PD-L1 diagnostics in the neoadjuvant setting: implications of intratumoral heterogeneity of PD-L1 expression in triple negative breast cancer for assessment in small biopsies.

PURPOSE: PD-L1 expression is a predictive biomarker for anti-PD-L1 immunotherapy in triple negative breast cancer (TNBC). In the neoadjuvant setting, immunohistochemical (IHC) evaluation of PD-L1 expression can only be performed on small tissue biopsies. In our study we investigated heterogeneity of PD-L1 expression in TNBC, and how reliably PD-L1 expression in small tissue samples reflects PD-L1 expression in larger tumor sections in TNBC. METHODS: Tissue microarrays (TMAs) were constructed from surgical specimens of 110 patients with TNBC. TMAs contained 4 cores (1 mm in diameter) per patient. To evaluate PD-L1 expression, TMAs were stained with PD-L1 IHC 22C3 PharmDx. Single-core PD-L1 expression was compared to overall PD-L1 expression of each patient's tumor, to ascertain how often small samples of tumor tissue show the same PD-L1 expression as larger tumor samples. RESULTS: Our study found substantial heterogeneity of PD-L1 expression between different TMA cores from the same patient. Heterogeneity was greater in immune cells (ICs) than in tumor cells, in large part due to the uneven distribution of ICs in the tumor. For IC PD-L1 expression, we found that sensitivity can be as low as 0.81 for detecting PD-L1 expression at the 1% threshold most commonly used in breast cancer. Negative predictive value for ICs was 0.7. CONCLUSIONS: There is substantial heterogeneity of PD-L1 expression between small tissue samples from the same TNBC tumor, especially for IC expression. This poses challenges for evaluation of PD-L1 expression in the neoadjuvant setting. Negative biopsies should prompt further investigation, and multiple biopsies might be necessary.

Multicentre study of the risk of invasive cancer and use of sentinel node biopsy in women with a preoperative diagnosis of ductal carcinoma in situ.

BACKGROUND: Ductal carcinoma in situ (DCIS) in the breast that is diagnosed by biopsy implies a risk of upstaging to invasive carcinoma (IC) on final pathology. These patients require a sentinel lymph node biopsy (SLNB) for axillary staging. A two-stage procedure is not always feasible and precise selection of patients who should be offered SLNB is crucial. The aims were: to determine the rate of upstaging, and use of redundant and required SLNB in women with a preoperative diagnosis of DCIS; and to identify patient and tumour characteristics that increase the risk of upstaging. METHODS: Patients with DCIS treated between 2008 and 2016 were identified using Orbit operation planning system software, and those suitable for the study were selected based on review of the medical records. Upstaging rates and proportions of redundant and required SLNBs were calculated. Associations between clinicopathological characteristics and upstaging were analysed using univariable and multivariable logistic regression analyses. RESULTS: Of 1368 patients initially identified, 975 women with a preoperative diagnosis of DCIS were included in the study. Tumours in 246 of these patients (25·2 per cent) were upstaged to IC. Redundant SLNB was performed in 392 of 975 women (40·2 per cent). Forty-four patients (4·5 per cent) with a final diagnosis of IC were not offered SLNB and thus potentially undertreated. In adjusted analysis, DCIS size, palpability and mass formation identified by breast imaging were associated with increased risk of upstaging. The Van Nuys classification was not associated with upstaging. CONCLUSION: Most patients with IC on final pathology underwent SLNB, but a considerable number of patients with DCIS had a redundant SLNB. Lesion size, palpability and mass formation, but not Van Nuys classification group, are suggested risk factors for upstaging.

ANTECEDENTES: El carcinoma ductal in situ (ductal carcinoma in situ, DCIS) de mama que se diagnostica mediante biopsia implica un riesgo de infraestadiaje de un carcinoma invasivo (invasive carcinoma, IC) en la anatomía patológica final. Estas pacientes requieren una biopsia del ganglio linfático centinela (sentinel lymph node biopsy, SLNB) para la estadificación axilar. Dado que un procedimiento en dos etapas no siempre es factible, la selección precisa de pacientes a las que se debe ofrecer SLNB es crucial. El objetivo del estudio era determinar la tasa de infraestadiaje inicial y el uso repetido/requerido de SLNB en mujeres con un diagnóstico preoperatorio de CDIS. Además, se identificarán las características del paciente y del tumor que aumentan el riesgo de necesidad de re-estadificación. MÉTODOS: Un total de 1.368 mujeres con DCIS tratadas entre 2008-2016 fueron identificadas utilizando el programa informático de la planificación de las intervenciones hospitalarias. Después de la revisión de los registros médicos, se incluyeron 975 pacientes en la cohorte del estudio. Se calcularon las tasas de infraestadiaje y la proporción del uso repetido/requerido de SLNB. Las asociaciones entre las características clinicopatológicas y la necesidad de re-estadificación se analizaron mediante análisis de regresión logística univariable y multivariable. RESULTADOS: De 975 pacientes diagnosticados inicialmente de DCIS, 246 (25,2%) fueron re-estadiados a IC. Se realizó SLNB repetidas en 392 (40,2%) de estos pacientes. En 44 pacientes (4,5%) con un diagnóstico final de IC no se les ofreció la SLNB y, por lo tanto, pudieron estar potencialmente infratratados. En el análisis ajustado, el tamaño del DCIS, la palpabilidad y la presencia de una masa en las imágenes radiológicas de la mama se asociaron con un mayor riesgo de necesidad de re-estadificación por infraestadiaje inicial. La clasificación de Van Nuys no se asoció con la re-estadificación. CONCLUSIÓN: La mayoría de pacientes con IC en la patología final se sometieron a SLNB, sin embargo, un número considerable de pacientes con DCIS se sometieron a SLNB repetidas. El tamaño de la lesión, la palpabilidad y la presencia de masa, aunque no el grupo de clasificación de Van Nuys, se consideran factores de riesgo relacionados con infraestadiaje inicial y necesidad de re-estadificación final.

A randomised study of tailored toxicity-based dosage of fluorouracil-epirubicin-cyclophosphamide chemotherapy for early breast cancer (SBG 2000-1).

STUDY AIM: Retrospective studies have demonstrated a worse outcome in breast cancer patients not developing leukopenia during adjuvant chemotherapy. The SBG 2000-1 is the first randomised trial designed to compare individually dosed chemotherapy without G-CSF support based on grade of toxicity to standard-dosed chemotherapy based on body surface area (BSA). METHODS: Patients with early breast cancer were included and received the first cycle of standard FEC (fluorouracil 600 mg/m2, epirubicin 60 mg/m2, cyclophosphamide 600 mg/m2). Patients with nadir leukopenia grade 0-2 after first cycle were randomised between either 6 additional courses of tailored FEC with increased doses (E 75-90 mg/m2, C 900-1200 mg/m2) or fixed treatment with 6 standard FEC. Patients with grade 3-4 leukopenia were registered and treated with 6 standard FEC. Primary end-point was distant disease-free survival (DDFS). RESULTS: The study enrolled 1535 patients, of which 1052 patients were randomised to tailored FEC (N = 524) or standard FEC (N = 528), whereas 401 patients with leukopenia grade 3-4 continued standard FEC and formed the registered cohort. Dose escalation did not statistically significantly improve 10-year DDFS (79% and 77%, HR 0.87, CI 0.67-1.14, P = 0.32) or OS (82% and 78%, respectively, HR 0.89, CI 0.57-1.16, P = 0.38). Corresponding estimates for the registered group of patients were DDFS 79% and OS 82%, respectively. CONCLUSIONS: The SBG 2000-1 study failed to show a statistically significant improvement of escalated and tailored-dosed chemotherapy compared with standard BSA-based chemotherapy in patients with low haematological toxicity, although all efficacy parameters showed a numerical advantage for tailored treatment.

Loss-of-function mutations in filaggrin gene and malignant melanoma: a case-control study.

BACKGROUND: Loss-of-function mutations in filaggrin gene (FLG) have been suggested to increase the susceptibility of skin malignancies due to reduced levels of epidermal filaggrin and its degradation products, urocanic acid, which may be protective against ultraviolet irradiation. OBJECTIVE: We aimed to investigate the association between FLG mutation status and the occurrence of malignant melanoma (MM) in Danish adults. METHODS: The prevalence of FLG mutations in a sample of MM biopsies was compared with a FLG-genotyped cohort from two general population studies. Pearson's chi-squared and Fisher's exact tests were used to compare the two groups. RESULTS: A total of 867 MM biopsies and 9965 general population controls were genotyped, respectively. In the MM sample, two (0.23%) individuals were homozygous and 80 (9.4%) were heterozygous mutation carriers. In the general population controls, the prevalence of FLG mutations was 18 (0.18%) and 835 (8.4%) for homozygous and heterozygous mutations, respectively. Fisher's exact test and Pearson's chi-squared test yielded non-significant P-values when the groups were compared. CONCLUSION: FLG mutation was not associated with MM in the studied populations. This finding indicates that epidermal deficiency of filaggrin and its degradation products does not influence the risk of MM significantly.

Filaggrin loss-of-function mutations, atopic dermatitis and risk of actinic keratosis: results from two cross-sectional studies.

BACKGROUND: Common loss-of-function mutations in filaggrin gene (FLG) represent a strong genetic risk factor for atopic dermatitis (AD). Homozygous mutation carriers typically display ichthyosis vulgaris (IV) and many have concomitant AD. Previously, homozygous, but not heterozygous, filaggrin gene mutations have been associated with squamous cell carcinomas. OBJECTIVE: The first objective was to examine the association between FLG mutations and actinic keratosis (AK). The second objective was to investigate the occurrence of AK in patients with IV and AD, respectively. METHODS: FLG mutation status in patients with AK was compared with controls from the general population. Furthermore, based on nationwide data from Danish registers, we compared the risk of AK in patients with IV, AD and psoriasis, respectively. RESULTS: The prevalence of homozygous FLG mutations was significantly higher in the AK group (n = 4, 0.8%) in comparison with the control group (n = 18, 0.2%), whereas the prevalence of heterozygous FLG mutations was lower. In hospital registry data, patients with AD exhibited an increased risk of AK than did psoriasis controls (adjusted OR 1.46; [95% CI 1.12-1.90]), whereas no difference in risk was observed between patients with IV and AD. CONCLUSIONS: This study indicates an increased susceptibility to AK in individuals with homozygous, but not heterozygous, FLG mutations and in patients with AD compared to psoriasis. Whether a reduction or absence of epidermal filaggrin could contribute to the susceptibility to AK in patients with IV and AD is unknown and additional research is needed to further explore this relationship.

Is 0.6T magnetic resonance mammography adequate in the detection of breast cancer?

PURPOSE: To evaluate whether relevant diagnostic information can be achieved when using magnetic resonance mammography (MRM) on mid-field as a supplement to conventional imaging and clinical examination in women with primary breast cancer. MATERIAL AND METHODS: 30 women (55 breasts containing 49 malignant tumors) planned for uni- or bilateral mastectomy were examined with dynamic MRM on mid-field, 0.6T. The women were examined with mammography (M) and ultrasonography (US) prior to MRM. The descriptions of the conventional examinations were evaluated retrospectively, whereas the MRM was evaluated prospectively, with knowledge of the M+US findings. Imaging findings suggesting malignancy were registered and correlated with pathology after mastectomy. A home-made rating system for evaluation of the detected lesions was tested. RESULTS: MRM detected seven additional malignant tumors, failed to detect three lesions and characterized four as gray-zone lesions according to the rating system. Sensitivity of finding the tumors with M+US was 79.0%, with a PPV for malignant tumors of 84.4%. One breast in which MRM found a malignant tumor had not initially been examined with US. Sensitivity with MRM was 91.6%, with a positive predictive value of malignant tumors of 97.7%. CONCLUSION: MRM on mid-field seems to improve the detection of cancers when used as a supplement to M+US in women with primary breast cancer. We believe that the results are fair compared to MRM on high-field, although further research and refinement are needed.

Leydig cell tumour--a rare testicular tumour.

Although Leydig cell tumour is a rare tumour which constitutes only 1-3% of all testicular tumours, still it is in the focus of interest because of the difficulties in determining its exact nature and subsequently the type of treatment and follow-up. We report a case of Leydig cell tumour with a review of the related literature.

Crohn's disease presenting as a breast abscess: a case report.

A case of cutaneous Crohn's disease (CD) involving the areola-nipple region of both breasts in a 45-year-old woman is reported. The lesion had initially been diagnosed and treated as a simple abscess. Histopathological examination, however, showed granulomatous inflammation with eosinophils suggesting extraintestinal CD. The patient also had a minor area of cutaneous CD localized at the umbilicus and severe anogenital lesions. Twenty-six years previously the woman had undergone a course of sulphasalazine treatment together with steroid enema for chronic inflammatory colitis, but the colitis had been inactive for 25 years. Treatment with sulphasalazine, metronidazole, prednisolone and azothioprine had only minor effect on the skin lesions. CD is a systemic disorder and the most prominent manifestations may be extraintestinal.

Vasculitis occurring during leflunomide therapy.

Leflunomide has recently been introduced for systemic treatment of rheumatoid arthritis. It has both immunosuppressive effects and anti-inflammatory properties. We report a patient treated with leflunomide who developed vasculitis as an adverse side effect. As far as we are aware, this is the first published report of vasculitis associated with leflunomide therapy.

US-guided fine needle aspiration versus coarse needle biopsy of thyroid nodules.

OBJECTIVE: The aim of this study was retrospectively to evaluate ultrasound (US) guided fine-needle aspiration (FNA), in combination with US-guided coarse-needle biopsies, (CNB) from solitary or dominant thyroid nodules routinely performed during a 2 year period. METHODS: Seventy seven patients were biopsied using US-guided FNA and CNB. FNA was performed using a 21-Gauge needle and CNB using a 18-Gauge single action spring-activated needle biopsy system. The biopsies were performed with local anaesthesia. The Department of Pathology routinely examined the biopsy specimens. The retrieval rate in obtaining material for diagnostic evaluation was FNA (97%), CNB (88%), FNA and CNB (100%). RESULTS: In all, 41 of the 77 patients underwent neck-surgery. The surgical specimens were used to determine the results of diagnosing neoplasia. The accuracy, sensitivity and specificity for FNA were 80, 83, and 77%. For CNB 86, 78, and 94%. For both FNA and CNB 80, 89 and 73%. The diagnostic value of the two methods showed no significant difference (P < 0.05). CNB revealed contrary to FNA, however, one additional cancer. Also a higher number of false positive findings was noticed using FNA. No serious complications were registered. Adequate biopsies were obtained in all the patients using the combination of US-guided FNA and CNB. No patient underwent rebiopsy. CONCLUSIONS: The study demonstrated that neither US-guided CNB nor the combination of US-guided FNA and CNB were superior to US-guided FNA. US-guided CNB is only recommended in few selected patients.

Treatment of perforating collagenosis of diabetes and renal failure with allopurinol.

We present a case of widespread reactive perforating collagenosis in a 63-year-old woman undergoing haemodialysis after diabetic nephropathy, who was treated successfully with allopurinol. The patient responded well and rapidly to a dose of 100 mg allopurinol daily. It is suggested that more patients with reactive perforating collagenosis may benefit from allopurinol therapy.

The terminal complement complex is generated in chronic leg ulcers in the absence of protectin (CD59).

Loss of membrane complement regulators accompanied by complement activation is suggested to be involved in the pathophysiological processes leading to tissue damage in myocardial ischaemia. In the present study we have investigated whether the same phenomenon may occur in ischaemic and/or venous hypertension leg ulcers. The deposition of complement, plasma complement regulators and expression of membrane regulators were detected by immunohistochemical methods, including immunofluorescence with antibodies against C3d, the terminal complement complex (TCC), vitronectin, clusterin, decay-accelerating factor (CD55) and protectin (CD59). Eleven frozen biopsies from ischaemic leg ulcers, 10 biopsies from venous hypertension leg ulcers, and 10 biopsies from normal skin were studied. In 9 of 11 ischaemic and in 5 of 10 venous hypertension leg ulcers, marked staining for TCC was found around the capillaries, most often at the ulcer margin. No TCC staining was found in normal skin. Staining for TCC was always accompanied by staining for clusterin and vitronectin and C3d. In normal skin, CD59 was found on the elastic fibers in the dermis, on the muscle coat, the Schwann sheath and acinar cells. Semiquantitative measurement of CD59 showed marked increased staining intensity in the endothelium in venous hypertension ulcers and diminished intensity in ischaemic ulcers compared to normal skin. No such difference could be observed for CD55. When TCC was positive in the capillary walls, weak or no staining for CD59 was found. A significantly higher ratio of TCC/CD59 was found in the ischaemic compared to venous ulcers (p = 0.018). This was due to a marked difference between the ulcer margins (p = 0.013). Localized areas in the venous ulcers had the same pattern as that seen in the ischaemic ulcers. Our results suggest that loss of CD59 may enhance deposition of TCC and that complement-dependent inflammation may be an important factor in the tissue-damaging processes seen in chronic leg ulcers.

Estrogen receptors in the human male prostatic urethra and prostate in prostatic cancer and benign prostatic hyperplasia.

Estrogen receptors (ERs) in the prostate and prostatic urethra were examined in 33 men with benign prostatic hyperplasia (BPH) and in 11 with prostate cancer (PC). The Abbot monoclonal ER-ICA assay was used for immunohistochemical investigation. In the BPH group, ERs were revealed in the prostatic stroma in eight cases and in the glandular epithelium in one. In four cases ERs were seen in the prostatic stroma and in the glandular epithelium. In the prostatic urethra, ERs were found in 19 cases located in the urothelium, lamina propria and/or periurethral glands. In the PC group, ERs were demonstrated in the prostatic stroma and/or prostatic urethra in 6 out of 11 cases. In both BPH and PC patients, immunoreactivity was weak and confined to few cells, indicating low ER content in the prostate as well as in the prostatic urethra. Dextran-coated charcoal (DCC) analysis was used for detection and quanticization of cytosolic and nuclear ERs. In the BPH group, ERs were detected once in the prostate and prostatic urethra in the nuclear and cytosol, and additionally in the prostatic urethra in the cytosol fraction in three cases. In all cases, ER content was low, ranging from 10-15 fmol/mg protein. In the PC group, ERs were detected in the prostatic urethra and/or prostate in the cytosol fraction from two patients. The contents were low, ranging from 10-13 fmol/mg protein. We conclude that in human BPH and PC, ERs can be present in the prostate and prostatic urethra. In the prostate, ERs are mainly located in the stroma, but in BPH specimens they can also be found in the glandular epithelium. Biochemically, the use of the DCC analysis is of limited value, since ER content in the human prostate and prostatic urethra is at the limit of detection with this method.

Ulcer bed infection. Report of a case of enlarging venous leg ulcer colonized by Pseudomonas aeruginosa.

We report a case of ulcer bed infection in an enlarging venous leg ulcer without clinical signs of cellulitis in the surrounding tissues. Signs of infection in the leg ulcer were: 1) cocci-like structures and bacteria-like rods around vessel walls in the viable ulcer bed, 2) vasculitis-like inflammation of deeply situated vessels of the viable tissue, 3) Pseudomonas aeruginosa-specific antibodies in the serum (other than against exotoxin A), 4) extensive epidermolysis of normal human skin by the wound exudate in vitro, and 5) P. aeruginosa exotoxin A in the wound exudate (23 ng/ml). In an in vitro cell assay, the wound exudate was cytotoxic and rabbit antibodies to exotoxin A, but not a serine proteinase inhibitor, inhibited this cytotoxicity. P. aeruginosa exotoxin A might contribute to the pathogenesis of the ulcer enlargement. The ulcer improved after the third skin graft, probably mainly due to effective treatment with a long-stretch compression bandage.

The expression of receptors for estrogen and epithelial growth factor in the male rabbit prostate and prostatic urethra following castration.

In the lower urinary tract of the male rabbit, estrogen receptors (ERs) are restricted to the urethra and the prostatic stroma. At present, the function of ERs in these tissues is not known. Epithelial growth factor (EGF) stimulates proliferation of epidermal and epithelial tissues, and several animal studies have indicated that EGF is regulated by estrogen. On this background, we have studied the effect of castration on the expression of ERs and EGF receptors in the rabbit prostatic urethra and prostate. Twelve male rabbits were studied fourteen days after castration, and eight normal rabbits were included as controls. In the control group, ERs were found in the urothelial lining and lamina propria of the prostatic urethra, and in the prostatic stroma. EGF receptors were demonstrated in the epithelial lining of the prostatic urethra and the glandular epithelium of the prostate. Following castration, the expression of ERs, assessed as the increase in the number of positively stained specimens, increased significantly in the lamina propria of the prostatic urethra and the prostatic stroma. EGF receptor expression increased significantly in the epithelial lining of the prostatic urethra. In the prostate, the increase was not significant. The results give no support to the view that ERs play role in the regulation of EGF receptors in the rabbit prostatic urethra nor the prostate.

Estrogen receptors in the human male bladder, prostatic urethra, and prostate. An immunohistochemical and biochemical study.

The distribution and quantity of estrogen receptors (ERs) in the human male bladder, prostatic urethra and the prostate were studied in eight males with recurrent papillomas of the bladder or monosymptomatic hematuria (median age 61 years), 14 men undergoing transurethral resection due to benign prostatic hyperplasia (median age 70 years), and nine men undergoing cystectomy due to malignant tumour of the bladder (median age 70 years). In the first group of patients, biopsies for immunohistochemical examination were obtained from the bladder vault, bottom, both side-walls, the trigone area, and the mid-portion of the prostatic urethra, and in the second group from three locations of the prostatic urethra (bladder neck, mid-portion and veramontanum). In the third group, tissue specimens were taken from the vault of the bladder, prostatic urethra, and the prostate, for immunohistochemical as well as biochemical analysis. In the first group, ERs were found in three out of eight specimens of the prostatic urethra, and in one of these, ERs were confined to periurethral glands. ERs could not be demonstrated in any of the bladder-biopsies. In the second group, ERs were not found in the bladder neck, but were seen in four preparations from the veramontanum and in two from the midportion of the urethra. ERs were located in the urothelium and periurethral glands. In the third group, ERs were seen immunohistochemically in the prostatic urethra (two cases) and the prostatic stromal tissue (two cases). ERs could be demonstrated in the bladder neither by immunohistochemistry nor biochemically.(ABSTRACT TRUNCATED AT 250 WORDS)

Non-hazardous organic solvents in the paraffin-embedding technique: a rational approach. Aliphatic monoesters for clearing and dewaxing: butyldecanoate.

The aim of this study was to substitute hazardous compounds, used in tissue processing and dewaxing, with compounds having lowest possible toxicity and inflammability without impairing the morphology, staining characteristics, or diagnostic value of the tissue sections. All aromatic compounds and aliphatic hydrocarbons (e.g. alkanes, isoparaffins, petroleum distillates, etc.) were rejected, primarily due to their high vapour pressure. Based on a theoretical study of compounds used for clearing, a number of non-hazardous potential substitutes were chosen. The following experimental study narrowed the group to three unbranched, saturated, aliphatic monoesters containing 12-14 carbon atoms. On large-scale testing of these compounds, we found butyldecanoate to be the closest to an ideal substitute for aromatic and aliphatic hydrocarbons in the histology department: the section quality is at least equal to that obtained with xylene. For dewaxing, it is used at 30-35 degrees C. Butyldecanoate is not suitable as a pre-mounting agent. In practice, this is no problem as modern mounting agents permit mounting of coverslips directly from ethanol without impairing the appearance of the section in the microscope. Butyldecanoate has only a slight odour, insignificant vapour pressure (< 0.01 kPa at 20 degrees C), and does not present a fire hazard (flash point 134 degrees C). The introduction of this compound in the laboratory poses no health hazard, and the substance is biodegradable.