Trastuzumab-Emtansine and Osimertinib Combination Therapy to Target HER2 Bypass Track Resistance in EGFR Mutation-Positive NSCLC.

Introduction: EGFR tyrosine kinase inhibitor improved the survival of patients with metastatic EGFR mutation-positive (EGFRm+) NSCLC. Despite high response rates, resistance develops inevitably in every patient. In up to 13%, HER2 protein overexpression is found on progression. We hypothesized that dual blockade of EGFR and HER2 by osimertinib combined with trastuzumab-emtansine (T-DM1) could reinduce tumor responses. Methods: In this multicenter, single-arm, phase 1-2 study (NCT03784599), patients with EGFRm+ NSCLC, progressing on osimertinib and HER2 overexpression were included. Patients were treated with T-DM1 3.6 mg/kg (intravenously) every 3 weeks and osimertinib 80 mg once a day. Primary end points were objective response rate (ORR) at 12 weeks and safety. Responses were assessed every 6 weeks (Response Evaluation Criteria in Solid Tumors 1.1). Sample size was calculated using Simon's two-stage minimax design (H0 = 41%, H1 > 55%, 80% power, one-sided type I error 10%: a ORR 16 of 36 was needed to proceed to 58 patients). Results: From January 2019 to April 2021, 27 patients were enrolled. ORR after 12 weeks of treatment was 4% (1 of 27). Median progression-free survival was 2.8 months (95% confidence interval: 1.4-4.6 mo). Most frequent treatment-related adverse events of any grade were fatigue, diarrhea, and nausea, among these, grade 3 in four patients. There were no grade 4 or 5 therapy-related adverse events. Conclusions: TRAEMOS (Trastuzumab-Emtansine and Osimertinib) is the first trial combining T-DM1 and osimertinib in patients with EGFRm+ NSCLC to target HER2 overexpression at osimertinib resistance. Safety profile was favorable compared with cytotoxic chemotherapy; but treatment revealed limited efficacy. Further clinical evaluation of this regimen is not warranted.

Trastuzumab and pertuzumab combination therapy for advanced pre-treated HER2 exon 20-mutated non-small cell lung cancer.

INTRODUCTION: In 1-3% of non-small cell lung cancer (NSCLC) human epidermal growth factor 2 (HER2) mutations are identified as a genomic driver. Nevertheless, no HER2-targeted treatment is approved for NSCLC. In the Drug Rediscovery Protocol (DRUP), patients are treated with off-label drugs based on their molecular profile. Here, we present the results of the cohort 'trastuzumab/pertuzumab for HER2 exon20 mutation positive (HER2m+) NSCLC'. METHODS: Patients with treatment refractory, advanced HER2m+ NSCLC with measurable disease (RECISTv1.1) were eligible. Treatment with intravenous trastuzumab combined with pertuzumab every 3 weeks was administered. The primary end-point was clinical benefit (CB: either objective response or stable disease ≥ 16 weeks). Patients were enrolled using a Simon-like 2-stage design, with 8 patients in stage 1 and up to 24 patients in stage 2 if at least 1 patient had CB in stage 1. At baseline, a biopsy for biomarker analysis, including whole genome sequencing, was obtained. RESULTS: Twenty-four evaluable patients were enrolled and treated between May 2017 and August 2020. CB was observed in 9 patients (38%); including an objective response rate of 8.3% (2 patients had a partial response) and 7 patients with stable disease ≥ 16 weeks. The most frequently observed HER2 mutation was p.Y772_A775dup (71%, n = 20). Median follow-up was 13 months, median progression-free survival and overall survival 4 (95% CI 3-6) and 10 months (95% CI 4 - not reached), respectively. Whole genome sequencing data (available for 67% of patients) confirmed the inclusion mutation in all cases. No unexpected toxicity was observed. CONCLUSION: Despite the fact that the study did meet its primary end-point, trastuzumab/pertuzumab was only marginally active in a subset of patients with heavily pre-treated HER2m+ NSCLC.

The force of HER2 - A druggable target in NSCLC?

Since several years targeted therapy has been part of treatment in NSCLC in subsets of patients with specific genetic alterations. One of these alterations involves HER2, a member of the ERBB family of tyrosine kinase receptors. Despite that HER2 alterations in NSCLC have been studied for years, there is still no consensus about subgroup definitions. In this review HER2 alterations in NSCLC are discussed, including diagnostic challenges and treatment strategies. Three principal mechanisms of HER2 alterations can be identified: HER2 protein overexpression, HER2 gene amplification and HER2 gene mutations. There are several methods for the detection of HER2 "positivity" in NSCLC, but no gold standard has been established. Laboratory methods for assessment of HER2 positivity in NSCLC include immunohistochemistry (IHC) for protein overexpression and fluorescent in situ hybridization (FISH) and next generation sequencing (NGS) for genetic alterations. Many trials testing HER2 targeted therapy in HER2 altered NSCLC has not lead to a renewed standard of care for this group of patients. Therefore, today the (re)search on how to analyse, define and treat HER2 alterations in NSCLC continues. Still there is no consensus about HER2 subgroup definitions and results of the many trials studying possible treatment strategies are inconclusive. Future research should focus on the most important missing link, whether all HER2 alterations are relevant oncogenic drivers and whether it should be considered as a therapeutic target in NSCLC.

Trastuzumab and paclitaxel in patients with EGFR mutated NSCLC that express HER2 after progression on EGFR TKI treatment.

BACKGROUND: HER2 expression and amplification are observed in ~15% of tumour biopsies from patients with a sensitising EGFR mutation who develop EGFR TKI resistance. It is unknown whether HER2 targeting in this setting can result in tumour responses. METHODS: A single arm phase II study was performed to study the safety and efficacy of trastuzumab and paclitaxel treatment in patients with a sensitising EGFR mutation who show HER2 expression in a tumour biopsy (IHC ≥ 1) after progression on EGFR TKI treatment. Trastuzumab (first dose 4 mg/kg, thereafter 2 mg/kg) and paclitaxel (60 mg/m2) were dosed weekly until disease progression or unacceptable toxicity. The primary end-point was tumour response rate according to RECIST v1.1. RESULTS: Twenty-four patients were enrolled. Nine patients were exon 21 L858R positive and fifteen exon 19 del positive. Median HER2 IHC was 2+ (range 1-3). For 21 patients, gene copy number by in situ hybridisation could be calculated: 5 copies/nucleus (n = 9), 5-10 copies (n = 8), and >10 copies (n = 4). An objective response was observed in 11/24 (46%) patients. Highest response rates were seen for patients with 3+ HER2 IHC (12 patients, ORR 67%) or HER2 copy number ≥10 (4 patients, ORR 100%). Median tumour change in size was 42% decrease (range -100% to +53%). Median duration of response was 5.6 (95% confidence interval [CI], 3.8 to 7.3) months. Treatment toxicity was mild with four patients experiencing grade ≥3 toxicity, including fatigue, neuropathy, neutropaenia, urinary tract infection, and pneumonitis. CONCLUSIONS: Trastuzumab-paclitaxel induces objective tumour responses in 46% of EGFR TKI pretreated patients with an activating EGFR mutation and HER2 expression. The treatment was well tolerated. The relation between response rate and HER2 expression level and copy number suggests effective HER2 targeting by trastuzumab, although the combination with paclitaxel does not allow to determine the relative contribution of the individual drugs in terms of treatment efficacy.

The prognostic value of the neoadjuvant response index in triple-negative breast cancer: validation and comparison with pathological complete response as outcome measure.

The Neoadjuvant response index (NRI) has been proposed as a simple measure of downstaging by neoadjuvant treatment in breast cancer. It was previously found to predict recurrence-free survival (RFS) in triple-negative (TN) breast cancer. It was at least as accurate as the standard binary system, the absence or presence of a pathological complete remission (pCR), which is the commonly employed outcome measure. The NRI was evaluated in an independent consecutive series of patients to validate the previous findings. Univariable and multivariable analyses were done to assess the predictive value of clinical parameters and of the NRI for RFS. We combined the original and validation series of patients to build a multivariable predictive model for RFS after neoadjuvant chemotherapy in TN breast cancer. The validation set (N = 108) confirmed that patients with a higher-than-median NRI (>0.7) had excellent RFS (P = 0.002), similar to that of patients who had achieved a pCR. Multivariable analysis in 191 patients showed that the NRI was a strong independent predictor of RFS (P = 0.0002), with N-stage (P = 0.001) and T-stage (P = 0.014) ranking second and third, respectively. Importantly, among patients who did not achieve a pCR (NRI values below 1), higher NRI values were still associated with better RFS. The NRI is a simple method and a practical tool to predict RFS in TN breast cancer patients treated with neoadjuvant chemotherapy. It adds prognostic information to the presence or absence of pCR and could be useful to compare the efficacies of different chemotherapy regimens.

Evolutionary relationships within a subgroup of HERV-K-related human endogenous retroviruses.

The prototype endogenous retrovirus HERV-K10 was identified in the human genome by its homology to the exogenous mouse mammary tumour virus. By analysis of a short 244 bp segment of the reverse transcriptase (RT) gene of other HERV-K10-like sequences, it has become clear that these elements represent an extended family consisting of multiple groups (the HML-1 to HML-6 subgroups). Some of these elements are transcriptionally active and contain an intact open reading frame for the RT protein, raising the possibility that this family is still expanding through retrotransposition. To better define the relationship of these endogenous retroviruses, we identified ten new members of the HML-2 subgroup. PCR was used to amplify reverse-transcribed RNA of a 595 bp region of the RT gene in a variety of human cell samples, including normal and leukaemic bone marrow and peripheral blood, placenta cells and a transformed T cell line. We provide an extensive phylogenetic analysis of the relationships for this cluster of HERV-K-related endogenous retroviral elements. Nucleotide diversity values for nonsynonymous versus synonymous codon positions indicate that moderately strong selection is or was operating on these retroviral RT gene segments. The evolution of this class of endogenous retroelements is discussed.

Effect of antrectomy and truncal vagotomy on erythromycin induced pancreatic polypeptide secretion.

Erythromycin, a motilin agonist, enhances gastrointestinal motility but also stimulates endogenous pancreatic polypeptide (PP) secretion. We investigated whether the effect of erythromycin on PP release is dependent on (1) prokinetic activity of erythromycin generated from the antrum and (2) the long vagus nerve since erythromycin acts via cholinergic neurons. Erythromycin induced PP secretion was determined in 14 patients with antrectomy (6 patients with Billroth I type anastomosis, 8 patients with Billroth II type anastomosis), in 6 patients with truncal vagotomy and pyloroplasty but without gastric resection and in 8 healthy controls. Plasma PP levels in response to erythromycin (3 mg/kg i.v.) were determined at regular intervals for 180 min. Erythromycin induced a significant increase in plasma PP in the control subjects from 22 +/- 4 pmol/l (basal) to 49 +/- 4 pmol/l at 10 min. In the patients with truncal vagotomy plasma PP secretion after erythromycin was significantly (P < 0.05) increased (peak increment vs. basal: 98 +/- 10 pmol/l vs. 27 +/- 2 pmol/l) and prolonged compared to controls. In the patients with antrectomy no significant increases in plasma PP over basal were observed after erythromycin infusion. It is concluded that erythromycin stimulates PP secretion in healthy controls. The PP response to erythromycin is exaggerated after truncal vagotomy but absent after antrectomy indicating that the antrum is essential for erythromycin induced PP secretion.

Application of the NASBA nucleic acid amplification method for the detection of human papillomavirus type 16 E6-E7 transcripts.

Using a human papillomavirus type 16 (HPV-16) E6-E7 specific primer set in a nucleic acid sequence-based amplification (NASBA) reaction, detection of HPV-16 transcripts was accomplished in a single enzymatic reaction at 41 degrees C. The NASBA reaction product was visualized either by Northern bolt analysis with an HPV-16 E6-E7-specific 32P-labelled oligonucleotide probe or by a non-radioactive enzyme-linked gel assay (ELGA). In combination with a rapid nucleic acid extraction procedure this method appears to be very suitable for the sensitive and specific detection of HPV-16 transcripts on small amounts of HPV-16-expressing cells of various sources, including cervical smears.

Identification of a unique group of human papillomavirus type 16 sequence variants among clinical isolates from Barbados.

The naturally occurring sequence variation of human papillomavirus type 16 (HPV-16) was analysed by direct sequence analysis of the PCR products of the long control region (LCR), the E5 and E7 open reading frames (ORFs), a segment of the L2 ORF overlapping the early viral poly(A) signal and a small segment of the L1 ORF or clinical isolates from Barbados and The Netherlands. Despite the widely different geographical and ethnic origin of the two groups of specimens, sequence analysis revealed relatively few mutational differences. Analysis of the LCR and the E5 ORF appeared to be the minimum requirement for the correct positioning of these variants in the HPV-16 phylogenetic tree. Most of the Barbadian variants appeared to be located at a unique position in the HPV-16 phylogenetic tree, at the internal branch close to the point where the European and Asian branches diverge. In contrast, most of the Dutch samples were located on the European branch.

Nutrient-specific effects of modified sham feeding on pancreatic polypeptide release.

OBJECTIVE: To study the effect of meal composition on pancreatic polypeptide release during modified sham feeding. DESIGN: In random order and on separate occasions, isocaloric, isothermic, isoosmotic, homogenized meals (1050 kJ; 250 kcal) either rich in fat (walnuts; 64 g fat, 7 g protein, 15 g starch per 100 g), protein (codfish, 1 g fat, 23 g protein per 100 g) or carbohydrates (bananas; 22 g starch, 1 g protein per 100 g) were sham-fed for 30 min by tasting and spitting out the meal. The plasma pancreatic polypeptide response was monitored by radioimmunoassay at 10 min intervals from 20 min before to 120 min after modified sham feeding. SETTING: Department of Gastroenterology and Hepatology of a University Hospital. SUBJECTS: Seven healthy volunteers: 3 male, 4 female; age 45 (range 30-77) years. RESULTS: Integrated plasma pancreatic polypeptide responses to modified sham feeding of codfish (1088 +/- 395 pM*120 min; P < 0.05) and walnuts (1200 +/- 542 pM*120 min) were distinctly higher (P < 0.05) than to modified sham feeding of bananas (-390 +/- 291 pM*120 min). CONCLUSIONS: These results demonstrate that the pancreatic polypeptide response to modified sham feeding is dependent on the composition of the meal.

Effect of acute hyperglycaemia on gall bladder contraction induced by cholecystokinin in humans.

This study examined the effect of acute hyperglycaemia, induced by intravenous glucose, on gall bladder motility. Six healthy volunteers were studied in random order on three occasions during normoglycaemia and hyperglycaemia with blood glucose concentrations stabilised at 8 and 15 mmol/l. Gall bladder volumes, measured with ultrasonography, were studied before and during infusion of stepwise increasing doses of cholecystokinin (CCK-33; 0.25, 0.5, and 1.0 IDU.kg-1.h-1). Each dose was given for 30 minutes. Pancreatic polypeptide (PP) secretion was determined as an indirect measure of cholinergic tone. Infusion of CCK-33 resulted in significant dose dependent reductions in gall bladder volume in all three experiments. Compared with normoglycaemia the gall bladder contraction was significantly (p < 0.05) reduced during infusion of 0.25 and 0.5 IDU kg-1.h-1 CCK-33 in the 8 mmol/l hyperglycaemic experiment, and during infusion of 0.25, 0.5, and 1.0 IDU kg-1.h-1 CCK-33 in the 15 mmol hyperglycaemic experiment. During hyperglycaemia basal plasma PP concentrations and PP secretion in response to CCK-33 were significantly (p < 0.05) reduced. It is concluded that blood glucose concentrations affect gall bladder motility, that an acute hyperglycaemia at 8 and 15 mmol/l reduces the gall bladder responsiveness to CCK-33 in a dose dependent manner, and that hyperglycaemia reduces basal and CCK-33 stimulated plasma PP concentrations, suggesting impaired cholinergic activity during hyperglycaemia.

Detection of cutaneous and genital HPV types in clinical samples by PCR using consensus primers.

Two sets of consensus PCR primers consisting of a common 3' primer CP-I and two 5'-primers, CP-IIG (primer set A) and CP-IIS (primer set B), in the E1 open reading frame of the human papillomavirus (HPV) genome are presented. These two primer sets enabled the detection of a 188 base pair (bp) fragment of HPV 1, 2, 3, 4, 5, 6b, 7, 8, 9, 10a, 11, 12, 14a, 16, 17, 18, 19, 20, 21, 22, 24, 25, 31, 33, 36, 37, 38, 39 and 46. HPV types 15, 23, 49 and 50 were poorly amplified and HPV type 41 was not amplified. The method is suitable for the detection of HPV DNA sequences in clinical samples of both cervical and cutaneous lesions.

Detection and typing of human papillomaviruses present in fixed and stained archival cervical smears by a consensus polymerase chain reaction and direct sequence analysis allow the identification of a broad spectrum of human papillomavirus types.

DNA well suited for polymerase chain reaction (PCR) amplification was purified from archival Papanicolaou smears. The detection of a wide range of human papillomavirus (HPV) types was made possible using a HPV-specific consensus primer pair, and typing was conveniently done by direct sequence analysis of the PCR product. The method could be of unique value in longitudinal and cross-sectional studies aimed at answering a number of fundamental pathological and epidemiological questions regarding HPV infection of the genital tract.

Effect of loxiglumide and atropine on erythromycin-induced reduction in gallbladder volume in human subjects.

This study was undertaken to investigate the effect of erythromycin, a motilin agonist with prokinetic activity, on fasting gallbladder volume. To evaluate the mechanism of action of erythromycin on gallbladder motility, erythromycin (3.5 mg/kg.20 min, intravenously) was infused on three separate occasions: during cholinergic blockage with atropine (0.005 mg/kg.hr), during cholecystokinin receptor blockade with loxiglumide (10 mg/kg.hr) and during saline solution infusion (control). Atropine, loxiglumide and saline solution infusions were started 3 hr before administration of erythromycin and were continued for 3 hr thereafter. Gallbladder volumes (measured by ultrasonography), plasma cholecystokinin levels (radioimmunoassay) and plasma pancreatic polypeptide levels (radioimmunoassay) were determined at regular intervals for 6 hr in six healthy volunteers. During the 3-hr infusion before administration of erythromycin, both loxiglumide and atropine significantly increased gallbladder volumes--from 18 +/- 2 to 37 +/- 3 cm3 (p less than 0.05) and from 17 +/- 3 to 24 +/- 2 cm3 (p less than 0.05), respectively--whereas saline solution did not significantly affect gallbladder volume. During control saline solution infusion, erythromycin induced prolonged gallbladder contraction that was significant (p less than 0.05) between 60 and 180 min and reached a maximum of 45% +/- 8% at 150 min. Plasma cholecystokinin levels were not affected by erythromycin. Erythromycin induced a significant (p less than 0.05) increase in plasma pancreatic polypeptide levels, from 12 +/- 1 pmol/L to 34 +/- 3 pmol/L. Loxiglumide did not prevent the erythromycin-induced reduction in gallbladder volume. Atropine markedly reduced the effect of erythromycin, causing slight but significant (p less than 0.05) gallbladder volume reductions (18% +/- 4%) between 150 and 180 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Gallbladder disease in cystic fibrosis.

Among the various gastrointestinal manifestations observed in patients with cystic fibrosis (CF), gallbladder abnormalities occur frequently. These include a high prevalence of nonfunctioning gallbladders (30%), micro-gallbladders (8-30%), and gallstones (4-30%). The underlying pathophysiology for this increased prevalence in patients with CF is not completely understood, due to contradictory findings. These findings concern: (1) abnormalities in bile acid metabolism resulting in bile that is supersaturated with cholesterol, (2) an impaired nucleation time, and (3) biliary stasis, due to bile duct abnormalities and/or impaired gallbladder motility. The diagnosis of gallbladder disease in CF may be obscured by other common gastrointestinal complications, resulting in a long delay between onset of symptoms and the diagnosis. Cholecystectomy in CF is the treatment of choice, provided they are carefully managed in the pre- and perioperative period. The operative morbidity and mortality, even with intensive management of pulmonary disease, amounted to 10% and 5%, respectively. Therefore, alternative options, like laparoscopic cholecystectomy are of interest and require further investigation, especially for CF patients with severe pulmonary disease.

Detection of human papillomavirus types by the polymerase chain reaction and the differentiation between high-risk and low-risk cervical lesions.

By means of a consensus polymerase chain reaction (PCR) method, the prevalence of HPV types was determined in cervical biopsies from 137 women referred to the gynecological outpatient clinic for colposcopy because of an abnormal cervical smear. The prevalence of HPV was 80.3%. There was a statistically highly significant rise in the prevalence of the oncogenic HPV types (16, 18, 31, 33) with increasing severity of cervical intraepithelial neoplasia (CIN I to III), indicating a role for these HPV types in the pathogenesis of cervical cancer. The prevalence of other HPV types decreased significantly with the severity of the lesion, suggesting that these HPV types play a less significant role in this process. These data indicate that HPV typing with PCR may be a valuable tool for distinguishing between high-risk and low-risk cervical lesions. Furthermore, our results suggest that the detection of HPV types by consensus PCR in the cervix of patients with an abnormal smear but without histologically detectable CIN is a useful tool for predicting which of these patients will eventually develop CIN. Finally, a relatively low percentage (3%) of HPV double infections is reported in this study.

Human papillomavirus type 16 transcripts expressed from viral-cellular junctions and full-length viral copies in CaSki cells and in a cervical carcinoma.

We have mapped using the RNA PCR the viral-cellular junctions of HPV16 viral-cellular cotranscripts expressed in CaSki cells and a cervical carcinoma to nt 3728 and 881, respectively. Both junctions were located within the E1-E2 region. Examination of the cellular sequences of the cotranscripts showed the presence of a polyadenylation signal in each of the transcripts. In CaSki cells and in the cervical carcinoma transcripts derived from the full-length early region including the E2 transcript were also detected. Our results suggest that the utilization of a cellular polyadenylation site could be important in the development of cancer by HPV.

The short arm of chromosome 11 likely is involved in the regulation of the human papillomavirus type 16 early enhancer-promoter and in the suppression of the transforming activity of the viral DNA.

Loci on the short arm of chromosome 11 between 11p11 and 11p15 likely are involved directly or indirectly in the regulation of the HPV-16 enhancer-promoter strength and this may contribute to the control of the level of viral early gene expression. Transient expression assays have shown that the HPV-16 enhancer-promoter functions much stronger in fibroblasts in which this region of chromosome 11 (del-11 cells) is deleted than in normal diploid human embryonic fibroblasts. The differential regulation of the HPV-16 long control region may be due either to the presence or activity of a cellular transcription factor which up-regulates HPV-16 early gene expression in del-11 cells or to a factor which down-regulates expression in diploid cells. Since the HPV-16 enhancer containing fragment (nt 7003 to nt 57) in combination with the SV40 promoter functions equally well in del-11 cells as in diploid cells a target of this factor likely is located in the putative HPV-16 early promoter region. The relative resistance of diploid human embryonic fibroblasts to HPV-16 induced transformation could be explained by the inactivity of the HPV-16 early promoter as these cells could be transformed by HPV-16 DNA constructs in which the early gene expression was driven from a heterologous enhancer-promoter. These results may indicate that loci on the short arm could suppress HPV-16-induced transformation by down-regulating the activity of the viral early promoter.

Localization of human papillomavirus type 16 DNA using the polymerase chain reaction in the cervix uteri of women with cervical intraepithelial neoplasia.

The localization of human papillomavirus type 16 (HPV-16) DNA throughout the cervix uteri of women with cervical intraepithelial neoplasia (CIN) was studied by utilizing the polymerase chain reaction technique directly on histologically defined sections of paraffin-embedded cervical tissue obtained by conizations. HPV-16 DNA was detected only in the sections that contained CIN lesions and/or koilocytes. No HPV-16 DNA was detected in sections that contained only normal epithelium. This is in accordance with HPV-16 playing a role in the development of CIN lesions.