Final outcomes analysis of the cell product SQZ-PBMC-HPV Phase 1 trial in incurable HPV16+ solid tumors shows improved overall survival in patients with increased CD8+ T cell tumor infiltration.

Cancer vaccines strive to induce robust, antigen-targeted, T-cell-mediated immune responses but have struggled to produce meaningful regression in solid tumors. An autologous cell vaccine, SQZ-PBMC-HPV, was developed by SQZ Biotechnologies using microfluidic squeezing technology to load PBMCs with HPV16 E6 and E7 antigens in HLA-A*02+ patients. The SQZ-PBMC-HPV-101 Phase 1 trial (NCT04084951) enrolled patients with incurable HPV16+ cancers. Here, we present a post hoc analysis of the relationship between Posttreatment CD8+ T cell infiltration and patient outcomes. SQZ-PBMC-HPV was administered as monotherapy every 3 weeks. Tumor samples were collected pre-dose and post-dose 4 weeks after treatment start. Biomarkers including CD8, MHC-I, E6, E7, GZMB, and Ki67 were evaluated by immunohistochemistry, immunofluorescence, and RNA in situ hybridization, and were correlated with clinical response, survival, and drug product composition. Eighteen patients had paired pre- and post-dose biopsies. Six (33%) had an increase in CD8+ T cell density in tumor parenchyma between screening and C2D8. Patients with increased CD8+ T cell density had improved disease control rate (66.7% vs 16.7%) and median overall survival (606.5 days vs 170.0 days, p = 0.0078). Drug product was significantly enriched for higher T cells and lower monocytes in the increased CD8+ T cell density group. In patients with incurable HPV16+ solid tumors treated with SQZ-PBMC-HPV, an increase in CD8+ T cell density within the tumor parenchyma was associated with superior disease control rate and overall survival. The product composition for patients with increased CD8+ T cell density was enriched for T cells.

Clinical and Analytical Performance of the Onclarity HPV Assay Using the VALGENT Framework.

As the demand for human papillomavirus (HPV)-related cervical screening increases, emerging HPV tests must be evaluated robustly using well-annotated samples, such as those generated in the Validation of HPV Genotyping Tests (VALGENT) framework. Through VALGENT, we assessed the performance of the BD Onclarity HPV assay, which detects 14 high-risk (HR) types and resolves six individual types and three groups of types. Consecutive samples from a screening population (n = 1,000), enriched with cytologically abnormal samples (n = 300), that had been tested previously with the GP5+/6+ PCR enzyme immunoassay (EIA) and the GP5+/6+ PCR LMNX assay (Diassay) were tested with the Onclarity assay. Type-specific HPV prevalences were analyzed according to age and cytological result. The accuracy of the Onclarity assay for the detection of cervical intraepithelial neoplasia grade 2+ (CIN2+) and CIN3+ was assessed relative to the GP5+/6+ EIA results by using noninferiority criteria. Overall agreement and type-specific agreement between the Onclarity assay and the GP5+/6+ LMNX assay were assessed. The prevalence of HPV types 16, 18, 31, and 45 increased with the severity of cytological results (P for trend, <0.05). For the detection of CIN2+, the Onclarity assay had a relative sensitivity of 1.02 (95% confidence interval [CI], 0.99 to 1.05; P < 0.001 for noninferiority) and a relative specificity of 0.99 (95% CI, 0.97 to 1.00; P = 0.186 for noninferiority). The kappa for agreement between the Onclarity assay and the GP5+/6+ LMNX assay for HR-HPV was 0.92 (95% CI, 0.89 to 0.94), and values for the six individual types ranged from 0.78 (95% CI, 0.68 to 0.87) for HPV-52 to 0.96 (95% CI, 0.93 to 0.99) for HPV-16. These data suggest that the Onclarity assay offers applications for clinical workstreams while providing genotyping information that may be useful for risk stratification beyond types 16 and 18.

Effect of glacial acetic acid pre-treatment of cervical liquid-based cytology specimens on the molecular detection of human papillomavirus.

OBJECTIVE: Cytology laboratories routinely treat cervical liquid-based cytology (LBC) specimens that are heavily contaminated with blood with glacial acetic acid (GAA) in order to lyse red blood cells and facilitate assessment. However, the impact on downstream human papillomavirus (HPV) detection is not well understood. This study examines the effect of GAA pre-treatment of ThinPrep(®) Preservcyt(®) specimens on the molecular detection of HPV. METHODS: A panel of 150 routinely collected cervical LBC specimens was tested with two commercial HPV tests, the Abbott RealTime High Risk HPV test (rtHPV) and the Qiagen Hybrid Capture 2 High Risk HPV DNA test (HC2), as aliquots before and after GAA treatment. Statistical analysis was performed using McNemars test and Bland and Altman plots. RESULTS: Agreement between the results of the rtHPV test on GAA-untreated and GAA-treated specimens was 95.7%, with no evidence of a significant difference in the distribution of the discrepant results (P = 0.414). HC2 test agreement on GAA-untreated and GAA-treated specimens was 91% at a cut-off of 1 relative light unit index (RLUI) and 92% at a cut-off of 2 RLUI. There was no evidence of a difference in the distribution of discordant results at a cut-off of 1 (P = 0.405) and 2 RLUI (P = 0.564). CONCLUSIONS: GAA pre-treatment of cervical ThinPrep Preservcyt LBC specimens had little effect on the two commercial HPV tests used in this study. The impact of GAA treatment on HPV testing should, however, be validated for all HPV tests and all LBC collection media used in each particular diagnostic setting.

A single ThinPrep slide may not be representative in all head and neck fine needle aspirate specimens.

OBJECTIVES: Ideally, head and neck aspiration should be performed by trained aspirators within the setting of a one-stop clinic, where smeared material is available for immediate assessment. However, this may not always be possible for practical reasons and the use of liquid-based techniques in head and neck cytology is increasing. Although liquid-based cytology has been extensively validated for use in gynaecological cytology, no studies have investigated whether or not a single ThinPrep slide is representative for head and neck aspirate specimens. We performed a prospective audit of head and neck fine needle aspiration specimens processed by the ThinPrep method to investigate whether a single ThinPrep slide was representative. METHODS: A prospective audit of 115 consecutive head and neck aspirates was carried out. A single ThinPrep slide was prepared and a diagnosis recorded. The remainder of the specimen was then spun down and prepared as a cell block. The ThinPrep and cell block diagnoses were compared. RESULTS: In 36 cases (31%), the cell block provided additional information that contributed to the diagnosis. In 14 (12%), the cell block was regarded as essential to the diagnosis. CONCLUSIONS: A single ThinPrep slide may not provide representative diagnostic material in all head and neck aspirates. This should be taken into consideration when contemplating the use of liquid-based methods for non-gynaecological cytology.

The use of the 'borderline' category in the reporting of cervical cytopathology in the UK: results of a survey conducted under the aegis of the British Society for Clinical Cytology.

OBJECTIVE: To determine how the 'borderline' category was used by cytopathologists in the UK when reporting cervical smears. METHODS: A questionnaire was sent by email to members of the British Society for Clinical Cytology. RESULTS: There is wide variation in the use of the 'borderline' category in the UK but the majority of respondents (77.6%) used it when reporting smears that were either on the borderline between negative and low grade squamous dyskaryosis ('borderline ?low grade'), or on the borderline between negative and high grade squamous dyskaryosis ('borderline ?high grade'), or on the borderline between negative and glandular dyskaryosis 'borderline ?glandular dyskaryosis'). A significant minority (15.7%), however, did not use 'borderline' when reporting smears that showed an abnormality that was possibly high grade squamous dyskaryosis. A majority (79.1%) of respondents thought that it would be useful to have separate reporting categories for 'borderline ?low grade' and 'borderline ?high grade'. CONCLUSIONS: There is diversity in the use of the category 'borderline' in the UK. The proposed revised BSCC terminology with separate categories for borderline ?low grade, borderline ?high grades and borderline ? glandular dyskaryosis reflects the opinion of the majority of respondents to the questionnaire.

Verapamil restoration of daunorubicin responsiveness in daunorubicin-resistant Ehrlich ascites carcinoma.

We have studied the influence of verapamil hydrochloride on the in vitro and in vivo effects of daunorubicin in Ehrlich ascites carcinoma. Daunorubicin-sensitive tumor was rendered resistant to daunorubicin by the continuous treatment of sequential generations of tumor-bearing BALB/c mice. The ability of daunorubicin to inhibit [(3)H]uridine and [(3)H]thymidine incorporation and the effect of daunorubicin on the mean survival time of host animals bearing daunorubicin-sensitive and daunorubicin-resistant Ehrlich ascites carcinoma were compared. The addition of verapamil to daunorubicin in vitro reduced the concentration of daunorubicin required to inhibit 50% of DNA and RNA synthesis in the daunorubicin-resistant tumor to that required in the daunorubicin-sensitive tumor, from 6 and 4.4 mug/ml to 1.5 and 1.3 mug/ml, respectively. Verapamil also restored drug sensitivity to daunorubicin-resistant Ehrlich ascites carcinoma in vivo. The 21.7+/-0.7 d mean survival time (MST) of BALB/c mice bearing daunorubicin-resistant tumor treated with daunorubicin alone rose to 44.0+/-0.7 d when the same tumor was treated with verapamil and daunorubicin, P < 0.001. This in vivo effect is specific for daunorubicin-resistant Ehrlich ascites carcinoma, since there is no alteration in MST of BALB/c mice bearing daunorubicin-sensitive or daunorubicin-resistant tumor when they are treated with verapamil alone or when BALB/c mice bearing daunorubicin-sensitive tumor are treated with daunorubicin and verapamil.

Transcription termination signal for the cat-86 indicator gene in a Bacillus subtilis promoter-cloning plasmid.

Plasmid pPL703 is a promoter-cloning plasmid for Bacillus subtilis consisting of the promoter-less cat-86 gene inserted between the EcoRI and BamHI sites of pUB110. The orientation of cat-86 in pPL703 is opposite to that of two major transcript species that occur within the pUB110 vector portion of pPL703. Therefore, transcripts initiated in cloned promoters which activate cat-86 expression presumably must terminate prior to entering the vector portion of pPL703 to permit stable maintenance of promoter-containing derivatives in host cells. We have identified an apparent Rho-independent transcription terminator 35 bp 3' to the cat-86 coding sequence. A restriction fragment spanning the terminator is 90% efficient in terminating transcription in both B. subtilis and Escherichia coli. The structure of the cat-86 transcription termination site is similar to Rho-independent termination sites identified in E. coli.

Method for blot-hybridization analysis of mRNA molecules from Bacillus subtilis.

By modifying hybridization techniques which are currently available to analyze RNA molecules we have developed a sensitive and reproducible method for 'Northern' analysis of RNA from Bacillus subtilis. The use of a thin (1 mm) vertical 2% agarose-6% formaldehyde gel seems to allow more efficient transfer and higher resolution of RNA upon hybridization analysis than does the use of thicker horizontal slab gels. Our improved hybridization method results in greatly reduced background upon autoradiography regardless of whether or not 32P-labelled nick-translated probes or probes synthesized on M13 vectors were purified from the unincorporated radionucleotides.

Expression of Escherichia coli trp genes and the mouse dihydrofolate reductase gene cloned in Bacillus subtilis.

The mouse dihydrofolate reductase gene and a segment of the Escherichia coli trp operon are expressed in Bacillus subtilis when cloned in the "expression plasmid" pPL608. The cloned mouse gene confers trimethoprim resistance on B. subtilis and the cloned trp fragment complements mutations in the B subtilis trpD, C and F genes Expression of both cloned fragments is dependent on a promoter present in the vector plasmid. The E. coli trp fragment is cloned in a HindIII site within a chloramphenicol acetyltransferase gene present on pPL608, and as a result, expression of the E. coli trpC gene product is inducible by chloramphenicol. The mouse gene is inserted at a PstI site preceding the chloramphenicol acetyltransferase gene and its expression is not chloramphenicol inducible. The replication functions and neomycin-resistance of pPL608 are derived from pUB110. Accordingly, pPL608 is stably maintained at high copy number in B. subtilis.

Chloramphenicol-inducible gene expression in Bacillus subtilis.

A cloned Bacillus pumilus cat gene expresses chloramphenicol-inducible chloramphenicol acetyltransferase activity in Bacillus subtilis. The chloramphenicol inducibility trait was shown to be determined by a 234-bp region of the cloned DNA. Nucleotide sequence analysis of this 234-bp segment indicated that the cat ribosome-binding site occurs within a 40-bp region containing 14-bp terminal inverted repeat sequences. Transcription of this region into RNA should sequester the cat ribosome-binding site in a stable stem-loop conformation. Chloramphenicol-mediated destabilization of the stem-loop is suggested as the basis for the chloramphenicol inducibility phenotype.

The relationship of high-intensity signals on magnetic resonance images to cognitive and psychiatric state in Alzheimer's disease.

In Alzheimer's disease (AD), the relationship between white-matter changes on magnetic resonance images and behavior are unclear. Therefore, magnetic resonance images, cognition, and psychiatric state were assessed in patients with AD with depression (AD/DEP; n = 18) and without depression (AD; n = 45), older depressed patients (n = 12) and older normal individuals (n = 25). High-intensity signals in the cortex and subcortical regions were similar in number and proportions among all groups, even when hypertensive patients were excluded. No correlations to cognitive or psychiatric state were found. Periventricular signals were categorized using a 1- (absent) to 6- (thick, irregular caps and stripes) point scale. The categories were similar among groups except that patients with AD exhibited more category 5 changes than did normal subjects, neuropsychological performance was significantly worse in patients with AD who had category 5 and 6 changes when compared to those in category 1. These results suggest that periventricular changes may predict poor neuropsychological performance in patients with AD. However, neither deep white-matter lesions nor periventricular changes are useful for diagnostic purposes.

Human papillomavirus type 18 associates with more advanced cervical neoplasia than human papillomavirus type 16.

A type-specific, sensitive, polymerase chain reaction-based assay for human papillomavirus (HPV) types 6b, 11, 16, 18, and 33 was applied to 47 cervical carcinomas, 60 cases of cervical intraepithelial neoplasia (CIN), and 24 samples of histologically normal cervix. As expected, the combined incidence of the common high-risk genital HPVs (types 16 and 18) was high in carcinomas (79%) and CIN 2/3 (60%), low in CIN 1 (25%), and nonexistent in the normal controls. Analysis of the data by viral type and pathology revealed statistically significant differences that consistently pointed to an association of HPV 18 with more advanced disease than HPV 16. This was exemplified by calculation of the relative HPV frequency in squamous cancers and CIN 2/3 lesions, which gave cancer to CIN prevalence ratios of 1.2 for HPV 16 and 2.3 for HPV 18, a twofold difference suggesting the possibility that there is a greater risk of progression or a more rapid transition to malignancy associated with HPV 18. Furthermore, HPV 16 was associated with 2.5-fold more cancers showing squamous differentiation (58%) than HPV 18 (23%), but both types showed an identical prevalence of 41% in the clinically more sinister adenocarcinomas, indicating that there may be an association between HPV type and cancer cell differentiation.

PCR analysis of the upstream regulatory region of human papillomavirus genomes in cervical intraepithelial neoplasia and cervical carcinoma.

AIMS: To test whether human papillomavirus (HPV) variants with large scale sequence alterations to the upstream regulatory region are present in cervical intraepithelial neoplasias (CIN) and cervical carcinomas. METHODS: New PCR based assays were designed specifically to detect large scale insertion/deletion alterations in the upstream regulatory region of HPV 16 and 18. The assays were applied to 24 cases of CIN and 34 cases of cervical carcinoma previously shown to contain these two high risk HPV types. RESULTS: No large scale sequence alterations were found in any of the HPV containing CIN or carcinomas. CONCLUSIONS: These negative findings suggest that major upstream regulatory region variants of HPV 16 and 18 do not contribute to most cervical neoplasms.

Pathologists dislike sound? Evaluation of a computerised training microscope.

AIM: To evaluate the use of multimedia enhancements, using a computerised microscope, in the training of microscope skills. METHODS: The HOME microscope provides facilities to highlight features of interest in conjunction with either text display or aural presentation. A pilot study was carried out with 10 individuals, eight of whom were at different stages of pathology training. A tutorial was implemented employing sound or text, and each individual tested each version. Both the subjective impressions of users and objective measurement of their patterns of use were recorded. RESULTS: Although both versions improved learning, users took longer to work through the aural than the text version; 90% of users preferred the text only version, including all eight individuals involved in pathology training. CONCLUSIONS: Pathologists appear to prefer visual rather than aural input when using teaching systems such as the HOME microscope and sound does not give added value to the training experience.

Interobserver variation in cell selection for DNA image cytometry.

AIMS: To describe a systematic investigation of interobserver differences in interpretation of nuclear morphology in preparations of small cell lung cancer (SCLC). METHODS: The screening/reviewing facility on the highly optimised microscope environment was used to individually tag 127 nuclei, chosen to reflect the spectrum of morphological appearances in nuclear preparations from three biopsy specimens of SCLC. Each nucleus was reviewed and labelled as control (lymphocyte), malignant or unsatisfactory by each of four observers. DNA histograms were plotted for each specimen using the nuclei identified as malignant by each participant. The histograms were compared in terms of identification of DNA stemlines and by calculation of a 5c exceeding rate (5cER). RESULTS: Interobserver variation in assessment of morphology was seen in 55.1% of nuclei. Disagreement occurred most frequently in the malignant/unsatisfactory category. Differences in morphological classification had little influence on histogram assessment by means of visual inspection but did show an effect on 5cER. CONCLUSIONS: There are significant interobserver differences in subjective assessment of nuclear morphology in cytometric preparations. This effect may seriously influence cytometric measurements.

Sensitivity of digoxigenin and biotin labelled probes for detection of human papillomavirus by in situ hybridisation.

The sensitivity of digoxigenin and biotin labelled DNA probes for the detection of human papillomavirus (HPV) by dot blotting and in situ hybridisation was compared in tissues from cervical, laryngeal, and anogenital neoplasia. Probes were either labelled with digoxigenin by the random primer technique and detected with anti-digoxigenin antibody, or labelled with biotin by nick translation and detected with streptavidin, both methods having a common final visualisation procedure using alkaline phosphatase. Digoxigenin labelled probes proved two to 10-fold more sensitive by quantitative dot blotting and four-fold more sensitive in detecting HPV 16 DNA in a series of 31 anal carcinomas, compared with biotinylated probes. The digoxigenin method also produced less non-specific background staining of tissue sections than biotin labelled probes. It is concluded that digoxigenin DNA labelling and detection provides a simple, reliable, and efficient alternative to the use of biotin or radioactive isotopes for the detection of HPV DNA by in situ hybridisation. Digoxigenin labelled probes also offer the possibility of double labelling in situ hybridisation procedures when used with biotin labelled probes to provide simultaneous identification of different DNA sequences.

Comparison of dopamine agonists in the treatment of hyperprolactinemic syndromes: a multicenter study.

Thirty-one patients with hyperprolactinemia were admitted for protocol study. Twenty-one of these patients had no findings of prolactinoma by computerized axial tomography (CAT) scanning; 10 had documented tumor by CAT scan. The patients were assigned to either Parlodel or Pergolide treatment on the basis of random numbers tables. They were treated for 6 months continuously and followed during this time with radiologic survey, hormonal evaluation, and blood chemistry determinations. Patients in both groups showed a decrease in prolactin levels, whether they were treated with Parlodel or Pergolide. The response was similar whether patients had hyperplasia or pituitary tumors. Patients with pituitary tumors tended to have a diminution in the size of their lesions regardless of the dopamine agonist used. The types of side effects experienced by various groups were similar regardless of the treatment. It is concluded that both Pergolide and Parlodel are useful in the treatment of hyperprolactinemic syndromes, although neither one appears to be superior to the other.

Neuroimaging features of neurenteric cysts: analysis of nine cases and review of the literature.

PURPOSE: To gain a better understanding of neurenteric (NE) cysts via correlation of imaging findings and surgical and pathologic data. METHODS: The medical records, imaging studies, surgical information, and pathologic material were retrospectively reviewed in nine patients with NE cysts, including seven proved and two very probable cases. RESULTS: NE cysts occurred in the cerebellopontine angle in one case and extended from the cerebellopontine angle to the C2 level in a second. In the latter patient and the remaining seven with intraspinal lesions, the NE cyst was always located anterior to the spinal cord. The most common myelographic and CT myelographic appearance was that of a lobulated intradural extramedullary (IDEM) mass. Two patients had an intramedullary NE cyst with a somewhat unusual appearing exophytic IDEM-appearing expansion that can be a characteristic feature of these lesions. MR imaging demonstrated the NE cyst to be isointense to hyperintense relative to cerebrospinal fluid on long TR sequences and isointense or slightly hyperintense to cerebrospinal fluid on T1-weighted images. These signal characteristics correlate with the high-protein-content fluid within the cysts, usually described at surgery as milky or mucinous in character. CONCLUSION: The diagnosis of NE cyst should be considered when imaging studies reveal the presence of a lobulated IDEM or an exophytic intramedullary cystic mass, especially in association with anterior spina bifida or other vertebral anomalies. MR can uniquely confirm the cystic nature of these masses and is the method of choice for their imaging investigation. Because cyst recurrence can occur, MR should also be used for long-term patient follow-up.

Computed tomography and magnetic resonance imaging in traumatic locked-in syndrome.

The first reported case of traumatic locked-in syndrome with computed tomographic and magnetic resonance scan confirmation of the brain stem lesion is presented. The lesion responsible for the patient's condition consisted of a hemorrhage in the ventral pontomedullary junction. The pathophysiology of the production of such lesions is discussed.