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The role of lymph node assessment/dissection (LND) in endometrial cancer (EC) has been debated for decades, with significant practice variation between centers. Molecular classification of EC provides prognostic information and can be accurately performed on preoperative endometrial biopsies. We assessed the association between molecular subtype and lymph node metastases (LNM) in order to determine if this tool could be used to stratify surgical decision making.All EC patients undergoing primary staging surgery with planned complete pelvic +/- para-aortic LND from a single institution in the 2015 calendar year were identified, with clinicopathological and outcome data assessed in the context of retrospectively assigned molecular classification.172 patients were included. Molecular classification of the total cohort showed 21 POLEmut (12.2%), 47 MMRd (27.3%), 74 NSMP (43.1%), and 30 p53abn (17.4%) ECs. Complete pelvic +/- para-aortic LND was performed in 171 of 172 patients, and LNM were found in 31/171 (18.1%). This included macrometastases (19/31), micrometastases (5/31), and isolated tumour cells (ITCs) (7/31). LNM were pelvic only in 83.9%, and pelvic plus para-aortic in 16.1%. There were no isolated para-aortic LNM. Molecular subtype was significantly associated with LNM (p = 0.004). There was a strong association between the presence of LNM and p53abn EC (nodal involvement in 44.8% of cases), with LNM detected in 14.2% of POLEmut, 14.9% of MMRd, and 10.8% of NSMP EC. On multivariate analysis, molecular subtype and preoperative CA 12525 were significantly associated with LNM (p = 0.021 and p = 0.022 respectively) but preoperative grade and histotype were not (p = 0.24).EC molecular subtype is significantly associated with the presence of LNM. As molecular classification can be obtained on preoperative diagnostic specimens, this information can be used to guide surgical treatment planning and may reduce the cost and morbidity of unnecessary lymph node staging in EC care.

We measured the variation in practice across all aspects of endometrial cancer (EC) management and assessed the potential impact of implementation of molecular classification.Centers from across Canada provided representative tumor samples and clinical data, including preoperative workup, operative management, hereditary cancer program (HCP) referrals, adjuvant therapy, surveillance and outcomes, for all EC patients diagnosed in 2016. Tumors were classified into the four ProMisE molecular subtypes.A total of 1336 fully evaluable EC patients were identified from 10 tertiary cancer centers (TC; n = 1022) and 19 community centers (CC; n = 314). Variation of surgical practice across TCs was profound (14-100%) for lymphadenectomy (LND) (mean 57% Gr1/2, 82% Gr3) and omental sampling (20% Gr1/2, 79% Gr3). Preoperative CT scans were inconsistently obtained (mean 32% Gr1/2, 51% Gr3) and use of adjuvant chemo or chemoRT in high risk EC ranged from 0-55% and 64-100%, respectively. Molecular subtyping was performed retrospectively and identified 6% POLEmut, 28% MMRd, 48% NSMP and 18% p53abn ECs, and was significantly associated with survival. Within patients retrospectively diagnosed with MMRd EC only 22% had been referred to HCP. Of patients with p53abn EC, LND and omental sampling was not performed in 21% and 23% respectively, and 41% received no chemotherapy. Comparison of management in 2016 with current 2020 ESGO/ESTRO/ESP guidelines identified at least 26 and 95 patients that would have been directed to less or more adjuvant therapy, respectively (10% of cohort).Molecular classification has the potential to mitigate the profound variation in practice demonstrated in current EC care, enabling reproducible risk assessment, guiding treatment and reducing health care disparities.

Introduction/Background* The role of lymph node (LN) assessment in endometrial cancer (EC) has been a subject of debate for decades, with significant variation in use between centres. Molecular classification of EC provides objective, prognostic information and be performed on diagnostic endometrial biopsy specimens. The Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE) uses targeted next-generation sequencing to detect pathogenic POLE exonuclease domain mutations (POLEmut) and immunohistochemistry to evaluate for the presence of mismatch repair deficiency (MMRd), TP53 mutations (p53abn) or tumours with no specific molecular profile (NSMP/p53wt). Herein, we assessed the association between EC molecular subtype and LN metastases in a single institutional cohort with a uniform approach to LN assessment. Methodology All ECs treated with primary surgery from a single institution in 2015 underwent ProMisE molecular subtyping and collection of clinicopathologic and outcomes data. Result(s)* Complete pelvic and para-aortic lymphadenectomies were performed in 171 of 172 consecutive cases of EC. The distribution of ProMisE subtypes and clinicopathologic features associated with molecular subtype are outlined in table 1. The p53abn subtype was observed across a range of EC histotypes, including low grade endometrioid endometrial carcinoma. LN metastases were found in 31/171 (18.1%) patients: pelvic only in 83.9% and pelvic plus para-aortic in 16.1%. LN metastases included macrometastases (19/31), micrometastases (5/31), and isolated tumour cells (ITCs) (7/31). Molecular subtype was significantly associated with LN metastases (p=0.004); there was a strong association between LN metastases and p53abn EC (nodal involvement in 44.8% of cases). LN metastases were observed in 14.2% of POLEmut, 14.9% of MMRd, and 10.8% of NSMP EC. By multivariate analysis, molecular subtype and CA 125 >25 kU/L were significantly associated with LN metastases (p=0.021 and p=0.022 respectively) compared to histotype, which showed no significant association with LN status (p=0.24). Conclusion* EC molecular subtype significantly associates with LN metastases and offers objective, reproducible, and prognostic information from diagnostic specimens. Pre-operative knowledge of molecular subtype can guide biologically-informed approaches to LN sampling, particularly for patients with high molecular risk (p53abn) EC.

With the recent elucidation of gastric-type lesions in the female genital tract (especially in the cervix), occasional cases of endometrial adenocarcinoma displaying gastric (gastrointestinal) differentiation have been reported, but they are currently not recognized as a distinct pathologic entity. We report 9 cases of endometrial mucinous lesions which exhibit gastric (gastrointestinal)-type features by morphology and immunohistochemistry, including 4 adenocarcinomas and 5 benign mucinous lesions, in patients aged 32 to 85. The adenocarcinomas showed gastric-type morphology in all 4 cases and goblet cells in 1, with a component of benign gastric-type mucinous glands in 1 case. Immunohistochemically, the adenocarcinomas were positive for CK7 (4/4), CEA (4/4), MUC6 (3/3), PAX8 (3/4), CK20 (2/4), CDX2 (2/4), and estrogen receptor (1/4). They were negative for Napsin A (0/3), with mutation-type p53 staining in 2/4 cases, block-type p16 positivity in 1/4, and scattered chromogranin-positive cells in 1/2. Targeted next-generation sequencing revealed nonsense mutation in RB1 gene for the case with block-positive p16. Follow-up was available in all adenocarcinoma cases and indicated aggressive behavior; 2 patients were dead of disease at follow-up of 7 months to 3 years, 1 was alive with progression at 9 months, and 1 was alive without disease at 7 months. The benign mucinous lesions (including the benign component in 1 adenocarcinoma) exhibited gastric-type morphologic features in 5/6 cases, goblet cells in 5/6, and Paneth-like neuroendocrine cells in 1/6. These benign mucinous lesions were associated with an endometrial polyp in 5/6 cases. Cytologic atypia was present in 2/6 cases and a lobular architecture resembling cervical lobular endocervical glandular hyperplasia in 4/6. Immunohistochemically, the benign mucinous lesions were positive for CK7 (5/5), CDX2 (5/6), estrogen receptor (4/5), MUC6 (4/5), CK20 (3/5), PAX8 (3/5), and CEA (2/4), with scattered chromogranin-positive cells in 4/4 cases; in all cases tested Napsin A was negative, p53 was wild-type and p16 was negative. We propose the term "endometrial gastric (gastrointestinal)-type adenocarcinoma" for this distinctive group of rare aggressive endometrial carcinomas. We believe that benign or atypical gastric (gastrointestinal)-type mucinous lesions are putative precursors for these adenocarcinomas, comparable to recognized premalignant gastric-type lesions in the cervix and the vagina. Future recognition and reporting of these gastric-type endometrial mucinous lesions will help delineate their pathogenesis and clinical significance.

Objective To determine the validity of sentinel lymph node (SLN) biopsy with ICG in endometrial cancer and to evaluate the factors associated with poor mapping or false negative. Methods We reviewed all patients who underwent primary surgery for endometrial carcinoma with SLN mapping using ICG followed by pelvic lymphadenectomy from February 2014 to December 2015. SLNs were ultrastaged on final pathology. Patients' demographics, surgical approach and histopathological factors were prospectively collected. Detection rate, sensitivity and negative predictive value (NPV) were calculated and univariate analysis was performed to evaluate factors associated with failed bilateral detection of SLNs. Results A total of 119 patients were included. The overall and bilateral detection rates were 93% and 74%. Sensitivity and NPV were 100% in patients with bilateral detection; 95% and 99% respectively in cases with at least unilateral detection. Advanced FIGO stage (III or IV) was the only factor related to failed bilateral detection (p = 0.01). In 14 hemi-pelvis, the specimen labelled as SLN did not contain nodal tissue on final pathology (only lymphatic channels), which represented 37% of the “failed detection” cases. One false negative occurred in a patient with an ipsilateral clinically suspicious enlarged lymph node. Conclusion ICG is an excellent tracer for SLN mapping in endometrial cancer. Advanced FIGO stage correlated with failed bilateral detection (p = 0.01). Suspicious lymph nodes should be removed regardless of the mapping. Care should be taken to ensure that SLN specimen actually contains nodal tissue and not only swollen lymphatic channels, as this represents a significant cause of failed SLN mapping.

The Cancer Genome Atlas recently identified a genomic-based molecular classification of endometrial carcinomas, with 4 molecular categories: (1) ultramutated (polymerase epsilon [POLE] mutated), (2) hypermutated (microsatellite instability), (3) copy number abnormalities-low, and (4) copy number abnormalities-high. Two studies have since proposed models to classify endometrial carcinomas into 4 molecular subgroups, modeled after The Cancer Genome Atlas, using simplified and more clinically applicable surrogate methodologies. In our study, 151 endometrial carcinomas were molecularly categorized using sequencing for the exonuclease domain mutations (EDM) of POLE, and immunohistochemistry for p53 and mismatch repair (MMR) proteins. This separated cases into 1 of 4 groups: (1) POLE EDM, (2) MMR-D, (3) p53 wildtype (p53 wt), or (4) p53 abnormal (p53 abn). Seven gynecologic pathologists were asked to assign each case to one of the following categories: grade 1 to 2 endometrioid carcinoma (EC), grade 3 EC, mucinous, serous carcinoma (SC), clear cell, dedifferentiated, carcinosarcoma, mixed, and other. Consensus diagnosis among all 7 pathologists was highest in the p53 wt group (37/41, 90%), lowest in the p53 abn group (14/36, 39%), and intermediate in the POLE EDM (22/34, 65%) and MMR-D groups (23/40, 58%). Although the majority of p53 wt endometrial carcinomas are grade 1 to 2 EC (sensitivity: 90%), fewer than half of grade 1 to 2 EC fell into the p53 wt category (positive predictive value: 42%). Pure SC almost always resided in the p53 abn group (positive predictive value: 96%), but it was insensitive as a marker of p53 abn (sensitivity 64%) and the reproducibility of diagnosing SC was suboptimal. The limitations in the precise histologic classification of endometrial carcinomas highlights the importance of an ancillary molecular-based classification scheme.

ObjectiveUterine serous carcinoma (USC) is an aggressive histologic subtype of endometrial cancer that shares similarities to serous ovarian cancer, with a propensity for spread to the upper abdomen, a high recurrence rate, and a poor prognosis. The aim of this study was to determine whether the traditional surgical staging procedure for endometrial cancer was adequate for USC or whether a more extensive surgery, similar to the staging procedure for ovarian cancer, needs to be performed. Specifically, the roles of omentectomy and sentinel lymph node (SLN) mapping were evaluated.MethodsWe retrospectively identified cases of presumed clinical stage I USC at our institution from April 2005 to March 2014. Medical records were reviewed for the following information: age at diagnosis, preoperative imaging, operative findings, surgical procedure, and final histology with definitive International Federation of Gynecology and Obstetrics stage.ResultsA total of 39 patients with presumed clinical stage I USC were identified. According to the final pathology report, the surgical stage was as follows: 17 stage IA (44%), 8 stage IB (20%), 3 stage II (8%), 2 stage IIIA (5%), 6 stage IIIC1 (15%), 1 IIIC2 (3%), and 2 stage IVB (5%). Therefore, 14 patients (36%) were surgically upstaged, but none of the patients had their clinical disease upstaged by virtue of finding microscopic metastatic disease in an otherwise normal-looking omentum. Sentinel lymph node mapping was performed in 19 patients (42%). Sensitivity and negative predictive value of SLN mapping were 100% when at least 1 SLN was identified.ConclusionsThe detection of microscopic disease in radiologically and clinically normal-appearing omentum seems to be rare in USC. Sentinel lymph node mapping seems to be valuable in the serous subtype of endometrial cancer. A less extensive surgery may be possible in patients with USC as it seems to provide the same information as a more extensive surgery.

Objectives Indocyanine green (ICG) with near-infrared (NIR) fluorescence imaging is a new tracer modality used for lymphatic mapping. We report our initial experience with ICG for SLN mapping in cervical and endometrial cancer using a new endoscopic fluorescence imaging system. Methods We reviewed all patients who underwent primary surgery for early-stage endometrial and cervical carcinoma with SLN mapping using fluorescence imaging followed by pelvic lymphadenectomy from February to July 2014. Intracervical injection of ICG at 3 and 9 o'clock was performed in all cases. SLNs were ultrastaged on final pathology. Sensitivity and specificity values were calculated. Results A total of 50 patients were included in the study (42 endometrial and 8 cervical cancers). The median age was 62 (24–88) and median BMI 29 (19–56). The median SLN count was 3.1 (0–7) and median lymph node count was 15 (2–37). The overall and bilateral detection rate was 96% (48/50) and 88% (44/50). Positive SLNs were identified in 22% of patients (11/50), including 8 isolated tumor cells (ITC), 2 micrometastasis and 1 macrometastasis. There was one side-specific false negative case. Sensitivity, specificity and NPV were 93.3%, 100% and 98.7% respectively per side. Paraaortic node dissection was performed in 22% of cases. Two had paraaortic node metastasis both in patients with positive pelvic SLN. There were no allergic reactions to the ICG. Conclusions Based on our pilot experience, NIR fluorescence imaging with ICG is an excellent and safe tracer modality for SLN mapping with a very high overall (96%) and bilateral (88%) detection rate.

Standardized terminology has proven benefits in cancer reporting; in contrast, reporting of benign diagnoses in endometrial biopsy currently lacks such standardization. Unification and update on the lexicon can provide the structure and consistency needed for optimal patient care and quality assurance purposes. The Special Interest Group in Gynecologic Pathology of the Canadian Association of Pathologists-Association Canadienne des Pathologistes (CAP-ACP) embarked in an initiative to address the current need for consensus terminology in benign endometrial biopsy pathology reporting. Nine members of the Special Interest Group developed a guideline for structured diagnosis of benign endometrial pathology through critical appraisal of the available peer-reviewed literature and joint discussions. The first version of the document was circulated for feedback to a group of professionals in akin fields, the CAP-ACP Executive Committee and the CAP-ACP general membership. The final 1-page document included 17 diagnostic terms comprising the most common benign endometrial entities, as well as explanatory notes for pathologists. The proposed terminology was implemented in the practice of 5 pathologists from the group, who applied the guideline to all benign endometrial biopsies over a 2-wk period. A total of 212 benign endometrial biopsies were evaluated in this implementation step; the recommended terminology adequately covered the diagnosis in 203 cases (95.8%). A list of terminology for benign endometrial biopsy reporting, based on expert consensus and critical appraisal of the available literature, is presented. On the basis of our results of implementation at multiple centers, the proposed guideline can successfully cover the large majority of diagnostic scenarios. The document has the potential to positively impact patient care, promote quality assurance, and facilitate research initiatives aimed at improving histopathologic assessment of benign endometrium.

Objective To evaluate the outcome and the role of adjuvant treatment in the management of patients with endometrial cancer and isolated tumor cells (ITCs) identified by SLN mapping. Methods This single center study identified all patients undergoing hysterectomy, salpingo-oophorectomy, lymphadenectomy and SLN mapping for endometrial cancer between November 2010 and December 2015. Data was prospectively collected. Progression-free survival was analyzed according to the Kaplan-Meier method and compared using the log-rank test. Results A total of 519 patients were included. Overall, 85 patients (16.4%) were found to have SLN metastases of which 43 (51%) were macrometastasis, 11 (13%) micrometastasis (MM) and 31 (36%) ITC. Eleven (35%) of patients with ITCs received adjuvant chemotherapy±whole pelvic radiation therapy (WPRT), 10 (32%) received WPRT, and 10 (32%) received either no adjuvant treatment or vault brachytherapy (VBT) only. ITC patients received significantly less chemotherapy (p=0.0001) and WPRT (p=0.007) compared to patients with macrometastasis. Of note, ITC were not considered node positive in our study. With a median follow-up of 29months (range: 0–67), the progression free survival (PFS) at 3-years for the ITC patients was 95.5%, similar to node negative (87.6%) and micrometastasis patients (85.5%), but statistically better than patients with macrometastasis (58.5%) (p=0.0012). Only 1/31 patient with ITC recurred (IB, 7cm carcinosarcoma) despite adjuvant treatments. None of the ITC patients with endometrioid histology recurred (0/28) and none of the ITC patients who did not receive adjuvant treatment or VBT recurred (0/10). Conclusions Patients with endometrial cancer found to have SLN ITCs have an excellent outcome. The use of adjuvant treatment should be tailored to uterine factors and histology and not solely based on the presence of ITCs. Patients with ITCs and otherwise low-risk uterine disease probably derive little benefit from receiving additional treatments. More studies are needed to confirm our results.

ObjectiveThis study aimed to evaluate the feasibility of simple vaginal trachelectomy and node assessment in patients with low-risk early-stage cervical cancer (MethodsFrom May 2007 to November 2012, 16 women with low-risk small-volume cervical cancer underwent a simple vaginal trachelectomy preceded by laparoscopic sentinel node mapping plus or minus pelvic node dissection. Data were collected prospectively in a computerized database. Descriptive statistics and Kaplan-Meyer estimate were used for analysis.ResultsPatients’ median age was 30 years and 12 (75%) were nulliparous. Six had a diagnostic cone, 6 had a loop electrocautery excision procedure, 3 had cervical biopsies, and 1 had polyp excision. All patients underwent a preoperative pelvic magnetic resonance imaging. Four patients had stage IA1 with lymph vascular space invasion (LSVI), 6 IA2, and 6 IB1. Ten (62.5%) had squamous lesions, 7 had adenocarcinoma. LVSI was present in 4 cases, suspicious in 2, and absent in 10. There were 2 surgical complications: a trocar site hematoma and a vaginal laceration. The median OR time was 150 minutes (range, 120–180 minutes) and median blood loss was 50 mL (range, 50–150 mL). On final pathology, lymph nodes were negative in all patients. Thirteen (81%) patients had either no residual disease (6) or residual dysplasia only (7) in the trachelectomy specimen. Margins were negative in all cases. With a median follow-up of 27 months (range, 1–65 months), there have been no recurrences. The recurrence-free survival at 24 months is 100%. Eight patients have conceived: 3 were term deliveries and 4 are ongoing.ConclusionsSimple trachelectomy and nodes seems to be a safe alternative in well-selected patients with early-stage low-risk cervical cancer. Our data will need to be confirmed in larger series.

BackgroundWomen with germ line BRCA1 or BRCA2 mutations have a marked increased risk of breast and ovarian cancer compared with the general population, whereas risk-reducing salpingo-oophorectomy (RRSO) significantly lowers the incidence of these cancers. The objective of this study was to review the clinical and pathological characteristics of a French Canadian population undergoing RRSO. Surgical morbidity was also evaluated.Materials and MethodsFrom December 1999 to December 2009, all women who underwent RRSO at our institution were identified. Medical records were retrospectively reviewed. Descriptive statistics, the Fischer exact test, and the Student t test were used for analysis.ResultsDuring the study period, RRSO was performed on 119 women. Mean age at surgery was 49 years (35–72 years), and 63 patients (53%) were premenopausal. Sixty-two women (52%) had a history of in situ or invasive breast cancer. BRCA1 and BRCA2 mutations were present in 34 patients (29%) and 42 patients (35%), respectively, whereas 43 patients (36%) were considered to have an increased risk of breast and ovarian cancer, despite a personal genetic test, which was either negative (n = 23) or unknown because the patient declined genetic testing (n = 20). Most patients with a uterus in place had a complementary hysterectomy (65%). Six complications occurred (3 hematomas, 2 cardiac arrhythmias, and 1 cystotomy). In one patient (0.8%), a high-grade stage II ovarian cancer was discovered at the time of surgery. Fallopian tube atypias were identified on final pathology in 8 cases (6.7%). After a median follow-up of 22 months, 4 women (3.4%) developed breast cancer and one woman (0.8%) developed peritoneal cancer.ConclusionsRisk-reducing salpingo-oophorectomy is highly effective in preventing ovarian, fallopian tube, and breast cancers in a high-risk French Canadian population; and the surgical morbidity is low.

Vaginal radical trachelectomy (VRT) is a fertility-sparing surgical technique used as an alternative to radical hysterectomy in early stage cervical carcinoma. With the advent of VRT, preoperative evaluation of the surgical margin has become imperative, because if the tumor is found within 5 mm of the endocervical margin, additional surgical resection is required. In a study published earlier from our center, we came to the conclusion that a frozen section should be conducted only when a cancerous lesion is grossly visible, and that it could be omitted in normal-looking specimens or VRT with nonspecific lesions. Since then, 53 VRT have been performed in our center, and frozen sections were conducted according to these recommendations. Fifteen VRT were grossly normal, 24 had a nonspecific lesion and 14 showed a grossly visible lesion. Final margins were satisfactory on all 15 grossly normal specimens. Of the 24 VRT with nonspecific lesions, 2 cases for which no frozen section was performed had unsatisfactory final margins (

The aim of this study was to determine the risk of metastasis in the remaining non-SLNs when the SLN is positive and to identify the factors that can predict this risk.We reviewed all patients who underwent primary surgery for endometrial carcinoma with lymphadenectomy and SLN mapping (November 2010-November 2013) in our center. SLNs were ultra-staged on final pathology.A total of 268 patients were included. Overall, 43/268 patients (16%) were found to have SLN metastasis: macro-metastasis in 24 patients, micro-metastasis in 7 and ITC in 12. Non-SLN metastases were found in 15 of the 43 patients (34.8%) with positive SLN. Size of the SLN metastasis was the only factor associated with an increased likelihood of non-SLN metastasis (p=0.005). When the size of the SLN metastasis was ≤2mm, the risk of having another positive lymph node was only 5%, conversely, when the size of the SLN metastasis was2mm, the risk of having another positive lymph node was 60.8% (p0.0001). Histologic type, grade, depth of myometrial invasion, LVSI, cervical stromal invasion and CA-125 were not predictive.When the SLN is positive, the risk of metastasis in the remaining non-SLNs was 34.8%. Size of the metastasis within the SLN was the only factor that could predict the risk of non-SLN metastasis; 2mm seems to be the cutoff size below which the risk of non-SLN metastasis is low.

Gene amplification is frequently observed in Leishmania cells selected for drug resistance. By gene targeting we have tagged both alleles of the H locus of Leishmania tarentolae with the neomycin and hygromycin phosphotransferase genes ( neo and hyg ). Selection of these recombinant parasites for low level methotrexate resistance led to amplification of the H locus as part of linear amplicons. The availability of tags has permitted us to determine that both alleles can be amplified in the same cell and that chromosomal deletions are frequent. When methotrexate concentration was increased in subsequent selection steps, circles were observed in several mutants. We have introduced a hyg marker into linear amplicons to test whether the circles originated from linear amplicons. After selection with a high methotrexate concentration, circles with the hyg marker were observed, showing that circles can indeed be formed from linear amplicons. The tagging of H locus alleles permits appreciation of the extent of genetic rearrangements leading to amplicon formation in Leishmania cells selected for drug resistance.

The mechanism of resistance to the metal arsenite has been studied and compared in L. mexicana, L. tropica, and L. tarentolae selected in a step by step manner for arsenite resistance. Amplification of the ABC transporter gene pgpA was found to be a frequent resistance mechanism in all species. Transfection of pgpA genes into different species indicated that both the origin of the pgpA gene and the recipient strain into which the gene is transfected seem important for resistance. An increase in the levels of trypanothione was also correlated with metal resistance in different Leishmania species. The mechanism used to increase the levels of trypanothione seems to differ, however, between the different species. This study points to a key role of transporters and thiol levels in metal resistance in Leishmania.

Resistance to the oxyanion arsenite in the parasite Leishmania is multifactorial. We have described previously the frequent amplification of the ABC transporter gene pgpA , the presence of a non‐PgpA thiol–metal efflux pump and increased levels of glutathione and trypanothione in resistant cells. Other loci are also amplified, although their role in resistance is unknown. By gene transfection, we have characterized one of these novel genes. It corresponds to gsh1 , which encodes γ‐glutamylcysteine synthetase, an enzyme involved in the rate‐limiting step of glutathione biosynthesis. Transfection of gsh1 in wild‐type cells increased the levels of glutathione and trypanothione to levels found in resistant mutants. These transfectants were not resistant to metals. However, when gsh1 was transfected in partial revertants, it conferred resistance. As pgpA is frequently co‐amplified with gsh1 , we co‐transfected the two genes into both wild‐type and partial revertants. Arsenite resistance levels in wild‐type cells could be accounted for by the contribution of PgpA alone. In the partial revertant, the gsh1 and pgpA gene product acted synergistically. These results support our previous suggestion that PgpA recognizes metals conjugated to thiols. Furthermore, amplification of gsh1 overcomes the rate‐limiting step in the synthesis of trypanothione, contributing to resistance. In addition, the results suggest that at least one more factor acts synergistically with the gsh1 gene product.

Publisher Summary This chapter describes the amplicons that can be detected and isolated. It also explains how the function of heavy metal resistance genes in Leishmania can be studied by gene transfection and gene disruption. Resistance to oxyanions in Leishmania is a complex phenomenon. The ease of detection of gene amplification has permitted the isolation of genes involved in resistance including one encoding for the ABC transporter PgpA and for a gene collaborating with it. Several other amplified genes in L. tarentolae , resistant to oxyanions have been isolated using the methodology described in the chapter. Studies of drug resistance mechanisms in parasites are correlative with the ability to introduce genes by electroporation. By disrupting the genes by homologous recombination, the role of genes in resistance can be directly studied. With the increased understanding of resistance mechanisms in laboratory strains, the resistance mechanisms prevailing in field strains isolated from patients who are unresponsive to pentavalent antimony treatment are tested. This is beneficial for the millions of people suffering from Leishmania infections.

Selection for methotrexate resistance in Leishmania spp. is often associated with amplification of the H locus short-chain dehydrogenase-reductase gene ptr1 as part of extrachromosomal elements. Extensive sequences are always coamplified and often contain inverted duplications, most likely formed by the annealing of inverted repeats present at the H locus. By gene targeting mediated by homologous recombination, several repeated sequences were introduced in the vicinity of ptr1. Selection for methotrexate resistance in these transfectants led to ptr1 amplification as part of small circles with direct or inverted duplications whether the integrated sequences consisted of direct or inverted repeats. Hence, for a region to he amplified in L. tarentolae during drug selection, a drug resistance gene is required and must be flanked by (any) homologous repeated sequences. The distance between these repeats and their orientation will determine the length of the amplicon and whether it contains direct or inverted duplications.

Leishmania tarentolae cells selected for resistance to the oxyanions pentavalent or trivalent antimonials or to trivalent arsenicals exhibited cross-resistance to the other oxyanions. The basis for resistance in these mutants was studied by transport experiments using radioactive arsenite. All mutants exhibiting high level resistance to arsenite showed a marked decrease in the steady-state accumulation of arsenite. Decreased accumulation was also observed in antimonials-resistant mutants cross-resistant to various concentrations of arsenite. Cells depleted of endogenous energy reserves with metabolic inhibitors were loaded with radioactive arsenite; following addition of glucose, rapid efflux of arsenite was observed from arsenite mutant cells. Mutants resistant to high levels of arsenicals exhibited amplification of the P-glycoprotein related gene ltpgpA or of a linear amplicon of unknown function. However, the efflux-mediated arsenite resistance did not correlate with the amplification of the ltpgpA gene or with the presence of the linear amplicon. The calcium channel blocker verapamil and arsenite act in synergy in cells exhibiting the efflux system. Overall the oxyanion efflux system in Leishmania shares several properties with other resistance efflux systems mediated by transporters.

The protozoan parasite Leishmania often responds to drug pressure by amplifying part of its genome. At least two loci derived from the same 800 kb chromosome were amplified either as extrachromosomal circles or linear fragments after sodium arsenite selection. A 50 kb linear amplicon was detected in six independent arsenite mutants and revertants grown in absence of arsenite rapidly lost the amplicon and part of their resistance. The circular extrachromosomal amplicons, all derived from the H locus of Leishmania, were characterized more extensively. In all cases, direct repeated sequences appeared to be involved in the formation of circular amplicons. Most amplicons were generated after homologous recombination between two linked P-glycoprotein genes. This recombination event was, in two cases, associated with the loss of one allele of the chromosomal copy. A novel rearrangement point was found in a mutant where the amplicon was created by recombination between two 541 bp direct repeats surrounding the P-glycoprotein gene present at the H locus. It is also at one of these repeats that an H circle with large inverted duplications was formed. We propose that the presence of repeated sequences in the H locus facilitates the amplification of the drug resistance genes concentrated in this locus.