provides a mechanistic link between primary oncogenic mutations and the induction of autophagy, a central mechanism of metabolic reprogramming, during PDA progression.

Autophagy in intra-hepatic cholangiocarcinoma.

Intra-hepatic cholangiocarcinoma (IHCC) is a primary cancer of the liver that shares histological features with the hepatic bile ducts from which it is thought to arise. The incidence of this disease is increasing, possibly related to increased inflammatory liver diseases such as viral hepatitis and steatohepatitis (commonly called "fatty liver"), induced by obesity, diabetes, and other metabolic derangements. Its prognosis is generally poor with early metastasis and presently there are limited effective treatments. A basic understanding of the disease has long been hampered by limited tumor cell lines and good model systems. Similarly, such limitations have obstructed new therapeutic inroads.

Kras(G12D) and p53 mutation cause primary intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (IHCC) is a primary cancer of the liver with an increasing incidence and poor prognosis. Preclinical studies of the etiology and treatment of this disease are hampered by the relatively small number of available IHCC cell lines or genetically faithful animal models. Here we report the development of a genetically engineered mouse model of IHCC that incorporates two of the most common mutations in human IHCC, activating mutations of Kras (Kras(G12D)) and deletion of p53. Tissue-specific activation of Kras(G12D) alone resulted in the development of invasive IHCC with low penetrance and long latency. Latency was shortened by combining Kras(G12D) activation with heterozygous or homozygous deletion of p53 (mean survival of 56 weeks vs. 19 weeks, respectively), which also resulted in widespread local and distant metastasis. Serial analysis showed that the murine models closely recapitulated the multistage histopathologic progression of the human disease, including the development of stroma-rich tumors and the premalignant biliary lesions, intraductal papillary biliary neoplasms (IPBN), and Von Meyenburg complexes (VMC; also known as biliary hamartomas). These findings establish a new genetically and histopathologically faithful model of IHCC and lend experimental support to the hypothesis that IPBN and VMC are precursors to invasive cancers.

Immunoglobulin heavy chain exclusion in the shark.

The adaptive immune system depends on specific antigen receptors, immunoglobulins (Ig) in B lymphocytes and T cell receptors (TCR) in T lymphocytes. Adaptive responses to immune challenge are based on the expression of a single species of antigen receptor per cell; and in B cells, this is mediated in part by allelic exclusion at the Ig heavy (H) chain locus. How allelic exclusion is regulated is unclear; we considered that sharks, the oldest vertebrates possessing the Ig/TCR-based immune system, would yield insights not previously approachable and reveal the primordial basis of the regulation of allelic exclusion. Sharks have an IgH locus organization consisting of 15-200 independently rearranging miniloci (VH-D1-D2-JH-Cmu), a gene organization that is considered ancestral to the tetrapod and bony fish IgH locus. We found that rearrangement takes place only within a minilocus, and the recombining gene segments are assembled simultaneously and randomly. Only one or few H chain genes were fully rearranged in each shark B cell, whereas the other loci retained their germline configuration. In contrast, most IgH were partially rearranged in every thymocyte (developing T cell) examined, but no IgH transcripts were detected. The distinction between B and T cells in their IgH configurations and transcription reveals a heretofore unsuspected chromatin state permissive for rearrangement in precursor lymphocytes, and suggests that controlled limitation of B cell lineage-specific factors mediate regulated rearrangement and allelic exclusion. This regulation may be shared by higher vertebrates in which additional mechanistic and regulatory elements have evolved with their structurally complex IgH locus.